Many aspects of the chemistry of carbohydrates are not specific to this class of compounds but are merely examples of the simple chemical reactions we have already met.

Therefore, against the usual practice, we have not attempted a full treatment of carbohydrate chemistry and biochemistry in this chapter. We want to avoid giving the impression that the reactions described here are something special to this group of compounds.

• Instead, we have deliberately used carbohydrates as examples of reactions in earlier chapters, and you will find suitable cross-references.
• Carbohydrates are among the most abundant constituents of plants, animals, and microorganisms. Polymeric carbohydrates function as important food reserves and as structural components in cell walls.
• Animals and most microorganisms are dependent upon the carbohydrates produced by plants for their very existence.
• Carbohydrates are the first products formed in photosynthesis and are the products from which plants synthesize their food reserves, as well as other chemical constituents.
• These materials then become the foodstuffs of other organisms. The main pathways of carbohydrate biosynthesis and degradation comprise an important component of intermediary metabolism that is essential for all organisms.

The name carbohydrate was introduced because many of the compounds had the general formula C_x(H₂O) y, and thus appeared to be hydrates of carbon. The terminology is now commonly used in a much broader sense to denote polyhydroxy aldehydes and ketones, and their derivatives.

Sugars or saccharides are other terms used in a rather broad sense to cover carbohydrate materials. Though these words link directly to compounds with sweetening properties, the application of the terms extends considerably beyond this. A monosaccharide is a carbohydrate usually in the range C₃ –C₉ whereas an oligosaccharide covers small polymers comprised of 2–10 monosaccharide units. The term polysaccharide is used for larger polymers.

Monosaccharides

Six-carbon sugars (hexoses) and five-carbon sugars (pentoses) are the most frequently encountered monosaccharide carbohydrate units in nature. Primary examples of these two classes are the hexoses glucose and fructose, and the pentose ribose. Note the suffix -ose as a general indicator of carbohydrate nature.

The structures above show some of the fundamental features of carbohydrates. Initially, we have drawn these compounds in the form of Fischer projections, a depiction developed for these compounds to indicate conveniently the stereochemistry at each chiral centre.

• The Fischer projection is drawn as a vertical carbon chain with the group of highest oxidation state, i.e. the carbonyl group, closest to the top, and numbering takes place from the topmost carbon.
• The carbonyl group in glucose and ribose is an aldehyde; such compounds are termed aldoses.
• Fructose, by contrast, has a ketone group and is therefore classified as a ketose. Glucose could also be termed an aldohexose and fructose a ketohexose, whereas ribose would be an aldopentose, names which indicate both the number of carbons and the nature of the carbonyl group.
• Another aspect of nomenclature is the use of the suffix -ulose to indicate a ketose. Fructose could thus be referred to as a hexulose, though we are more likely to see this suffix in the names of specific sugars,

Example:

Ribulose is a ketose isomer of the aldose ribose.

Each of these compounds has a prefix D- with the name. As we saw, this indicates that the configuration at the highest numbered chiral centre is the same as that in D-( R)-(+)-glyceraldehyde; the alternative stereochemistry would be related to L-( S)-(−)-glyceraldehyde and consequently be part of an L-sugar

Structures of the various D-aldoses in the range C₃ –C₆ are shown below. These compounds are multifunctional structures, having a carbonyl group and several hydroxyls, usually with two or more chiral centres.

You will notice that we are comparing the stereochemistry in the different possible diastereoisomers for compounds containing several chiral centres. There is a corresponding series of enantiomeric L-sugars; only a few of these are shown.

Synthesis of ¹⁴C -labelled glucose

A sequence known as the Kiliani–Fischer synthesis was developed primarily for extending an aldose chain by one carbon and was one way in which configurational relationships between different sugars could be established.

A major application of this sequence nowadays is to employ it for the synthesis of ¹⁴C-labelled sugars, which in turn may be used to explore the role of sugars in metabolic reactions. The synthesis of ¹⁴C-labelled D-glucose starts with the pentose D-arabinose and ¹⁴C-labelled potassium cyanide, which react together to form a cyanohydrin.

Since cyanide can attack the planar carbonyl group from either side, the cyanohydrin product will be a mixture of two diastereoisomers that are epimeric at the new chiral centre. The two epimers are usually formed in unequal amounts because of a chiral influence from the rest of the arabinose structure during the attack of the nucleophile.

The nitrile groups in the product mixture are then hydrolysed to carboxylic acids ( Upon heating, the acids readily form cyclic esters (lactones) through the reaction of the hydroxyl group on C-4 with the carboxylic acid, the five-membered ring being most favoured (see

The pair of lactones is then reduced using sodium amalgam under acidic conditions to yield aldehydes, though it has been found that this reaction can also be achieved using aqueous sodium borohydride.

Sodium borohydride reacts readily with lactones, though it is not usually effective in reducing esters. It is also normally difficult to stop at an aldehyde intermediate (see Section 7.11), but the reduction of a lactone gives initially a hemiacetal; ring opening of the hemiacetal then leads to the aldehyde.

The product will be a mixture of the two epimeric sugars D-glucose and D-mannose, which will be labelled with 14C in the aldehyde function. Separation of the diastereoisomeric products may be achieved via fractional crystallization or by chromatography and may be carried out at either the cyanohydrin stage or the final product stage.

Note how the process may be modified to extend its versatility. Thus, using 14C-labelled potassium cyanide with D-erythrose yields a mixture of [1-¹⁴C]-D-ribose and [1-¹⁴C]-D-arabinose. The sequence could then be repeated on the latter product, using unlabelled KCN, to give [2-¹⁴C]-D-glucose.

Enolization and isomerization

In common with other aldehydes or ketones that have hydrogen on the α-carbon, enolization is possible, especially when sugars are treated with base. The additional presence of a hydroxyl on the α-carbon causes further isomerization. Thus, treatment of D-glucose with dilute aqueous sodium hydroxide at room temperature leads to an equilibrium mixture also containing D-mannose and D-fructose.

Removal of the α-hydrogen in D-glucose leads to enolization (we have omitted the enolate anion in the mechanism). Reversal of this process allows epimerization at C-2, since the enol function is planar, and a proton can be acquired from either face, giving D-mannose as well as D-glucose.

Alternatively, we can get isomerization to D-fructose. This is because the intermediate enol is an enediol; restoration of the carbonyl function can, therefore, provide either a C-1 carbonyl or a C-2 carbonyl. The equilibrium mixture using dilute aqueous sodium hydroxide at room temperature consists mainly of D-glucose and D-fructose, with smaller amounts of D-mannose. The same mixture would be obtained if either D-mannose or D-fructose were treated similarly.

Note that harsher conditions may lead to further changes, e.g. epimerization at C-3 in fructose, plus isomerization, or even reverse aldol reactions. In general, basic conditions must be employed with care if isomerizations are to be avoided. To preserve stereochemistry, it is usual to ensure that free carbonyl groups are converted to acetals or ketals (glycosides, see Section 12.4) before basic reagents are used. Isomerization of sugars via enediol intermediates features prominently in the glycolytic pathway of intermediary metabolism.

Cyclic hemiacetals and hemiketals

Monosaccharide structures may be depicted in openchain forms showing their carbonyl character, or in cyclic hemiacetal or hemiketal forms. Alongside the Fischer projections of glucose, ribose, and fructose shown earlier, we included an alternative representation of the compound in its cyclic form. The compounds exist predominantly in cyclic forms, which result from the nucleophilic attack of an appropriate hydroxyl onto the carbonyl.

Both six-membered pyranose and five-membered furanose structures are encountered, a particular ring size usually being characteristic of any one sugar. Thus, although glucose has the potential to form both six-membered and five-membered rings, an aqueous solution consists almost completely of the six-membered hemiacetal form; five-membered rings are usually formed more rapidly, but six-membered rings are generally more stable and predominate at equilibrium. The names pyranose and furanose are derived from the oxygen heterocycles pyran and furan. Shown below is a reminder of how we can transform a Fischer projection of sugar into a cyclic form

The pentose ribose is also able to form six-membered pyranose and five-membered furanose rings. In solution, ribose exists mainly (76%) in the pyranose form; interestingly, however, when we meet ribose in combination with other entities, e.g. nucleosides, it is almost always found in furanose form.

Fructose is a ketose and, therefore, forms hemiketal ring structures. Like ribose, it is usually found in combination as a five-membered furanose ring, though the simple sugar in solution exists primarily in the pyranose form.

The anomeric centre

Since the carbonyl group is planar and may be attacked from either side, two epimeric structures (anomers) are possible in each case, and in solution, the two forms are frequently in equilibrium because hemiacetal or hemiketal formation is reversible. The two anomers are designated α or β by comparison of the chiralities at the anomeric centre and the highest-numbered chiral centre. If these are the same (RS convention), the anomer is termed β, or α if they are different.

In practice, this translates to the anomeric hydroxyl being ‘up’ in the case of β-D-sugars and α-L-sugars. It is interesting to note that the descriptors α or β were originally assigned to the two forms of glucose based on the order in which they crystallized out from the solution. Without changing the nomenclature for these two compounds, α or β are now assigned on a much more rigid stereochemical basis.

By convention, the ring form of sugars is drawn with the ring oxygen to the rear and the anomeric carbon furthest right. Wedges and the bold bond help to emphasize how we are looking at the chair-like pyranose ring. However, to speed up the drawing of structures we tend to omit these, and then the lower bonds always represent the nearest part of the ring.

Since there are two anomeric forms, and these are often in equilibrium via the acyclic carbonyl compound, we can use a new type of bond to indicate that the configuration is not specified and could be of either stereochemistry. This is the wavy or wiggly bond, and to display our indecision further we usually site it halfway between the two possible positions.

It follows that, when we dissolve a sugar such as glucose or ribose in water, we create a mixture of various equilibrating structures. The relative proportions of pyranose and furanose forms, and their respective anomers for the eight aldohexoses, are shown in  In each case, the proportion of non-cyclic form is very small.

The most stable conformation of the cyclic sugar is mainly determined by a minimization of steric interactions, i.e. the maximum number of equatorial substituents (see Section 3.3.2). It follows that the preferred conformation for β-D-glucose will be that with all substituents equatorial; the alternative has all substituents axial. Carbohydrate chemists have introduced a neat way of referring to the two.

Equilibrium proportions of pyranose and furanose forms ofaldohexoses in water:

Ring size and a numeric form of common sugars

Sugars exist predominantly in cyclic hemiacetal or hemiketal forms, and whilst both six-membered pyranose and five-membered furanose structures are encountered, a particular ring size is usually characteristic for any one sugar, especially when it is found in combination with other entities in natural structures. The most commonly encountered monosaccharides and their usual anomers are shown here. By convention, the ring form is drawn with the ring oxygen to the rear and the anomeric carbon furthest right. Also shown are the accepted abbreviations for these sugars.

The two anomers are designated α or β by comparison of the chiralities at the anomeric centre and the highest-numbered chiral centre. If these are the same (RS convention), the anomer is termed β, or α if they are different.

Note that the D and L prefixes are assigned based on the chirality (as depicted in Fischer projections) at the highest numbered chiral centre and its relationship to D-( R)-(+)-glyceraldehyde or L-( S)-(−)-glyceraldehyde The stereochemistries of the various substituents may be deduced by considering the implications of the Fischer projection conformers, in that the left-hand conformer of glucose is termed ⁴C₁, and the right-hand one ¹C₄.

The ‘C’ indicates chair conformation, the superscript numeral is the carbon atom that is above the plane of the ring, and the subscript numeral is which carbon atom is below the plane of the ring. For this description, we consider the pyranose ring as originally planar, distorted to a chair by pushing carbons 1 and 4 out of the plane.

At first glance, the preferred conformation for L-hexoses,

Example:  α-L-rhamnose, appears different from that of the D-hexoses.

This is readily rationalized by considering the preferred conformation of α-L-glucose – the α-anomer is chosen simply because we can easily follow the anomeric substituent. Since α-L-glucose is the enantiomer of α-D-glucose, we can draw the mirror image representation, and then rotate this so that the heterocyclic oxygen comes to the required position.

Note that you may also encounter another version of the cyclic form referred to as the Haworth representation.

• This shows the ring as a planar system and is commonly used in biochemistry books. However, we know that five-membered and six-membered rings are certainly not planar.
• The Haworth representation nicely reflects the up–down relationships of the various substituent groups, but is quite uninformative about the shape of the molecule, and whether the substituents are equatorial or axial.

In really bad cases, authors omit the hydrogen atoms, giving an ambiguous structure – do lines mean methyl or hydrogen? Haworth representations may be easier to draw, but you are strongly encouraged to use the more informative conformational structures.

One of the consequences of forming a cyclic hemiacetal or hemiketal is that the nucleophilic hydroxyl adds to the carbonyl group and forms a new hydroxyl. This new group is susceptible to many normal chemical reactions of hydroxyls,

Example:

Esterification and this type of reaction effectively freezes the carbohydrate into one anomeric form, since the ring-opening and equilibration can now no longer take place.

Consider esterification of glucose with acetic anhydride β-D-Glucose will be acetylated to give the β-acetate, whereas α-D-glucose will specifically give the α-acetate. These two forms do not equilibrate merely by dissolving in a solvent, although they can be interconverted by some other means,

Example: Nucleophilic substitution reactions with acetate.

If we wish to consider esterification of the α– β mixture, we could use the unspecified wavy bond representation shown on the right.

Alditols

Reduction of the aldehyde or ketone group in sugar is readily achieved using a variety of reducing agents. Reduction occurs on the small amount of open-chain form present at equilibrium. As the openchain form is removed, the equilibrium is disturbed until total reduction is achieved. The products are polyhydroxy compounds termed alditols.

Reduction of aldoses is the more satisfactory reaction, in that a single product is formed. On the other hand, the reduction of ketoses generates a new chiral centre, and two epimeric alditols will result. Thus, treatment of D-glucose with sodium borohydride gives D-glucitol, also known as D-sorbitol.

It should be noted that LAH is not a satisfactory reducing agent for this reaction because of the several hydroxyl groups present

On the other hand, borohydride reduction of the ketose D-fructose will give a mixture of D-glucitol and its epimer, D-mannitol. A better approach to D-mannitol would be a reduction of the aldose D-mannose. D-glucitol (sorbitol) is found naturally in the ripe berries of the mountain ash (Sorbus aucuparia) but is prepared semi-synthetically from glucose.

It is half as sweet as sucrose, is not absorbed orally, and is not readily metabolized in the body. It finds particular use as a sweetener for diabetic products. D-Mannitol also occurs naturally in manna, the exudate of the manna ash Fraxinus ornus. This material has similar characteristics to sorbitol but is used principally as a diuretic. It is injected intravenously, is eliminated rapidly into the urine, and removes fluid by an osmotic effect.

Glycosides

The cyclic hemiacetal and hemiketal forms of monosaccharides are capable of reacting with an alcohol to form acetals and ketals. The acetal or ketal product is termed a glycoside, and the non-carbohydrate portion is referred to as an aglycone. In the nomenclature of glycosides we replace the suffix -ose in the sugar with -oside.

Simple glycosides may be synthesized by treating an alcoholic solution of the monosaccharide with an acidic catalyst, but the reaction mixture usually then contains a mixture of products.

This is an accepted problem with many carbohydrate reactions; it is often difficult to carry out selective transformations because of their multifunctional nature.

The reaction of glucose with methanol and gaseous HCl yields four acetal products, the α- and β-pyranosides and α- and β-furanosides, which may be separated. The pyranosides are the predominant components, and the major product is the α-pyranoside.

This is perhaps unexpected, in that the β-pyranoside has all its substituents equatorial, whereas the α-anomer has its anomeric substituent axial. This so-called anomeric effect arises from a favourable electronic stabilization in the axial anomer that is not possible in the equatorial anomer. It involves overlap from the ring oxygen lone pair, and to achieve this the lone pair and the substituent must be antiperiplanar.

The anomeric effect is rather complex and will not be considered in any detail. It occurs when we have a heterocyclic ring (O, N, or S), with an electronegative substituent (halogen, OH, OR, OCOR, etc.) adjacent to the heteroatom, and favours the isomer in which the substituent is axial.

Thus, with the first of the simple acetals shown below, where we need to consider only conformational isomerism, some 75% of the axial conformer is present at equilibrium. Without the ring oxygen, we would see an equatorial isomer predominating. In the second example, the additional stability conferred by the equatorial methyl group increases even further the proportion of the conformer with the axial methoxyl.

We have noted that an aqueous solution of glucose exists as an equilibrium mixture containing some 64% of the β-anomer

Based simply on steric effects, this proportion appears somewhat low, whereas because of the anomeric effect just described the proportion now seems rather high. Anomeric effects are observed to be solvent-dependent, and hydroxy compounds experience considerable solvation with water through hydrogen bonding.

This significantly increases the steric size of the substituent and reinforces the steric effects By considering the reversibility of the acetalforming reactions, treatment of either of the two methyl pyranosides with acidic methanol will produce the same equilibrium mixture. A related equilibration occurs with the anomers of glucose, as seen earlier.

It should also be noted that hydrolysis of glycosides (acetals or ketals) will occur under acid-catalysed conditions if we have an excess of water present. This is a reversal of the process for glycoside formation, with the equilibrium favouring the aglycone plus sugar rather than the glycoside. The sugar product will again be the equilibrium mixture of anomers.

Acid-catalysed hydrolysis of glycosides

Hydrolysis of glycosides can also be achieved by the use of specific enzymes, e.g. β-glucosidase for β-glucosides and β-galactosidase for β-galactosides. These enzymes mimic the acid-catalysed processes, are commercially available, and may be used just like a chemical reagent.

Some examples of natural O-,S-,C- ,and N – glycosides

Many different types of glycoside structures are found in nature, especially in plants. Since the presence of a sugar unit in the structure provides polarity, glycosylation is likely a means by which an organism makes an aglycone water-soluble and transportable. Most of the natural glycosides are compounds in which the aglycone is an alcohol or a phenol, and such derivatives are termed O-glycosides. O-glycosides are thus acetals or ketals.

Less commonly, one encounters compounds in which a thiol (RSH) has been bonded to the sugar unit resulting in a thioacetal. These compounds are termed S-glycosides. Some examples of O- and S-glycosides are shown below.

Salicin is an O-glycoside of a phenol, namely salicyl alcohol. Salicin is a natural antipyretic and analgesic found in willow bark and is the template from which aspirin (acetylsalicylic acid, was developed.

Prunasin from cherry laurel is an example of a cyanogenic glycoside, the hydrolysis of which leads to the release of toxic HCN ). It is the O-glucoside of the alcohol mandelonitrile, the trivial name for the cyanohydrin of benzaldehyde. It is the further hydrolysis of mandelonitrile that liberates HCN.

S-glycosides in nature are quite rare, but there is an important group called glucosinolates. These compounds are responsible for the pungent properties of mustard, horseradish and members of the cabbage family. One example is sinigrin, found in black mustard seeds. When seeds are crushed, enzymic hydrolysis liberates the aglycone, which subsequently rearranges to the pungent principle allylisothiocyanate.

A related glucosinolate glucoraphanin is found in broccoli and is associated with the beneficial medicinal properties of this vegetable. This is hydrolysed to the isothiocyanate sulforaphane, which is believed to induce carcinogen-detoxifying enzyme systems.

Other natural glycosides are not acetals or ketals, but analogues in which the nucleophilic species has been an amine ( N-glycosides), or even some carbanionic species so that the sugar becomes attached to carbon ( C-glycosides). It should be noted that the presence of a C–C bond between the sugar and the aglycone means that C-glycosides are not cleaved by simple hydrolysis, but require an oxidative process.

C-glycosides are typified by barbaloin, a component of the natural purgative drug cascara, but, as a group, the N-glycosides are perhaps the most important to biochemistry. N-glycosidic linkages are found in the nucleosides, components of DNA and RNA. In addition, nucleosides are essential parts of the structures of crucial biochemicals such as ATP, coenzyme A, NAD⁺, etc. The amine in these types of compounds is part of a purine or pyrimidine base.

Perhaps the most significant group of glycoside derivatives are polysaccharides. In these structures, the aglyconeis itself another sugar, so that the polymer chain is composed of a series of sugar units joined by acetal or ketal linkages. Short carbohydrate polymers may also be found in some of the more complex O-glycosides,

Example: The heart drug digoxin from Digitalis lanata

Making a methyl glucopyranoside is relatively straightforward in that we can use the alcohol methanol as solvent, and, since it is thus present in large excess, this helps to disturb the equilibrium. The process is much less attractive for a more complex alcohol that is probably not available in excess and is unlikely to function as a suitable solvent.

Trying to join together two or more sugars would also be fraught with problems since each sugar contains several hydroxyl groups capable of acting as the nucleophile. These problems have been overcome by exploiting nucleophilic substitution for glycoside synthesis rather than the hemiacetal to acetal conversion we have been looking at, combined with the use of protecting groups to avoid unwanted couplings. A valuable reagent for adding a glucose unit onto a suitable nucleophile is acetobromoglucose.

Glucose is first esterified to penta- O-acetylglucose using acetic anhydride. Note that the hemiacetal hydroxyl is also esterified, and thus any equilibration with an aldehyde form is now not possible. When this penta-acetate is treated with HBr, the anomeric acetate is preferentially lost under the acidic conditions, due to the stabilization conferred by the heterocyclic oxygen.

Note that this is the same type of intermediate we implicated in the conversion of hemiacetals into acetals. Acetobromoglucose then results from the nucleophilic attack of bromide onto the cationic system; in acetal formation, the nucleophile would be an alcohol. The anomeric effect is considerably larger when the substituent is halide than it is with alkoxy groups, so the product formed is almost exclusively the α-anomer.

In the conversion of hemiacetals into acetals. Acetobromoglucose then results from the nucleophilic attack of bromide onto the cationic system; in acetal formation, the nucleophile would be an alcohol. The anomeric effect is considerably larger when the substituent is halide than it is with alkoxy groups, so the product formed is almost exclusively the α-anomer. The product is consequently the esterified β-glucoside derivative.

Further base treatment then hydrolyses the ester functions, liberating the glucoside salicin. As we shall, this type of substitution process is similar to the way glucosides (and polysaccharides) are produced in nature, though the enzymic reactions do not require any ester-protecting groups for the sugars.

Biosynthesis of glycosides via U DPsugars The widespread occurrence of glycosides and polysaccharides in nature demonstrates there are processes for attaching sugar units to a suitable atom of an aglycone to give a glycoside, or to another sugar to give a polysaccharide. Linkages tend to be through oxygen, although they are not restricted to oxygen, since S-, N-, and C-glycosides are also well-known. The agent for glycosylation is a uridine diphosphosugar,

Example: UDP-glucose.

Of course, the uridine portion is itself a glycoside, an N-riboside of the pyrimidine base uracil. The glucosylation process can be envisaged as a simple S_N2 nucleophilic displacement reaction, with an alcohol or phenol nucleophile, and a phosphate derivative as the leaving group. This S_N2 displacement is analogous to that seen in the chemical synthesis of glycosides using acetobromoglucose

S_N2 processes occur with inversion of configuration so since UDPglucose has its leaving group in the α-configuration, the product formed by the S_N2 process has the β-configuration. This is the configuration most commonly found in natural O-glucosides. Some natural products do possess an α-linkage, however. It appears that such compounds originate via a double S_N2 process, in which a nucleophilic group on the enzyme reacts first with the UDPglucose and then the hydroxy nucleophile displaces the enzymic group.

Other UDPsugars,

 Example: UDP-galactose or UDPxylose, are utilized in the synthesis of glycosides containing different sugar units.

The S-, N-, and C-glycosides are formed by a similar process with the appropriate nucleophile. This type of reaction is also used in the biosynthesis of polysaccharides ), and in the metabolism of drugs and other foreign compounds via glucuronides.

Cyclic Acetals And Ketals Protecting Groups

We have just seen that intramolecular reactions between the carbonyl group and one or other of the hydroxyl functions readily lead to the formation of cyclic hemiacetal or hemiketal forms. Further, these products may then be converted into acetals or ketals by an intermolecular reaction with another alcohol molecule, giving us glycosides.

We could also form an acetal or ketal by supplying a carbonyl compound and exploiting the hydroxyl groups of the sugar. This provides a particularly useful means of protecting some of the hydroxyl groups whilst other reactions are carried out; the protecting group is then easily removed by effectively reversing the acetal/ketal reaction using hydrolytic conditions.

In principle, several different types of acetal or ketal might be produced. In this section, we want to exemplify a small number of useful reactions in which two of the hydroxyl groups on the sugar are bound up by forming a cyclic acetal or ketal with a suitable aldehyde or ketone reagent.

Aldehydes or ketones react with 1,2- or 1,3-diols under acidic conditions to form cyclic acetals or ketals. If the diol is itself cyclic, then the two hydroxyl groups need to be cis-oriented to allow the thermodynamically favourable fused-ring system to form.

Thus, cis-cyclohexanes-1,2- diol reacts with acetone to form a cyclic ketal, a 1,2- O-isopropylidene derivative usually termed, for convenience, an acetonide

When required, the original diol may be regenerated by acid hydrolysis. Sugars are polyhydroxy compounds, and it is not always easy to predict which of the hydroxyls will react in this way. There are other complicating factors too.

The ring size (pyranose/furanose) of the product may differ from that of the starting sugar. It may be that a more stable pyranose form does not have disoriented hydroxyl groups, whereas a less favoured furanose form does so that the latter can form cyclic acetals/ketals. The equilibration of pyranose/furanose forms allows this type of change to occur.

Thus, D-galactose reacts with acetone to give a diketal: the less-favoured α-form has two pairs of cis-oriented hydroxyls that can react. It thus yields a diacetonide. Only the primary alcohol group is left unprotected and is available for further modification if desired.

D-glucose provides a rather more complicated picture, unfortunately. Whilst the pyranose α-anomer could yield a mono-acetonide, there is no other pair of cis-hydroxyls that can react. However, it turns out that the furanose form has two sets of hydroxyls that can react; the product obtained is an acetonide of α-D-glucofuranose.

Note that a six-membered ketal ring involving the hydroxyls at 4 and 6 is not favoured; this is because such a ring would necessarily force one of the two methyls into an axial position. On the other hand, these two hydroxyls can be employed in forming a cyclic acetal with benzaldehyde. Benzaldehyde shows a tendency to form six-membered ring acetals, and because the two substituents are phenyl and hydrogen, we can have a favourable chair system with the phenyl equatorial.

It is not the intention to explain all such variations and add to potential confusion. The behaviour of most sugars for cyclic acetal and ketal formation is well documented for those wishing to work with these compounds. The objective here is merely to illustrate the potential for selective protection of the hydroxyl groups.

Oligosaccharides

The term oligosaccharide is frequently used to classify a small polysaccharide comprised of some two to five monomer units, a name derived from the Greek oligos, meaning a few. A pre-eminent example is the disaccharide sucrose, which we commonly call ‘sugar’ and is utilised widely as a sweetening agent and as the raw material for sweets and other confectionary. Other important disaccharides are maltose, a hydrolysis product from starch, and lactose, the main sugar component of cow’s milk.

If we inspect these structures, we can see that they are acetals or ketals equivalent to the glycosides described above, though the alcohol portion is one of the hydroxyl groups of a second monosaccharide structure.

The linkages are conveniently defined by a shorthand system of nomenclature; this indicates the carbons that are joined by the acetal/ketal bond through the use of numbers and an arrow, together with the configuration α or β at the anomeric carbon. Note that each monosaccharide is numbered separately and there is no unique numbering system for the combined structure.

Thus, maltose becomes D-Glc( α1 → 4)D-Glc, which conveys the information that two molecules of D-glucose are bonded between carbon-1 of one molecule and carbon-4 of the second and that the configuration at the anomeric centre (C-1 of the first glucose residue) is α.

Similarly, lactose, a combination of D-galactose and D-glucose, is D-Gal( β1 → 4)D-Glc, the configuration at the anomeric centre of galactose being β. Note that the configuration at the hemiacetal anomeric centre in the second sugar (glucose) is not indicated; it could be α or β, as with a monosaccharide Longhand systematic nomenclature that treats one sugar as a substituent on the other can also be used.

In the systematic names, the ring size (pyranose or furanose) is also indicated. Thus maltose is 4- O-( α-D-glucopyranosyl)-D-glucopyranose, and lactose becomes 4- O-( β-D-galactopyranosyl)-Dglucopyranose.

Lactulose

Lactulose is a semi-synthetic disaccharide prepared from lactose and is composed of galactose linked β1 → 4 to fructose. Galactose is an aldose and exists as a six-membered pyranose ring, whereas fructose is a ketose and forms a five-membered furanose ring.

Systematically, lactulose is called 4- O-( β- D-galactopyranosyl)-D-fructofuranose; again, the configuration at the anomeric centre of fructose is unspecified. In abbreviated form, this becomes DGal( β1 → 4)D-Fru. Lactulose is widely employed as a laxative. It is not absorbed from the gastrointestinal tract, is predominantly excreted unchanged, and helps to retain fluid in the bowel by osmosis.

Sucrose

Sucrose is composed of glucose and fructose, and again we have a six-membered pyranose ring coupled to a five-membered furanose ring. However, there is a significant difference when we compare its structure with that of lactulose: in sucrose, the two sugars are both linked through their anomeric centres. In the shorthand representation, we thus have to indicate the configuration at each anomeric centre, so the linkage becomes α1 → β2. Sucrose is thus abbreviated to D-Glc(α1 → β2)D-Fru. The systematic nomenclature for sucrose is α-D-glucopyranosyl- (1 → 2)-β-D-fructofuranoside, which also includes the arrow to avoid confusion.

Since the two sugars in sucrose are both linked through their anomeric centres, this means that both the hemiacetal/hemiketal structures are prevented from opening; and, in contrast to maltose, lactose, and lactulose, there can be no open-chain form in equilibrium with the cyclic form. Therefore, sucrose does not display any of the properties usually associated with the masked carbonyl group. In nature, the formation of oligosaccharides, and also of polysaccharides, is dependent upon the generation of an activated sugar bound to a nucleoside diphosphate, typically a UDPsugar.

As outlined above ), nucleophilic displacement of the UDP leaving group by a suitable nucleophile generates the new sugar derivative. This will be a glycoside if the nucleophile is a suitable aglycone molecule, or an oligosaccharide if the nucleophile is another sugar molecule.

This reaction, mechanistically of S_N2 type, should give an inversion of configuration at C-1 in the electrophile, generating a product with the β-configuration in the case of UDP-glucose, as shown. Many of the linkages formed between glucose monomers have the α-configuration, and it is believed that a double S_N2 mechanism operates, which initially involves a nucleophilic group on the enzyme.

Polysaccharides

Structural Aspects

Polysaccharides fulfil two main functions in living organisms, as food reserves and as structural elements. Plants accumulate starch as their main food reserve, a material that is composed entirely of glucopyranose units, but in two different types of polymer, namely amylose and amylopectin. Amylose is a linear polymer containing some 1000–2000 glucopyranose units linked α1 → 4.

Amylopectin is a much larger molecule than amylose (the number of glucose residues varies widely, but may be as high as 106), and it is a branched-chain molecule. In addition to α1 → 4 linkages, amylopectin has branches at about every 20 units through α1 → 6 linkages. These branches then also continue with α1 → 4 linkages but may have subsidiary α1 → 6 branching, giving a tree-like structure.

The mammalian carbohydrate storage molecule glycogen is analogous to amylopectin in structure but is larger and contains more frequent branching, about every 10 residues. The branching in amylopectin and glycogen is achieved by the enzymic removal of a portion of the α1 → 4-linked straight chain containing several glucose residues, then transferring this short chain to a suitable 6-hydroxyl group. A less common storage polysaccharide found in certain plants is inulin, which is a relatively small polymer of fructofuranose, linked through β2 → 1 bonds.

Cellulose is reputedly the most abundant organic material on Earth, being the main constituent in plant cell walls. It is composed of glucopyranose units linked β1 → 4 in a linear chain. Alternate residues are ‘rotated’ in the structure, allowing hydrogen bonding between adjacent molecules, and construction of the strong fibres characteristic of cellulose, as in cotton.

Hydrolysis of polysaccharides

Hydrolysis of polysaccharides (and oligosaccharides) follows the comments under glycosides above. Thus, treatment of amylose, amylopectin, or cellulose with hot aqueous acid will result in the formation of glucose as the sole product, through hydrolysis of acetal linkages. Under milder, less forcing conditions, it is possible to isolate short-chain oligosaccharides as a result of random hydrolysis of linkages.

• More specific hydrolysis may be achieved by the use of enzymes. Thus, the enzyme α-amylase in saliva and the gut can catalyse hydrolysis of α1 → 4 bonds throughout the starch molecule to give mainly maltose, with some glucose and maltotriose, the trisaccharide of glucose.
• Amylose is hydrolysed completely by this enzyme, but the α1 → 6 bonds of amylopectin are not affected. Another digestive enzyme, α-1,6-glucosidase, is required for this reaction. Finally, pancreatic maltase completes the hydrolysis by hydrolysing maltose and maltotriose.
• The milk of mammals contains the disaccharide lactose as the predominant carbohydrate, to the extent of about 4–8%. Lactose, therefore, provides the basic carbohydrate nutrition for infants, who metabolize it via the hydrolytic enzyme lactase.

Lactase enzyme activity in adult humans is usually considerably lower than in infants. Lactose intolerance is a condition in certain adults who are unable to tolerate milk products in their diet.

• This is a consequence of very low lactase levels, such that ingestion of lactose can lead to adverse reactions, typically gastric upsets. Cellulose differs from amylose principally in the stereochemistry of the acetal linkages, which are α in amylose but β in cellulose.
• α-Amylase is specific for α1 → 4 bonds and is not able to hydrolyse β1 → 4 bonds. An alternative enzyme, termed cellulase, is required. Animals do not possess cellulase enzymes, and thus cannot digest wood and vegetable fibres that are predominantly composed of cellulose.
• Ruminants, such as cattle, are equipped to carry out cellulose hydrolysis, though this is dependent upon cellulase-producing bacteria in their digestive tracts.

Oxidation Of Sugars: Uronic Ccids

Sugars may be oxidized by a variety of reagents, and the most susceptible group in aldoses is the aldehyde. The use of aqueous bromine as a mild oxidizing agent achieves oxidation of the aldehyde group in D-glucose, and the product is the corresponding carboxylic acid D-gluconic acid.

The general term used for such a polyhydroxy carboxylic acid is an aldonic acid. These are named by substituting -one acid for -ose of the sugar. Polyhydroxy carboxylic acids have the potential to form lactones (cyclic esters,).

And D-gluconic acid readily forms a 1,4-lactone in solution. In principle, both five- and six-membered rings might be produced, but the five-membered system is favoured.

• More vigorous oxidation results in the oxidation of one or more hydroxy groups, with the primary alcohol group being attacked most readily.
• Thus, oxidizing either D-glucose or D-gluconic acid with aqueous nitric acid leads to a dicarboxylic acid, D-glucaric acid.
• Dicarboxylic acids of this type are termed aldaric acids. Again, aldaric acids readily form five-membered lactones, which may be the 1,4- or 3,6- lactones, or the dilactone.

Determination of blood glucose levels

The peptide hormone insulin is produced by the pancreas and plays a key role in the regulation of carbohydrate, fat, and protein metabolism. In particular, it has a hypoglycaemic effect, lowering the levels of glucose in the blood.

A malfunctioning pancreas may produce a deficiency in insulin synthesis or secretion, leading to the condition known as diabetes mellitus.

• This results in increased amounts of glucose in the blood and urine, diuresis, depletion of carbohydrate stores, and subsequent breakdown of fat and protein. Incomplete breakdown of fat leads to the accumulation of ketones in the blood, severe acidosis, coma, and death.
• Where the pancreas is still functioning, albeit less efficiently, the condition is known as type 2 diabetes (non-insulin-dependent diabetes, NIDDM)and can be managed satisfactorily by a controlled diet or oral antidiabetic drugs.
• In type 1 diabetes (insulin-dependent diabetes, IDDM), pancreatic cells no longer function, and injections of insulin are necessary, one to four times daily, depending on the severity of the condition.
• These treatments need to be combined with a controlled diet and regular monitoring of glucose levels but do not cure the disease, so treatment is lifelong. Quick and easy methods have been developed so that patients can monitor their blood glucose levels regularly. One such method depends upon the oxidation of glucose to gluconic acid in a reaction catalysed by the enzyme glucose oxidase.

This enzyme can be obtained from several microorganisms,

Example: Aspergillus and Penicillium species, and for convenience are usually immobilized onto a suitable support. The microbial enzyme converts glucose into gluconic acid utilizing molecular oxygen as an oxidant, but detection of the process is dependent upon the simultaneous production of hydrogen peroxide.

Hydrogen peroxide may be detected by exploiting a secondary chemical reaction that produces a coloured product; this is compared with a standard colour chart to indicate the colour intensity and, therefore, give a measure of the glucose concentration.

Alternatively, it may be scanned in a colourimeter to give a more accurate assay. Even more accuracy can be obtained by using a voltammetric sensor, in which the hydrogen peroxide is oxidized to oxygen on an electrode surface, thus generating an electrical current that is directly proportional to the glucose concentration

⇒ \(\mathrm{H}_2 \mathrm{O}_2+2 \mathrm{HO}^{-} \rightleftharpoons \mathrm{O}_2+\mathrm{H}_2 \mathrm{O}+2 \mathrm{e}^{-}\)

The method is highly specific for glucose. Related sugars, such as mannose, xylose and galactose, are not oxidized by this enzyme or react only in trace amounts.

Uronic acids:

Uronic acids are produced from aldoses when just the terminal –CH₂OH group has been oxidized to a carboxylic acid. They are named after the parent sugar, substituting -uronic acid for -use; thus, D-glucuronic acid is the 6-carboxylic acid analogue of D-glucose. It should be apparent from the preceding comments that it will not be possible to oxidize the primary alcohol function selectively in the presence of the more reactive aldehyde group, so it becomes necessary to protect the aldehyde by an appropriate means.

It may also be desirable to protect other hydroxyls.

For example:

The formation of the acetonide of galactose protects the aldehyde and all hydroxyls except that at position 6 . It now remains to oxidize the primary alcohol and remove the protecting groups.

An alternative approach is to oxidize both the carbonyl and primary alcohol functions to carboxylic acids, then selectively reduce that corresponding to the required aldehyde. This may be achieved by reducing the 1,4-lactone of D-glucaric acid, using the same reaction as where it was employed in the synthesis of labelled glucose.

Uronic acids are found in nature, but they are formed enzymatically by selective oxidation of the primary alcohol function of a sugar. Oxidation takes place not on the free sugar, but on UDPsuga derivatives, as utilized in glycoside biosynthesis. UDP-glucuronic acid is an important carrier in the metabolism of drug molecules.

Some examples of natural uronic a cid d derivatives

Polymers of uronic acids are encountered in nature in structures known as pectins, which are essentially chains of D-galacturonic acid residues linked α1 → 4, though some of the carboxyl groups are present as methyl esters. These materials are present in the cell walls of the fruit, and the property that aqueous solutions under acid conditions form gels is the basis of jam-making.

Alginic acid is a polymer of D-mannuronic acid residues joined by β1 → 4 linkages. It is the main cell wall constituent of brown algae (seaweed). Salts of alginic acid are valuable thickening agents in the food industry, and the insoluble calcium salt is the basis of absorbable alginate surgical dressings.

The intensely sweet constituent in the root of liquorice (Glycyrrhiza glabra) is glycyrrhizin, a mixture of potassium and calcium salts of glycyrrhizic acid. It is said to be 50–150 times as sweet as sucrose. Glycyrrhizic acid is a glycoside of the triterpene aglycone glycyrrhizic acid. The sugar portion is a disaccharide comprised of two molecules of D-glucuronic acid, so is termed a diglucuronide. Liquorice is used in confectionery and as a flavouring agent for beers and stouts. It also finds considerable use in drug formulations to mask the taste of bitter drugs and for its emulsifying surfactant properties.

Glucuronides in drug metabolism

One of the principal ways by which foreign compounds are removed from the body is to conjugate them into glucuronic acid. This conjugation process not only binds the unwanted compound but also converts it into a highly polar material that is water soluble and can be excreted in aqueous solution, typically via the kidneys. The polarity is provided both by the hydroxyl groups and by the ionizable carboxylic acid group. Typical chemicals that may become conjugated with glucuronic acid include alcohols, phenols, carboxylic acids, amines, and thiols.

Drugs must also be considered as foreign compounds, and an essential part of drug treatment is to understand how they are removed from the body after their work is completed. Glucuronide formation is the most important of so-called phase II metabolism reactions. Aspirin, paracetamol, morphine, and chloramphenicol are examples of drugs excreted as glucuronides.

Glucuronides

Glucuronides are formed in mammals by reaction with uridine diphosphoglucuronic acid (UDPglucuronic acid; UDP-GA) in processes catalysed by uridine diphosphoglucuronyltransferase enzymes. This reaction is entirely analogous to the enzymic glycosylation process we looked at above. The reaction with UDP-GA can be envisaged as a simple S_N2 nucleophilic displacement reaction, with an appropriate nucleophile,

Example: An alcohol or amine, and a phosphate derivative as the leaving group.

UDP glucuronyltransferase enzymes have very broad substrate specificity and can catalyse reactions with a wide variety of foreign molecules and drugs.

UDP-GA is formed from UDP-glucose by enzymic oxidation of the primary alcohol group. We have already noted that UDP-glucose is also the biochemical precursor of glucose-containing polysaccharides,

Example: Starch and glycogen

The opium alkaloid morphine is one of the most valuable analgesics for the relief of severe pain. It is known to be metabolized in the body to O-glucuronides, by reaction at the phenolic and alcoholic hydroxyls.

The glucuronides formed are water soluble and readily excreted. An interesting feature is that the two monoglucuronides have significantly different pharmacological activities. Although morphine 3-O-glucuronide is antagonistic to the analgesic effects of morphine, morphine 6-O-glucuronide is a more effective and longer-lasting analgesic than morphine itself, and with fewer side effects.

Vitamin C

Vitamin C, also known as L-ascorbic acid, clearly appears to be of a carbohydrate nature. Its most obvious functional group is the lactone ring system, and, although termed ascorbic acid, it is certainly not a carboxylic acid. Nevertheless, it shows acidic properties, since it is an enol, in fact, an enediol. It is easy to predict which enol hydroxyl group is going to ionize more readily.

It must be the one β to the carbonyl, the ionization of which produces a conjugate base that is nicely resonance stabilized. Indeed, note that these resonance forms correspond to those of an enolate anion derived from a 1,3-dicarbonyl compound. Ionization of the α-hydroxyl provides less favourable resonance, and the remaining hydroxyls are typical non-acidic alcohols. Thus, the pKa of vitamin C is 4.0 and is comparable to that of carboxylic acid.

Vitamin C is essential for the formation of collagen, the principal structural protein in skin, bone, tendons, and ligaments, being a cofactor in the hydroxylation of the amino acids proline to 4-hydroxyproline, and of lysine to 5-hydroxylysine. These hydroxy amino acids account for up to 25% of the collagen structure. Vitamin C is also associated with some other hydroxylation reactions,

Example:

The hydroxylation of tyrosine to dopa (dihydroxyphenylalanine) in the pathway to catecholamine Deficiency leads to scurvy, a condition characterized by muscular pain, skin lesions, fragile blood vessels, bleeding gums, and tooth loss. Vitamin C also has valuable antioxidant properties, and these are exploited commercially in the food industries.

Most animals can synthesize vitamin C, though humans and primates cannot and must obtain it via their diet. Citrus fruits, peppers, guavas, rose hips, and blackcurrants are especially rich sources, but it is present in most fresh fruits and vegetables.

In animals, ascorbic acid is synthesized in the liver from D-glucose, by a pathway that initially involves specific enzymic oxidation of the primary alcohol function, giving D-glucuronic acid. This is followed by a reduction to L-gluonic acid, which is effectively a reduction of the carbonyl function in the ring-opened hemiacetal.

Lactone formation in gluonic acid leads to the favourable five-membered system, and then oxidation of the secondary alcohol to a carbonyl effectively gives ascorbic acid. However, the more favourable structure of ascorbic acid is the enol tautomer with the conjugated α, β-unsaturated lactone.

Ascorbic acid formation in plants follows an analogous pathway, starting from either D-glucose or D-galactose. Man and other primates appear deficient in the enzyme that oxidizes gluconolactone to keto-lactone, and we are thus dependent on a dietary source of vitamin C.

An unfortunate twist is the apparent configurational change from D to L in going from glucuronic to gluconic acid. This is a consequence of renumbering. In gluonic acid, the carboxylic acid group has the higher oxidation state, becomes the topmost substituent in the Fischer projection, and is numbered carbon-1.

As a result, the D descriptors for glucuronic acid and the L descriptors for gluconic acid now refer to two different chiral centres. You can see why we were rather unenthusiastic about the value of D and L. We are also uncomfortable that the –CH₂OH → –CO₂H change inferred in the glucose → glucuronic acid by convention maintains the same configuration in the two compounds. From the gluonic acid example, we might reasonably expect to apply the same type of renumbering in glucuronic acid.

Aminosugars

Aminosugars are the result of the replacement of one or more hydroxyl groups in sugar by amino groups. They are formed in nature by transamination processes on appropriate keto sugars, which are themselves the product of regiospecific enzymic oxidation processes.

Thus, D-glucosamine (2-amino-2-deoxy-D-glucose) is readily appreciated as a metabolic product from D-glucose. Note here the convenient way we can name an aminosugar by relating it to a normal sugar via the removal of a hydroxyl (2-deoxy) and then the addition of an amino (2-amino)

D-glucosamine and D-galactosamine, usually as N-acetyl derivatives, are part of the structures of several natural polysaccharides, whilst other uncommon aminosugars are components of the aminoglycoside antibiotics. We have also noted the occurrence of N-glycosides, where the nitrogen substitution is at the anomeric centre.

A simple chemical approach to aminosugars is to use S_N2 displacement by ammonia of a suitable leaving group, such as a tosylate (toluene p-sulfonate, see

This process can be made selective for position 6 since the less-hindered primary alcohol group is more readily esterified than the secondary alcohol.

2-Aminosugars such as glucosamine may be synthesized by a modified Kiliani–Fischer process. The starting aldose, here D-arabinose, is treated with ammonia, producing an imine, and then with HCN to yield epimeric 2-aminonitriles.

The remaining steps lead to a mixture of D-glucosamine and D-mannosamine, which will need to be separated. Protecting groups such as cyclic acetals and ketals may also be employed to achieve selective reaction.

Aminosugars and aminoglycoside antibiotics

The aminoglycosides form an important group of antibiotic agents and are immediately recognizable as modified carbohydrate molecules. Typically, they have two or three uncommon sugars attached through glycoside linkages to an aminocyclitol, i.e. an amino-substituted hydroxycyclohexane system.

The first of these agents to be discovered was streptomycin from Streptomyces griseus. Its structure contains the aminocyclitol streptamine, though both amino groups are bound as guanidino substituents in the derivative streptidine.

Other medicinally useful aminoglycoside antibiotics are based on the aminocyclitol 2-deoxystreptamine,

Example:

Gentamicin C1 from Micromonospora purpurea. Although streaming and 2-deoxystreptamine are cyclohexane derivatives, they are both of carbohydrate origin and derived naturally from glucose

The other parts of streptomycin, namely L-streptose and the aminosugar 2-deoxy-2-methylaminoL-glucose ( N-methyl-L-glucosamine), are also ultimately derived from D-glucose. Gentamicin C1 contains two aminosugars, L-garosamine and D-purpurosamine.

The aminoglycoside antibiotics have a wide spectrum of activity, including activity against some Gram-positive and many Gram-negative bacteria. However, their widespread use is limited by nephrotoxicity, which results in impaired kidney function, and by ototoxicity, which is a serious side-effect that can lead to irreversible loss of hearing.

These antibiotics are thus reserved for the treatment of serious infections where less-toxic drugs have proved ineffective. The aminoglycoside antibiotics interfere with protein biosynthesis by acting on the smaller 30S subunit of the bacterial ribosome.

Aminosugars are also components of many macrolide antibiotics. These are macrocyclic lactones with a ring size typically of 12–16 atoms. Two or more sugar units are attached through glycoside linkages, these sugars tending to be unusual 6-deoxy structures often not found outside of this class of compounds,

Example: L-cladinose.

At least one sugar is an amino sugar, e.g. D-desosamine. These antibiotics have a narrow spectrum of antibacterial activity, principally against Gram-positive microorganisms. Their antibacterial spectrum resembles but is not identical to, that of the penicillins, so they provide a valuable alternative for patients allergic to the penicillins.

Erythromycin produced by cultures of Saccharopolyspora erythraea is the principal macrolide antibacterial currently used in medicine. It exerts its antibacterial action by inhibiting protein biosynthesis, binding to the larger 50S subunit of bacterial ribosomes and blocking the translocation step.

Spiramycin is another macrolide, recently introduced into medicine for the treatment of toxoplasmosis, infections caused by the protozoan Toxoplasma gondii. This contains a 16-membered lactone ring (erythromycin has a 14-membered ring), and two aminosugars, D-mycaminose and D-forosamine. D-Forosamine is remarkable in having only one hydroxyl group, and that is bound up in the hemiacetal ring system.

Polymers Containing Aminosugars

The structure of chitin is rather similar to that of cellulose, though it is composed of β1 → 4- linked N-acetylglucosamine residues. Chitin is a major constituent in insect skeletons and the shells of crustaceans,

Example: Crabs and lobsters; as with cellulose, its strength again depends on hydrogen bonding between adjacent molecules, producing rigid sheets. Chemical deacetylation of chitin provides chitosan, a valuable industrial material used for water purification because of its chelating properties, and in wound-healing preparations

Bacterial cell walls contain peptidoglycan structures in which the carbohydrate chains are composed of alternating β1 → 4-linked N-acetylglucosamine and O-lactyl-N-acetylglucosamine (also called N-acetylmuramic acid) residues. These chains are cross-linked via peptide structures. Part of the peptidoglycan of Staphylococcus aureus is shown here

This shows the involvement of the lactyl group of the N-acetylmuramic acid in linking the peptide with the carbohydrate via an amide/peptide bond. The biological activities of the β-lactam antibiotics,

Example:

Penicillins and cephalosporins stem from an inhibition of the cross-linking mechanism during the biosynthesis of the bacterial cell wall. The mammalian blood anticoagulant heparin is also a carbohydrate polymer in which amino sugars (glucosamine) alternate with uronic acid residues.

Polymers of this kind are known as anionic mucopolysaccharides or glycosaminoglycans. Heparin consists of two repeating disaccharide units, in which the amino functions and some of the hydroxyls are sulfated, producing a heterogeneous polymer. The carboxyls and sulfates together make heparin a strongly acidic water-soluble material.

Carbohydrate determinants of blood groups

Most people are aware that blood is classified into several types, the blood groups. These are termed A, B, O, etc. It is essential in blood transfusions that the donor blood matches that of the recipient, otherwise, antibodies are produced in the new blood. This leads to aggregation of red blood cells, with potentially fatal results through blockage of blood vessels.

The blood group antigens are glycoproteins, carbohydrates having an attached protein chain, and the various blood groups can be correlated with a single monosaccharide residue in the carbohydrate portion. At the end of the carbohydrate section in type O blood antigens, there is a D-galactopyranose ring to which is attached an L-fucopyranose sugar through an α1 → 2 linkage.

In type B blood antigens, the galactose residue has a second D-galactose residue attached, through an α1 → 3 linkage. In type A blood antigens, the second sugar residue is now N-acetyl-D-galactosamine, again attached through an α1 → 3 linkage. It has been found that enzymic removal of the terminal galactose residue from type B or of the N-acetyl-galactosamine residue from type A converts the B or A antigens into O antigens. It is also known that individuals with type B or type A antigens possess additional enzyme systems that specifically add the extra terminal carbohydrate unit to the type O antigen.

The post Carbohydrates appeared first on BDS Notes.

Numerous amino acids are found in nature, we are concerned primarily with those that make up the structures known as peptides and proteins. Peptides and proteins are both polyamides composed predominantly of α- amino acids linked through their carboxyl and α- amino functions.

In biochemistry, the amide linkage is traditionally referred to as a peptide bond. Whether the resultant polymer is classified as a peptide or a protein is not clearly defined; generally, a chain length of more than 40 residues confers protein status, whereas the term polypeptide can be used to cover all chain lengths.

Proteins in all organisms are made up of the same set of 20 α-amino acids, though the organism is not necessarily capable of synthesizing all of these.

Some amino acids are obtained from the diet.

• The amino acids are combined in a sequence that is defined by the genetic code, the sequence of bases in DNA.  gives the structures of these 20 amino acids together with the standard three-letter and one-letter abbreviations used to represent them.
• Proline is strictly an imino acid rather than an amino acid, but it is normally included as one of the 20 amino acids.
• The amino acids are also subclassified according to the chemical and physical characteristics of their R substituent.
• Since the polypeptide structure combines both the amino and carboxylic acid functions of an amino acid into amide linkages, the overall properties of the polypeptide are going to be defined predominantly by the characteristics of these R substituents.

The amino acid components of proteins have the L configuration, but many peptides are known that contain one or more D-amino acids in their structures.

D-Amino acids are not encoded by DNA, and peptides containing them are produced by what is termed ‘non-ribosomal peptide biosynthesis’. D-Amino acids generally arise by epimerization of L-amino acids. All the protein L-amino acids have the S configuration, except for glycine, which is not chiral, and L-methionine which is R, a consequence of the priority rules for systematic descriptors of configuration.

Amino acids structure and standard abbreviations:

Two of the protein amino acids, threonine, and isoleucine, have two chiral centers; therefore, diastereoisomeric forms are possible. In proteins, each of these amino acids exists in a single diastereoisomeric form.

The pK_a of the carboxylic acid group of amino acids is around 2, and that of the amino group (as conjugate acid) is around 9. As we saw this means that the carboxylic acid group (a stronger acid than the ammonium cation) will protonate the amino group (a stronger base than the carboxylate anion). At pH 7, therefore, amino acids with neutral R groups will exist mainly as the overall neutral, but doubly charged zwitterionic form (the weaker acid and weaker base).

The carboxylate group becomes protonated as the pH decreases, whereas at higher pH the ammonium ion becomes deprotonated, in both cases yielding a singly charged species. The uncharged amino acid (as we almost always draw it!) is a negligible contributor at any pH

When the R group contains another ionizable group, the amino acid will have more than two dissociation constants. The carboxylic acid groups of aspartic acid and glutamic acid, the amine of lysine, and the guanidino group of arginine will all be ionized at pH 7, and the imidazole nitrogen of histidine will be partially protonated. However, neither the phenolic group of tyrosine nor the thiol group of cysteine will be ionized at this pH

The ionization properties of side-chain substituents will usually carry through into the peptide or protein and influence the behavior of the polymer. However, the actual pKa values of the amino acid side-chains in the protein are modified somewhat by the position of the amino acid in the chain, and the environment created by other substituents. Typical pK_a values

Note that the side chains of glutamine and asparagine are not basic; these side chains contain amide functions, which do not have basic properties. The heterocyclic ring in tryptophan can also be considered as non-basic since the nitrogen lone pair electrons form part of the aromatic π electrons and are unavailable for bonding to a proton

In addition to the 20 amino acids described, there are also a few amino acids quite frequently encountered that are not encoded by DNA. These are mainly found in peptides and are typically slightly modified versions of the common amino acids, such as N-methyl amino acids. These components are represented by an appropriate variation of the normal abbreviation,

Example:

N-methyl amino acids such as Tyr(Me) or Leu(Me), though N-dimethylglycine is often referred to as sarcosine (Sar)

pK_a values for free and protein-bound amino acids:

A frequently encountered modification is the conversion of the C-terminal carboxyl into an amide. This is represented as Phe–NH₂, for example, which must be considered carefully, and not be interpreted as an indication of the N-terminus.

Some other variants are shown below, with their abbreviations. Pyroglutamic acid may be found where a terminal glutamic acid residue, linked to the chain through its carboxyl, forms a cyclic amide (lactam)

Some common amino acids not encoded by DNA

Peptides And Proteins

Although superficially similar, peptides and proteins display a wide variety of biological functions, and many have marked physiological properties. For example, they may function as structural molecules in tissues, as enzymes, as antibodies, or as neurotransmitters. Acting as hormones, they can control many physiological processes, ranging from gastric acid secretion and carbohydrate metabolism to growth itself. The toxic components of snake and spider venoms are usually peptides in nature, as are some plant toxins.

These different activities arise as a consequence of the sequence of amino acids in the peptide or protein (the primary structure), the three-dimensional structure that the molecule then adopts as a result of this sequence (the secondary and tertiary structures), and the specific nature of individual side-chains in the molecule. Many structures have additional modifications to the basic polyamide system shown, and these features may also contribute significantly to their biological activity.

The tripeptide formed from L-alanine, L-phenylalanine and L-serine by two condensation reactions is alanyl–phenylalanyl–serine, considering each additional amino acid residue as a substituent on the previous. This would be more commonly represented as Ala–Phe–Ser, using the standard three-letter abbreviations for amino acids

By convention, the left-hand amino acid in this sequence is the one with a free amino group, the N-terminus, and the right-hand amino acid has the free carboxyl, the C-terminus. Thus, Ser–Phe–Ala is different from Ala–Phe–Ser, and represents a quite different molecule. Sometimes, the termini identities are emphasized by showing H– and –OH; H– represents the amino group and –OH the carboxyl group. Some peptides are cyclic, and this convention can have no significance, so arrows are incorporated into the sequence to indicate peptide bonds in the direction CO→NH. As sequences become longer, one-letter abbreviations for amino acids are commonly used instead of the three-letter abbreviations, thus Ala–Phe–Ser becomes AFS.

Abbreviations assume the L-configuration applies throughout, and any D-amino acids would be specifically noted,

Example: Ala–D-Phe–Ser.

Glutathione:

Glutathione is an important tripeptide; but it is a slightly unusual one, in that it has an amide linkage that involves the γ- carboxyl of glutamic acid rather than a normal amide bond utilizing the C-1 carboxyl group. To specify this bonding, the glutathione structure is written as γ-Glu–Cys–Gly

Peptides and proteins may be hydrolyzed to their constituent amino acids by either acid or base hydrolysis . However, because of its nature, the amide bond is quite resistant to hydrolytic conditions, a very important feature for natural proteins. Hydrolysis of peptides and proteins, therefore, requires heating with quite concentrated strong acid or strong base. Neither acid nor base hydrolysis provides the ideal hydrolytic conditions, however, since some of the constituent amino acids are found to be sensitive to the reagents.

Acid hydrolysis is preferred, but the indole system of tryptophan is largely degraded in strong acid, and the sulfur-containing amino acid cysteine is also unstable. Serine, threonine, and tyrosine may also suffer partial degradation. Those amino acids containing amide side-chains,

Example:

Asparagine and glutamine, will be hydrolyzed further, giving the corresponding structures with acidic side-chains, namely aspartic acid and glutamic acid

The Molecular Shape Of Proteins: Primary Secondary And Tertiary Structures

Peptides and proteins are composed of amino acids linked together via amide (peptide) bonds, the amino group of one condensing with the carboxylic acid of another. The sequence of amino acids in a peptide or protein is closely controlled by genetic factors.

Some peptides are synthesized via a multi-functional enzyme complex (non-ribosomal peptide synthesis), whereas others, including the larger proteins, are produced on the ribosome, and the sequence can be related directly to the nucleotide sequence of DNA.

This amino acid sequence provides what we term the primary structure of the protein, although this term also includes the position of disulfide bridges, the result of covalent bonding between pairs of cysteine residues. Disulfide bridges produce cross-linking in the polypeptide chain.

This covalent bonding arises as a result of biochemical oxidation of the thiol groups in two cysteine residues, and it may also be achieved chemically with the use of mild oxidizing agents. This modification of thiol groups may thus loop a polypeptide chain or cross-link two separate chains.

It also significantly modifies the properties of a protein by removing two polar and potentially acidic (pK_a 10.3) groups, replacing them with a nonpolar disulfide function. Under suitable hydrolytic conditions, a protein containing one or more disulfide bridges will yield cysteine residues still joined by this type of bonding. This amino acid ‘dimer’ is called cystine (Cys–Cys).

Because of the similarity in names, it is usual practice to differentiate them in speech by pronouncing cysteine as sis-tay-een, whereas cysteine is pronounced sis-teen. The disulfide bridge is easily formed and is just as easily broken. It may be cleaved to thiol groups by reduction with reagents such as sodium borohydride or by the use of other thiol reagents.

For example:

Mercaptoethanol (HSCH₂CH₂OH) is routinely used in protein analysis to help locate disulfide bridges through an equilibration reaction.

The mechanism for this reaction is shown below:

Oxidation with stronger oxidizing agents,

Example:

Potassium permanganate or performic acid, converts the disulfide to two molecules of a sulfonic acid, namely cysteic acid.

This reaction may be of value in sequence analysis, to determine the position of disulfide bridges (as opposed to unmodified cysteine residues) in the primary structure.

Disulfide bridges in insulin

The peptide hormone insulin is produced by the pancreas and plays a key role in the regulation of carbohydrate, fat, and protein metabolism.

• In particular, it has a hypoglycaemic effect, lowering the levels of glucose in the blood. A malfunctioning pancreas leads to a deficiency in insulin synthesis and the condition known as diabetes.
• This results in increased amounts of glucose in the blood and urine, diuresis, depletion of carbohydrate stores, and subsequent breakdown of fat and protein. Incomplete breakdown of fat leads to the accumulation of ketones in the blood, severe acidosis, coma, and death.
• Diabetes treatment requires daily injections of insulin; since insulin is a peptide, it would be degraded by stomach acid if taken orally. Insulin does not cure the disease, so treatment is lifelong.

Human insulin:

Human insulin is composed of two straight-chain polypeptides joined by disulfide bridges.

This structure is known to arise from a single straight-chain polypeptide, preproinsulin, containing 100 amino acid residues. This loses a 16-residue portion of its chain and forms proinsulin, in which disulfide bridges connect the terminal portions of the chain in a loop.

A central portion of the loop (the C chain) is then cleaved out, leaving the A chain (21 residues) bonded to the B chain (30 residues) by two disulfide bridges. There is also a third disulfide bridge interconnecting two cysteine residues in the A chain. This is the resultant insulin.

Mammalian insulins from different sources are very similar and may be used to treat diabetes. The compounds show variations in the sequence of amino acid residues 8–10 in chain A, and at amino acid 30 in chain B.

Porcine insulin and Bovine insulin for drug use are extracted from the pancreas of pigs and cattle respectively. More frequently, human insulin is now employed. This is produced by the use of recombinant DNA technology to obtain the two polypeptide chains, and then linking these chemically to form the disulfide bridges

Peroxides, including hydrogen peroxide (H₂O₂), can damage cells by causing unwanted oxidation reactions. The tripeptide glutathione (GSH) is able to participate in a cellular protection mechanism via its ability to form disulfide bridges.

In an enzymic reaction catalysed by glutathione peroxidase, GSH reacts with peroxides and becomes oxidized to form a dimer (GSSG) linked by a disulfide bridge.

In so doing, it reduces the peroxide. In the case of H₂O₂, this generates water, whereas an organic peroxide would yield water and an alcohol

H₂O₂ + 2GSH →  GSSG + 2H₂O

ROOH + 2GSH →  GSSG + ROH + H₂O

In order maintain adequate levels of GSH, the oxidized dimer is then reduced back to the original thiol components. This is achieved using the enzyme GSH reductase in a reaction involving NADPH and FAD cofactors

Protein chains are not the sprawling, ill-defined structures that might be expected from a single polypeptide chain. Most proteins are compact molecules, and the relative positions of atoms in the molecule contribute significantly to its biological role. A particularly important contributor to the shape of proteins is provided by the peptide bond itself. Drawn in its simplest form, one might expect free rotation about single bonds, with a variety of conformations possible.

However there is resonance stabilization in an amide, via electron movement from the lone pair on the nitrogen to the carbonyl oxygen. We have already noted this type of resonance stabilization in amides , and also in esters. It was invoked in explaining reactivity and pKa values compared with other types of carbonyl compounds, and the non-basic behavior of the nitrogen atom in amides

To achieve this stabilization, the p orbital on nitrogen needs to be lined up with the carbonyl π bond. The immediate consequence of this are that five bonds in the peptide linkage must be coplanar. There is no free rotation about the N–C bond, because it is involved with a partial double-bond system. Of course, there are potentially two configurations with respect to this N–C bond, corresponding to cis and trans versions if it were a true double bond. It is not surprising that the transform is energetically favored, where we have the large groups, i.e. the rest of the chain, arranged to give minimum interaction.

Zig-Zag conformation with main chain trans oriented

Rotational freedom about single bonds

Rotation about the C–N bond is restricted

We now see good reasons for drawing a polypeptide chain in the accepted zigzag form. Note, however, that the remaining single bonds in the chain do allow rotation, and this is why we see a wide variety of different shapes in proteins. We can also appreciate that, in general, the carbonyl groups and N–H groups are all going to be coplanar. This leads to the secondary structure of proteins, a consequence of hydrogen bonding possible because of the regular array of carbonyl and N–H groups.

The most easily appreciated example of this is the β-pleated sheet, one of the ways in which a polypeptide chain can be arranged in an ordered fashion. Polypeptide chains align themselves side-by-side, stabilized by multiple hydrogen bonding, allowed by the regular array of carbonyl and N–H bonds.

The alignment may be parallel, such that all the carbonyl to amino peptide linkages are in the same direction, or antiparallel where carbonyl to amino peptide linkages run in opposite directions. Although there are going to be groups of atoms that are planar, the whole chain is not planar. Instead, these arrangements take up a pleated array, which helps to minimize interaction between the large R groups.

Parallel sheets may involve different polypeptide chains via intermolecular hydrogen bonds, or the same chain via intramolecular hydrogen bonds. For intramolecular interactions, the chain length needs to be substantial, i.e. proteins rather than peptides, and it will be necessary for the polypeptide chain to bend back upon itself. The commonest type of arrangement for bending back a chain is called the β-turn, resulting in hydrogen bonding between residues n and n + 3.

Note also that the imino acid proline must distort the regular zigzag array and introduce a bend into the chain; two configurations may be considered,

And both are possible since there is little difference in energy between them. Further, there is no N–H in proline, so hydrogen bonding involving this residue is not possible.

Alpha (α) Helix:

Whereas the β-pleated sheet provides a particularly nice and easily appreciated example of regular hydrogen bonding in polypeptide chains, the most common arrangement found in proteins is the α-helix. Do not worry about the α or β used in the nomenclature; this merely signifies that the helical structure ( α) was deduced earlier than that of the pleated sheet ( β).

• The α-helix is a right-handed helix, an ordered coil array stabilized by hydrogen bonding between carbonyl and N–H groups in the same chain. In a right-handed helix, movement along the chain involves a clockwise or right-handed twist, just like an ordinary screw – you turn the screwdriver clockwise.
• Hydrogen bonds link carbonyl and N–H bonds in amino acids that are separated by three other residues, and each turn of the helix is found to take up 3.6 amino acid residues. Note that all of the R groups, which in the majority of amino acids are quite bulky, are accommodated on the outside of the helix. Only the imino acid proline cannot fit into the regular array of the α-helix.
• We have just seen that proline must distort the regular array and introduce a bend into the chain, and that there is no N–H for hydrogen bonding. The secondary structure is responsible for some of the physical properties of proteins.

For example:

Structural proteins such as α-keratins in skin and hair are fibrous in nature and have good elastic properties. This elasticity can be traced back to the α-helix structure, in which weak hydrogen bonds are parallel to the direction of stretching, i.e. a spring-like structure.

On the other hand, proteins such as α-fibroin in silk are relatively inelastic since they contain the β-pleated sheet structure, where extension is resisted by the full strength of covalent bonds.

• In the β-pleated sheet, the weaker hydrogen bonds would be perpendicular to the direction of stretching.
• However, most proteins have a roughly spherical shape and are thus termed globular proteins. Globular proteins are likely to contain portions of the polypeptide chain that adopt both helical and sheet structures.
• In contrast to structural proteins like α-keratin or α-fibroin, the helical or sheet fragments in globular proteins are rather short and do not extend far without a change in direction. The overall folding of the polypeptide chain and the three-dimensional arrangement produced provide the tertiary structure of the protein.

The globular shape is facilitated, however, by some other non-covalent interactions.

Terity structure intramolecular interaction

The conformation of a protein is determined and maintained by a range of intramolecular interactions that arise from some of the amino acid sidechain substituents. These are non-covalent interactions, although we must also remember that a disulfide bond formed between pairs of cysteine residues also contributes to the three-dimensional shape of the protein by providing cross-chain links.

Non-covalent interactions are relatively weak when compared with covalent bonds, but there are usually many such interactions in a protein, and, overall, a substantial degree of stabilization is attained.

1. Hydrophobic interactions:

Hydrophobic interactions. Many of the amino acids contain side-chains that are hydrocarbon in nature, either aliphatic or aromatic

• In an aqueous environment, such groups are hydrophobic, and any folding in the protein that concentrates these hydrophobic areas together, and away from water, is going to be favored.
• This hydrophobic effect tends to encourage the burying of hydrophobic side chains in the interior of the protein and provides a significant part of the driving force for protein folding.
• Because so many amino acids have hydrocarbon side chains, not all can be accommodated in the interior, and hydrophobic groups tend to be about equally distributed between the interior and the surface of the molecule. On the other hand, hydrophilic sidechains are more likely to be found on the surface of a protein.

Non-polar, hydrophobic side-chains:

Polar, hydrophilic side-chains:

2. Hydrogen bonds:

Hydrogen bonds are responsible for the fundamental characteristics of the α-helix and β-pleated sheet; in addition, they contribute to the final shape of a globular protein. Hydrogen bonds can form in a variety of ways, involving the peptide backbone, polar amino acid side-chains, and also water molecules. Some of the hydrogen bonding situations are shown below; others can be deduced.

It should be appreciated that amino acids such as serine, threonine, tyrosine, and cysteine all contain side-chain alcohol or thiol groups that may participate in hydrogen bonding and stabilize a particular protein conformation.

3. Ionic bonds:

Ionic bonds. Carboxylic acid groups in amino acid side chains (aspartic acid, glutamic acid) will be ionized at pH 7, and nitrogen-containing groups (lysine, arginine) will similarly be protonated. Isolated hydrophilic groups such as these will never be found in the hydrophobic interior of a globular protein but will be positioned on the outer surface in proximity to water molecules.

• However, pairs of oppositely charged ions may be found in the interior since electrostatic interactions can provide the necessary attractive forces.
• Thus, non-covalent hydrophobic interactions, hydrogen bonds, and electrostatic bonds all contribute to the overall shape of a protein .
• As we shall see, many pertinent properties of a protein are then provided by the appropriate combination of the remaining amino acid side chains that reside on the surface, allowing specific binding to various molecules. This is the essence of enzymic activity and drug-receptor interactions.
• With some proteins, there is a further level of structure, i.e. quaternary structure, which may need to be considered. This arises because two or more protein chains aggregate to form the normal functional protein.
• Typically, the separate subunits, often the same, are held together by non-covalent interactions, as seen in the consideration of tertiary structure. Not all proteins have quaternary structure.

Protein binding sites

From our considerations above, we can see just how important the interactions of various amino acid side chains are to the structure and shape of proteins.

• These interactions tend to be located inside the protein molecule, stabilizing a particular conformation and generating the overall shape as in a globular protein.
• However, it is obvious that there are also going to be many amino acid side chains located on the surface of a protein, and these in turn will be capable of interacting with other molecules.
• These interactions will be intermolecular, rather than the intramolecular interactions that contribute to protein structure.
• Because there will also be several amino acid side-chains close, a combination of interactions may generate a site that has a specific shape, and a specific array of forces. The site will then be able to bind a particular molecule or part of a molecule.
• These amino acid side chains, therefore, allow strong binding to specific molecules, and the particular molecule can be regarded as being a perfect fit both in terms of the forces involved and in a geometric sense.

As a result, even a small change in the structure of the molecule could well spoil the fit and upset the interplay of forces – the binding is powerful and reasons by specific.

The sole function of many proteins is simply to bind other molecules. Thus, immunoproteins can bind to alien molecules (antigens) and destroy their activity by complexation. Some proteins act as hormones, influencing metabolic rates by binding to another molecule or structure. Binding to a protein may be the way an organism transports a molecule or even an ion to a different part of the organism

For example:

Hemoglobin transports molecular oxygen around the body, and cytochromes transport electrons within a cell.

• Drug–receptor interactions and enzymic activity are also a consequence of the binding of molecules to a protein.
• A substance that elicits a particular biological response by interaction at a receptor site is termed an agonist.
• An antagonist is a substance that inhibits the action of an agonist, often by competing for the same receptor site. The binding site on an enzyme is usually termed the active site.

Pain relief: morphine mimics natural peptides called endorphins

Although the pain-killing properties of the opium alkaloid morphine and related compounds have been known for a considerable time, the existence of endogenous peptide ligands for the receptors to which these compounds bind is a more recent discovery. It is now appreciated that the body produces a family of endogenous opioid peptides that bind to a series of receptors in different locations.

These peptides include enkephalins, endorphins, and dynorphins, and are produced primarily, but not exclusively, in the pituitary gland. The pentapeptides Metenkephalin and Leu-enkephalin were the first to be characterized. The largest peptide is β-endorphin (‘endogenous morphine’), which is several times more potent than morphine in relieving pain.

Although β-endorphin at its N-terminus contains the sequence for Met-enkephalin, the latter peptide and Leu-enkephalin are derived from a larger peptide, namely proenkephalin A, and β-endorphin itself is formed by cleavage of the peptide pro-opiomelanocortin. The proenkephalin. A structure contains four Met-enkephalin sequences and one of Leu-enkephalin. The dynorphins,

Example: Dynorphin A, is also produced by cleavage of a larger precursor, namely proenkephalin B (prodynorphin), and all contain the Leu-enkephalin sequence.

• Some 20 opioid ligands have now been characterized.
• When released, these endogenous opioids act upon specific receptors, inducing analgesia and depressing respiratory function and several other processes.
• The individual peptides have relatively high specificity towards different receptors.
• It is known that morphine, β-endorphin, and Met-enkephalin are agonists for the same site. The opioid peptides are implicated in analgesia brought about by acupuncture since opiate antagonists can reverse the effects.
• The hope of exploiting similar peptides as ideal, non-addictive analgesics has yet to be attained; repeated doses of endorphin or enkephalin produce addiction and withdrawal symptoms.

A common structural feature required for centrally acting analgesic activity in opioids is the combination of an aromatic ring and a piperidine ring that maintains the stereochemistry at the chiral center, as shown below.

The three-dimensional disposition of the nitrogen function to the aromatic ring allows morphine and other analgesics to bind to a pain-reducing receptor in the brain. The terminal tyrosine residue in the natural agonist’s Met-enkephalin and Leu-enkephalin is mimicked by portions of the morphine structure

The Chemistry Of Enzyme Action

Enzymes are proteins that act as biological catalysts. They not only bind molecules but also provide a special environment in which the molecules are chemically modified.

• Enzymes cannot promote a reaction that is not energetically favorable, but by binding the reagents in the necessary orientation and nearby, they significantly reduce the activation energy for the transformation.
• By the amino acid side chains, enzymes can provide a highly specific binding site for their substrates, anchoring these reagents in an appropriate manner and suitable proximity so that a reaction can occur, as well as providing any necessary acid or base catalyst for the reaction. In some cases, a further reagent, a coenzyme, must also be bound for the reaction to occur.
• After the reaction is completed, the products are then released from the enzyme, so that the reaction can be repeated on further molecules of the substrates. These amino acid side chains confer immense catalytic power to the enzyme, giving it the ability to carry out reactions that organic chemists can only dream of.

Several types of bonding might be utilized to bind substrates to enzymes.

These are analogous to the bondings that contribute to the secondary and tertiary structures of a protein and include the following noncovalent interactions:

1. Electrostatic bonding, via acids, bases, phosphates
2. Hydrophobic interactions, via alkyl groups, aromatic rings

Hydrogen bonding, via NH, OH, SH, C=O. In addition, we can meet examples of covalent bonding that are responsible for binding a substrate, where a functional group in the substrate reacts chemically with a protein side-chain functional group.

Two important reactions are:

1. Imine formation, via NH₂ and C=O;
2. Thioester formation, via SH and C=O.

A large proportion of the substrates used in intermediary metabolism are in the form of phosphates.

Phosphates are favored in nature since they usually confer water solubility on the compound, and they provide a functional group that can bind to enzymes through simple electrostatic bonding.

In many cases, the phosphate group may also feature as a chemically reactive functional group – phosphates are good leaving groups. We have considered the structures of the various amino acids in terms of polarity, basicity, acidity, etc.

Here is a useful reminder of the important functionalities that are pertinent to enzyme action

• Amino acids with hydroxyl groups: Ser, Thr, Tyr
• Amino acids containing thiol (sulfhydryl) groups: Cys
• Amino acids with acidic groups: Asp, Glu
• Amino acids with basic groups: Lys, Arg, His

It is not sensible to try to cover a wide range of enzymic reactions to demonstrate how amino acid side chains are responsible for the chemical changes the enzyme brings about. Instead, one or two suitable examples will suffice to illustrate the general principles.

Acid-base catalysis

Let us first remind ourselves of the two mechanisms for hydrolysis of an ester, namely acid-catalysed hydrolysis and base-catalyzed hydrolysis

Acid-catalyzed hydrolysis of esters:

Base-catalyzed hydrolysis of esters:

Since enzymic reactions proceed in aqueous solution at pH 7 or thereabouts, where the concentrations of hydronium and hydroxide ions are both approximately 10−7 M, we need alternatives to strong acids and strong bases to formulate comparable enzymic mechanisms.

• Such reagents are provided by those amino acid side chains that are ionized at pH 7. Thus, the capacity for acid or base catalysis is built into the active site of many enzymes.
• Furthermore, the effective concentration of these groups at the active site is high, making them very effective acid and base catalysts. These donors and acceptors of protons are called general acid catalysts and general base catalysts respectively.
• The amino acids in question are the basic amino acids lysine, arginine, and histidine, and the acidic amino acids aspartic acid and glutamic acid.
• The side-chain functions of these amino acids, ionized at pH 7, act as acids or bases.

In a reverse sequence, protons may be acquired or donated to regenerate the conjugate acids and conjugate bases.

The most effective acid-base catalyst is one whose pKa is 7.0, since at pH 7.0 the concentrations of acid and conjugate base are equal. With just a slight decrease in pH it would become protonated and function as a general acid catalyst, whereas with a slight increase in pH, it would become unprotonated and, therefore, a general base catalyst.

Enzymes are active over a limited pH range; the pH value of maximum activity is known as the pH optimum, and this is characteristic of the enzyme. It typically reflects the pH necessary to achieve the appropriate ionization of amino acid side chains at the active site.

• The side-chain of histidine has a pKa value of 6.0; above pH 6.0, the imidazole ring acts as a proton acceptor or general base catalyst, whereas below pH 6.0 it is protonated and can act as a proton donor or general acid catalyst.
• At pH 7.0, the imidazole ring can be considered as partially protonated, since both ionized and non-ionized forms are present in the ratio of about 1: 10. Consequently, we find that the imidazole ring of histidine participates in acid-base catalysis in many enzymes.
• As we mentioned earlier, the pKa values of histidine side chains in a protein are modified by the neighboring amino acid residues and are typically in the range 6–7. These values provide a level of ionization somewhere between 9 and 50%, depending upon the protein.
• Remember that tautomerism can occur in imidazole rings When we meet structures for the amino acid histidine, we may encounter either of the tautomeric forms shown. Do not think there is a discrepancy in structures.

On the other hand, we can write resonance structures for the protonated ring, the imidazolium cation

Now let us see how the imidazole grouping of histidine can be involved in the general acid-catalyzed and general base-catalyzed hydrolysis of esters by enzymes. In essence, the chemical and enzymic reactions are very similar.This is to be expected since enzymes can only catalyze an energetically favorable reaction. The role of acid or base is largely achieved in the enzyme reactions by implicating the imidazolium/imidazole system

General Acid-catalyzed ester hydrolysis:

General Base-catalyzed ester hydrolysis:

In the general acid-catalyzed mechanism, the imidazolium ion acts as a proton donor to protonate the carbonyl oxygen, thus producing a better electrophile. The protonated ester is then attacked by a water nucleophile, after which the imidazole nitrogen removes the now unwanted proton from the nucleophile; the imidazole nitrogen consequently becomes reprotonated.

The carboxylic acid is then formed via the loss of the leaving group, and this is facilitated by protonation so that the leaving group is an alcohol rather than an alkoxide.

The imidazolium proton is again a participant in this process.

• Finally, abstracting the proton from the protonated carbonyl regenerates the imidazolium ion. As in the chemical reactions, the general base-catalyzed process is mechanistically rather simpler. The imidazole nitrogen acts as a base to remove a proton from water, generating hydroxide that attacks the carbonyl.
• Subsequently, the alkoxide leaving group is reprotonated by the imidazolium ion
• However, not included in the above mechanisms are other amino acid side chains at the active site, whose special role will be to help bind the reagents in the required conformation for the reaction to occur.
• Examples of such interactions are found with acetylcholinesterase and chymotrypsin, representatives of a group of hydrolytic enzymes termed serine hydrolases, in that a specific serine amino acid residue is crucial for the mechanism of action.
• The proposed enzyme mechanisms just described, and those that follow, are depicted to show how certain amino acid residues become involved. Remember that the enzyme active site is three-dimensional, whereas our representation is only two-dimensional.

This means that bond angles and bond lengths sometimes look a little odd and distorted. However, such imperfect representations are easier to follow than if we had provided pictures that tried to emulate three-dimensional views

Acetylcholinesterase, a serine esterase

Acetylcholine:

Acetylcholine is a relatively small molecule that is responsible for nerve-impulse transmission in animals. As soon as it has interacted with its receptor and triggered the nerve response, it must be degraded and released before any further interaction at the receptor is possible. Degradation is achieved by hydrolysis of acetate and choline by the action of the enzyme acetylcholinesterase, which is located in the synaptic cleft. Acetylcholinesterase is a serine esterase that has a mechanism similar to that of chymotrypsin

Hydrolysis involves nucleophilic attack by the serine hydroxyl onto the ester carbonyl. This leads to the transfer of the acetyl group from acetylcholine to the enzyme’s serine hydroxyl, i.e. formation of a transient acetylated enzyme, and the release of choline. . Hydrolysis of the acetylated enzyme then occurs rapidly, releasing acetate and regenerating the free enzyme.

The active site of the enzyme contains two distinct regions: an anionic region that contains a glutamic acid residue, and a region in which a histidine imidazole ring and a serine hydroxyl group are particularly important.

It is easy to see that the glutamic acid side-chain, ionized at pH 7, can attract the positively charged acetylcholine using ionic interactions. This allows binding and locates the ester function close to the serine side chain and the imidazole ring.

• Serine itself would be insufficiently nucleophilic to attack the ester carbonyl, so the reaction is facilitated by the participation of the imidazole ring of histidine. The basic nitrogen in this residue is oriented so that it can remove a proton from the serine hydroxyl, increasing nucleophilicity and allowing an attack on the ester carbonyl.
• This leads to the formation of the transient acetylated enzyme and the release of choline.
• Hydrolysis of the acetylated enzyme utilizes water as a nucleophile, but again involves the imidazole ring, and regenerates the free enzyme.
• Not included in this simplified description of the active site is the important role played by another amino acid residue.
• The basicity of the histidine nitrogen is increased because of the proximity of a neighboring aspartate residue. This facilitates the removal of a proton from the active site serine.
• The relationship of these three residues provides a charge-relay network, in which a charge, here a proton, is effectively passed from one molecule to another.
• In due course, the groups can be restored to their original nature by a reversal of the sequence. We shall see this feature again with chymotrypsin below.

Aspartate−histidine−serine charge relay network

Acetylcholinesterase:

Acetylcholinesterase is a remarkably efficient enzyme; turnover has been estimated as over 10 000 molecules per second at a single active site. This also makes it a key target for drug action, and acetylcholinesterase inhibitors are of considerable importance. Some natural and synthetic toxins also function by inhibiting this enzyme

Chymotrypsin and other serine proteases

The serine proteases cleave amide (peptide) bonds in peptides and have a wide variety of functions, including food digestion, blood clotting, and hormone production. They feature as one of the best-understood groups of enzymes as far as the mechanism of action is concerned.

We can ascribe a function to many of the amino acid residues in the active site, and we also understand how they determine the specificity of the various enzymes in the group.

Example:

Chymotrypsin cleaves peptides on the C-terminal side of aromatic amino acid residues phenylalanine, tyrosine, and tryptophan,

And to a lesser extent some other residues with bulky side chains,

Example:

Leu, Met, Asn, Gln. On the other hand, trypsin cleaves peptides on the C-terminal side of the basic residues arginine and lysine.

Elastase usually catalyzes the hydrolysis of peptide bonds on the C-terminal side of neutral aliphatic amino acids, especially glycine or alanine. These three pancreatic enzymes are about 40% identical in their amino acid sequences, and their catalytic mechanisms are nearly identical.

Cleavage sites for serine proteases:

Because of their known specificity, these enzymes, especially trypsin, and chymotrypsin, have been widely utilized in helping to determine the amino acid sequences of peptides (see Hydrolysis using these enzymes generate smaller peptide fragments via hydrolysis at specific amino acid residues. The shortened chains can then be sequenced and, with a little logic and reasoning, the order in which they are attached in the larger peptide can be deduced.

The differences in specificity are known to be a consequence of the amino acid sequences at the binding sites of the enzymes; these sequences are almost identical.

• Thus, trypsin and chymotrypsin differ in only one residue at the binding site.
• This residue is located in a so-called ‘pocket’ in the binding site and allows the binding of substrates containing specific amino acids in their structure.
• The pocket in chymotrypsin contains a serine residue, and the pocket provides a hydrophobic environment allowing the binding of aromatic amino acid side-chains.
• On the other hand, the trypsin pocket has an aspartate residue and binds substrates with the positively charged amino acid residues lysine and arginine.

For simplicity, the additional binding resulting from the pocket residues has not been included in the mechanistic interpretation below.

The mechanism of action of chymotrypsin can be rationalized as follows.

• The enzyme-substrate complex forms, with the substrate being positioned correctly through hydrogen bonding and interaction with the ‘pocket’ as described above.
• The nucleophilicity of a serine residue is only modest, but here it is improved by

The participation of the histidine group, with the basicity of the histidine nitrogen also being increased because of the proximity of a neighboring aspartate residue – a charge-relay network, as seen with acetylcholinesterase

• This allows nucleophilic attack on the peptide carbonyl, giving an initial tetrahedral transition state.
• We also know that specific amino acid residues are positioned so that they help to stabilize this anionic transition state.
• The formation of the carbonyl group is followed by cleavage of the peptide bond.
• The proton required to form the amino group is acquired from the imidazole. The product is now an acyl–enzyme intermediate, actually an ester involving the serine hydroxyl.
• This ester is hydrolysed by a water nucleophile, and deprotonation is achieved via the aspartate–histidine system once again.
• This generates another tetrahedral transition state, which collapses and allows the release of the carboxylic acid and regeneration of the serine hydroxyl by protonation from the imidazole system.

Note that penicillins and structurally related antibiotics are frequently deactivated by the action of bacterial β-lactamase enzymes.

• These enzymes also contain a serine residue in the active site, and this is the nucleophile that attacks and cleaves the β-lactam ring.
• The β-lactam (amide) linkage is hydrolyzed, and then the inactivated penicillin derivative is released from the enzyme by further hydrolysis of the ester linkage, restoring the functional enzyme.
• The mode of action of these enzymes thus closely resembles that of the serine proteases; . Whilst chymotrypsin and trypsin are especially useful in peptide sequence analysis, they also have medicinal applications.
• Their ability to hydrolyze proteins makes them valuable for wound and ulcer cleansing (trypsin) or during cataract removal (chymotrypsin).

Enoliztionand enolate anion biochemistry

Let us now look at an example of how nature exploits the equivalent of enol and enolate anion chemistry. Enolization provides another application of acid-base catalysis. We saw that the chemical process for enolization could be either acid- or base-catalyzed, and the following scheme should remind us of the mechanism for base-catalyzed enolization of acetone.

Base-catalysed enolization:

The enzymic processes appear exactly equivalent, except that protons are removed and supplied through the involvement of peptide side chains. It is unlikely that a distinct enolate anion is formed; instead, we should consider the process as concerted with a smooth flow of electrons. Thus, as a basic group removes a proton from one part of the molecule, an acidic group supplies a proton at another.

The example of triose phosphate isomerase in provides us with an easily understood analogy.

Enzyme-catalysed enolization:

Triose phosphate isomerase Enolization via acid-base catalysis

Triose phosphate isomerase is one of the enzymes of glycolysis and is responsible for converting dihydroxyacetone phosphate into glyceraldehyde 3-phosphate by a two-stage enolization process. An intermediate enediol is involved – this common enol can revert to a keto form in two ways, thus providing the means of isomerization

The active site of the enzyme contains a glutamic acid residue that is ionized at pH 7 and supplies the base. A histidine residue, partially protonated at pH 7, in turn, supplies the proton necessary to form the common enol

The process continues, in that the now uncharged histidine is suitably placed to remove a proton from the second of the two hydroxyls, and tautomerization is achieved by abstraction of a proton from the now non-ionized

We have seen many examples of chemical reactions involving enolate anions, and should now realize just how versatile they are in chemical synthesis.

We have also seen several examples of how equivalent reactions are utilized in nature.

• For the triose phosphate isomerase mechanism above, we did not invoke a distinct enolate anion intermediate in the enolization process but proposed that there was a smooth flow of electrons.
• For other reactions, we shall also need to consider whether enolate anions are actually involved, or whether a more favorable alternative exists. The aldol-type reaction catalysed by the enzyme aldolase is an excellent illustration of nature’s approach to enolate anion chemistry.
• Aldolase catalyzes both aldol and reverse aldol reactions according to an organism’s needs. In glycolysis, the substrate fructose 1,6-diphosphate is cleaved by a reverse aldol reaction to provide one molecule of glyceraldehyde 3-phosphate and one molecule of dihydroxyacetone phosphate.

In carbohydrate synthesis, these two compounds can be coupled in an aldol reaction to produce fructose 1,6- diphosphate

It is conceptually easier to consider initially the aldol reaction rather than the reverse aldol reaction. This involves generating an enolate anion from the dihydroxyacetone phosphate by removing a proton from the position α to the ketone group.

• This enolate anion then behaves as a nucleophile towards the aldehyde group of glyceraldehyde 3-phosphate, and an addition reaction occurs, which is completed by abstraction of a proton, typically from solvent.
• In the reverse reaction, the leaving group would be the enolate anion of dihydroxyacetone phosphate.
• Now let us consider the difficulties associated with this reaction, should we attempt it using chemical reagents. In contrast to the chemical aldol reaction,

The enzymic reaction has several remarkable advantages:

• The reaction is conducted at room temperature;
• It is conducted at pH 7 without the need for a strong base to generate the enolate anion
• Although it is a mixed aldol reaction, it is quite specific, giving a single product
• Both substrates have the potential to form an enolate anion
• Both substrates have the potential to act as an electrophile
• Dihydroxyacetone phosphate has the potential to form two enolate anions
• Other functional groups in the substrates remain unchanged;

The reaction is reversible and can be employed in either direction under similar conditions. How this is achieved with the enzyme and the role played by some of the amino acid side-chains can now be considered

Chemical aldol reaction:

Enzymic aldol reaction:

A particularly important interaction with the enzyme is that dihydroxyacetone phosphate is bound to the protein using an imine linkage between the ketone group and an amino group on the enzyme.

This produces a two fold advantage:

• First, it anchors the substrate to the enzyme through a covalent linkage; second, it allows the formation of an enamine by removal of the proton originally α to the ketone.
• An enamine is the equivalent of an enolate anion, but enamine formation is much easier than enolate anion formation and can occur without the need for a strong base.
• Proton removal is achieved by the participation of one of the basic groups on the enzyme.
• With the second substrate glyceraldehyde 3- phosphate appropriately positioned, the aldol addition can then take place, the completion of which requires a supply of a proton from the enzyme.
• The product can then be released from the enzyme by hydrolysis of the imine bond, restoring the original ketone of the substrate and the amino group on the enzyme. The reverse aldol reaction can be rationalized similarly.

The active site of an aldolase

The active site of the aldolase enzyme is believed to be as shown. Although several amino acid residues are involved with bonding the substrates at the active site, the critical amino acid residues are a lysine and an aspartic acid residue.

• The lysine forms a substrate–enzyme bond via an imine linkage, and the aspartic acid residue functions as a general acid-base.
• Basic amino acid residues are involved in binding the phosphate substrates; to simplify the overall picture, these are not specified here.
• A lysine residue reacts with the carbonyl of dihydroxyacetone phosphate, forming first an addition product that dehydrates to give an imine linkage.
• An aspartate residue is suitably positioned to function as the active site base that removes a proton from the imine and generates the enamine.
• The resultant aspartic acid residue is then involved again in providing a proton to complete the aldol addition.
• The active site also facilitates the ketone–hemiketal interconversion, so that the product liberated is the hemiketal form of fructose 1,6-diphosphate.
• In the reverse reaction, aspartate removes a proton from the alcohol, which allows the formation of a transient carbanion or enamine.

The carbanion/enamine is subsequently protonated via aspartic acid

Citrate synthase catalyzes an aldol reaction rather than a Clasien reaction

The reaction is catalyzed by citrate synthase in the Krebs cycle. is primarily an aldol reaction, but the subsequent step, hydrolysis of a thioester linkage, is also catalyzed by the same enzyme. This is shown below

Mechanistically, we can consider it as an attack of an enolate anion equivalent from acetyl-CoA onto the ketone group of oxaloacetate. However, if we think carefully, we conclude that this is not what we would predict Of the two substrates, oxaloacetate is the more acidic reagent, in that two carbonyl groups flank a methylene.

According to the enolate anion chemistry, we would predict that oxaloacetate should provide the enolate anion and that this might then attack acetyl-CoA in a Claisen reaction The product expected in a typical base-catalyzed reaction would, therefore, be an acetyl derivative of oxaloacetate

That this is not the case for the enzyme citrate synthase suggests we must look at the enzyme binding site to rationalize the different reaction sequence. It becomes clear that the enzyme binding site positions the substrates so that there are acidic and basic amino acid residues available to produce the enolate anion equivalent of acetyl-CoA (shown here as the enol), but not for the oxaloacetate

Imidazole rings of histidine residues are suitably oriented to participate in the aldol reaction. A histidine residue is also involved in the next step, the hydrolysis of citryl-CoA, and release of citric acid as the final product. It is the hydrolysis of the thioester that disturbs the equilibrium and drives the reaction to completion.

As with other examples of enzyme mechanisms, we can see that the exact array of amino acid residues in the binding site dictates binding of substrates and their chemical interaction to yield products.

Thioesters as intermediates

The reaction of an amino group with an aldehyde or ketone leads to an imine, which, as we have just seen with aldolase, provides a splendid example of how to bond a carbonyl substrate to an enzyme, and yet maintain its chemical reactivity in terms of enolate anion chemistry.

• Another type of covalent interaction is quite commonly encountered, and this exploits the thiol group of cysteine.
• Thiols are more acidic than oxygen alcohols, sulfur is a better nucleophile than oxygen, and sulfur derivatives provide better-leaving groups than the corresponding oxygen ones (see
• It is not surprising that nature makes very good use of these properties.

We shall meet several examples of this type of process, and so only the general mechanism will be considered at this stage.

The thiol group of a cysteine residue acts as a nucleophile towards a suitable carbonyl system, which may frequently be a coenzyme A ester.

• Normal addition–elimination occurs, and the leaving group is expelled. This process effectively anchors the acyl residue to the enzyme through a thioester linkage.
• This now allows a nucleophilic substrate to approach the enzyme active site, and become acylated by reacting with the bound acyl group.
• This results in regeneration of the cysteine thiol group. Protons are likely removed and supplied as necessary by the participation of general acids or bases at the active site.
• We have shown the cysteine thiol group as uncharged.

The pK_a for this group in cysteine is an application of the Henderson–Hasselbach equation indicating there will be negligible ionization at pH 7. Nevertheless, under the influence of a suitable basic group,

Example:

Arginine pK_a 12.5, ionization to thiolate may be possible. In such an environment, thiolate may act as the nucleophile in the mechanism

Enzyme inhibitors

Nature has designed enzymes to carry out modest chemical modifications on a specific substrate. In certain cases, a small number of related substrates may be modified similarly, though not always with the same efficiency, i.e. the enzyme shows broad substrate specificity.

• The chemical change catalyzed is usually small, and several enzymes will be required to change the structure of the substrate significantly. This is made clear when we consider the pathways of intermediary metabolism.
• In a few of these pathways, we shall meet examples of where several enzyme activities are combined, either as a multi-functional enzyme or as an enzyme complex where the individual components may be separated.
• This allows a significant chemical change to be catalyzed by a single protein system. Whatever the arrangement of enzymes, it is clear to see that a single enzyme activity functions as a link in a chain and, therefore, can be used to control whether or not a sequence of reactions proceeds. We can thus exploit a chain’s weakest link
• Enzyme inhibitors are chemicals that may serve as a natural means of controlling metabolic activity by reducing the number of enzyme molecules available for catalysis. In many cases, natural or synthetic inhibitors have allowed us to unravel the pathways and mechanisms of intermediary metabolism.

Enzyme inhibitors may also be used as pesticides or drugs. Such materials are designed so that they inhibit a specific enzyme that is peculiar to an organism or a disease state. For example, a good antibiotic may inhibit a bacterial enzyme, but it should not affect the host person or animal.

We may consider enzyme inhibitors as either irreversible or reversible inhibitors. Some inhibitors become covalently linked to the enzyme and are bound so strongly that they cannot be removed. As a result, the enzyme activity decreases and eventually becomes zero.

• Irreversible inhibitor: E + → EI
• Reversible inhibitor: E +  → EI

Irreversible inhibition

Irreversible inhibition in an organism usually results in a toxic effect.

Examples of this type of inhibitor are the organophosphorus compounds that interfere with acetylcholinesterase.

• The organophosphorus derivative reacts with the enzyme in the normal way, but the phosphorylated intermediate produced is resistant to normal hydrolysis and is not released from the enzyme
• The enzyme becomes inactivated, and a toxic level of acetylcholine builds up. Organophosphorus compounds provide a range of insecticides and nerve gases.

Reversible inhibitors:

Reversible inhibitors are potentially less damaging. In the presence of a reversible inhibitor, the enzyme activity decreases, but to a constant level as equilibrium is reached.

• The enzyme activity reflects the lower level of enzyme available for catalysis. We can subdivide the reversible inhibition into three types, i.e. competitive, non-competitive, and allosteric inhibition.

Competitive inhibitors

Competitive inhibitors bind to specific groups in the enzyme active site to form an enzyme–inhibitor complex. The inhibitor and substrate compete for the same site, so that the substrate is prevented from binding.

• This is usually because the substrate and inhibitor share considerable structural similarity. Catalysis is diminished because a lower proportion of molecules have a bound substrate.
• Inhibition can be relieved by increasing the concentration of substrate. Some simple examples are shown below.
• Thus, sulfanilamide is an inhibitor of the enzyme that incorporates p-aminobenzoic acid into folic acid, and has antibacterial properties by restricting folic acid biosynthesis in the bacterium.
• Some phenylethylamine derivatives,

Example:

Phenelzine provides useful antidepressant drugs by inhibiting the enzyme monoamine oxidase.

The cis-isomer maleic acid is a powerful inhibitor of the enzyme that utilizes the trans-isomer fumaric acid in the Krebs cycle.

Non-competitive inhibitors do not bind to the active site but bind at another site on the enzyme and distort the shape of the protein, resulting in a lowering of activity. Both inhibitor and substrate can bind simultaneously to the enzyme.

A non-competitive inhibitor decreases the activity of the enzyme rather than lowering the proportion of molecules with a bound substrate. In contrast to competitive inhibition, increasing the concentration of substrate has no effect on the level of inhibition. The chemical structures of non-competitive inhibitors frequently bear no similarity to the natural substrate structures

For example:

Heavy metal ions, such as Pb²⁺ and Hg²⁺, inhibit the activity of some enzymes by binding to thiol groups, and cyanide reacts with and inhibits iron–porphyrin enzymes

• The third type of inhibition is called allosteric inhibition and is particularly important in the control of intermediary metabolism.
• This refers to the ability of enzymes to change their shape (tertiary and quaternary structure when exposed to certain molecules.
• This sometimes leads to inhibition, whereas in other cases it may activate the enzyme.
• The process allows subtle control of enzyme activity according to an organism’s demands. Further consideration of this complex phenomenon is outside our immediate needs.

Angiotensin-converting enzyme (ACE) inhibitors: captopril

Captopril was the first of a range of orally active drugs to counter high blood pressure, a group known collectively as ACE inhibitors. ACE is the abbreviation for angiotensin-converting enzyme, a protein that converts the decapeptide angiotensin I into the octapeptide angiotensin 2 by hydrolytic removal of a pair of amino acids.

• Angiotensin 2 has a powerful vasoconstrictor effect, so increases blood pressure. By inhibiting the action of ACE, angiotensin 2 levels are limited, blood vessels dilate, and blood pressure is reduced.
• This is of particular value in reducing the risk of heart attacks in patients prone to high blood pressure.

ACE is a carboxypeptidase enzyme that splits off a pair of amino acids from the C-terminal end; its active site is known to contain a zinc atom.

The development of captopril was one of the first examples of successful drug design based upon knowledge of the active site of the target enzyme. It was designed to fit the known active site of carboxypeptidase A, an enzyme very similar to ACE.

Captopril resembles the terminal dipeptide cleaved from angiotensin I, in that the proline carboxylate can bind to a positive center, the amide carbonyl can hydrogen bond, and the thiol group is a good ligand for the Zn²⁺ component. Captopril is thus a competitive inhibitor of the enzyme; it can bind to the enzyme, but, in so doing, inhibits its hydrolytic action. Several captopril-like drugs are now available, their main advantage over captopril being their increased duration of action, Example:  Enalapril

Peptide Biosynthesis

Synthesis and biosynthesis of peptides and proteins requires the combination of amino acids via amide bonds. We have seen earlier that the chemical reaction of amines and acids to produce a simple amide is severely hindered by initial salt formation and that

a more efficient way of making amides is to employ a carboxylic derivative that is non-acidic and has a better-leaving group. Thus, acyl halides, anhydrides, or even esters provide better substrates. In nature, we find that esters or thioesters are the reactive species employed.

⇒\(\mathrm{RCOCl}+\mathrm{H}_2 \mathrm{NR}^{\prime} \longrightarrow \mathrm{RCONHR}^{\prime}+\mathrm{HCl}\)

⇒ \((\mathrm{RCO})_2 \mathrm{O}+\mathrm{H}_2 \mathrm{NR}^{\prime} \longrightarrow \mathrm{RCONHR}^{\prime}+\mathrm{RCO}_2 \mathrm{H}\)

⇒ \(\left.\begin{array}{l}
\mathrm{RCO}_2 \mathrm{R}+\mathrm{H}_2 \mathrm{NR}^{\prime} \longrightarrow \mathrm{RCONHR}^{\prime}+\mathrm{ROH} \\
\mathrm{RCOSR}+\mathrm{H}_2 \mathrm{NR}^{\prime} \longrightarrow \mathrm{RCONHR}^{\prime}+\mathrm{RSH}
\end{array}\right\} \begin{aligned}
& \begin{array}{l}
\text { esters and thioesters } \\
\text { are } \text { used in nature }
\end{array}
\end{aligned}\)

A further requirement for the chemical synthesis of peptides would be to take steps to avoid any sidechain functional groups reacting under the conditions used for amide bond formation.

• This can be accomplished by the use of appropriate protecting groups, though these will then have to be removed at a later stage in the synthesis.
• Nature employs enzymic reactions that position the functional groups in an appropriate orientation to react.
• Consequently, any side-chain functionalities are kept well away and do not interfere with the processes of amide bond formation.
• The final consideration is to assemble the amino acids in the correct order. In all cases, we need to choose the correct amino acid at each step, but we shall see that nature uses quite sophisticated techniques, and specificity is conferred by nucleic acids and enzymes. In the laboratory, we must pick up reagent bottles in the correct sequence.

Peptides are produced in nature by one of two methods, termed ribosomal peptide biosynthesis and non-ribosomal peptide biosynthesis.

In the former process, peptide biosynthesis takes place on the ribosomes, and the amino acid precursors are combined in a sequence that is defined by the genetic code, the sequence of bases in DNA. In nonribosomal peptide biosynthesis, peptides are synthesized by a more individualistic sequence of enzyme-controlled reactions. Despite the differences in programming the sequence, the chemical linkage of amino acid residues is achieved in a rather similar fashion.

Ribosomal peptide biosynthesis

A simplified representation of peptide biosynthesis, as characterized in the bacterium Escherichia coli. The major aspect to be considered here relates to the bond forming processes involved in linking the amino acids.

Initially, the amino acid is activated by an ATP-dependent process, producing an aminoacyl-AMP. This may be considered to be a nucleophilic attack of the amino acid carboxylate group onto the P=O system of ATP with the expulsion of diphosphate as the leaving group.

Carboxylate is not an especially good nucleophile, but we have seen it used in SN2 reactions to synthesize esters. Here, the attack is Carboxylate is not an especially good nucleophile, but we have seen it used in S_N2 reactions to synthesize esters. Here, the attack is on a reactive anhydride; a similar type of reaction is seen in fatty acid degradation

The intermediate aminoacyl-AMP can also be seen to be an anhydride but in this case a mixed anhydride of carboxylic and phosphoric acids.

This can react with a hydroxyl group in ribose, part of a terminal adenosine group of transfer-RNA (tRNA). This then binds the amino acid via an ester linkage, giving an aminoacyl-tRNA.

The tRNA involved will be specific for the particular amino acid.

Peptide bond formation is the result of two such aminoacyl-tRNA systems interacting, the amino group in one behaving as a nucleophile and displacing the tRNA from the second, i.e. simply amide formation utilizing an ester substrate. The process is repeated as required. The sequence of amino acids is controlled by messenger RNA (mRNA), the message being stored as a series of three-base sequences (codons) in its nucleotides.

Elongation of the peptide continues until a termination codon is reached, and the peptide or protein is then hydrolyzed and released from the tRNA carrier.

Non-ribosomal peptide biosynthesis

In marked contrast to the ribosomal biosynthesis of peptides and proteins where a biological production line interprets the genetic code of mRNA, many natural peptides are known to be synthesized by a more individualistic sequence of enzyme-controlled processes, in which each amino acid is added as a result of the specificity of each enzyme involved.

• The many stages of the whole process appear to be carried out by a multi-functional enzyme nonribosomal peptide synthase (NRPS) comprised of a linear sequence of modules.
• Each module is responsible for inserting a particular amino acid to generate the sequence in the peptide product. The amino acids are first activated to aminoacyl-AMP derivatives as for ribosomal peptide biosynthesis.
• These are then converted into thioesters, by reaction with thiol functions in the enzyme.

The process is exactly analogous to forming aminoacyl-tRNA units but utilizes SH rather than OH as a nucleophile.

The residues are held so as to allow a sequential series of peptide bond formations gives a simplified representation), until the peptide is finally released from the enzyme A typical module consists of an adenylation (A) domain, a peptidyl carrier protein (PCP) domain, and a condensation (C) or elongation domain.

• The A domain activates a specific amino acid as an aminoacyl-AMP mixed anhydride, which is then transferred to the PCP domain to form an aminoacyl thioester.
• The thioester linkage is not to a cysteine residue in the protein,
•  Instead, it involves pantothenic acid (vitamin B5) bound to the enzyme as pantetheine, and this is used to carry the growing peptide chain via its thiol group.

The important significance of this is that the long ‘pantheinyl arm’ allows different active sites on the multi-functional enzyme to be reached in the chain assembly process

Nucleophilic attack by the amino group of the neighboring aminoacyl thioester is catalyzed by the C domain, and this results in amide (peptide) bond formation.

• Enzyme-controlled biosynthesis in this manner is a feature of many microbial peptides, especially those containing unusual amino acids not encoded by DNA and where post-translational modification is unlikely.
• As well as activating the amino acids and catalyzing the formation of the peptide linkages, the enzyme may possess other domains that are responsible for epimerizing L-amino acids to D-amino acids probably through enol-like tautomers in the peptide.
• A terminal thioesterase domain is also required. This is responsible for terminating the chain extension process by hydrolyzing the thioester and releasing the peptide from the enzyme.

Many medicinally useful peptides have cyclic structures. Cyclization may result if the amino acids at the two termini of a linear peptide link up to form another peptide bond. Alternatively, ring formation can very often be the result of ester or amide linkages that utilize side-chain functionalities (CO₂H, NH₂, OH) in the constituent amino acids, probably through enol-like tautomers in the peptide.

A terminal thioesterase domain is also required. This is responsible for terminating the chain extension process by hydrolyzing the thioester and releasing the peptide from the enzyme.

Many medicinally useful peptides have cyclic structures. Cyclization may result if the amino acids at the two termini of a linear peptide link up to form another peptide bond. Alternatively, ring formation can very often be the result of ester or amide linkages that utilize side-chain functionalities (CO₂H, NH₂, OH) in the constituent amino acids.

Ciclosporin, a cyclic peptide composed mainly of unusual amino acids

The cyclosporins are a group of cyclic peptides produced by fungi such as Cylindrocarpon lucidum and Tolypocladium inflatum. These agents show a rather narrow range of antifungal activity, but high levels of immunosuppressive and anti-inflammatory activities. The main component from the culture extracts is cyclosporin A, but some 25 naturally occurring cyclosporins have been characterized

Cyclosporin

Cyclosporin A contains 11 amino acids, joined in a cyclic structure by peptide bonds. The structure is also stabilized by intramolecular hydrogen bonds. Only two of the amino acids, i.e. alanine and valine, are typical of proteins.

• The compound contains several N-methylated amino acid residues, together with the even less common L- α-aminobutyric acid and an N-methylated butenyl methyl threonine. There is one D-amino acid, i.e. D-alanine, and the assembly of the polypeptide chain is known to start from this residue.
• Many of the other natural cyclosporin structures differ only concerning a single amino acid (the α-aminobutyric acid residue) or the number of amino acids that have the extra N-methyl group.
• Of all the natural analogs, and many synthetic ones produced, cyclosporin A is the most valuable for drug use, under the drug name ciclosporin.

It is now widely exploited in organ and tissue transplant surgery, to prevent rejection following bone marrow, kidney, liver, and heart transplants.

It has revolutionized organ transplant surgery, substantially increasing survival rates in transplant patients. It is believed to inhibit T-cell activation in the immunosuppressive mechanism by first binding to a receptor protein, giving a complex that then inhibits a phosphatase enzyme called calcineurin.

The resultant aberrant phosphorylation reactions prevent appropriate gene transcription and subsequent T-cell activation.

Penicillins and cephalosporins are modified tripeptides

Penicillin and cephalosporin antibiotics are usually classed as β-lactam antibiotics, since their common feature is a lactam function in a four-membered ring, typically fused to another ring system. This second ring takes in the β-lactam nitrogen atom and also contains sulfur.

In the case of penicillins,

Example:  Benzylpenicillin, the second ring is a thiazolidine,

And in the cephalosporins,

Example:  Cephalosporin C, this ring is a dihydrothiazine.

What is not readily apparent from these structures is that they are both modified tripeptides and their biosyntheses share a common tripeptide precursor.

The tripeptide precursor is called ACV, an abbreviation for δ-(L- α-aminoacyl)-L-cysteinyl-D-valine. ACV is an acronym and does not refer to the systematic abbreviations for amino acids described in. ACV is the linear tripeptide that leads to isopenicillin N, the first intermediate with the fused-ring system found in the penicillins.

ACV is produced by the modular system for non-ribosomal peptide biosynthesis. The amino acid precursors are L- α-aminoadipic acid (an unusual amino acid derived by modification of L-lysine), L-cysteine, and L-valine; during tripeptide formation, the L-valine is epimerized to D-valine

Medicinally useful penicillins are formed by replacing the acyl group of the side-chain amide in isopenicillin N with an alternative acyl group. This is sometimes achieved biochemically in the fungal culture, but more frequently it is accomplished through semi-synthetic procedures.

Isopenicillin N is also the precursor of the cephalosporins, the formation of which requires a ring expansion. The five-membered thiazolidine ring of the penicillin is expanded, taking in one of the methyl groups, to produce a six-membered heterocycle

Bacterial peptidoglycans D-amino acids and the antibacterial action of penicillins

Bacterial cell walls contain peptidoglycan structures in which carbohydrate chains (composed of alternating β1 → 4-linked N-acetylglucosamine and O-lactyl-N-acetylglucosamine residues) are cross-linked via peptide structures.

Part of the peptidoglycan of Staphylococcus aureus is shown here, illustrating the involvement of the lactyl group of the O-lactyl-N-acetylglucosamine (also called N-acetylmuramic acid) in linking the peptide with the carbohydrate via an amide/peptide bond. The peptide cross-links include some D-amino acids, namely D-alanine and D-glutamic acid.

At the start of the cross-linking process, the peptide chains from the N-acetylmuramic acid residues have a terminal –Lys–D-Ala–D-Ala sequence. The lysine from one chain then becomes bonded to the penultimate Dalanine of another chain through five glycine residues, at the same time displacing the terminal D-alanine. The mechanism involves a serine residue at the active site of the enzyme. This residue is used to convert an amide linkage into an ester, and a reversal of this sequence provides the new peptide bond

Cross-linking in peptidoglycan biosynthesis:

The biological activities of the β-lactam antibiotics,

Example:  Penicillins and cephalosporins

• Stem from an inhibition of the cross-linking mechanism during the biosynthesis of the bacterial cell wall.
• The β-lactam drugs bind to enzymes (penicillin-binding proteins) that are involved in the late stages of the biosynthesis of the bacterial cell wall.
• During the cross-linking process, the peptide–D-Ala–D-Ala intermediate in its transition state conformation closely resembles the penicillin molecule.

 Enzyme inhibition by β-lactams:

As a result, the penicillin occupies the active site of the enzyme and becomes bound via the active-site serine residue.

• This binding causes irreversible enzyme inhibition and stops cell-wall biosynthesis. Growing cells are killed due to rupture of the cell membrane and loss of cellular contents.
• The binding reaction between penicillinbinding proteins and penicillins is chemically analogous to the action of β-lactamases however, in the latter case, penicilloic acid is subsequently released from the β-lactamase, and the enzyme can continue to function.
• Inhibitors of acetylcholinesterase also bind irreversibly to the enzyme through a serine hydroxyl.
• The penicillins are very safe antibiotics for most individuals. The bacterial cell wall has no counterpart in mammalian cells, and the action is thus very specific.
• However, a significant proportion of patients can experience allergic responses, ranging from a mild rash to fatal anaphylactic shock.
• Cleavage of the β-lactam ring through nucleophilic attack of an amino group in a protein is believed to lead to the formation of antigenic substances that then cause the allergic response.

Peptide Synthesis

Many different approaches have been developed for peptide synthesis, and it is not the intention to cover more than the basic principles here, with a suitable example.

• The philosophy to convert two amino acids into a dipeptide is to transform each difunctional amino acid into a monofunctional compound, one of which has the amino group protected, whilst the other has the carboxyl group protected.
• This allows the remaining amino and carboxyl groups to react, provided the carboxyl group is suitably activated to make it more reactive, as discussed above.
• After coupling and formation of the new amide bond, the product can be deprotected to yield the dipeptide. Alternatively, one or other of the protecting groups can be removed, allowing the sequence to be repeated, leading to larger peptides.

This is shown in the following general scheme.

Protecting groups

What is not included here is the need also to protect any vulnerable functional groups in the amino acid side-chains.

• A range of methods is available to protect amino, carboxyl, thiol, and hydroxyl groups and prevent them from reacting during the amide bond synthesis.
• Such groups also have to be removed after their job is done, using conditions that do not destroy the new amide bonds.
• Where amino acid sidechains have carboxylic acid or amino groups, you will readily appreciate that manipulating protecting groups on these groups separately from those related to making the peptide linkage can turn out to be a highly delicate operation.

Let us consider one method to synthesize the dipeptide Ala–Leu. It is necessary to protect the amino group of Ala and the carboxyl group of Leu.

Amino group protection:

Amino group protection may be achieved by converting the amine into its N-tert-butyloxycarbonyl (tBOC or just BOC) derivative, by reaction with di-tert-butyl dicarbonate. This reagent should be considered as a variant of a carboxylic acid anhydride; it reacts in just the same way. The product is termed BOC-Ala and is strictly a carbamate, a half ester–half amide of carbonic acid.

Protection of amino group: tBOC

Carbamates behave like amides; the amino group is no longer basic or nucleophilic. The BOC-protecting group can thus be removed readily by treating it with dilute aqueous acid.

The process involves protonation, loss of the tert-butyl cation, and then decarboxylation. On the other hand, the carbonyl group is too hindered to be attacked by the base.

Removal of tBOC protecting group:

Carboxyl protection:

Carboxyl protection of the second amino acid is usually achieved by conversion to an ester using an appropriate alcohol and acidic catalyst Although methyl and ethyl esters work perfectly well, their removal typically requires alkaline hydrolysis, which may be undesirable. More acceptable are esters that can be removed via catalytic hydrogenolysis,

Example: Benzyl esters.

Protection of carboxyl group: benzyl ester:

The dicyclohexyl carbodiinide coupling reaction:

Activation of the carboxyl and coupling may be achieved through the use of a single reagent, dicyclohexylcarbodiimide (DCC).

• This compound removes a proton from the carboxylic acid, producing a cation that is readily attacked by the carboxylate nucleophile across one of the C–N double bonds – the protonated imine behaves as a good electrophile.
• The product is now an activated ester (an O-acylisourea) that can be attacked by any available nucleophile. The amino group of the second amino acid derivative provides the nucleophile, resulting in the expulsion of a very stable urea as the leaving group, and production of the protected dipeptide.

DCC is a very attractive reagent, in that there is no need to generate the activated derivative separately. One merely mixes the two protected amino acid derivatives in an aprotic solvent such as CH₂Cl₂, adds DCC, and dicyclohexylurea is removed as an insoluble by-product. The desired dipeptide can then be obtained by removal of the protecting groups, as already outlined. Note that, in the example shown, we are extending the chain by adding new amino acid residues to the carboxyl terminus

Pepti de synthesis on polymeric supports

Synthesis of peptides in solution using the method outlined above, or alternative procedures, is laborious and often low-yielding since each intermediate needs isolating and purifying at each stage of the synthesis.

An alternative approach developed by Merrifield is to attach the growing peptide chain to a polymer, which renders it insoluble. This allows the use of excess reagents, and the removal of impurities merely by washing the polymer, which is usually in the form of beads. This approach is the basis of automated peptide synthesizers since the process can be fast, simple, and readily repeated.

In the initial step, the first BOC-protected amino acid is bound to the polymer, e.g. polystyrene in which a proportion of the phenyl rings have chloromethyl substitution. Attachment to these residues is through the carboxyl via an ester linkage.

This involves a simple nucleophilic substitution reaction, with the carboxylate as the nucleophile and chloride as the leaving group.  After each stage, the insoluble polymer–product combination is washed free of impurities

The BOC-protecting group is then removed from the amino acid, allowing the next protected amino acid to be bonded to the polymer-bound substrate via the DCC coupling reaction.

The processes of BOC removal and DCC coupling are then repeated with as many amino acid residues as required. This procedure extends the chain by adding new amino acid residues to the amino terminus.

Finally, the polypeptide is released from the polymer by treatment with HF. All the steps are carried out without isolating any intermediate. An early peptide synthesizer produced the 125 amino acid protein ribonuclease in an overall yield of 17%, a quite staggering achievement

Determination Of Peptide Sequence

Chemical methods for determining the amino acid sequence of a peptide or protein have been developed, and the normal approach is to exploit the properties of the amino group at the N-terminus. A long-established procedure for identifying the N-terminal amino acid is use of the Sanger reagent 2,4-dinitrofluorobenzene. This reacts with an amine by nucleophilic displacement of the fluorine.

Normally, substitution on a benzene ring is achieved by electrophilic attack, with subsequent loss of a proton. With the Sanger reagent, the presence of three strongly electron-withdrawing substituents allows nucleophilic attack and then displacement of fluoride as a leaving group.

• The initial addition of a nucleophile to the aromatic system generates a transient carbanion, which is stabilized by the nitro groups. Charge is then lost by expelling fluoride as a leaving group, restoring the aromatic ring system
• We have already noted that fluoride is not normally a very effective leaving group. Here, the nucleophilic addition is the rate-determining step, though it is favored by the very large inductive effect from the fluorine and the stabilization from the nitro groups.
• This allows the formation of the additional carbanion, and, even though fluoride is a poor leaving group, it can be lost from the anion to restore aromaticity.

This type of reaction is strictly an addition–elimination mechanism but is referred to as an S_NAr mechanism, or nucleophilic aromatic substitution.

After treatment of the peptide with the Sanger reagent, all peptide bonds are then cleaved by hydrolysis, giving a mixture of amino acids, with the N-terminal one carrying a 2,4-dinitrophenyl group. Being yellow, this compound is readily detected and can be characterized easily by chromatographic comparison with standards.

Although 2,4-dinitrofluorobenzene will also react with any free amino group in an amino acid side-chain,

Example: That in lysine, only the N-terminal amino acid will carry the

2,4-dinitrophenyl residue in its α-amino group. A more useful procedure, in that it allows sequential determination of the N-terminal amino acids in a peptide, is the Edman degradation. This process removes the N-terminal amino acid, but leaves the rest of the chain intact, so allowing further reactions to be applied. The reagent used here is phenyl isothiocyanate

Edman degradation

The carbon in the isothiocyanate grouping is highly susceptible to nucleophilic attack by the peptide’s free amino group. Overall addition to the C=N creates a thiourea derivative. Making the conditions strongly acidic then promotes nucleophilic attack by the sulfur of the thiourea onto the carbonyl of the first peptide bond, producing a five-membered thiazoline heterocycle.

Proton loss occurs from the nitrogen, and this creates an intermediate that is equivalent to the addition product in simple acid-catalyzed amide hydrolysis, though here we have employed a sulfur rather than an oxygen nucleophile. Bond cleavage follows, leaving the first amino acid as part of a thiazolinone system. The rest of the peptide chain is unaffected.

Thus, the N-terminal amino acid can be identified by analysis of the thiazolinone, and the process can be repeated on the one-unit-shortened polypeptide chain. Under the acidic conditions, the thiazolinone is actually unstable, and rearranges to a phenylthiohydantoin.

The reasons for the rearrangement need not concern us; a mechanism is shown merely to demonstrate that it can be rationalized. The phenylthiohydantoin derivative produced can be identified simply by chromatographic comparison with authentic standards

The repetitive cycle to identify a sequence of N-terminal amino acids has been automated. In practice, it is limited to about 20–30 amino acids, since impurities build up and the reaction mixture becomes too complex to yield unequivocal results. The usual approach is to break the polypeptide chain into smaller fragments by partial hydrolysis, preferably at positions relating to specific amino acid residues in the peptide chain.

There are ways of doing this chemically, and the enzymes chymotrypsin and trypsin are also routinely used for this purpose. The shortened chains can then be sequenced and, with a little logic and reasoning, the order in which they are attached can be deduced, leading us to the entire amino acid sequence. The process can be exemplified using a simple hypothetical example containing 12 amino acid residues, although, in practice, this is small enough to be achieved by an automatic amino acid sequencer.

The N-terminal amino acid can be ascertained by the Sanger method. Enzymic cleavage using either chymotrypsin or trypsin will break the peptide into smaller fragments. The fragments obtained will be different, depending on the enzyme and its specificity. The smaller fragments are then each sequenced by the Edman technique.

C-terminal residues in the smaller peptides can be related to knowledge of the enzyme cleavage sites; this may point to the C-terminal residue of the full peptide if it does not correspond to an enzymic cleavage site. The full sequence can be deduced from these fragments by lining up matching sequences of overlapping portions.

The post Amino Acids Peptides And Proteins appeared first on BDS Notes.

A reaction mechanism is a detailed step-by-step description of a chemical process in which reactants are converted into products.

It consists of a sequence of bond-making and bond-breaking steps involving the movement of electrons and provides a rationalization for chemical reactions. Above all, following a few basic principles allows one to predict the likely outcome of a reaction.

On the other hand, it must be appreciated that there will be times when it can be rather difficult to actually ‘prove’ the proposed mechanism, and in such instances, we are suggesting a reasonable mechanism consistent with experimental data.

The basic layout of this book classifies chemical reactions according to the type of reaction mechanism involved, not by the reactions undergone within any specific group of compounds. As we proceed, we shall meet several types of general reaction mechanisms.

Initially, however, reactions can be classified as ionic or radical, according to whether bond-making and bond-breaking processes involve two electrons or one electron respectively.

Ionic Reactions

As the name implies, ionic reactions involve the participation of charged entities, i.e. ions. Bond-making and bond-breaking processes in ionic reactions are indicated by curly arrows that represent the movement of two electrons.

The tail of the arrow indicates where the electrons are coming from, the arrowhead where they are going.

Lone pairs, originally nonbonding electrons, can also be used in bond-making processes.

These simple examples illustrate the basic rules for mechanism and the use of curly arrows. The concepts are no different from those we have elaborated for drawing resonance structures:

1. Curly arrows must start from an electron-rich species. This can be a negative charge, a lone pair, or a bond.
2. Arrowheads must be directed towards an electron-deficient species. This can be a positive charge, the positive end of a polarized bond, or a suitable atom capable of accepting electrons, for example, an electronegative atom or Lewis acid.

If we are to draw sensible mechanisms, putting in the correct number of bonds and assigning the correct charges, then we must know the number of electrons around any particular atom.

We have already considered how to assess the formal charge on an atom; the following resume´ e´ covers those occasions that we are most likely to meet.

Carbon has four bonding electrons and can attain a stable octet of electrons by bonding to four other atoms, i.e. it has a valency of four.

Carbon can also bond to just three other atoms by donating a pair of electrons from the octet to one of the atoms originally bonded, in so doing breaking the bond.

It will then carry a positive charge; it has effectively donated its single electron contribution from the shared pair comprising the single bond. This positively charged carbon is called a carbocation (in older nomenclature a carbonium ion).

Note that, with only six electrons involved in bonding, the carbocation is a planar entity, having two electrons in each of three sp² orbitals and with an unfilled p orbital.

Alternatively, carbon can carry a negative charge if it accepts both electrons from one of the original bonds, leaving the other group electron-deficient and positively charged.

It has effectively gained a single electron and is termed a carbanion. In this case, carbon carries a full octet of electrons and is tetrahedral, as if it had four single bonds.

The lone pair of electrons occupy the fourth sp³ orbital. Remember, carbon cannot form more than four bonds! Nitrogen has three bonding electrons and a lone pair; it can bond to three atoms, i.e. it has a valency of three.

However, it can also bond to four atoms by donating its lone pair, in which case it will then carry a positive charge.

Nitrogen can also bond to just two atoms. Here, it carries a negative charge, since the octet is made up by acquiring one electron. Oxygen has two bonding electrons and two lone pairs. It can bond to two other atoms and is usually divalent.

It can also bond to one atom in a negatively charged form, or to three atoms in a positively charged form. The oxonium cation produced still carries a lone pair, but these electrons will not participate in further bonding, since this would necessitate an unfavourable double-charged oxygen.

Hydrogen has one bonding electron and can bond to one other atom; it is monovalent. The electrons in this bond can be donated to hydrogen, giving the hydride anion, or can be donated to the other atom, generating a proton.

The proton thus contains no electrons. This seems a rather unnecessary statement, but it means a proton can only be an acceptor of electrons, and can never donate any. Curly arrows may be directed towards protons, but can never start from them! This would be a serious mechanistic error. Nevertheless, most students seem to make this error at some time or other.

Counting the number of electrons on a particular atom becomes even more important when mechanisms become a little more complex and involve the making and breaking of bonds at the same atom.

This is going to be routine at carbon atoms, and the statement above, that ‘carbon cannot form more than four bonds’, becomes an important guiding principle.

Any mechanism that adds electrons to a carbon atom that is already carrying its full octet of electrons will also require the breaking of a bond and the removal of the excess electrons. Initially, it is a good idea to show nonbonding electrons in a mechanism, so that the number of electrons can be assessed and the correct charges defined.

In due course, it is quicker to draw mechanisms without all the lone pairs, and it is normal practice to use representations showing just charges and only the lone pairs involved in subsequent bonding. The following mechanisms omit the lone pairs not involved in bonding but are perfectly acceptable.

Bond polarity

The concept of bond polarity has been discussed in some detail in Chapter 2 (see Section 2.7). Because different atomic nuclei have a particular ability to attract electrons, bonds between unlike atoms may not be shared equally.

This leads to a charge imbalance, with one of the atoms taking more than its share of the electrons. We refer to this as bond polarity.

An atom that is more electronegative than carbon will thus polarize the bond, and we can consider the atoms as being partially charged.

This is indicated in a structure by putting partial charges ( δ+ and δ−) above the atoms. It can also be represented by putting an arrowhead on the bond in the direction of electron excess.

The relatively small difference in electronegativities between hydrogen and carbon means there is not going to be much polarity associated with a C–H bond. Most atoms other than hydrogen and carbon when bonded to carbon are going to be electron-rich and bonds may therefore display considerable polarity.

This is illustrated by carbon-oxygen and carbon-nitrogen single bonds. Double bonds show even greater polarity. This polarity helps us to predict chemical behaviour and is crucial to our prediction of chemical mechanisms.

Nucleophiles, Electrophile Les, And Leaving Groups

Reagents are classified as nucleophiles or electrophiles. Nucleophiles are electron-rich, nucleus-seeking reagents, and typically have a negative charge (anions) or a lone pair.

Compounds with multiple bonds, for example, alkenes, alkynes, and aromatics can also act as nucleophiles in so-called electrophilic reactions.

Electrophiles are electron-deficient, electron-seeking reagents, and typically have a positive charge (cations) or are polarizable molecules that can develop an electron-deficient centre.

Many reactions will involve both nucleophiles and electrophiles. These may then be classified as nucleophilic if the main change to the substrate involves the attack of a nucleophile, or electrophilic if the principal change involves an attack of the substrate onto an electrophile. This distinction will become clearer in due course. The electron-rich species is always regarded as the attacking agent

Leaving the group is the terminology used for ions or neutral molecules that are displaced from a reactant as part of a mechanistic sequence. Frequently, this displacement is the consequence of a nucleophile attacking an electrophile, and where the electrophile carries a suitable leaving group.

Good leaving groups are those that form stable ions or neutral molecules after they leave the substrate. We shall frequently need to write mechanisms involving general nucleophiles, electrophiles or leaving groups.

Standard abbreviations are Nu− or Nu: for a nucleophile (charged or uncharged), E+ for an electrophile, and L− or L: for a leaving group. In many instances, an electrophile containing a leaving group would simply be represented by C–L.

Radical Reactions

Radicals (sometimes termed free radicals) are uncharged high-energy species with an unpaired electron, and may contain one or more atoms:

For clarity, nonbonding electrons are usually omitted, though to propose meaningful mechanisms it is important to remember how many electrons are associated with each atom. The unpaired electron must always be shown.

In the formation of radicals, a bond is broken and each atom takes one electron from the pair constituting the bond. Bond-making and bond-breaking processes are indicated by single-headed (fishhook) curly arrows representing the movement of one electron.

A radical mechanism sequence requires three distinct types of process: initiation, propagation, and termination.

Initiation is the formation of two radical species by bond fission, whereas propagation involves the reaction of a radical with a neutral molecule, a process that leads to the generation of a new radical. Because radicals are so reactive, the propagation process may continue as long as reagent molecules are available.

Finally, the reaction is brought to a conclusion by the combination of two radical species, so that the unpaired electrons, one from each species, are combined into a new single bond. The radical pairing termination step is analogous to a reversal of the initiation step.

It occurs readily because of the reactivity of radicals; it follows, therefore, that the initiation step will require the input of a considerable amount of energy to dissociate the single bond. In the propagation steps shown above, the radical propagates a further radical by causing the fission of a single bond in the substrate. Many important radical reactions involve compounds with double bonds as substrates, and the π bond is cleaved during the radical addition reaction.

It makes good sense to draw free-radical mechanisms in the manner shown by these examples. However, shorter versions may be encountered in which not all of the arrows are drawn.

These versions bear considerable similarity to two-electron curly arrow mechanisms, in that a fishhook arrow is shown attacking an atom, and a second fishhook arrow is then shown leaving this atom. The other electron movement is not shown but is implicit.

This type of representation is quite clear if the complement of electrons around a particular atom is counted each time; but, if in any doubt, use all the necessary fishhook arrows.

Reaction Kinetics And Mechanism

One of how we can obtain information about a mechanistic sequence is to study the rate of reaction.

The dependence of the reaction rate on the concentration of reagents and other variables indicates the number and nature of the molecules involved in the rate-determining step of the reaction.

The rate-determining step is defined as the slowest transformation in the sequence, with all other transformations proceeding much faster than this. Consider a turnstile at a football match.

This limits the rate at which spectators enter the ground. How rapidly people walk towards the turnstile or away from it once they are in the ground cannot influence the rate at which they get through the turnstile.

The rate of reaction is given by the equation in which k is the rate constant, and A, B, etc. are the variables on which the rate depends. Square brackets are used to indicate concentrations. It is rare for more than two variables to be involved, and often it is only one.

The most common types of rate expression. In first-order reactions, the rate expression depends upon the concentration of only one species, whereas second-order reactions show dependence upon two species, which may be the same or different.

The molecularity, or number of reactant molecules involved in the rate-determining step, is usually equivalent to the kinetic reaction order, though there can be exceptions.

For instance, a bimolecular reaction can appear to be first-order if there is no apparent dependence on the concentration of one of the reagents. Such a situation might occur when the solvent was also one of the reagents.

Despite occasional apparent anomalies such as this, the rate expression gives us valuable information about the likely reaction mechanism. If the reaction is unimolecular, the rate-determining step involves just one species, whereas the rate-determining step involves two species if it is bimolecular.

We can then deduce the probable reaction, and our proposed mechanism must reflect this information. The kinetic rate expressions will be considered further as we meet specific types of reactions.

Intermediates And Transition States

Any realistic mechanism will include several postulated structures, perhaps charged structures or radicals, which lie on the pathway leading from reactants to products. Some of these intervening structures are termed intermediates, and others transition states.

These are differentiated by their stability, and whether they can be detected by appropriate analytical methods. A diagram that follows the energy change during the reaction can illustrate their involvement.

The xcoordinate is usually termed the ‘reaction coordinate’, and in many cases equates to time, though the possibility that the reaction is reversible prevents us from showing this as a simple time coordinate.

In which reactants are converted into products. The difference between the energy of the reactants and products is called the standard free energy change for the reaction. As shown, the change in energy is negative, so that the reaction liberates energy and is potentially favourable.

It does not occur spontaneously, however, since the reactants need to acquire sufficient energy to collide and react.

This energy is termed the activation energy – even gunpowder needs a match to set off the explosion! The high-energy peak in the curve is termed the transition state or sometimes, activated complex. This material cannot be isolated, or even detected.

In an alternative scenario, again with a negative energy change, the energy profile may appear different, In this case, there is again an activation energy required to set the reaction off, but this energy maximum is then followed by an energy minimum.

The energy minimum represents an intermediate in the reaction pathway. It is converted into the products by overcoming a second activation energy, though this is likely to be considerably less than the first activation energy.

Because the intermediate is at an energy minimum, this material may be stable and can be isolated, or it may be reactive and short-lived, but detectable.

The two energy maxima represent different transition states. The energy diagrams shown here are merely generalized examples. We shall meet some specific examples as we consider various reaction mechanisms.

Types Of Reaction

At first glance, there appear to be an infinite number of different chemical reactions, all of which will have to be remembered.

A cursory look through any textbook of organic chemistry does little to dispel this fear. However, the beauty and strength of the mechanism is that it allows us to predict chemical behaviour without having to remember lots of chemical reactions.

A further reassuring fact is that virtually all of the chemical reactions can be classified according to a reaction type, and the number of distinct reaction types is rather few.

We only need to consider reaction types according to what is achieved in the conversion, namely substitution, elimination, addition, or rearrangement. In general terms, these may be represented as follows.

We will then subdivide these reaction types according to the type of reagent that brings about the change, to rationalize typical reactions further.

For example, addition reactions can be subdivided into nucleophilic addition, electrophilic addition, or radical addition.

Whilst this does increase the number of permutations, we shall see that it is necessary to do this, and it is also perfectly logical for our understanding of how reactions occur.

Arrows

We have now encountered several different types of arrows routinely used in chemistry to convey particular meanings. We have met curly arrows used in mechanisms, double-headed resonance arrows, equilibrium arrows, and the simple single arrows used for reactions.

This is a convenient point to bring together the different types and provide a checklist for future reference. We are also showing how additional information about a reaction may be presented with the arrow.

Some Common Mistakes In Drawing Mechanisms

Experience tells us that whilst many students find mechanisms easy and logical, others despair and are completely bewildered. We cannot guarantee success for all, but we hope that by showing a few of the common mistakes we may help some of the latter group join the former.

To make the examples chosen as real as possible, these have all been selected from students’ examination answers.

The mechanisms relate to reactions we have yet to meet, but this is not important. At this stage, it is the manipulation of curly arrows that is under consideration. You may wish to return to this section later.

Mistakes With Valencies

As electrons are moved around via curly arrows, it is imperative to remember how many electrons are associated with a particular atom, and not to exceed the number of bonds permitted.

The usual clanger is five-valent carbon, typically the result of making a new bond to a fully substituted carbon (four bonds, eight electrons) without breaking one of the old bonds. This is the case in the example shown.

Mistake with formal charges

It is also important when counting electrons to assign any formal charge as necessary. It is all too common to see hydroxide presented with a lone pair but without any charge. Unfortunately, subsequent ionic reactions then just do not ‘balance’.

If one considers that hydroxide is derived by ionization of NaOH, or by loss of a proton from H2O, this problem should not arise.

Arrows From Protons

Ask yourself how many electrons are there in a proton. We trust the answer is none, and you will thus realize that arrows representing the movement of electrons can never start from a proton.

It seems that this mistake is usually made because, if one thinks of protonation as the addition of a proton, it is tempting to show the proton being put on via an arrow. With curly arrows, we must always think in terms of electrons.

We were even less keen on the second example, where, in the resonance delocalization step, an arrow is shown taking electrons away from a positive charge and creating a new positive centre.

Vague arrows

Some mechanisms have arrows going in all sorts of directions. Arrows must ‘flow’ from start to finish; they should not veer off in different directions. Many of the arrows do not represent electron movements, and it would appear that, as a last resort, students have tried to memorize the mechanism rather than rationalizing it.

This is both dangerous and rather unnecessary. The logical approach gets the right answer, requires relatively little effort, and cuts out the need to learn the mechanism. Mechanisms should not be learnt; they should be deduced.

In this example, the student remembered that a series of curly arrows was required, and they are generally in the right places, but not coming from electron-rich species, and not flowing in the right direction. This is typical of trying to remember a mechanism, which then fails to obey the general rules.

Too many steps at once

It is tempting to draw a mechanism with a series of curly arrows leading to the product via the minimum number of structures. We can often use several curly arrows in the same structure, but only provided we do not destroy the rationale for the mechanism.

In the example shown, the two curly arrows suggest a concerted interaction of three entities. This is improbable and does not tell us why the reaction should take place. Using the longer sequence, we see that the acid catalyst activates the carbonyl group towards nucleophilic attack, and is later regenerated.

The second example also emphasizes that base is needed to generate the nucleophile, the charged phenoxide being a better nucleophile than the phenol.

In the third example, arrows veer off in different directions, rather than flowing smoothly from start to finish. This mechanism is wrong in that an intermediate has been omitted.

Unrealistic izations

It is often necessary to ionize one of the reagents to initiate a reaction, and this requires careful consideration if the mechanism is to be realistic.

For example, we should not attempt to protonate substrates under basic conditions, and we are unlikely to generate anionic species under acidic conditions. These are fairly obvious limitations but are frequent mistakes.

Some thought about relative acidity and basicity is also sensible; ionization of alcohols does not occur without a strong base, as suggested in the example.

Primary Carbocations

Should you wish to use carbocations in a reaction mechanism, you must consider the relative stability of these entities. Tertiary carbocations are OK, and in many cases so are secondary carbocations.

Primary carbocations are just not stable enough, unless there is the added effect of resonance, as in benzylic or allylic systems.

Arrows curled the wrong way Can arrows curl the wrong way? Yes, they can, as the example shows. You should always understand that the arrowhead is depositing electrons between the start of the arrow and the atom it is approaching so that the new bond is formed at the inside of the curl.

The electrophilic addition to alkenes is one of the occasions when the direction of the curl matters and can convey the formation of different products. Although the product shown is correct, the curly arrow is wrong.

Making bonds to O+ or N+

It is tempting to consider O+ and N+ as electron-deficient species and, therefore, open to attack by nucleophiles. Here, we must count electrons to appreciate the true nature of these charged systems.

Both O+ bonded to three atoms and N+ bonded to four atoms are isoelectronic with tetravalent carbon, in other words, they have a full octet of electrons. Despite the positive charge, these atoms are not electron-deficient and are unable to make a new bond with the electron-rich nucleophiles.

The post Reaction Mechanisms appeared first on BDS Notes.

As the term suggests, a substitution reaction is one in which one group is substituted for another. For nucleophilic substitution, the reagent is a suitable nucleophile and it displaces a leaving group. As we study the reactions further, we shall see that mechanistically related competing reactions, eliminations, and rearrangements, also need to be considered.

The SN2 reaction: bimolecular nucleophilic substitution The abbreviation SN2 conveys the information ‘substitution–nucleophilic–bimolecular’.

The reaction is essentially the displacement of one group, a leaving group, by another group, a nucleophile. It is a bimolecular reaction since kinetic data indicate that two species are involved in the rate-determining step

Rate = k[RL][Nu]

Nu is the nucleophile, RL is the substrate containing the leaving group L, and k is the rate constant. In general terms, the reaction can be represented as below.

Differences in electronegativities between carbon and the leaving group atom lead to bond polarity. This confers a partial positive charge on the carbon and facilitates the attack of the nucleophile.

As the nucleophile electrons are used to make a new bond to the carbon, electrons must be transferred away to a suitable acceptor to maintain the carbon’s octet.

The suitable acceptor is the electronegative leaving group. The nucleophile attacks from the side opposite the leaving group – electrostatic repulsion prevents attack in the region of the leaving group.

This results in an inversion process for the other groups on the carbon center under attack, rather like an umbrella turning inside out in a violent gust of wind.

The process is concerted, i.e. the bond to the incoming nucleophile is made at the same time as the bond to the leaving group is being broken.

As a consequence, the mechanism involves a high-energy transition state in which both the nucleophile and leaving group are partially bonded, the Nu–C–L bonding is linear, and the three groups X, Y, and Z around carbon are in a planar array.

This is the natural arrangement to minimize steric interactions if we wish to position five groups around an atom, and will involve three sp2 orbitals and a p orbital as shown.

The p orbital is used for the partial bonding; note that we cannot have five full bonds to a carbon atom. The energy profile for the reaction proceeds from reactants to products via a single high-energy transition state.

The rate of an SN2 reaction depends upon several variables. These are:

1. The nature of the substituents bonded to the atom attacked by the nucleophile;
2. The nature of the nucleophile;
3. The nature of the leaving group;
4. Solvent effects.
5. We can consider these in turn.
6. The effect of substituents

The SN2 mechanism requires the attack of a nucleophile at the rear of the leaving group, and consequently, the size of the groups X, Y, and Z will influence the ease of approach of the nucleophile.

Experimental evidence shows the relative rates for SN2 reactions of halides. This is primarily a result of steric hindrance increasing as one goes from primary to secondary to tertiary compounds.

With the tert-butyl group, the approach of the nucleophile is hindered by three methyl groups, so much so that the SN2 reaction is not normally possible.

SN2 reactions: The racemization of 2-iodobutane

The inversion in an SN2 reaction can be demonstrated in a rather simple experiment. If (+)-(R)-2-iodobutane is heated in acetone solution, it is recovered unchanged.

However, when sodium iodide is added to the mixture, there is no apparent chemical change, but the optical activity gradually diminishes until it becomes zero, i.e. racemic (±)-(RS )-2-iodobutane has been formed.

In this reaction, an equilibrium is set up. The nucleophile, iodide, is the same as the leaving group. Therefore, inversion of configuration merely converts the (+)-isomer into the (−)-isomer.

As a result, the optical activity gradually disappears and ultimately becomes zero as the mixture becomes the racemic (±)-form.

We are never going to get a complete conversion of the (+)- into (−)-enantiomer because the reverse reaction will also occur.

This is mechanistically identical to the forward reaction, so either (+)- or (−)-2-iodobutane as starting material would give a racemic product, i.e. it is a racemization reaction.

This is an unusual reaction, in that the energy of the products will be identical to the energy of the reactants, though the interconversion of isomers involves activation energy that must be overcome by the application of heat.

Should a reaction be attempted with tertiary substrates, one does not usually get a substitution, but alternative side reactions occur.

If the potential leaving group is attached to unsaturated carbon, as in vinyl chloride or phenyl chloride, attack by nucleophiles is also extremely difficult, and these compounds are very unreactive in SN² reactions compared with simple alkyl halides.

In these cases, the reason is not so much steric but electrostatic, in that the nucleophile is repelled by the electrons of the unsaturated system.

In addition, since the halide is attached to carbon through a sp2-hybridized bond, the electrons in the bond are considerably closer to carbon than in a sp3-hybridized bond of an alkyl halide.

Lastly, resonance stabilization in the halide gives some double bond character to the C–Hal bond. This effectively strengthens the bond and makes it harder to break. This lack of reactivity is also true for SN¹ reactions.

Nucleophiles: nucleophile city and basicity

The SN²-type reaction can be considered simply as being initiated by the attack of a nucleophile onto the electron-deficient end of a polarized bond X–Y.

If X = H, then this equates to the removal of a proton and we would consider the nucleophile to be a base. It follows that there is going to be a close relationship between a group’s capacity to act as a nucleophile, i.e. nucleophilicity, and its ability to act as a base, i.e. basicity. Thus, the hydroxide ion can act as a nucleophile or as a base.

In many cases, nucleophilicity can be correlated with basicity, and this forms a helpful way of predicting how good a potential nucleophile may be. The sequences of relative basicity are also reflected in relative nucleophilicities.

The approximation works best for comparisons where the identity of the attacking atom is the same, for Example N, or O, as illustrated. The correlation is useful but not exact.

This is because basicity is a measure of the position of equilibrium between a substrate and its conjugate acid, whereas nucleophilicity relates to the rate of reaction.

The above relationship breaks down when one looks at atoms in the same column of the periodic table.

As atomic number increases, basicity decreases, whilst nucleophilicity increases so that electrons associated with larger atoms become less localized, consequently forming weaker bonds with protons the other hand, electrons in the larger atoms are more easily polarizable, and it becomes easier for them to be donated to an electrophile; this leads to greater nucleophilicity. Despite these inconsistencies, there are two important features worth remembering

1. An anion is a better nucleophile than an uncharged conjugate acid;
2. Strong bases are good nucleophiles.

Selective alkylation of m morphine to codeine and pholcodine Opium is a crude exudate obtained from the opium poppy Papaver somniferum, and it provides several medicinally useful alkaloids. One of these is codeine, which is widely used as a moderate analgesic.

Opium contains only relatively small amounts of codeine (1–2%), however, most of the codeine for drug use is obtained by semi-synthesis from morphine, which is the major component (12–20%) in opium. Conversion of morphine into codeine requires selective methylation of the phenolic hydroxyl. This can be achieved by an SN2 reaction under basic conditions.

Morphine has two hydroxyls, but one is a phenol and the other is an alcohol. Because phenols (pKa about 10) are considerably more acidic than alcohols (pKa about 16), only the phenol will become ionized under mild basic conditions.

Since the phenolate anion (charged) will then be a much better nucleophile than the alcohol hydroxyl (uncharged), the SN2 reaction will selectively involve the phenolate group. The alcohol group does not react under these conditions.

The methylating agent (electrophile) used in this reaction is dimethyl sulfate; the leaving group is the anion of a sulfuric acid ester and is the conjugate base of a strong acid.

The same type of reasoning allows the production of pholcodine, an effective cough suppressant, from morphine. In this semi-synthesis, the electrophile is N-(chloroethyl)morpholine, and the leaving group is chloride.

Solvent effects

Nucleophilicities are affected by solvents, and any correlations with basicity can break down in protic solvents like methanol or ethanol.

This is because anions are stabilized by hydrogen bonding, and become solvated. These solvation molecules must be lost before the anion can attack as a nucleophile.

Accordingly, better solvents for nucleophilic substitution reactions are the so-called aprotic polar solvents, which contain no protons that allow hydrogen bonding to occur. Anions, consequently, become more nucleophilic in aprotic polar solvents than they are in protic solvents.

As an example, the SN² reaction of chloride with methyl iodide leading to methyl chloride is some 106 times faster in dimethylformamide (DMF) than in methanol. This is because there is no hydrogen bonding possible in DMF.

In sharp contrast, the reaction in the structurally similar solvent N-methyl formamide (HCONHMe), which still contains an N–H that can participate in hydrogen bonding, is only 45 times as fast as in methanol.

Chloride ions actually form stronger hydrogen bonds with methanol than with Nmethylformamide, so there is an increase in reactivity, but hardly as dramatic as with the aprotic solvent DMF.

Leaving groups

The nature of the leaving group is a further important feature of nucleophilic substitution reactions. For the SN² reaction to proceed smoothly,

We need to generate strong bonding between the nucleophile and the electrophilic carbon, at the same time as the bonding between this carbon and the leaving group is weakened.

The high-energy transition state may thus be considered to require the general characteristics shown in the scheme below.

Good leaving groups are those that form stable ions or neutral molecules after they leave the substrate. Consequently, the capacity of a substituent to act as a leaving group can also be related to basicity.

Strong bases (the conjugate bases of weak acids) are poor leaving groups; but, as we have seen above, they are good nucleophiles. On the other hand, weak bases (the conjugate bases of strong acids) are good leaving groups, but they make poor nucleophiles.

We can now understand and predict why some nucleophilic substitution reactions are favored and others are not. Thus, it is easy to convert methyl bromide into methanol by the use of hydroxide as a nucleophile.

On the other hand, it is not feasible to convert methanol into methyl bromide merely by using bromide as the nucleophile.

The difference here is primarily due to the nature of the leaving groups. Bromide is a weak base and a good leaving group, whereas hydroxide is a strong base and, therefore, a poor leaving group.

Nevertheless, the latter transformation can be achieved by improving the ability of the leaving group to depart by carrying out the reaction under acidic conditions.

Thus, protonation of the substrate via the oxygen lone pair produces the conjugate acid. This now has greater polarization favoring nucleophilic attack and, most importantly, changes the leaving group from hydroxide (a strong base) to water (a weak base).

The reaction is now facilitated and proceeds readily. Chemical modification of poor leaving groups into good leaving groups may also be considered as a way of enhancing the ease of substitution reactions.

Two important reagents that may be used with alcohols are p-toluenesulfonyl chloride (tosyl chloride) and methanesulfonyl chloride (mesyl chloride). Both anions p-toluenesulfonate (tosylate) and methanesulfonate (mesylate) are excellent leaving groups, being the conjugate bases of strong acids (pKa < 0).

SN2 reactions in cyclic systems

The inversion process accompanying SN2 reactions may have particular significance in cyclic compounds. Thus, if we consider the disubstituted cyclopentane derivative shown undergoing an SN2

Reaction, we observe that the substituents were arranged in a cis relationship in the original compound and the consequence of inversion is the formation of a trans product. However, it is found that cyclic substrates tend to react much more slowly than similar acyclic compounds.

In small rings this is a consequence of ring strain; the SN2 transition state requires the three groups other than the nucleophile and the leaving group to be spaced 120◦ apart. This would be a severe problem for three- and four-membered rings (angles 60◦ and 90◦ respectively).

It is not a problem for five-membered rings, where this is the normal bond angle in the ring, and such compounds react just as readily as acyclic compounds.

Cyclohexyl derivatives react some 100-fold less readily than acyclic compounds, however, ring strain cannot be an important factor: the 109◦ tetrahedral angles are the same as in an acyclic compound. In cyclohexyl compounds, the rate of reaction is slowed by steric interactions with axial hydrogens.

A consequence of the low rate of reaction in SN2 reactions is that side reactions in cyclohexane derivatives, especially elimination reactions (see Section 6.4.1), may often dominate over substitution.

The SN1 reaction: unimolecular nucleophilic substitution

The abbreviation SN1 conveys the information ‘substitution–nucleophilic–unimolecular’. The reaction achieves much the same result as the SN2 reaction, i.e. the replacement of a leaving group by a nucleophile, but is mechanistically different.

It is unimolecular, since kinetic data indicate that only one species is involved in the rate-determining step: Rate = k[RL] where RL is the substrate containing the leaving group L and k is the rate constant. Note that the nucleophile Nu does not figure in the rate equation. In general terms, the reaction can be represented as below.

The first step of the reaction is the loss of the leaving group, transforming the initial polarization ( δ + / δ−) in the molecule into complete charge separation.

To achieve this, we need a good leaving group as with SN² reactions, but also a structure in which the positively charged carbon, a carbocation, is suitably stabilized.

This ionization step constitutes the slow part of the sequence, the rate-determining step, and, since only one molecular species is involved, it is responsible for the observed kinetic data.

Once the reactive carbocation is formed, it is rapidly attacked by a suitable nucleophilic species, thus generating the final product. In SN1 reactions, the nucleophilicity of the nucleophile is relatively unimportant.

Because of the high reactivity of the carbocation, any nucleophile, charged or uncharged, will rapidly react. Therefore, as the rate equation shows, the nucleophile plays no part in controlling the overall reaction rate.

We have shown carbocation formation as reversible; it would be if the leaving group recombined with the carbocation.

If there is an excess of an alternative nucleophile, however, we shall get the required product. The carbon atom of the carbocation has only six bonding electrons and is a planar entity. The bonding electrons are in sp² orbitals, and there is also an unoccupied p orbital.

The attacking nucleophile can attack from either face of this planar species; so, when X, Y, and Z are different, the product will turn out to be a mixture of two possible stereoisomers.

As there is usually an equal probability of attack at each face, the product will be a racemic mixture. This is in marked contrast to the product from an SN² reaction, where there would be an inversion of configuration and formation of a single enantiomer.

The carbocation is an intermediate in the reaction sequence and corresponds to a minimum in the energy profile.

Its formation depends upon overcoming an activation energy, corresponding to that required for the fission of the bond to the leaving group. Since the carbocation is very reactive, there will be a much smaller activation energy for reaction with the nucleophile.

Thus, tert-butanol reacts readily with HBr to give the corresponding bromide. This reaction could not proceed via the SN² mechanism because steric crowding prevents access to the nucleophile. Instead, an SN¹ mechanism can be formulated.

The initial step would be protonation of the alcohol group to improve the nature of the leaving group, i.e. water rather than hydroxide, and allow the formation of the carbocation.

Loss of the leaving group would be the slow, rate-determining step, but the following step, the attack of the nucleophile onto the carbocation, would then be rapid.

Why some SN1 reactions do not lead to racemic products

Notwithstanding the remarks above concerning the equal probability of a nucleophile attacking either face of the planar carbocation and, therefore, producing a racemic product, many SN1 reactions result in varying degrees of inversion and racemization.

This can be rationalized in terms of preferential attack of the nucleophile from the face opposite the leaving group simply because, as the leaving group departs, it hinders attack from that side.

In the example shown, there is slightly more of the ‘inverted’ product in the reaction mixture, though the effect is not especially large. In other recorded examples, up to about 80% of the product might be the inverted form.

It follows that the SN² process is accompanied by complete inversion, whereas an SN¹ process will involve racemization or partial inversion.

The effect of substituents

The SN1 mechanism requires the initial loss of the leaving group to generate a reactive carbocation.

Experimental evidence concerning the relative rates for SN1 reactions of halides. The differences in reactivity reflect structural features that stabilize the intermediate carbocation. Carbocations are stabilized by the electron-donating effect of alkyl groups, which help to disperse the positive charge.

We have noted that alkyl groups have a modest electron-donating effect (see Section 4.3.3). In carbocations, this is not a simple inductive effect, but results from overlap of the σ C–H (or C–C) bond into the vacant p orbital of the carbocation. This leads to a favorable delocalization of the positive charge.

Accordingly, tertiary carbocations benefit from three such effects and are favored over secondary carbocations with two effects, whilst the single effect in primary carbocations is insufficient to provide significant stabilization.

Thus, SN1 reactions are highly favored at tertiary carbon and very much disfavoured at primary carbon. However, in addition, carbocations may be stabilized by resonance.

Simple examples of this are met with the allyl and benzyl cations, so that allyl chloride and benzyl chloride react via SN1 reactions, although superficially these appear to involve primary carbocations.

Note that with allyl derivatives there is potential for the nucleophile to react with the different resonance forms, perhaps leading to a mixture of products.

This is not the case with the benzylic substrates, since only the benzylic product is formed; addition to the ring would destroy the stability conferred by aromaticity.

One of the most stable carbocation structures is the triphenylmethyl cation (trityl cation). In this structure, the positive charge is stabilized by resonance employing all three rings. Trityl chloride ionizes readily and can capture an available nucleophile.

SN1 reactions in cyclic systems

We noted above that the inversion of configuration that accompanied SN2 reactions was particularly apparent in cyclic systems, and that cis derivatives would be converted into trans products in disubstituted rings, and vice versa. Should The net result is that the product mixture consists of two diastereoisomers?

Sn1 Or Sn²?

As we have just seen, SN1 reactions are highly favored at tertiary carbon, and very much disfavoured at primary carbon. This is in marked contrast to SN2 reactions, which are highly favored at primary carbon and not at tertiary carbon.

With SN² reactions, consideration of steric hindrance rationalized the results observed. This leads to the generalizations for nucleophilic substitutions with secondary substrates being able to participate in either type of process.

The most distinguishing feature of the SN1 mechanism is the intermediate carbocation. Formation of the carbocation is the rate-determining step, and this is more favorable in polar solvents that can assist in facilitating the charge separation/ionization.

A useful, though not always exact, SN1 reaction occurs in a similar sort of cyclic system, then there may be stereochemical consequences, though these are easily predicted.

Thus, should the dimethylcyclohexanol shown below participate in an SN1 reaction, then we can deduce that the carbocation will be attacked from either face by the nucleophile, but not necessarily to the same extent.

The guide is that SN1 reactions are going to be favored by an acidic/positive environment, and are less likely to occur under basic/negative conditions. Since good nucleophiles are often also strong bases, this does tend to limit the applicability of SN¹ reactions.

Indeed, under strongly basic conditions, side reactions such as elimination are more likely to occur than nucleophilic substitution reactions.

However, all is not lost, because the carbocation is a particularly good electrophile and can be used with relatively poor nucleophiles. This is illustrated in the following examples.

Finally, do appreciate that, depending upon conditions, both SN1 and SN2 mechanisms might be operating at the same time, with each contributing its stereochemical characteristics to the product.

Biological SN1 reactions involving allylic cations

The living groups most commonly employed in nature are phosphates and diphosphates. These good leaving groups are anions of the strong acids phosphoric (pKa 2.1) and diphosphoric (pKa 1.5) acids respectively. The pKa values given refer to the first ionization of these polyfunctional acids.

The compound dimethylallyl diphosphate provides an excellent example of a natural product with a diphosphate leaving group that can be displaced in a nucleophilic substitution reaction.

Suitable nucleophiles are hydroxyl groups, Example a phenol, though frequently an electron-rich nucleophilic carbon is employed.

Dimethylallyl diphosphate is a precursor of many natural products that contain in their structures branched-chain C5 subunits termed isoprene units.

Although both SN2 and SN1 mechanisms might be formulated for such reactions, all the available evidence favors an SN1 process. This is rationalized in terms of the formation of a favorable resonance-stabilized allylic cation by loss of the leaving group.

In the majority of natural product structures, the nucleophile has attacked the allylic system on the same carbon that loses the diphosphate, but there are certainly examples of nucleophilic attack on the alternative tertiary carbon.

Geranyl diphosphate and farnesyl diphosphate are analogs of dimethylallyl diphosphate that contain two and three C5 subunits respectively; they can undergo the same SN1 reactions as dimethylallyl diphosphate. In all cases, a carbocation mechanism is favored by the resonance stabilization of the allylic carbocation.

Dimethylallyl diphosphate, geranyl diphosphate, and farnesyl diphosphate are precursors for natural terpenoids and steroids. The possibility of nucleophilic attack on different carbons in the resonance-stabilized carbocation facilitates another modification exploited by nature during terpenoid metabolism.

This is a change in double-bond stereochemistry in the allylic system. The interconversions of geranyl diphosphate, linalyl diphosphate, and neryl diphosphate provide neat but satisfying examples of the chemistry of simple allylic carbocations.

Thus, geranyl diphosphate ionizes to the resonance-stabilized geranyl carbocation; in nature, this can recombine with the diphosphate anion in two ways, reverting to geranyl diphosphate or forming linalyl diphosphate.

In linalyl diphosphate, the original double bond from geranyl diphosphate has now become a single bond, and free rotation is possible. Ionization of linalyl diphosphate occurs, giving a resonance-stabilized neryl carbocation, one form of which now has a Z double bond. Recombination of this with diphosphate leads to neryl diphosphate, a geometric configurational isomer of geranyl diphosphate.

It is normally very difficult to change the configuration of a double bond. Nature achieves it easily in this allylic system via carbocation chemistry.

Nucleophilic Substitution Reactions

Halide as a nucleophile: alkyl hali des Halide can be employed as a nucleophile in either SN2 or SN1 reactions to generate an alkyl halide. However, note that, in the general example shown, protonation by the acidic reagent HBr is required to improve the leaving group.

The utility of this simple transformation is often to increase the reactivity of the substrate, in that halide is a good leaving group and so can participate in other nucleophilic substitution reactions.

Oxygen and sulfur as nucleophiles: Ethers, Esters, Thioethers, Epoxides

Alkyl halides can react with water or alcohols by either SN2 or SN1 mechanisms to give alcohols or ethers respectively.

It is often preferable to use basic conditions with hydroxide or alkoxide as a better nucleophile, though this may lead to elimination and alkene formation as a competing reaction.

Although a carboxylate anion is only a relatively modest nucleophile, it is possible to exploit an SN² reaction to prepare esters from carboxylic acids as an alternative to the usual esterification methods. Such methods might be useful, depending upon the nature and availability of starting materials.

Sulfur nucleophiles behave similarly to oxygen compounds. Again, the anion will be a better nucleophile than the thiol; and since thiols are more acidic than alcohols, the conjugate bases are more easily generated.

Note also that ring-opening nucleophilic substitution reactions may be possible, and that these will give a product with two functional groups since the leaving group is still attached to the original molecule through another bond.

A simple example of a ring-opening substitution reaction is the acid-catalyzed hydrolysis of epoxides. In the example shown, protonation of the epoxide oxygen improves the leaving group, and an SN2 reaction may then proceed using water as the nucleophile.

Three-membered rings must of necessity be cis-fused and the inversion process, therefore, generates a trans-1,2-diol.

This is true even if the other end of the epoxide ring system is attacked, though it will produce the enantiomeric product. Since both reactions can occur with equal probability, the product here is racemic.

S -Adenosylmethionine in biological methylation reactions

In biological methylation, the S-methyl group of the amino acid L-methionine is used to methylate suitable O, N, S, and C nucleophiles. First, methionine is converted into the methylating agent S-adenosylmethionine (SAM).

SAM is a nucleoside derivative. Both the formation of SAM and the subsequent methylation reactions are nice examples of biological SN2 reactions.

L-methionine is a thioether that acts as a sulfur nucleophile to react with adenosine triphosphate (ATP);. Sulfur is a good nucleophile, and ATP contains a good leaving group, the triphosphate moiety.

The leaving group is in a primary position, favoring an SN² reaction, the product of which is SAM. This can be regarded as similar to a protonated alcohol in nucleophilic substitution, in that it now contains a good leaving group that is a neutral molecule, in this case, the thioether S-adenosylhomocysteine.

Subsequent SN² reactions with appropriate nucleophiles (alcohols, phenols, amines, etc.) produce the methylated compounds.

Note that in nature, these are all enzyme-catalyzed reactions. This makes the reactions specific.

It means possible competing SN2 reactions involving attack at either of the two methylene carbons in SAM are not encountered. It also means that where the substrate contains two or more potential nucleophiles, a reaction occurs at only one site, dictated by the enzyme.

The enzymes are usually termed methyltransferases. Thus, in animals, an N-methyltransferase is responsible for SAM-dependent N-methylation of noradrenaline (norepinephrine) to adrenaline (epinephrine), whereas an O-methyltransferase in plants catalyzes the esterification of salicylic acid to methyl salicylate.

Glutathione a s a sulfur nucleophile in the metabolism of foreign compounds

Glutathione is a tripeptide containing a thiol grouping, which is part of the amino acid cysteine.

This SH group plays an important role as a nucleophile in the metabolism of potentially dangerous foreign compounds taken in by the body.

The potential of SH as a nucleophile is exploited in metabolic reactions catalyzed by enzymes termed glutathione S-transferases, which conjugate the foreign compounds, i.e. bind them to glutathione.

Conjugation markedly reduces the biological activity of the compound, and most conjugates are inactive. In addition, conjugation usually increases the polarity of the substrate, thus increasing its water solubility and its potential to be excreted.

There may be further modification to the glutathione part of the conjugate before the foreign compound is finally excreted.

Care: this is not the structural “conjugation”. Glutathione can react with many potentially toxic electrophiles, including halides and epoxides that react via simple SN2 reactions.

A specific example involving aflatoxins is shown in Box 6.8. We shall see other examples of glutathione reacting as a nucleophile in detoxification reactions, where conjugation is not the result of nucleophilic substitution.

For example, it might be a nucleophilic addition to an electrophile such as an unsaturated carbonyl compound.

Nitrogen as a nucleophile: ammonium salts, amines

Amines react with alkyl halides to give initially ammonium salts, from which an amine product is liberated in the presence of a base, typically an excess of the amine.

However, this is not always a useful reaction, in that the product amine is usually just as nucleophilic as the starting amine, allowing further SN² reactions to occur. Depending upon conditions, mixtures of amines together with the quaternary salt may be produced.

Nevertheless, it offers a convenient route to amino acids, both natural and unnatural, since the amino group in amino acids is less basic (pKa about 9.8) than a simple amine (pKa about 10.6) and is consequently rather less nucleophilic. a about 9.8) than a simple amine (pKa about 10.6) and is consequently rather less nucleophilic.

Curare-like muscle relaxants: quaternary ammonium salts

The production of a quaternary ammonium salt from a tertiary amine and an alkyl halide forms the synthetic route to decamethonium, the first of a range of synthetic muscle relaxants having an action like the natural materials found in the arrow-poison curare.

Decamethonium is a di-quaternary salt, as are more modern analogs, such as suxamethonium.

Suxamethonium superseded decamethonium as a drug because it has a shorter and more desirable duration of action in the body. This arises because it can be metabolized by ester-hydrolysing enzymes (esterases)

Curare-like muscle relaxants act by blocking acetylcholine receptor sites, thus eliminating the transmission of nerve impulses at the neuromuscular junction.

There are two acetylcholine-like groupings in the molecules, and the drugs, therefore, probably span and block several receptor sites.

The neurotransmitter acetylcholine is also a quaternary ammonium compound. The natural material present in curare is tubocurarine, a complex alkaloid that is a mono-quaternary salt.

Under physiological conditions, the tertiary amine will be almost completely protonated, and the compound will similarly possess two positively charged centers.

A Flatoxins And DNA Damage

The aflatoxins are rather unpleasant fungal toxins. At high levels, they can cause severe liver damage in animals and humans, and at lower levels, they are implicated in liver cancer.

These toxins are produced by the fungus Aspergillus flavus, a common contaminant on nuts and grains. Aflatoxin B1 is the most commonly encountered example, and it is also one of the most toxic.

We now know that the toxicity is initiated by the oxidative metabolism of the toxin in the body, converting aflatoxin B1 into an electrophilic epoxide.

This epoxide is attacked in an SN2 reaction by a nitrogen atom in a guanine residue of DNA. This leads to irreversible binding of the toxin to DNA, inhibition of DNA replication and RNA synthesis, and initiation of mutagenic activity.

Fortunately, nature provides an alternative nucleophile whose role is to mop up dangerous electrophiles such as aflatoxin B1 epoxide before they can do damage, and to remove them from the body. This compound is glutathione, a tripeptide composed of glutamic acid, cysteine, and glycine.

It is the thiol grouping that acts as a nucleophile, attacking the epoxide function of the toxin.

In this way, the toxin becomes irreversibly bound to glutathione, and the additional polar functionalities in the adduct mean that the product becomes water-soluble. The glutathione–toxin adduct can thus be excreted from the body.

Carbon as a nucleophile: nitriles, Gri guard reagents, acetylides

Nucleophilic substitution reactions employing carbon as a nucleophile are important in synthetic chemistry in that they create a new C–C bond.

A carbon nucleophile, of course, must be in the form of anionic carbon, or its equivalent. One of the simplest sources of anionic carbon is the cyanide anion. HCN is a weak acid (pKa 9.1) and forms a series of stable salts.

Sodium and potassium cyanides are convenient sources of cyanide, which in many reactions behaves similarly to a halide nucleophile. Thus, the reaction of an alkyl halide with cyanide creates a nitrile and extends the carbon chain in the substrate by one carbon.

It is easy to rationalize why cyanide can displace a halide such as bromide: HCN is a weak acid (pKa 9.1), so cyanide is a good nucleophile, whereas HBr is a strong acid (pKa − 9), and bromide is a good leaving group.

As we shall see later, other reactions of nitriles extend the usefulness of this reaction. Thus, reduction of nitriles gives amines, whereas hydrolysis generates carboxylic acid.

Organometallic reagents also provide carbon nucleophiles that can be considered to behave as carbanions. Although there are a variety of organometallic reagents available, we include here only two types of reagents, namely Grignard reagents and acetylides.

Reacting an alkyl or aryl halide, usually the bromide, with metallic magnesium in ether solution produces Grignard reagents. An exothermic reaction takes place in which the magnesium dissolves, and the product is a solution of the Grignard reagent RMgBr or ArMgBr.

The formation of this product need not concern us, but its nature is important. We can deduce from the ions Mg2+ and Br− that it contain the equivalent of R− or Ar−, i.e. the alkyl or aryl group has been transformed into its carbanion equivalent. This carbanion equivalent can behave as a nucleophile in typical nucleophilic substitution reactions.

In the example shown, the reaction of a Grignard reagent with the epoxide electrophile ethylene oxide proceeds as expected, and after acidification results in the formation of an alcohol that is two carbons longer than the original nucleophile The carbanion equivalent from a Grignard reagent is also a strong base.

pKa values for alkanes are typically about 50 and for aromatics about 44. Not surprisingly, a Grignard reagent reacts readily with Acetylides are formed by treating terminal acetylenes with a strong base, sodium amide in liquid ammonia being the one most commonly employed.

Acetylenes with a hydrogen atom attached to the triple bond are weakly acidic (pKa about 25) due to the stability of the acetylide anion water to form the hydrocarbon, so these reactions must be conducted under anhydrous conditions and this anion can thus act as a nucleophile.

It reacts with appropriate electrophiles, e.g. alkyl halides, in the manner expected. This reaction extends a carbon chain by two or more atoms, depending on the acetylide used.

Probably the most significant examples of carbon nucleophiles are enolate anions. These can participate in a wide variety of important reactions, and simple nucleophilic substitution reactions are included among these.

However, we shall consider these reactions at a later stage, when the nature and formation of enolate anions are discussed we shall consider hydride as a nucleophile that participates in a typical SN2 process. This achieves the replacement of a leaving group by hydrogen and, therefore, is a reduction of the substrate.

Hydride as nucleophilicle: lithium aluminium hydride and sodium borohydride de reduction ons Many complex metal hydrides such as lithium aluminium hydride (LiAlH4, abbreviated to LAH) and sodium borohydride (NaBH4) can deliver hydride in such a manner that it appears to act as a nucleophile.

We shall look at the nature of these reagents later under the reactions of carbonyl compounds, where we shall see that the complex metal hydride never actually produces hydride as a nucleophile, but the aluminium hydride anion can effect a transfer of hydride.

Hydride itself, for Example from sodium hydride, never acts as a nucleophile; owing to its small size and high charge density it always acts as a base. Nevertheless, to understand the transformations In the example shown overleaf where hydride attacks the epoxide function, the product is an alcohol, and the reaction is completed by supplying a proton source, usually water.

Lithium aluminium hydride reacts violently with water, liberating hydrogen, and the heat of the reaction usually ignites the hydrogen.

LAH must, therefore, be used in rigorously anhydrous conditions, usually in ether solution. Any solvent containing OH or NH groups would destroy the reagent by acting as a proton donor for hydride.

The addition of water as a proton source has to be carried out with considerable caution since any unreacted LAH will react violently with this water. In the laboratory, safe removal of excess LAH may be achieved by adding small amounts of an ester such as ethyl acetate.

Note that LAH is a powerful reducing agent and will attack many other functional groups, especially carbonyl groups.

An analogous series of reactions is involved when sodium borohydride is used as the reducing agent.

Sodium borohydride is considerably less reactive than LAH, and reactions proceed much more slowly. This reagent may be used in alcoholic or even aqueous solutions, so there are no particular hazards associated with its use.

Simple examples shown above are the base-catalysed formation of oxygen- and nitrogen-containing ring systems. We have shown base-initiated ionization of the alcohol to an alkoxide anion in epoxide formation; the anion is a better nucleophile than the alcohol.

For pyrrolidine synthesis, the amino group is sufficiently nucleophilic for a reaction to occur, but a base is required to remove a proton from the first formed intermediate.

Formation of cyclic compounds

In substrates where there is a good leaving group in the same molecule as the nucleophile, one may get an intramolecular process and create a ring system.

It is usually necessary to find conditions that favour an intramolecular process over the alternative intermolecular reaction. This is typically achieved by carrying out the reaction at relatively higher dilutions, thereby minimizing the intermolecular processes.

Competing Reactions Eliminations And Rearrangements

When nucleophilic substitution reactions are attempted, the expected product may often be accompanied by one or more additional products that arise from competing reactions.

Since these competing reactions share features of the nucleophilic substitution mechanism, they are readily rationalized, and it is possible to devise conditions to minimize or maximize the formation of such products.

The most common alternative reactions are eliminations and rearrangements, which we shall consider in turn.

Elimination reactions

The E2 reaction: bimolecular elimination

The abbreviation E2 conveys the information ‘elimination–bimolecular’. The reaction is a concerted process in which a nucleophile removes an electrophile at the same time as a leaving group departs. It is bimolecular since kinetic data indicate that two species are involved in the rate-determining step:

Rate = k[RL][Nu]

Nu is the nucleophile, RL is the substrate containing the leaving group L, and k is the rate constant. The electrophile removed is usually hydrogen, so we can consider that the nucleophile is acting as a base.

We have seen above the close relationship between basicity and nucleophilicity, so the E2 mechanism provides an example of how the alternative property of nucleophiles may come into play and lead to different products. To achieve an SN2 reaction, the nucleophile must approach to the rear of the leaving group and then displace it.

If a rear-side approach is hindered by adjacent groups, or perhaps because the nucleophile is rather large, it becomes energetically easier for the nucleophile to act as a base and remove a proton from the substrate.

As the proton is removed, electrons that were involved in bonding the proton to the substrate are then used to form the double bond; however, to maintain the octet of electrons on the neighbouring carbon, the electrons will have to be transferred to a suitable acceptor, in this case, the leaving group.

As with the SN2 mechanism, the reaction is concerted and proceeds through a high-energy transition state, in which partial bonds have been established. The energy profile will look the same as that of an SN2 reaction The elimination reaction generates a new π bond in a planar alkene.

Since the π bond is perpendicular to the plane of the alkene, we can predict that the most favourable way to achieve the new π bonding is to start with the H–C–C–L atoms in a planar array. This will line up the orbitals and allow easy development of the π bond.

The nucleophile approaches from the side opposite the electronegative leaving group – electrostatic repulsion discourages attack in the region of the leaving group. With the substrate in the favoured staggered conformation, we describe this arrangement of atoms as anti-periplanar.

The requirement for the proton electrophile and the leaving group involved in the elimination to be anti to each other is demonstrated by the nature of the product obtained from a suitable substrate, Example (1R,2R)-1-bromo-1,2-diphenylpropane, when treated with base. The only product formed is (Z)-1,2- diphenylprop-1-ene.

This is the product of an anti-elimination of H and Br when the substrate is in a staggered conformer.

If H and Br were positioned on the same side of the conformer, then it would need to be in an unfavourable eclipsed conformer to line up the orbitals.

Elimination of H and Br in this fashion is termed a syn elimination and would lead to the E-product. However, this is not the product formed, and, in general, syn eliminations are very rare.

The anti-stereochemical relationship is obligatory by observing elimination reactions in suitable cyclohexane derivatives.

The only way to achieve a planar arrangement of the H–C–C–L atoms is when H and L are both axial and, consequently, trans to each other (transaxial). Thus, consider menthyl chloride and neomenthyl chloride, which are stereoisomers differing in configuration at just one centre.

Treatment of neomenthyl chloride with base rapidly produces two different alkenes, i.e. 2- 2-menthene and 3-menthene.

If one considers the three-dimensional shape of neomenthyl chloride, it can be seen that, in the preferred conformer with the two alkyl groups equatorial, the chlorine is an axial substituent.

This means there are two different hydrogen atoms adjacent that are also axial and anti-periplanar to the chlorine.

As a consequence, two different E2 eliminations can occur; hence the two observed products. That the two products are not formed in equal amounts will be considered in the next section.

On the other hand, menthyl chloride is only slowly converted by treatment with base, and into a single product, i.e. 2-menthene. In the preferred conformation of menthyl chloride, all three substituents are equatorial, and no adjacent hydrogen is in a planar relationship to the chlorine leaving group.

The fact that slow elimination occurs at all is a result of conformational isomerism into the less-favoured conformer that has all three substituents axial.

In this conformer, there is a single hydrogen anti-periplanar with the chlorine, so elimination occurs giving just one product. The conformational equilibrium is slowly disturbed because the elimination removes the small concentration of unfavoured conformers.

Direction of elimination

The E2 elimination of HCl from neomenthyl chloride described above produced two products, namely 2- 2-menthene and 3-menthene in a ratio of about 1 : 3.

It is a general observation that, where different alkene products can arise through E2 elimination, the more-substituted alkene predominates.

2-Menthene contains a double bond with two alkyl substituents, whereas the double bond in 3-menthene has three substituents.

The more-substituted alkene is termed the Saytzeff product; the less-substituted alkene is termed the Hofmann product. We recommend you disregard the proper names and think of the products in terms of ‘more-substituted alkene’ and ‘less-substituted alkene’.

A further example of the more-substituted alkene predominating is found in the elimination of HBr from 2-bromobutane. The major product is the more-substituted alkene but-2-ene, which predominates over the less-substituted alkene but-1-ene by a factor of 4: 1.

The reasoning for this direction of elimination is twofold. The more-substituted alkene is actually of lower energy than the less-substituted alkene because of the stabilizing electron-donating effect of alkyl groups, and a similar effect will occur in the transition state where the double bond is developing. This is seen in the energy profile for the reaction.

The stabilizing effect of alkyl groups appears to involve the overlap of σ C–H (or C–C) orbitals with the π system of the alkene, rather as we have seen with carbocations (see Section 6.2.1). The more alkyl groups attached, the more stabilization the alkene derives.

This effect is relatively small and both products are formed, usually with one predominating. The more substituted Saytzeff product typically predominates when the leaving group is small, for example, halide.

On the other hand, when there is a large leaving group present, Example quaternary ammonium, then steric effects become more important than the stabilizing effects of alkyl groups.

This is exemplified by the heatinitiated decomposition of the quaternary ammonium salt below. The elimination is now governed by which is the more favourable conformer of the substrate where a hydrogen atom is positioned anti to the quaternary ammonium substituent.

Two such possibilities can be considered. The conformer set up for 1,2-elimination is more favourable than the conformer for 2,3-elimination since the latter conformer would necessitate a less favourable gauche interaction.

An alternative conformer for 2,3-elimination has two unfavourable gauche interactions. Thus, it is the large leaving group that now dictates the direction of elimination, and the less-substituted alkene (Hofmann product) predominates.

Again, it should be noted that both products are obtained – the effect is not sufficiently great to produce one product exclusively.

Note that with some cyclic substrates, the leaving group may remain as part of the product alkene. Elimination reactions played an important role in the early structural analysis of alkaloids (typically cyclic amines).

Combination of N-methylation followed by elimination may be used to open up nitrogen heterocycles, as shown with piperidine.

One further consideration relating to the nature of the products in eliminations is the stereochemistry of the double bond. For instance, base-catalysed elimination of HBr from 2-bromopentane gives three products.

This elimination involves a small leaving group, so the more substituted alkene predominates. However, E and Z isomers of this Saytzeff product are produced, and in unequal amounts.

The major product is the E-alkene can be rationalized in terms of minimizing steric repulsion during the transition state.

Note the terminology that can be used to describe product distribution in this type of reaction. Reactions are termed regiospecific where one product is formed exclusively, or regioselective where one product predominates.

Atracurium is a curare-like muscle relaxant that is metabolized via an elimination reaction We have seen above that the muscle relaxant properties of curare and synthetic analogues result from competing with acetylcholine at receptors, thus blocking nerve impulses at the neuromuscular junction.

As quaternary ammonium salts, there are two well-separated acetylcholine-like groupings in the molecules, and the drugs probably span and block several receptor sites.

These agents work rapidly and are of considerable value in surgery. However, artificial respiration is required until the agent is metabolized, and thus broken down by the patient. Recent developments have led to agents with a built-in functional group that allows more rapid metabolism.

Initially, the presence of ester groupings, as in suxamethonium, allowed fairly rapid metabolism in the body via esterase enzymes that hydrolyse these linkages. The enzyme involved appears to be a non-specific serum acetylcholinesterase. Even better is the inclusion of functionalities that allow additional degradation via an elimination reaction.

Such an agent is atracurium. In addition to enzymic ester hydrolysis, atracurium is also degraded in the body by a non-enzymic elimination reaction that is independent of liver or kidney function. Normally, this elimination would require strongly alkaline conditions and a high temperature, but the presence of the carbonyl group increases the acidity of the proton and thus facilitates its removal.

Elimination can proceed readily under physiological conditions, giving atracurium a half-life of about 20 minutes. This is particularly valuable where patients have low or atypical esterase enzymes.

Atracurium contains four chiral centres (including the quaternary nitrogens) and is supplied as a mixture of stereoisomers; a single isomer cisatracurium has now been introduced. This isomer is more potent than the mixture, has a slightly longer duration of action, and produces fewer cardiovascular side effects.

The E1 reaction: unimolecular elimination The abbreviation E1 conveys the information ‘elimination–unimolecular’. The reaction achieves the same result as the E2 reaction but is mechanistically different in that it involves a carbocation intermediate.

It is unimolecular since kinetic data indicate that only one species is involved in the rate-determining step where RL is the substrate containing the leaving group L and k is the rate constant.

The nucleophile Nu does not figure in the rate equation. Just as the E2 mechanism shares features of the SN2 mechanism, the E1 mechanism shares features of the SN1 reaction.

The initial step is the formation of a carbocation intermediate through the loss of the leaving group. This slow step becomes the rate-determining step for the whole reaction, i.e. the E1 mechanism is unimolecular. In general terms, the reaction can be represented as follows.

Once formed, the carbocation could be attacked by a nucleophile – the SN1 reaction. However, if the nucleophile acts as a base, then it removes a proton from a position adjacent to the positive centre and the original bonding electrons are used to discharge the positive charge and make a new double bond.

A stereochemical consequence of this is that the proton lost should be perpendicular to the plane of the carbocation to achieve maximum overlap with the unfilled p-orbital during the formation of the π bond.

We do not have the same strict stereochemical requirements as in the E2 mechanism, and isomeric alkenes may well be produced. If several hydrogens are available for elimination, then the preferred product formed is the more substituted Saytzeff alkene.

E1 elimination in the synthesis of tamoxifen

We have already employed the tamoxifen structure as an example of defining the configuration of double bonds.

Tamoxifen is a highly successful oestrogen-receptor antagonist used in the treatment of breast cancer. It may be synthesized by the following sequence.

The main skeleton of the drug is constructed by a Grignard addition reaction on the appropriate ketone using phenyl magnesium bromide. This produces a tertiary alcohol.

It now remains to eliminate water from this structure. This is achieved under acid conditions.

An E1 mechanism is involved: protonation of the tertiary alcohol allows loss of water as the leaving group and generation of a carbocation, which is favoured since it is both tertiary and benzylic.

However, completion of the elimination by proton loss gives a 1: 1 mixture of the E- and Z-alkenes, since there is no stereocontrol at this stage – free rotation about the C–C bond in the alcohol and subsequent structures until the double bond is formed means both stereochemistries will be produced. The drug material tamoxifen is the Z-isomer.

E1 or E2 ?

We have seen above that the structure of the substrate is the most important feature that dictates the mechanism of substitution reactions. Thus, the SN2 mechanism is favoured when the reaction takes place at a primary centre, whereas an SN1 mechanism is preferred at tertiary centres, or where stable intermediate carbocations can be produced.

We can use similar reasoning to predict that an E2 mechanism might be preferred when the leaving group departs from a primary centre and that an E1 mechanism is likely when structural features facilitate carbocation formation.

By structural features, we mean tertiary, allylic, or benzylic centres. When a secondary centre is involved, then either E1 or E2 might occur, depending upon reaction conditions.

In general, these predictions are found to be sound. However, there is an apparent anomaly, in that E2 reactions also frequently occur with tertiary substrates. If we think a little deeper, we shall discover that it is not unreasonable for this to be so.

The E2 reaction is initiated by the base removing a proton, and this is still possible even where there is a tertiary centre.

Although, for steric reasons, a nucleophile cannot approach a tertiary centre to displace a leaving group (SN2 reaction), it is still feasible for a base to remove a proton from an adjacent carbon.

Accordingly, the E2 mechanism becomes relatively favourable, even with tertiary substrates, when we use a strong base or more concentrated base. We are thus more likely to get an E1 mechanism when we have a tertiary centre, and weak bases or bases in low concentration.

Polar solvents are also going to be conducive to carbocation mechanisms. Just as acidic conditions help to favour SN1 reactions, they also going to favour E1 reactions.

Elimination or substitution? Elimination can be a troublesome side reaction during substitution reactions. In general terms:

• Strong Bases Favour Elimination;
• Large Bases Favour Elimination;
• Steric Crowding In The Substrate Favours Elimination;
• High Temperatures And Low Solvent Polarity Favour Elimination.

Carbocati on rearrangement reactions

Most organic reactions involve changes to functional groups whilst the fundamental molecular skeleton remains unchanged.

In molecular rearrangements, groups migrate within the molecule and the molecular skeleton is modified. In most rearrangements, the groups migrate to the next atom, a 1,2-shift, though 1,3-shifts and other migrations are known.

The most common examples of rearrangements involve an electron-deficient atom, and pre-eminent among these are carbocations.

Since carbocations are a feature of the SN1 and E1 mechanisms, it follows that rearrangements can be side-reactions of these types of transformation. The driving force in carbocation rearrangements is to form a more stable carbocation.

Consider a proposed nucleophilic substitution reaction on the secondary alcohol shown using aqueous HBr.

As a secondary alcohol, either SN2 or SN1 mechanisms are possible (see Section 6.2.3), but SN1 is favoured because of the acidic environment and the large tert-butyl group hindering the approach of the nucleophile. The expected SN1 bromide product is formed, together with a smaller amount of the E1-derived alkene in a competing reaction.

However, other products are also produced. These are isomers of the above products and have a rearranged carbon skeleton. Their formation is rationalized as follows:

The first-formed carbocation is secondary. This carbocation can become a more stable tertiary carbocation via rearrangement, in which a methyl group with its pair of electrons migrates from one carbon to the adjacent positive centre.

Now the rearranged tertiary carbocation can yield SN1- and E1-type products in much the same manner as the original secondary carbocation.

A rearranged bromide is formed, together with two alkenes from an E1 process, with both more-substituted Saytzeff and less-substituted Hofmann alkenes being produced. The formation of such rearranged products proves that this unexpected transformation must occur.

These carbocation rearrangements are termed Wagner–Meerwein rearrangements. They are most commonly encountered with secondary carbocations where rearrangement produces a more stable tertiary carbocation.

They are less common with tertiary carbocations, which are already stabilized by the maximum number of alkyl groups, and where any rearrangement would tend to produce only a less stable secondary carbocation.

Wagner–Meerwein rearrangements are not restricted to methyl migrations, and we may also see the transfer of hydrogen with an electron pair, i.e. a hydride migration.

This is observed in the case of the secondary alcohol illustrated, where a secondary carbocation would be generated.

A methyl migration would merely lead to another secondary carbocation, and this serves no stabilizing effect.

However, a hydride migration produces a tertiary carbocation, so this process will stabilize the system. This is what happens, and the major product is a bromide where the halogen appears to have attacked the wrong position, i.e. different from that which originally carried the leaving group. This is the pointer to something unusual occurring.

Again, the driving force is the conversion of a secondary carbocation into a more stable tertiary carbocation.

Hydride migration also accounts for one of the observed products from the treatment of the cyclohexanol tosylate with acetic acid.

Although the predominant product is the corresponding acetate (one could formulate either SN1 or SN2 mechanisms for the formation of this product), about 30% of the alternative acetate is formed.

This can be rationalized as arising from a carbocation that is rearranged by hydride migration. This is favoured because the resultant carbocation is an allylic cation, and stabilized by resonance.

In most cases, the driving force for a rearrangement is the conversion of a secondary carbocation into a more stable tertiary carbocation.

Surprisingly, there are examples of where a tertiary carbocation is transformed into a secondary carbocation, but there needs to be some more powerful driving force to achieve this. Relief of ring strain is a particular case.

The cyclobutane-ring-containing alcohol can yield a tertiary carbocation, but the product from an SN1 reaction with HBr contains a cyclopentane ring.

Its formation is rationalized via a Wagner–Merwein rearrangement in which ring expansion occurs. This is represented as equivalent to a methyl migration, but the methylene group is part of the carbon chain.

There is significant relief of ring strain in going from a four-membered ring to a five-membered ring, which is more than enough to make up for the energy change in going from a tertiary carbocation to a less stable secondary carbocation. Carbocations also feature as intermediates in electrophilic addition reactions and Friedel–Crafts alkylations.

Rearrangements may also be observed in these carbocations if they have the appropriate structural features. It does not matter how the carbocation is produced, subsequent transformations will be the same as we have seen where rearrangements are competing reactions in nucleophilic substitution.

Thus, electrophilic addition of HCl to 3,3-dimethylbut-1- ene proceeds via protonation of the alkene, and leads to the preferred secondary rather than primary carbocation.

However, this carbocation may then undergo a methyl migration to produce the even more favourable tertiary carbocation. Finally, the two carbocations are quenched by a reaction with chloride ions. The product mixture is found to contain predominantly the chloride from the rearranged carbocation.

The enhanced stability of benzylic carbocations is nicely illustrated by the addition of HBr to the two alkenes shown below. In the case of 2-phenylbut-1- ene, protonation of the alkene leads to a carbocation that is both tertiary and benzylic and is significantly favoured over an alternative primary carbocation. Quenching with the bromide nucleophile gives the tertiary bromide.

On the other hand, 3-phenyl prop-1- ene is protonated to a secondary carbocation. In this case, rearrangement by hydride migration leads to a more favourable benzylic carbocation, and a benzylic bromide is the observed product.

Rearrangements seem to provide us with an unexpected complication to ruin our carefully thought-out plans for interconverting chemicals.

It is sometimes difficult to predict when they might occur, but we should recognize occasions when they might become a nuisance, e.g. look at the structure of any proposed carbocation intermediate.

In most cases, we shall be more concerned with rationalizing such transformations, rather than trying to predict their possible occurrence.

Carbocation rearrangements: synthesis of camphor from α-pinene

Although the monoterpene camphor occurs naturally, substantial amounts are produced semi-synthetically from α-pinene, a component in turpentine. Treatment of α-pinene with aqueous HCl protonates the double bond by an electrophilic addition (see Section 8.1.1) and generates the more favoured tertiary carbocation. Rather than simply being attacked by a nucleophile, this carbocation rearranges.

The tertiary carbocation contains a strained four-membered ring, and an alkyl shift allows relief of ring strain, generating five-membered rings and a secondary carbocation.

It would appear that the relief of ring strain more than compensates for the loss of tertiary character in the carbocation.

Thus, it is the secondary carbocation that interacts with a nucleophile. In this case, the nucleophile is water, the major component of the aqueous HCl. The product is thus isoborneol.

Camphor is then obtained from isoborneol by oxidation of the secondary alcohol to a ketone.

Carbocation rearrangements in nature: b biosynthesis of lanosterol Many examples of carbocation rearrangements can be found in nature, particularly in the biosynthesis of terpenoids and steroids.

Nature generates carbonation in three main ways. The first of these is the loss of a leaving group, with diphosphate being the most common leaving group.

Protonation of an alkene also produces a carbocation, and, as we would predict, this tends to form the more substituted and thus more stable carbocation. Also encountered is the ring opening of an epoxide group, which may be considered to be acid-initiated.

Perhaps the most spectacular of the natural carbocation rearrangements is the concerted sequence of 1,2-methyl and 1,2-hydride Wagner–Meerwein shifts that occur during the formation of lanosterol from squalene. Lanosterol is then the precursor of the steroid cholesterol in animals.

Carbocation formation is initiated by an epoxide ring opening in squalene oxide, giving a tertiary carbocation, and this is transformed into the four-ring system of the protosteryl cation by a series of electrophilic addition reactions.

The resultant protosteryl cation has a tertiary carbocation in the side chain, and a hydride shift generates another tertiary cation. A second hydride shift follows, then two methyl shifts, each time generating a new tertiary cation.

Lastly, the positive charge is neutralized via the loss of a proton, giving the alkene lanosterol.

There is no obvious energy advantage in such tertiary-to-tertiary cation changes, but it must be appreciated that this is an enzyme-catalysed reaction, and the enzyme plays a crucial role in the reactions that occur.

These hydride and methyl migrations do occur, as demonstrated by isotopic labelling studies.

Further, it is noted that most of them involve inversion of stereochemistry at the particular centre, a feature of the concerted nature of these rearrangements, so that as one group leaves another approaches from the rear. Thus, we have the features of SN2 reactions in a carbocation mechanism.

This is a complicated series of reactions but includes impressive examples of carbocation rearrangements. The electrophilic cyclization sequence is also quite striking.

The post Nycleophilic Reactions: Nucleophilic Substitution appeared first on BDS Notes.

Heterocycles:

Cyclic compounds in which one or more of the ring atoms are not carbon are termed heterocycles; the noncarbon atoms are referred to as heteroatoms.

• We shall limit our discussions to compounds in which the heteroatoms are nitrogen, oxygen, or sulfur.
• To study and understand their properties, heterocycles are conveniently grouped into two classes, i.e. non-aromatic and aromatic.

Non-Aromatic Heterocycles:

We have already met many examples of nonaromatic heterocycles in earlier chapters, for example., cyclic ethers, including epoxides, and cyclic amines, as well as lactones, lactams, and cyclic acetals and ketals.

• From the familiar examples shown below, it should be clear that the standard approach to generating heterocyclic systems requires a difunctional compound containing a leaving group or elec aerophilic center, together with a nucleophilic species that provides the heteroatom.
• The nomenclature of simple heterocyclic ring systems containing one heteroatom is indicated overleaf.

These form a useful reference, but there is little to be gained in committing them to memory.

Note, however, that the most important of these structures tend to have a trivial rather than systematic name, a consequence of long-standing common usage.

• Some of these, for example., tetrahydrofuran and tetrahydropyran, are derived from the name of the corresponding aromatic heterocycle by the concept of reduction.

A few examples of commonly encountered heterocycles with two heteroatoms are also shown.

Numbering always begins at the heteroatom; in the case of morpholine, numbering starts at oxygen, the heteroatom of a higher atomic number.

• Remember that an accepted alternative in nomenclature is to indicate a heteroatom by the prefix aza-, oxo, or thia in the appropriate carbocycle.
• Thus, we could name piperidine as azacyclohexane, and ethylene oxide tetrahydrofuran as oxacyclopentane.

The chemistry of these non-aromatic heterocycles differs little from the chemistry of their acyclic counterparts and we emphasize only the relative reactivity of the three-membered ring systems towards ring opening, thus achieving relief of ring strain.

We have already noted the ring opening of epoxides, and similar reactivity is found with aziridines and iranes.

• Four-membered systems are also considerably strained and reactive towards nucleophiles, though not as readily as the three-membered compounds.
• Some of these heterocycles provide us with valuable laboratory solvents, for example., the ethers tetrahy drofuran and dioxane (1,4-dioxane). Others are useful as organic bases, for example., piperidine, pyrrolidine, and morpholine.

The basicities of the nitrogen derivatives are comparable to those of similar acyclic amines, but physical properties, for example., higher boiling point, make them more versatile than the simple amines.

From the pK_a values shown, there is relatively little difference in basicities for diethylamine, pyrrolidine, or piperidine. Note, however, that morpholine and piperazine are weaker bases than piperidine.

• This is the result of an electron-withdrawing inductive effect from the second heteroatom, making the nitrogen atom both less basic and also less nucleophilic. This makes morpholine a useful base with basicity between that of piperidine and pyridine (pK_a 5.2).
• The second pK_a value for the diamine piperazine is substantially lower than the first since the inductive effect from the protonated amine will withdraw electrons away from the unprotonated amine.

Aromaticity And Heteroaromaticity:

Pyridine is structurally related to benzene: one CH unit has been replaced by N. If we consider the constitutions of the two compounds in more detail, we shall see even closer similarity.

• Thus, we have seen that the ring atoms in benzene are sp² hybridized. The remaining singly occupied p orbitals are oriented at right angles to the plane of the ring, and overlap to form a delocalized π system, extending to form a closed loop above and below the ring.
• Compared with what we might expect for the hypothetical cyclohexatriene, this results in a considerable stabilization, with significantly modified structure and reactivity in benzene. We termed this aromaticity.
• Benzene conforms to Huckel’s rule, which predicts that planar cyclic polyenes containing 4n + 2 π electrons show enhanced stability associated with aromaticity.
• Pyridine is also aromatic: nitrogen contributes one electron in a p orbital to the π electron system, and its lone pair is located in an sp² orbital that is in the plane of the ring and perpendicular to the π electron system.

It also conforms to Huckel’s rule, in that we still have an aromatic ¨ sextet of π electrons.

One of the structural features of benzene that derives from aromaticity is the equal length of the C–C bonds (1.40 A), which lies between the conformal single (1.54 A) and double (1.34˚A) bonds.

• Nevertheless, we continue to draw benzene with single and double bonds because this allows us to represent reaction mechanisms in terms of electron movements.

Pyridine does not have a perfect hexagon shape; the symmetry is distorted because the C–N bonds are slightly shorter (1.34 A) than the C–C bonds (1.39–1.40 A).

Nitrogen is more electronegative than carbon, and this influences the electron distribution in the π electron system in pyridine through inductive effects, such that nitrogen is electron-rich.

• In addition, the nitrogen will also become electron-rich through a resonance effect: several resonance forms may be drawn that have a negative charge on nitrogen. These effects thus reinforce each other.
• The heteroatom thus distorts the π electron cloud of the aromatic ring system, drawing electrons towards the nitrogen and away from the carbons.

The consequences of this are that we can predict that the pyridine nitrogen will react readily with electrophiles, whereas the remainder of the ring system will be resistant to electrophilic attack.

An experimental probe for aromaticity is the chemical shift of the hydrogen signals in NMR spectroscopy.

• The substantially greater δ values for benzene protons (δ 7.27 ppm) compared with those in alkenes (δ 5–6 ppm) have been ascribed to the presence of a ring current that creates its magnetic field opposing the applied magnetic field.
• This ring current is the result of circulating electrons in the π system of the aromatic ring.
• The hydrogen NMR signals for pyridine also appear at relatively large δ values, in the range of 7.1–8.5 ppm, typical of aromatic systems.

The signals do not all appear at the same chemical shift; the heteroatom distorts the π electron distribution and affects the 2/6, 3/4, and 5 positions to different extents.

• Now let us consider pyrrole, where we have a five-membered ring containing nitrogen. Pyrrole is also aromatic.
• This is somewhat unexpected: how can we get six π electrons from just five atoms? The answer is that each carbon contributes one electron as before, but nitrogen now contributes two electrons, its lone pair, to the π electron system.
• We can draw Frost circles to show the relative energies of the molecular orbitals for pyridine and pyrrole.

The picture for pyridine is essentially the same as for benzene, with six π electrons forming an energetically favorable closed shell.

• For pyrrole, we also get a closed shell, and there is considerable aromatic stabilization over electrons in the six atomic orbitals.
• However, the contribution of the nitrogen lone pair to the aromatic sextet in pyrrole makes the nitrogen atom relatively electron deficient. The nitrogen atom should create an inductive effect, as in pyridine, drawing electrons towards the heteroatom.
• However, a consideration of the resonance structures leads to several resonance forms with a positive charge on nitrogen. The resonance effect is opposite to the inductive effect and of greater magnitude.

Overall, the heteroatom distorts the π electron cloud of the aromatic ring system by pushing electrons away from the nitrogen and towards the carbons.

The difference in electron distribution in pyridine and pyrrole manifests itself via the measured dipole moments. More importantly, we shall see that this electron distribution influences the chemical reactivity of the two systems.

• In broad terms, ring systems where the carbons are electron deficient because of the electron-withdrawing effect of the heteroatom, for example., pyridine, are more reactive towards nucleophiles than benzene.
• On the other hand, ring systems where the carbons are electron-rich because of the electron-donating heteroatom, for example., pyrrole, are more reactive towards electrophiles than benzene.
• Note the deliberate choice of terminology here: ring systems where the carbons are electron deficient or electron rich.
• You may meet the older terminology of π-deficient heterocycles and π-excessive heterocycles, but these can give a false impression.
• Each heterocycle contains six π electrons, so it is not the heterocycle that is electron deficient or electron-rich, but the carbons that receive less or more than their equal share because of the effect of the heteroatom.
• Though we shall return to this again, one critical difference between pyridine and pyrrole to note here relates to basicity. Pyridine is a base because its nitrogen still carries a lone pair able to accept a proton.

Pyrrole is not basic: it has already used up its lone pair in contributing to the aromatic sextet.

Six-Membered Aromatic Heterocycles Pyridine:

From our discussions in the last section, we might expect pyridine to display properties associated with the nitrogen function and the aromatic ring.

Not surprisingly, it turns out that the aromatic ring affects the properties of the amine; but, more significantly, the aromatic properties are greatly influenced by the presence of the heteroatom.

Based on our earlier knowledge, and from a simple inspection of its structure, we might expect to observe three types of general reactivity in pyridine. We might expect to see:

Reaction at the heteroatom – the non-bonding electrons on the nitrogen might coordinate to H+ or another suitable electrophile.

Reaction of the aromatic π system – typical elec trophilic substitution as seen for benzene might be expected.

Reaction of the C=N ‘imine’ function – though this is not an isolated imine function but is part of the aromatic ring, its polarization might make it susceptible to nucleophilic attack.

Reassuringly, our predictions turn out to be well-founded.

Pyridine is a base (pK_a pyridinium cation 5.2), but it is a considerably weaker base than a typical non-aromatic heterocyclic amine such as piperidine (pK_a piperidinium cation 11.2).

• This is because the lone pair electrons in pyridine are held in an sp² orbital.
• The increased s character of this orbital, compared with the sp3 orbital in piperidine, means that the lone pair electrons are held closer to the nitrogen, and are consequently less available for protonation.
• The lower basicity of pyridine compared with piperidine is thus a hybridization effect.

Although pyridine is a weak base, it can form salts with acids and is widely used in chemical reactions as an acid scavenger and as a very good polar solvent.

Just as pyridine is a weaker base than piperidine, it is also a poorer nucleophile. Nevertheless, it reacts with electrophiles to form stable pyridinium salts.

• In the examples shown, primary alkyl halides form N-alkylpyridinium salts, whereas acyl halides and anhydrides react to give N-acylpyridinium salts.
• We have already seen the latter compounds involved in esterification reactions, and see the value of pyridine in removing acidic by-products, for example., HCl.

Of course, N-acyl pyridinium salts will easily be hydrolyzed under aqueous conditions.

Even better than pyridine in such reactions is the derivative 4-N, N-dimethylamino pyridine (DMAP), where a resonance effect from the dimethylamino substituent reinforces the nucleophilicity of the pyridine nitrogen.

It then also promotes the acylation step by improving the nature of the leaving group. The example shows its function in a typical esterification process, i.e. acylation of an alcohol.

There are other gains, as well. Pyridine as a solvent is difficult to remove from the products, and it smells quite awful.

• In this reaction, a catalytic amount of DMAP is all that is necessary, and a more acceptable solvent can be employed.
• The pre-eminent reactivity associated with aromatic compounds is the ease of electrophilic substitution.
• As we have already predicted, the pyridine ring is rather unreactive towards electrophilic reagents, and these tend to be attacked by nitrogen instead, making the ring even less reactive.

It is readily seen from the intermediate addition cations and their resonance structures that attack at C-2 or C-4 will be unfavorable, in that one of the resonance forms features an unstable electron-deficient nitrogen cation.

Attack at C-3 is the more likely, simply based on an inspection of resonance structures for the addition cation.

• However, elec trophilic attack still tends to be unfavourable, because many electrophilic reagents, for example., HNO₃ –H₂SO₄, are strongly acidic, and the first effect is protonation on nitrogen.
• The attack of E⁺ on a positively charged pyridinium cation is even less favorable. Under acidic conditions, we require an attack on free pyridine, the concentration of which will be very small.
• Thus, under equivalent conditions, pyridine undergoes electrophilic substitution very much more slowly than benzene, by a factor of about 106.

Even Friedel–Crafts acylations are inhibited, because the nitrogen complexes with the Lewis acid, again leading to a cationic nitrogen.

A striking demonstration of the reduced activity towards electrophiles for the pyridine ring compared with the benzene ring will be seen later when we consider the fused heterocycles quinoline and isoquinoline.

• These contain a benzene ring fused to a pyridine ring; electrophilic substitution occurs exclusively in the benzene ring.
• To facilitate electrophilic substitution, it is possible to first convert pyridine into pyridine N-oxide by the action of a peracid such as peracetic acid or m-chloroperbenzoic acid (MCPBA;).
• N-oxide formation is not peculiar to pyridine, but it is a general property of tertiary amines. There is no overall charge in the molecule, but it is not possible to draw the structure without charge separation.

Although the introduced oxygen atom causes electron withdrawal through an inductive effect, there is a greater and opposing resonance effect that donates electrons into the ring system.

This improves reactivity towards electrophiles. Consideration of resonance structures shows positions 2, 4, and 6 are now electron-rich.

Nitration of pyridine N-oxide occurs at C-4; very little 2-nitration is observed. The pyridine compound can then be regenerated by deoxygenation with triphenylphosphine.

Pyridine, on the other hand, is more reactive than benzene towards nucleophilic aromatic substitution

• This is effectively a reaction towards the C=N ‘imine’ function, as described above.
• The attack is attacked at principally at positions 2 and 4, as predictable from resonance structures of reaction intermediates.

Attack at the 3 position does not allow the nitrogen to help stabilize the negative charge.

However, for an unsubstituted pyridine, the leaving group to finish off this reaction is hydride, which is a strong base and thus a poor leaving group.

• It may be necessary to use an oxidizing agent to function as a hydride acceptor to the Chichibabin reaction to facilitate this type of hydride transfer.

Nevertheless, there is a classic example of this process, known as the Chichibabin reaction, in which pyridine is converted into 2-aminopyridine through heating with sodium amide.

The hydride released appears to abstract a proton from the product since the other product of the reaction is gaseous hydrogen. The aminopyridine anion is finally quenched with water.

• The product is mainly 2-aminopyridine, probably the result of the enhanced inductive effect on carbons immediately adjacent to the electronegative nitrogen.
• It is much more effective to have a better-leaving group in the pyridine system. Thus 2- or 4-chloropyridines react with several nucleophiles to generate substituted products.
• Note that one can predict from the resonance structures that 3-chloropyridine, despite having a satisfactory leaving group, would not be susceptible to nucleophilic substitution at position 3.

It is not possible in the addition of anion to share the charge with nitrogen.

Methylpyridines are called picolines. 2-picoline and 4-picoline may be deprotonated by treatment with a strong base, giving useful anions.

• The methyl acidity results because of resonance stabilization in the conjugate base, providing an enolate anion analog.

However, pK_a values for 2-picoline (32) and 4-picoline (34) show that they are somewhat less acidic than ketones.

These anions can now be used as nucleophiles in several familiar reactions, for example., S_N2 reactions with alkyl halides, or aldol reactions with carbonyl compounds.

It is worthwhile here to relate the behavior of 2-chloropyridine and 2-methylpyridine to carbonyl chemistry.

• If we consider the pyridine ring as an imine, and therefore a carbonyl analog, then with 2-chloropyridine we are seeing reactions that parallel nucleophilic substitution of an acyl halide through an addition–elimination mechanism.
• With 2-methylpyridine we are seeing typical aldol reactions with activated methyl derivatives.

Nicotine, Nicotinic Acid, And Nicotinamide:

Nicotine is an oily, volatile liquid and is the principal alkaloid found in tobacco (Nicotiana tabacum). It can be seen to be a combination of two types of heterocycle, i.e. the aromatic pyridine and the non-aromatic N methylpyrrolidine.

In small doses, nicotine can act as a respiratory stimulant, though in larger doses it causes respiratory depression. Nicotine is the only pharmacologically active component in tobacco, and it is highly addictive.

• On the other hand, tobacco smoke contains several highly carcinogenic chemicals formed by incomplete combustion.
• Tobacco smoking also contributes to atherosclerosis, chronic bronchitis, and emphysema, and is regarded as the single most preventable cause of death in modern society.
• Nicotine, in the form of chewing gum, nasal sprays, or trans-dermal patches, is available for use by smokers who wish to stop the habit.
• Nicotine affects the nervous system, interacting with the nicotinic acetylcholine receptors, and the tight binding is partially accounted for by the structural similarity between acetylcholine and nicotine.
• Curare-like antagonists also block nicotinic acetylcholine receptors. There are other acetylcholine receptors, termed muscarinic, that are triggered by the alkaloid muscarine.

The tropane alkaloid hyoscyamine (see Box 10.9) binds to muscarinic acetylcholine receptors.

Oxidation of nicotine with chromic acid led to the isolation of pyridine-3-carboxylic acid, which was given the trivial name nicotinic acid.

• We now find that nicotinic acid derivatives, especially nicotinamide, are biochemically important.
• Nicotinic acid (niacin) is termed vitamin B3, though nicotinamide is also included under the umbrella term vitamin B3 and is the preferred material for dietary supplements.
• It is common practice to enrich many foodstuffs, including bread, flour, corn, and rice products.

Deficiency in nicotinamide leads to pellagra, which manifests itself in diarrhea, dermatitis, and dementia.

Nicotinic acid and nicotinamide are precursors of the coenzymes NAD⁺ and NADP⁺, which play a vital role in oxidation-reduction reactions and are the most important electron carriers in intermediary metabolism.

We shall look further at the chemistry of NAD⁺ and NADP⁺ shortly, but note that, in these compounds, nicotinamide is bound to the rest of the molecule as an N-pyridinium salt.

An intriguing feature of nicotinic acid formation in animals is that it is a metabolite produced from the amino acid tryptophan.

This means the pyridine ring is formed by biochemical modification of the indole fused-ring system, and, as you might imagine, it involves a substantial sequence of transformations.

Nucleophilic Addition To Pyridinium Salts:

The reaction of nucleophiles with pyridinium salts leads to addition, giving dihydropyridines.

Attack is normally easier at positions 2 or 6, where the inductive effect from the positively charged nitrogen is greatest; however, if these sites are blocked, attack occurs at position 4. This is easily predicted from a consideration of resonance structures.

Thus, treatment of N-methyl pyridinium salts with cyanide produces a mixture of 2- and 4-cyanide hydropyridines, with the 2-isomer predominating.

It is quite difficult to reduce benzene or pyridine because these are aromatic structures. However, partial reduction of the pyridine ring is possible by using complex metal hydrides on pyridinium salts.

• Hydride transfer from lithium aluminum hydride gives the 1,2-dihydro derivative, as predictable from the above comments.
• Sodium borohydride under aqueous conditions achieves a double reduction, giving the 1,2,5,6-tetrahydro derivative, because protonation through the unsaturated system is possible.
• The final reduction step requires catalytic hydrogenation. The reduction of pyridinium salts is of considerable biological importance.

Note the way we can refer to the unsaturated heterocycle by considering it as a reduced pyridine, for example., a dihydropyridine (two double bonds) or tetrahydropyridine (one double bond).

Nicotinamide Adenine Dinucleotide: Reduction Of A Pyridinium Salt:

Nicotinamide adenine dinucleotide (NAD⁺) is a complex molecule in which a pyridinium salt provides the reactive functional group, hence the superscript + in its abbreviation.

• NAD⁺ acts as a biological oxidizing agent, and in so doing is reduced to NADH (reduced nicotinamide adenine dinucleotide).
• An enzyme, a dehydrogenase, catalyzes the process and NAD⁺ is the cofactor for the enzyme. The reaction can be regarded as directly analogous to the hydride reduction of a pyridinium system to a dihydropyridine, as described above.
• We have already seen that NADH can act as a reducing agent, delivering the equivalent of hydride to a carbonyl compound.

In the oxidizing mode, the enzyme can extract hydride from the substrate and use it to reduce the pyridinium salt NAD⁺, producing the dihydropyridine NADH.

The substrate in most reactions of this type is alcohol, which becomes oxidized to an aldehyde or ketone, for example., ethanol is oxidized to acetaldehyde.

• Some reactions employ the alternative phosphorylated cofactor NADP⁺; the phosphate does not function in the oxidation step but is merely a recognition feature helping to bind the compound to the enzyme. The full structures of NAD⁺ and NADP⁺.
• Note that the attack of hydride is at position 4 of the dihydropyridine ring. This is controlled by the enzyme, but it is also probably the only site accessible since the rest of the complex molecule hinders the approach to positions 2 and 6.

Tautomerism: Pyridones

The pyridine ring system may carry substituents, just as we have seen with benzene rings.

• We have encountered several such derivatives in the previous section. Hydroxy or amino heterocycles, however, may sometimes exist in tautomeric forms.
• We have met the concept of tautomerism primarily with carbonyl compounds, and have seen the isomerization of keto and enol tautomers.
• In certain cases, for example., 1,3-dicarbonyl compounds, the enol form is a major component of the equilibrium mixture. amides.
• This was used to explain why amides are fragile bases. Note that such resonance forms of pyridones are favorable, having a positive charge on the nitrogen and a negative charge on the more electronegative oxygen.
• In addition, the structure gains further stabilization from the carbonyl group. The pyridone forms are very much favored over the phenol forms, and typical C=O peaks are seen in the infrared (IR) spectra.

In the example shown, liquid acetylacetone contains about 76% of the enol tautomer.

2-Hydroxy- and 4-hydroxy-pyridines are in equilibrium with their tautomeric ‘amide’ structures containing a carbonyl. These tautomers are called 2- pyridone and 4-pyridone respectively.

This type of tautomerism does not occur with the corresponding benzene derivative phenol, since it would destroy the stabilization conferred by aromaticity.

So why can tautomerism occur with a hydroxypyridiet? It is because 2-pyridone and 4-pyridone still retain aromaticity, with the nitrogen atom donating its lone pair electrons to the aromatic sextet.

• This is more easily seen in the resonance structures and should remind us of the resonance stabilization in amides. This was used to explain why amides are fragile bases.
• Note that such resonance forms of pyridones are favorable, having a positive charge on the nitrogen and a negative charge on the more electronegative oxygen. In addition, the structure gains further stabilization from the carbonyl group.

The pyridone forms are very much favored over the phenol forms, and typical C=O peaks are seen in the infrared (IR) spectra.

Note, however, that we cannot get the same type of tautomerism with 3-hydroxypyridine. In polar solvents, 3-hydroxypyridine may adopt a dipolar zwitterionic form.

• This may look analogous to the previous structure, but appreciate that there is a difference. With 3-hydroxyproline, the zwitterion is a major contributor and arises simply from acid-base properties.
• The hydroxyl group acts as an acid, losing a proton, and the nitrogen acts as a base, gaining a proton.

The structure from 2-pyridone is a minor resonance form that helps to explain charge distribution; the compound is almost entirely 2-pyridone.

Like amides, 2- and 4-pyridones are also very weak bases, much weaker than amines.

• Like amides, they protonate on oxygen rather than nitrogen. This further emphasizes that the nitrogen lone pair is already in use and not available for protonation.
• On the other hand, the N–H can readily be deprotonated; pyridones are appreciably acidic (pK_aabout 11).
• The conjugate base benefits from considerable resonance stabilization, both via the carbonyl group and also via the ring.

The main structures in which charge is associated with the electronegative N or O atoms.

It is thus possible to N-alkylate a pyridone by exploiting its acidity. As with enolate anions, there is the possibility for O-alkylation and N-alkylation.

Although it depends upon the conditions and the nature of the electrophile, carbon electrophiles tend to react with nitrogen rather than oxygen.

A useful reaction of pyridones is conversion into chloropyridines by the use of phosphorus oxychloride POCl₃ in the presence of PCl₅. This appears to react initially on oxygen, forming a good leaving group, which is subsequently displaced by chloride.

Aminopyridines are also potentially tautomeric with corresponding imino forms.

However, 2-aminopyridine and 4-aminopyridine exist almost entirely as amino tautomers – indeed, we have just seen 2-aminopyridine as a product of the Chichibabin reaction.

• Which tautomer is preferred for hydroxy and amino heterocycles is not always easily explained; but, as a generalization, we find that the oxygen derivatives exist as carbonyl tautomers and amino heterocycles favor the amino tautomers.
• At this stage, we should just register the potential for tautomerism in aminopyridines; we shall see important examples with other heterocycles.

Aminopyridines protonate on the ring nitrogen, and 4-aminopyridine is a stronger base than 2-aminopyridine.

This may be rationalized by a consideration of resonance in the conjugate acids.

• The conjugate acids from ring protonation benefit from charge delocalization, which is greater in 4-amino pyridinium than in 2-amino pyridinium.
• This type of delocalization is not possible in 3-aminopyridinium; 3-aminopyridine (pK_a 6.0) is the weakest base of the three aminopy ridines and has basicity more comparable to that of pyridine (pK_a 5.2).

Pyrylium Cation And Pyrones

Pyrones are oxygen analogs of pyridones and are potentially aromatic. However, there is little evidence that the dipolar resonance forms of either 2-pyrone or 4-pyrone make any significant contribution.

• Their chemical behavior suggests they should be viewed more as conjugated lactones (2-pyrones) or vinylogous lactones (4-pyrones) rather than aromatic systems since many reactions lead to ring opening.

The pyrylium cation is isoelectronic with pyridine: it has the same number of electrons and, therefore, we also have aromaticity. Oxygen is normally divalent and carries two lone pairs.

If we insert oxygen into the benzene ring structure, then it follows that, by having one electron in a p orbital contributing to the aromatic sextet, there is a lone pair in an sp² orbital, and the remaining electron needs to be removed, hence the pyrylium cation.

However, oxygen tolerates a positive charge less readily than nitrogen and aromatic stabilization is less than with pyridine.

Flavonoids are natural phenolic systems containing pyrylium and pyrone rings and provide the most prominent examples.

We have met some of these systems under antioxidants. Coumarins contain a 2-pyrone system. Note that all of these compounds are fused to a benzene ring and are strictly benzopyran or benzopyrylium systems.

Dicoumarol And Warfarin:

Warfarin provides us with a slightly incongruous state of affairs: it is used as a drug and also as a rat poison.

• It was developed from a natural product, dicoumarol, and provides us with a nice example of how pyrone chemistry resembles that of conjugated lactones rather than aromatic systems.
• Many plants produce coumarins; coumarin itself is found in sweet clover and contributes to the smell of new-mown hay.
• However, if sweet clover is allowed to ferment, oxidative processes initiated by the microorganisms lead to the formation of 4-hydroxycoumarin rather than coumarin.

4-Hydroxycoumarin then reacts with formaldehyde, also produced via the microbial degradative reactions, and provides dicoumarol.

4-Hydroxycoumarin can be considered an enol tautomer of a 1,3-dicarbonyl compound; conjugation with the aromatic ring favors the enol tautomer. This now exposes its potential as a nucleophile.

• Whilst we may begin to consider enolate anion chemistry, no strong base is required and we may formulate a mechanism in which the enol acts as the nucleophile, in a simple aldol reaction with formaldehyde.

Dehydration follows and produces an unsaturated ketone, which then becomes the electrophile in a Michael reaction. The nucleophile is a second molecule of 4-hydroxycoumarin.

Animals fed spoiled sweet clover were prone to fatal hemorrhages. The cause was traced to the presence of dicoumarol.

• This compound interferes with the effects of vitamin K in blood coagulation, the blood loses its ability to clot, and minor injuries can lead to severe internal bleeding.
• Synthetic dicoumarol has been used as an oral blood anticoagulant in the treatment of thrombosis, where the risk of blood clots becomes life-threatening.

It has since been superseded by warfarin, a synthetic development based on the natural product.

Warfarin was initially developed as a rodenticide and has been widely employed for many years as the first-choice agent, particularly for the destruction of rats.

• After consumption of warfarin-treated bait, rats die from internal hemorrhage.
• Warfarin is synthesized from 4-hydroxycoumarin by a Michael reaction on benzalacetone, again exploiting the nucleophilicity of the hydroxypyrone.

Benzalacetone is the product of an aldol reaction between benzaldehyde and acetone.

Five-Membered Aromatic Heterocycles: Pyrrole

Pyrrole (azacyclopentadiene) is the ring system obtained if we replace the CH₂ group of cyclopenta diene with NH.

• Although cyclopentadiene is certainly not aromatic, pyrrole has an aromatic character because nitrogen contributes two electrons, its lone pair, to the π-electron system.
• We have also noted from resonance forms that nitrogen carries a partial positive charge, and the carbons are electron-rich.

This is stronger than the opposing inductive effect.

This is reflected in the basicity of pyrrole. Pyrrole is a particularly weak base, with pK_a of the conjugate acid −3.8.

First, we should realize that protonation of pyrrole will not occur on nitrogen: nitrogen has already used up its lone pair by contributing to the aromatic sextet, so protonation would necessarily destroy aromaticity.

It is possible to protonate pyrrole using a strong acid, but even then the protonation occurs on C-2 and not on the nitrogen.

• Although this still destroys aromaticity, there is some favorable resonance stabilization in the conjugate acid.
• Protonation on C-3 is not as favorable, in that there is less resonance stabilization in the conjugate acid.
• It turns out that, as opposed to acting as a base, pyrrole is potentially an acid (pKa 17.5); it is not a particularly strong acid, but stronger than we might expect for a secondary amine system (pK_a about 36).
• This is because the anion formed by losing the proton from nitrogen has a negative charge on the relatively electronegative nitrogen, but maintains its aromaticity.

Unlike in pyrrole, the anion resonance structures do not involve charge separation.

It is appropriate here to compare the acidity of cyclopentadiene, which has pK_a 16, is considerably more acidic than most hydrocarbon systems and comparable to water and alcohols.

• Removal of one of the CH₂ protons from the non-aromatic cyclopentadiene generates the cyclopentadienyl anion.
• This anion has an aromatic sextet of electrons, two electrons being contributed by the negatively charged carbon.
• The charge distribution in pyrrole leads us to predict that it will react readily with electrophiles; or, put another way, pyrrole will behave as a nucleophile.
• This is indeed the case, and the ease of elec trophilic substitution contrasts with the behavior of pyridine above, where charge distribution favored nucleophilic attack onto the heterocycle.

Although our resonance description of pyrrole shows negative charge can be dispersed to any ring carbon, pyrrole reacts with electrophiles preferentially at C-2 rather than C-3, unless the 2-position is already substituted.

This may reflect that there is more charge dispersion in the addition cation from attack at C-2 than there is from attack at C-3.

• This is, of course, the same argument as used above for C-protonation; protonation (pyrrole acting as a base) also occurs at C-2.
• As with protonation, electrophiles do not react at the nitrogen center. Pyrrole is very reactive towards electrophiles.

For example, treatment with bromine leads to the substitution of all four positions.

Indeed, it is often difficult to control electrophilic attack so that monosubstitution occurs.

A further problem is that pyrrole polymerizes in the presence of strong acids and Lewis acids, so that typical electrophilic reagents, for example., HNO₃ –H₂SO₄ and RCOCl–AlCl₃, cannot be used. Polymerization involves the conjugate acid functioning as the elec trophile.

To achieve useful monosubstitution it is necessary to employ relatively mild conditions, often without a catalyst.

• Nitration may be accomplished with the reagent acetyl nitrate, giving mainly 2-nitropyrrole.
• Acetyl nitrate is formed by reacting acetic anhydride with fuming nitric acid. Since the other product is acetic acid, there is no strong mineral acid present to cause polymerization.

It is also possible to synthesize 2-acetyl pyrrole simply by using acetic anhydride, and pyrrole can act as the nucleophile in the Mannich reaction.

Although pyrrole is a weak acid, it can be deprotonated by using a strong base, for example., sodium hydride, and the anion can be used in typical nucleophilic reactions.

This allows simple transformations such as N-alkylation, N-acylation, and N-sulfonation. Note particularly that whereas pyrrole reacts with elec trophiles at carbon, usually C-2, the pyrrole anion reacts at the nitrogen atom.

Porphyrins And Corrins:

Pyrrole reacts with aldehydes and ketones under acidic conditions to form polymeric compounds. In many cases these are intractable resin-like materials; however, with appropriate carbonyl compounds, interesting cyclic tetramers can be formed in very good yields.

Thus, pyrrole and acetone react as shown above. This involves pyrrole acting as the nucleophile to attack the protonated ketone in an aldol-like reaction.

• This is followed by the elimination of water, facilitated by the acidic conditions. This gives an intermediate alkylidene pyrrolium cation, a highly reactive electrophile that reacts with another molecule of nucleophilic pyrrole.
• We then have a repeat sequence of reactions, in which further acetone and pyrrole molecules are incorporated.
• The presence of the two methyl substituents from acetone forces the growing polymer to adopt a planar array, and this eventually leads to a cyclic tetramer, the terminal pyrrole attacking the alkylidene pyrrolium cation at the other end of the chain.
• The cyclic tetramer shown is structurally related to the porphyrins. The basic ring system in porphyrins is porphin, which is more oxidized than the tetramer from the pyrrole–acetone reaction, and has four pyrrole rings linked together by methine (–CH=) bridges.
• One of the features of porphin is that it is aromatic. It contains an aromatic 18 π-electron system, which conforms to Huckel’s rule, 4n + 2 with n = 4.
• The aromatic ring weaves around the porphin structure, and is composed entirely of double-bond electrons; it does not incorporate any nitrogen lone pairs.

Note that we can draw Kekule-like resonance structures for porphin. Do not be confused by seeing alternative structures with the double bonds arranged differently.

Porphyrin rings are formed in nature by a process that is remarkably similar to that shown above. Though the sequence contains some rather unusual features, the coupling process also involves nucleophilic attack onto an alkylidene pyrrolium cation. This may be generated from the precursor porphobilinogen by the elimination of ammonia.

One of the important properties of porphyrins is that they are complex with divalent metals, the pyrrole nitrogens being ideally spaced to allow this.

Of vital importance to life processes are the porphyrin derivatives chlorophyll and haem. Chlorophyll (a mixture of structurally similar porphyrins; chlorophyll a is shown) contains magnesium, and is, of course, the light-gathering pigment in plants that permits photosynthesis.

Plants and a few microorganisms use photosynthesis to produce organic compounds from inorganic materials found in the environment, whereas other organisms, such as animals and most microorganisms, rely on obtaining their raw materials in their diet, for example., by consuming plants.

• Haemoglobin, the red pigment in blood, serves to carry oxygen from the lungs to other parts of the body tissue.
• This material is made up of the porphyrin haem and the water-soluble protein globin.
• The haem component shares many structural features with chlorophyll, one of the main differences being the use of Fe²⁺ as the metal rather than Mg²⁺ as in chlorophyll.
• The oxygen-carrying ability of hemoglobin involves a six-coordinate iron, with an imidazole ring from the protein (a histidine residue) occupying the sixth position.
• Porphyrin rings containing iron are also a feature of the cytochromes. Several cytochromes are responsible for the latter part of the electron transport chain of oxidative phosphorylation that provides the principal source of ATP for an aerobic cell.
• Their function involves alternate oxidation-reduction of the iron between Fe²⁺ (reduced form) and Fe³⁺ (oxidized form).

The individual cytochromes vary structurally, and their classification (a, b, c, etc.) is related to their absorption maxima in the visible spectrum. They contain a haem system that is covalently bound to protein through thiol groups.

An especially important example is cytochrome P-450, a coenzyme of the so-called cytochrome P-450-dependent mono-oxygenases.

• These enzymes are frequently involved in biological hydroxylations, either in biosynthesis or in the mammalian detoxification and metabolism of foreign compounds such as drugs.
• Cytochrome P-450 is named after its intense absorption band at 450 nm when exposed to CO, which is a powerful inhibitor of these enzymes.
• A redox change involving the Fe atom allows binding and the cleavage of molecular oxygen to oxygen atoms, with subsequent transfer of one atom to the substrate.
• In most cases, NADPH features as a hydrogen donor, reducing the other oxygen atom to water. Many such systems have been identified, capable of hydroxylating aliphatic or aromatic systems, as well as producing epoxides from alkenes.
• A related ring system containing four pyrroles is seen in vitamin B₁₂, but this has two pyrroles directly bonded and is termed a corrin ring.
• Vitamin B₁₂ is extremely complex and features six-coordinate Co²⁺ as the metal component. Four of the six coordination are provided by the corrin ring nitrogens and a fifth by a dimethyl benzimidazole moiety.
• The sixth is variable, being cyano in cyanocobalamin (vitamin B₁₂), but other anions may feature in vitamin B₁₂ analogs.

Vitamin B₁₂ appears to be entirely of microbial origin, with intestinal flora contributing to human dietary needs. Insufficient vitamin B₁₂ leads to pernicious anemia, a disease that results in nervous disturbances and low production of red blood cells.

Furan And Thiophene:

Furan and thiophene are the oxygen and sulfur analogs respectively of pyrrole. Oxygen and sulfur contribute two electrons to the aromatic sextet but still retain lone pair electrons.

• There is one significant difference, however, in that oxygen uses electrons from a 2p orbital, whereas the electrons that sulfur contributes originate from a 3p orbital.
• In the case of furan H of thiophene, this reduces orbital overlap with the carbon 2p orbitals.
• Both compounds are thus aromatic, and their chemical reactivity reflects what we have learned about pyrrole.

• The most typical reaction is electrophilic substitution. However, we find that pyrrole is more reactive than furan towards electrophiles and thiophene is the least reactive; all are more reactive than benzene
• This relates to the relative stability of positive charges located on nitrogen, oxygen, and sulfur. We used similar electronegativity reasoning to explain the relative basic strengths of nitrogen, oxygen, and sulfur derivatives.

Furan is also the ‘least aromatic’ of the three, i.e. it has the least resonance stabilization, and undergoes many reactions in which the aromatic character is lost, for example., addition reactions or ring opening.

Note that the dipoles of furan and thiophene are opposite in direction to those in pyrrole.

• In furan and thiophene, there is a greater inductive effect opposing the resonance effect, whereas in pyrrole the resonance contribution was greater.

In the non-aromatic analogs, the heteroatom is at the negative end of the dipole in all cases.

Nevertheless, we can interpret the reactions of furan and thiophene by logical consideration as we did for pyrrole.

• In electrophilic substitutions, there is again a preference for 2- rather than 3-substitution, and typical electrophilic reactions carried out under acidic conditions are difficult to control.
• However, because of lower reactivity compared with pyrrole, it is possible to exploit Friedel-Crafts acylations, though using less-reactive anhydrides rather than acyl chlorides, and weaker Lewis acids than AlCl₃.
• Nitration can be achieved with acetyl nitrate rather than nitric acid. In the case of furan, this is slightly anomalous, in that it involves an addition intermediate by combination of the carbocation with acetate.

This subsequently aromatizes by loss of acetic acid. The less-reactive thiophene can even be nitrated with concentrated nitric acid when it yields a mixture of 2- and 3-nitrothiophene.

Six-membered rings with two heteroatoms

Diazines:

A diazabenzene, i.e. a benzene ring in which two of the CH functions have been replaced with nitrogen, is termed a diazine.

• Three isomeric variants are possible; these are called pyridazine, pyrimidine, and pyrazine.
• These structures are all aromatic, the nitrogen atoms functioning in the same way as the pyridine nitrogen, each contributing one p electron to the aromatic sextet, with a lone pair in an sp² orbital.

The diazines are much weaker bases than pyridine (pK_a 5.2).

If we consider the inductive and resonance effects in pyridine, we have seen that these both draw electrons toward the nitrogen.

• Therefore, a second nitrogen will have destabilizing effects on the conjugate acid formed by the protonation of the first nitrogen.
• The order of basicity in pyridazine, pyrimidine, and pyrazine is influenced by secondary effects, which will not be considered here.
• Deprotonation is very difficult and would require extremely strong acids; in the case of pyridazine, it is essentially impossible because of the need to establish positive charges on adjacent atoms.
• In general, we can consider that the extra nitrogen, through its combined effects, makes the other ring atoms more electron deficient than they would be in pyridine; as a result, the diazines are more susceptible to nucleophilic attack than pyridine.
• In pyrazines and pyridazines, the second nitrogen helps by withdrawing electrons from atoms that would carry a negative charge in the addition anion.

In pyrimidines, the two nitrogens share the negative charge of the addition anion, and pyrimidines are the more reactive towards nucleophiles.

Halodiazines react readily with nucleophiles with a displacement of the halide-leaving group.

• This follows what we have seen with halopyridines, but the halodiazines are more reactive because of the influence of the extra nitrogen.
• Thus, 2-chloropyrazine and 3-chloropyridazine easily yield the corresponding amino derivatives on heating with ammonia in an alcohol solution.
• The 2- and 4-halo pyrimidines are even more reactive and substitute at room temperature. This is because of the improved delocalization of negative charge in the addition anion.

5-Halopyrimidines are the least susceptible to nucleophilic displacement: the halogen is neither α nor γ to nitrogen, and cannot benefit from any favorable charge localization on nitrogen.

Diazines are generally resistant to electrophilic attack on carbon, and, as for pyridine, addition to nitrogen is observed.

• Alkyl halides give mono-quaternary salts; di-quaternary salts are not formed under normal conditions.
• Of course, if the diazine ring carries a substituent that makes the starting material non-symmetric, then the product will almost always be a mixture of two isomeric quaternary salts.

Steric and inductive effects rather than resonance effects appear to influence the reaction and formation of the major product.

Tautomerism In Hydroxy- And Amino-Diazines:

We have seen that 2- and 4-hydroxypyridines exist primarily in their tautomeric ‘amide-like’ pyridone forms.

• This preference over the ‘phenolic’ tautomer was related to these compounds still retaining their aromatic character, with further stabilization from the carbonyl group.
• 3- Hydroxypyridine cannot benefit from this additional stabilization. In contrast, 2-aminopyridine and 4-aminopyridine exist almost entirely as amino tautomers, although they are potentially tautomeric with imino forms.
• We also encounter tautomerism in hydroxy- and amino-diazines, and the preference for one tautomeric form over the other follows what we have seen with the pyridine derivatives.
• Thus, except for 5-hydroxy pyrimidine, all the mono-oxygenated diazines exist predominantly in the carbonyl tautomeric form.

We term these ‘amide-like’ tautomers diazinones. 5-Hydroxypyrimidine is analogous to 3- hydroxypyridine, in that the hydroxyl is wrongly positioned for tautomerism.

The diazinon tautomers are identified by using terminology such as 3(2H)-pyridazine for the car bonyl tautomer of 3-hydroxypyrazine.

• The 3(2H) prefix signifies the position of the oxygen (3- pyridazine) and specifies the NH is at position 2.
• Note that, in addition to the diazine–diazinone tau H tomerism, when the nitrogens have a 1,3-relationship there is further tautomerism possible, for example., 4(1H)- pyrimidone 4(3H)-pyrimidone.

Diazinones may be converted into chlorodiazines by the use of phosphorus oxychloride, just as pyridones yield chloropyridines.

Aminodiazines exist in the amino form. These compounds contain two-ring nitrogens and a primary amino group.

Interestingly, they are more basic than the unsubstituted diazine and always protonate on a ring of nitrogen.

• This allows resonance stabilization of the conjugate acid utilizing the lone pair of the amino substituent.
• It has been found that one can predict which nitrogen is protonated from the ring nitrogen–amino substituent relationship, which follows the preference sequence γ > α > β, as in the examples shown.

This can be related to achieving maximum charge distribution over the molecule.

Pyrimidines And Nucleic Acids:

The storage of genetic information and the transcription and translation of this information are functions of the nucleic acids deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

• They are polymers whose building blocks are nucleotides, which are themselves combinations of three parts, i.e. a heterocyclic base, a sugar, and phosphate.
• The bases are either monocyclic pyrimidines or bicyclic purines (see Section 14.1). Three pyrimidine bases are encountered in DNA and RNA, cytosine (C), thymine (T), and uracil (U).
• Cytosine is common to both DNA and RNA, but uracil is found only in RNA and thymine is found only in DNA.

In nucleic acid, the bases are linked through an N-glycoside bond to a sugar, either ribose or deoxyribose; the combination base plus sugar is termed a nucleoside. The nitrogen bonded to the sugar is shown.

We should note particularly that uracil and thymine are dioxypyrimidines, whereas cytosine is an amino oxypyrimidine. All three pyrimidines are thus capable of existing in several tautomeric forms.

The number of possible forms is reduced somewhat by the fact that one of the nitrogens is bonded to the sugar in the nucleic acid; it no longer carries hydrogen to participate in tautomerism.

• The tautomeric forms indicated are found to predominate in nucleic acids. The oxygen substituents exist almost entirely as carbonyl groups, whereas
• the amino group is preferred over possible imino forms. Although we are accustomed to thinking of nucleic acids containing ‘pyrimidine’ bases, this is not strictly correct. Cytosine exists as an aminopyrimidone, and thymine and uracil are pyrimidines. Further, they are not particularly basic.
• Cytosine is the most basic of the three (pK_a 4.6), in that the amino group by a resonance effect can stabilize the conjugate acid (compare 4-aminopyrimidine pK_a 5.7 above). Thymine and uracil are very weak bases, in that they are ‘amide-like’.
• The most far-reaching feature of nucleic acids is the ability of the bases to hydrogen bond to other bases.
• This property is fundamental to the double helix arrangement of the DNA molecule, and the translation and transcription via RNA of the genetic information present in the DNA molecule.
• Hydrogen bonding occurs between complementary purine and pyrimidine bases and involves either two or three hydrogen bonds.
• In DNA, the base pairs are adenine–thymine and guanine-cytosine. In RNA, base pairing involves guanine-cytosine and adenine–uracil.
• This property will be discussed in detail in Section 14.2, but it is worth noting at this stage that hydrogen bonding is achieved between amino substituents (N–H) and the oxygen of carbonyl groups.
• These functions in the pyrimidine bases arise directly from the tautomeric preferences.

Five-Membered Rings With Two Heteroatoms:

We have looked at the five-membered aromatic heterocycles pyrrole, furan, and thiophene. The introduction of a second heteroatom creates azoles. This name immediately suggests that nitrogen is one of the heteroatoms.

• As soon as we consider valencies, we discover that to draw a five-membered aromatic heterocycle with two heteroatoms, it must contain nitrogen.
• A neutral oxygen or sulfur atom can have only two bonds, and we cannot, therefore, have more than one of these atoms in any aromatic heterocycle. On the other hand, there is potential for having as many nitrogens as we like in an aromatic ring.
• Thus, in five-membered aromatic heterocycles with two heteroatoms, we can have two nitrogens, one nitrogen plus one oxygen, or one nitrogen plus one sulfur.

The heteroatoms can be positioned only 1,2 or 1,3. The numbering of the ring system starts from the heteroatom with the higher atomic number; nitrogen will always be the higher of the two numbers in the oxazole and thiazole systems. In imidazole, numbering begins at the NH.

We can visualize these heterocycles as similar to the simpler aromatic systems pyrrole, furan, and thiophene.

• For example, in imidazole, each carbon and nitrogen will be sp² hybridized, with p orbitals contributing to the aromatic π system. The carbon atoms will each donate one electron to the π system.
• Then, as in pyrrole, the NH nitrogen supplies two electrons, and, as in pyridine, the =N– supplies one electron and retains a lone pair.

Oxygen or sulfur would also supply two electrons, as we saw in furan and thiophene.

It also follows that a compound like imidazole has one pyridine-like nitrogen and one pyrrolelike nitrogen.

• We may thus expect to see imidazole having properties resembling a combination of either pyridine- or pyrrole-like reactivity.
• The availability and location of lone pair electrons are crucial to our understanding of imidazole chemistry, and it often helps to include these in the structure.

1,3-Azoles: Imi Dazole, Oxazole, And Thiazole:

Imidazole (pK_a 7.0) is a stronger base than either pyridine (pK_a 5.2) or pyrrole (pK_a − 3.8).

• When we compared the basicity of pyridine with that of the aliphatic amine piperidine (pK_a 11.1), we implicated the higher s character of the pyridine lone pair (sp²) compared with that in piperidine (sp³) to account for pyridine’s lower basicity.
• Even so, imidazole seems abnormally basic for a compound with sp²-hybridized nitrogen.
• Therefore, when we meet structures for the imidazole-containing amino acid histidine, we may encounter either of the tautomeric forms shown.
• Though there imidazole appears to stem from the symmetry of the conjugate acid, and the resonance stability conferred by this.

The 1,3-relationship allows the two nitrogen atoms to share the charge equally. Note that pK_a 7.0 means imidazole is 50% protonated in water.

Imidazole (pK_a 14.2) is also more acidic than pyrrole (pK_a 17.5); this, again, is a feature conferred by symmetry and enhanced resonance stabilization in the conjugate base.

Oxazole (pK_a 0.8) and thiazole (pK_a 2.5) are weak bases. The basicity of the nitrogen is reduced by the presence of the other heteroatom.

Oxygen and sulfur provide a stronger electron-withdrawing inductive effect, compared with nitrogen, but a much weaker electron-releasing resonance effect.

Tautomerism In Imidazoles:

A complicating factor in imidazoles is tautomerism. Imidazole tautomerizes rapidly in solution and consists of two identical tautomers.

• This becomes a problem, though, in an unsymmetrically substituted imidazole, and tautomerism means 4-methylimidazole is in equilibrium with 5-methylimidazole.
• Depending upon substituents, one tautomer may predominate. Tautomerism of this kind cannot occur with N-substituted imidazoles; it is dependent upon the presence of an N–H group.

Tautomerism is also not possible with oxazoles or thiazoles.

Therefore, when we meet structures for the imidazole-containing amino acid histidine, we may encounter either of the tautomeric forms shown. Though there will usually be no indication that tautomers exist, do not think there is a discrepancy in structures.

The Imidazole Ring Of Histidine: Acid-Base Properties

The amino acid histidine contains an imidazole ring. We have just seen that unsubstituted imidazole as a base has pK_a 7.0. From the Henderson–Hasselbalch equation

⇒ \(\mathrm{pH}=\mathrm{p} K_{\mathrm{a}}+\log \frac{\text { [base }]}{\text { [acid }]}\)

we can deduce that in water, at pH 7, the concentrations of acid and conjugate base are equal, i.e. imidazole is 50% protonated.

• The imidazole side-chain of histidine has a pK_a value of 6.0, making it a weaker base than the unsubstituted imidazole.
• This reflects the electron-withdrawing inductive effect of the amino group, or, more correctly the ammonium ion, since amino acids at pH values around neutrality exist as doubly charged zwitterionic forms.
• Using the Henderson–Hasselbalch equation, this translates to approximately 9% ionization of the heterocyclic side-chain of histidine at pH 7.

In proteins, pK_a values for histidine side chains are estimated to be in the range 6–7, so the level of ionization will, therefore, be somewhere between 9 and 50%, depending upon the protein.

This level of ionization is particularly relevant in some enzymic reactions where histidine residues play an important role.

• This means that the imidazole ring of a histidine residue can act as a base, assisting in the removal of protons, or that the imidazolium cation can act as an acid, donating protons as required.
• The terminology used for such donors and acceptors of protons is a general acid catalyst and general base catalyst respectively.
• A typical role for the histidine imidazole ring is shown below, in the enzyme mechanism for a general base-catalyzed hydrolysis of an ester.
• The imidazole nitrogen acts as a base to remove a proton from water, generating hydroxide that attacks the carbonyl. Subsequently, the alkoxide-leaving group is deprotonated by the imidazolium ion.

General Base-Catalysed Ester Hydrolysis:

The beauty of this is that we effectively have the same mechanism as in the hydrolysis of an ester using aqueous sodium hydroxide. However, with the enzyme catalyst, this is all taking place at pH 7 or thereabouts.

• Implicit in the above mechanism, though not emphasized, is the pronounced ability of imidazole rings to hydrogen bond.
• Imidazole resembles water, in that it is both a very good donor and a very good acceptor for hydrogen bonding.

Imidazole (and also pyrazole) has a higher-than-expected boiling point, ascribed to intermolecular hydrogen bonding. This leads to polymeric-like structures for imidazole and dimers for pyrazole.

In enzymic mechanisms, we are not usually going to get imidazole–imidazole hydrogen bonding, but the ability of imidazole to hydrogen bond to water, to other small molecules, and carboxylic acid side chains facilitates the enzyme reaction by correctly positioning the reagents.

Histamine And Histamine Receptors:

Most people have heard of antihistamines, even if they have little concept of the nature of histamine.

Histamine is the decarboxylation product from histidine and is formed from the amino acid by the action of the enzyme histidine decarboxylase.

The mechanism of this pyridoxal phosphate-dependent reaction will be studied in more detail later.

Histamine is released from mast cells during inflammatory or allergic reactions. It then produces its typical response by interaction with specific histamine receptors, of which there are several types.

• H₁ receptors are associated with inflammatory and allergic reactions, and H₂ receptors are found in acid-secreting cells in the stomach. Drugs to target both of these types of receptors are widely used.
• The term antihistamine usually relates to H₁ receptor antagonists. These drugs are valuable for pain relief from insect stings or for the treatment and prevention of allergies such as hay fever.
• Major effects of histamine include dilation of blood vessels, inflammation and swelling of tissues, and narrowing of airways.
• In serious cases, life-threatening anaphylactic shock may occur, caused by a dramatic fall in blood pressure. Remarkably, current H₁ receptor antagonists, for example., diphenhydramine, bear little if any structural similarity to histamine.
• The main clinical use of H₂ receptor antagonists is to inhibit gastric secretion in the treatment of stomach ulcers.

These agents all contain features that relate to the histamine structure, in particular the heterocyclic ring. Cimetidine and ranitidine are the most widely used in this class.

Cimetidine contains an imidazole ring comparable to histamine, a sulfur atom (thioether group) in the side chain, and a terminal functional group based upon a guanidine.

• Ranitidine bears considerable similarity to cimetidine, but there are some important differences.
• The heterocycle is now furan rather than imidazole, and the guanidine has been modified to an amidine. A newer drug, nizatidine, is a variant of ranitidine with a thiazole heterocyclic ring system.

Reactivity Of 1,3-Azoles:

Electrophiles can add to N-3, the azomethine =N–, of 1,3-azoles as they can to the pyridine nitrogen.

N Alkylation is complicated in the case of imidazole by the possibility of forming a dialkyl imidazolium salt; the first-formed protonated N-alkyl imidazole can be deprotonated by imidazole, then alkylated further.

N-acylation is mechanistically similar, and mono-acylation can be accomplished by using two molar equivalents of imidazole to one of the acylating agents, the second mole serving to deprotonate the first-formed N-3-acyl imidazolium salt.

• Note that in Ac dimethylimidazolium salt alkylation and acylation, it is the =N– that acts as the nucleophile; this carries the only lone pair.

However, proton loss occurs from the other nitrogen, giving the impression that the N–H has been alkylated or acylated.

The 1,3-diazoles are much less susceptible to electrophilic substitution than pyrrole, furan, and thiophene, but are more reactive than pyridine. Imidazole is the most reactive and may be nitrated readily.

• Substitution occurs at C-5, but tautomerism then leads to the 4(5) mixture.
• The position of substitution may be predicted from a consideration of resonance structures: attack at C-5 provides maximum delocalization with no particularly unfavorable resonance forms.

There is less delocalization after the attack at C-2; one of the resonance forms has an unfavorable electron-deficient nitrogen.

In general, the 1,3-diazoles do not react by nucleophilic substitution, although imidazole can participate in the Chichibabin reaction with substitution at C-2; the position of substitution is equivalent to that noted with pyridine.

• Nucleophilic species that are strong bases, like sodium amide, are more likely to remove the NH proton (pK_a 14).

However, oxazole and thiazole do not have any NH, and the most acidic proton is that at C-2. The electronegative oxygen and sulfur can support an adjacent negative charge.

It is found that quaternary salts of 1,3-azoles are deprotonated at C-2 in the same way.

• Rates of deprotonation are considerably faster because of the influence of the quaternary center that provides a favorable inductive effect.
• The conjugate base bearing opposite charges on adjacent atoms is termed a ylid (or ylide; pronounced il-ide). This ylid, with neg negative charge on carbon, is potentially a nucleophilic species.
• Thus, it is found that both oxazoline and thiazolium salts undergo H–D exchange at C-2 remarkably quickly under basic conditions, illustrating very simply this nucleophilic behavior.

The Thiazolium Ring In Thiamine:

Thiamine (vitamin B₁), in the form of thiamine diphosphate (TPP), is a coenzyme of considerable importance in carbohydrate metabolism.

• Dietary deficiency leads to the condition beriberi, characterized by neurological disorders, loss of appetite, fatigue, and muscular weakness.
• We shall study several TPP-dependent reactions. At this stage, we should merely examine the structure of thiamine, and correlate its properties with our knowledge of heterocycles.
• Thiamine contains two heterocyclic rings, a pyrimidine, and a thiazole, the latter present as a thiazolium salt.

The pyrimidine portion is unimportant for our understanding of the chemistry of TPP, though it may play a role in some of the enzymic reactions.

The proton in the thiazolium ring is relatively acidic (pK_a about 18) and can be removed by even weak bases to generate the carbanion or ylid; a ylid is a species with positive and negative charges on adjacent atoms.

• This ylid is an ammonium ylid with extra stabilization provided by the sulfur atom. The lid can act as a nucleophile and is also a reasonable leaving group.
• Prominent among TPP-dependent reactions is the oxidative decarboxylation of pyruvic acid to acetyl-CoA; this reaction links the glycolytic pathway to the Krebs cycle.
• The addition of the thiazolium ylid to the carbonyl group of pyruvic acid is the first reaction of this sequence, and this allows the necessary decarboxylation, the positive nitrogen in the ring acting as an electron sink.

In due course, the thiazolium ylid is regenerated as a leaving group. We shall look at this sequence in more detail.

1,2-Azoles: Pyrazole, Isoxazole, And Isothiazole

As in the 1,3-azoles, the =N–nitrogen carries a lone pair of electrons and 1,2-azoles are thus potentially basic.

• However, the direct linking of the two heteroatoms has a base-weakening effect.
• Thus, pyrazole has pK_a 2.5 and isoxazole pK_a − 3.0. The higher basicity in pyrazole is probably related to the symmetry of the contributing resonance structures.

The greater electron-withdrawing effect of oxygen compared with sulfur is reflected in the basicity of isothiazole (pK_a−0.5).

Heterocycles Fused To A Benzene Ring:

Many interesting and important heterocyclic compounds contain fused ring systems.

• Some of the common ones are the result of fusing a heterocycle to equivalent resonance structures in a benzene ring, and these have long-established trivial names, for example., indole, quinoline, and isoquinoline.
• Systematic names can be derived by relating to the parent heterocycle and using the prefix benzo to indicate its fusion with benzene.

It is necessary to define which of the bonds in the heterocycle is fused to benzene, and this is accomplished through the use of a bond descriptor, a lowercase italic letter in square brackets.

Thus, indole is benzo[b]pyrrole, quinoline is benzo[b]pyridine, and isoquinoline, an isomer of quinoline with a different type of fusion, becomes benzo[c]pyridine. A few other examples are shown below.

• Note that the bonds of the heterocycle are lettered starting from the heteroatom.
• Where we have two similar heteroatoms, lettering is chosen to produce the lower alternative. Thus, quinazoline is benzo[d]pyrimidine, not benzo[e]pyrimidine.

Where the heteroatoms are different, just as we number from the atom of higher atomic number, we also letter from the same atom. Hence, benzo[d]isoxazole is quite different from benzo[c]isoxazole.

The final fused ring system is then given a completely new numbering system, different from that of the heterocycle.

• Typically, this starts adjacent to the bridgehead atom and then proceeds around the fused ring. The major criterion is to generate the lowest number for the first heteroatom.
• Note that, in most cases, we have little regard for Kekulé forms of pyridine rings in which the Kekule form of a benzene or pyridine ring is drawn. The three versions of quinoline shown are simply contributing resonance forms.

However, some structures, such as isoindole, benzo[c]furan, or benzo[c]isoxazole above, can only be drawn in one way without invoking charge separation.

Quinoline And Isoquinoline

Quinoline and isoquinoline are benzopyridines. They behave by showing the reactivity associated with either the benzene or the pyridine rings.

• Quinoline is basic with a pK_a of 4.9, similar to that of pyridine (pK_a 5.2).
• As with pyridine, the nitrogen carries a lone pair in an sp² orbital. Alkyl halides and acyl halides also react with nitrogen to give N-alkyl- and N-acyl quinolinium salts.

The N-alkyl salts are stable, but the N-acyl salts hydrolyze rapidly in the presence of water.

Quinoline is much more reactive towards elec trophilic substitution than pyridine, but this is because substitution occurs on the benzene ring, not on the pyridine.

• We have already seen that pyridine carbons are unreactive towards electrophilic reagents, with strongly acidic systems protonating the nitrogen first, further inhibiting the reaction.
• This is again true in quinoline, so that the protonated system is involved in the reaction, and the benzene ring undergoes substitution.

With a nitrating mixture of HNO₃ –H₂SO₄, the products are 5- and 8-nitroquinoline in roughly equivalent amounts.

This may be rationalized by considering the stability of intermediate addition cations.

• When the electrophile attacks at C-5 or C-8, the intermediate cation is stabilized by resonance, each having two favorable forms that do not perturb the aromaticity of the pyridinium system.
• In contrast, for attack at C-6 or C-7, there is only one such resonance form. We used similar reasoning to explain why naphthalene undergoes preferential electrophilic substitution at the α-positions.

Whilst we may be a little unhappy about the protonation of a quinolinium cation to an intermediate that carries two positive charges, we find that N-methylquinolinium salts also undergo nitration at a similar rate to quinoline; so this mechanism appears correct.

Nucleophilic substitution occurs at C-2, and to a lesser extent C-4, as might be predicted from similar reactions with pyridine.

• Chichibabin amination occurs rather more readily than with pyridine, ggiving2-aminoquinoline.
• A typical hydride abstraction process occurs when quinoline is heated with sodium amide.

However, better yields have been achieved by performing the reaction at low temperatures in liquid ammonia solvent and then oxidizing the intermediate dihydroquinoline salt using potassium permanganate.

Quinolines carrying 2- or 4-halo substituents undergo nucleophilic substitution readily, in the same manner as 2- and 4-halo pyridines.

• Hydroxyquinolines with the hydroxyl at positions 2 or 4 exist mainly in the carbonyl form, i.e. 2-quinolone and 4-quinolone.

Note, however, that hydroxyls on the benzene ring would be typical phenols. Again, aminoquinolines follow the pyridine precedent, and the tautomeric imino forms are not observed.

Both 2-aminoquinoline and 4-aminoquinoline pro tonate first on the ring nitrogen, with 4-aminoquin oline being the more basic, the conjugate acid benefiting from increased charge distribution through H⁺ resonance.

No such resonance structures can be drawn for 3-aminoquinoline, which is much less basic (pK_a 4.9).

Quinolone Antibiotics:

The quinolone antibiotics feature as the one main group of antibacterial agents that is synthetic, and not derived from or based upon natural products, as are penicillins, cephalosporins, macrolides, tetracyclines, and aminoglycosides.

The first of these compounds to be employed clinically was nalidixic acid; more recent drugs in current use include ciprofloxacin, norfloxacin, and ofloxacin

‘Quinolone’ as a descriptor is an oversimplification, since nalidixic acid contains two fused pyridine rings rather than a benzopyridine, and ofloxacin has a morpholine ring fused to the quinolone.

• Nevertheless, the quinolone substructure is generally used when referring to this group of antibiotics.

The most important structural features for good antibacterial activity are a carboxylic acid at position 3, a small alkyl group at position 1, a 6-fluorine substituent, and a nitrogen heterocycle, often piperazine, at position 7.

Quinolones are good general antibiotics for systemic infections, and they are particularly useful for urinary tract infections because high concentrations are excreted into the urine.

• The mode of action involves interference with DNA replication by inhibiting DNA gyrase, a bacterial enzyme related to mammalian topoisomerases that breaks and reseals double-stranded DNA during replication.
• Isoquinoline (pK_a 5.4) has a similar basicity to quinoline and pyridine and also undergoes N-alkylation and N-acylation.

Nitration occurs smoothly to give predominantly 5-nitroisoquinoline; the isoquinolinium cation reacts more readily than the quinolinium cation.

Nucleophilic substitution occurs exclusively at position 1 in isoquinoline; the alternative position C-3 is quite unreactive.

This is explained by the loss of benzene resonance in the intermediate anion. Thus, Chichibabin amination gives 1-aminoisoquinoline.

Substitution with displacement of halide occurs readily at C-1 and much less readily at C-3 for the same reasons, i.e. the loss of benzene resonance if C-3 is attacked.

1-Isoquinolone exists completely in the carbonyl form, whereas 1-amino isoquinoline is the normal tautomer.

The basicity of 1-amino isoquinoline (pK_a 7.6) is similar to that of 2-aminoquinoline (pK_a 7.3).

Indole

Indole is the fusion of a benzene ring with a pyrrole. Like quinoline and isoquinoline, indole behaves as an aromatic compound.

• However, unlike quinoline and isoquinoline, where the reactivity was effectively part benzene and part pyridine, the reactivity in indole is modified by each component of the fusion.
• The closest similarity is between the chemistry of pyrroles and indoles. Indoles, like pyrroles, are very weak bases. The conjugate acid of indole has pK_a − 3.5; that of pyr role has pK_a − 3.8.

As in the case of pyrrole, nitrogen has already contributed its lone pair to the aromatic sextet, so N-protonation would necessarily destroy aromaticity in the five-membered ring.

Nevertheless, an equilibrium involving the N-protonated cation is undoubtedly set up, since acid-catalyzed deuterium exchange of the N hydrogen occurs rapidly, even under very mild acidic conditions.

• Protonation eventually occurs preferentially on carbon, as with pyrrole; but there is a difference, in that this occurs on C-3 rather than on C-2. This is the influence of the benzene ring.
• It can be seen that protonation on C-3 allows resonance in the five-membered ring and charge localization on nitrogen.

In contrast, any resonance structure from protonation at C-2 destroys the benzene ring aromaticity.

Indole is very reactive towards electrophiles, and it is usually necessary to employ reagents of low reactivity. Nitration with HNO₃ –H₂SO₄ is unsuccessful (compare pyrrole), but can be achieved using benzoyl nitrate.

It is also possible to brominate and methylate at C-3; however, conditions must be controlled carefully, since further electrophilic reactions may then occur.

Treatment with acetic anhydride leads to 1,3-diacetyl indole. Indole reacts readily as the nucleophile in Mannich reactions. This provides convenient access to other derivatives, as shown below.

Thus, quaternization at the side-chain nitrogen allows the ready elimination of trimethylamine. This is facilitated by the electron-releasing ability of the indole nitrogen and can be brought about by a mild base.

• By choosing KCN as the mild base, the transient 3-methyleneindoleninium salt can be trapped by cyanide nucleophiles, leading to indole acetonitrile.
• Reduction of the nitrile group with LAH provides a route to tryptamine. A simple addition to carbonyl compounds occurs under mild acidic conditions.
• Examples are given illus trate reaction with acetone, an aldol-like reaction, and conjugate addition to methyl vinyl ketone, a Michael-like reaction.

The first-formed alcohol products in aldol-like reactions usually dehydrate to give a 3- 3-alkylidene-3H-indolium cation.

We noted above that pyrrole, though a very weak base, is potentially acidic (pK_a 17.5).

• This was because the anion formed by losing the proton from nitrogen has a negative charge on the relatively electronegative nitrogen, but maintains its aromaticity.

The indole anion is also formed by the loss of the N–H proton (pK_a 16.2) using sodium amide sodium hydride, or even a Grignard reagent as a base.

The indole anion is resonance stabilized, with a negative charge localized mainly on nitrogen and C-3.

• It can now participate as a nucleophile, for example., in alkylation reactions. However, this can lead to N-alkylation or C-alkylation at C-3.
• Which is the predominant product depends upon several variables; but, as a general rule, if the associated metal cation is sodium, then the anion is attacked at the site of highest electron density, i.e. the nitrogen.
• Where the cation is magnesium, i.e. the Grignard reagent, then the partial covalent bonding to nitrogen prevents attack there, and the reaction occurs at C-3.

Indoles In Biochemistry:

Some rather important indole derivatives influence our everyday lives. One of the most common ones is tryptophan, an indole-containing amino acid found in proteins.

• Only three of the protein amino acids are aromatic, the other two, phenylalanine and tyrosine are simple benzene systems.
• None of these aromatic amino acids is synthesized by animals and they must be obtained in the diet. Despite this, tryptophan is surprisingly central to animal metabolism.

It is modified in the body by decarboxylation (see Box 15.3) and then hydroxylation to 5-hydroxytryptamine (5-HT, serotonin), which acts as a neurotransmitter in the central nervous system.

Serotonin mediates many central and peripheral physiological functions, including contraction of smooth muscle, vasoconstriction, food intake, sleep, pain perception, and memory, a consequence of it acting on several distinct receptor types.

• Although 5-HT may be metabolized by monoamine oxidase, platelets and neurons possess a high-affinity mechanism for reuptake of 5-HT.
• This mechanism may be inhibited by the widely prescribed antidepressant drugs termed selective serotonin reuptake inhibitors (SSRI), for example., fluoxetine (Prozac), thereby increasing levels of 5-HT in the central nervous system.
• Migraine headaches that do not respond to analgesics may be relieved by the use of an agonist of the 5-HT1 receptor since these receptors are known to mediate vasoconstriction.
• Though the causes of migraine are not clear, they are characterized by dilation of cerebral blood vessels.
• 5-HT1 agonists based on the 5-HT structure in current use include the sulfonamide derivative sumatriptan, and the more recent agents naratriptan, rizatriptan, and zolmitriptan.

These are of considerable value in treating acute attacks.

Several of the ergot alkaloids also interact with 5-HT receptors. Some are used medicinally, but the most notorious is the semi-synthetic derivative lysergic acid diethylamide (LSD).

• This is itself an indole derivative, though the indole is part of a more complex fused-ring system.
• Nevertheless, from the structural similarities, it is not difficult to see why LSD might trigger 5-HT receptors.
• It has the additional ability to interact with noradrenaline and dopamine receptors, thus generating a complex pharmacological response.
• LSD is probably the most powerful psychotomimetics known, intensifying and distorting perceptions.

Experiences can vary from beautiful visions to living nightmares, and no two ‘trips’ are alike.

Also known to be hallucinogenic are the indole derivatives psilocin and psilocybin found in the so-called magic mushrooms, Psilocybe species.

• Ingestion of these small fungi causes visual hallucinations with rapidly changing shapes and colors.
• Psilocybin is the phosphate of psilocin; although based on 4-hydroxytryptamine, it also acts on 5-HT receptors.

Melatonin is N-acetyl-5-methoxytryptamine, a simple derivative of serotonin. It is a natural hormone secreted by the pineal gland in the brain during the hours of darkness.

• It is involved in controlling the body’s day-night rhythm, the ability to sleep during the night, and to stay awake during the day.
• When given as a drug, melatonin induces sleep and adjusts the internal body clock. It is now used as a means of reducing the effects of jet lag
• Plants also require hormones to trigger their growth patterns. One of these is indole-3-acetic acid, which controls cell elongation and is produced in the growing shoot tips.
• One of the major subdivisions of plant alkaloids is termed the indole alkaloid group. All contain the basic indole heterocycle, and many have valuable pharmacological activity that can be exploited in drug materials.
• The indole portion is very often fused to another heterocycle; we shall see some typical structures, where we shall consider them under fused heterocycles.

Fused Heterocycles

There is ample scope for increasing structural complexity by fusing two or more heterocycles.

• Shown below are a few of the ring systems encountered in natural compounds, many of which have interesting, and potentially useful, biological properties.
• Note that in some cases the rings are fused so that the heteroatom can be at the ring junction and is thus common to both rings.

This gives us even more combinations.

We do not wish to consider these further, but instead, we shall concentrate on just two groups of fused heterocycles of particular importance, the purines and pteridines. Both purine and pteridine are parent heterocycles for nomenclature purposes.

• The systematic procedure for naming fused heterocycles is an extension of what we saw when we considered a benzene ring fused to a heterocycle. The main difference is that we have to identify bonds in two different rings to indicate the fusion.
• We use lettering for bonds in one heterocycle and numbers for bonds in the other. Numbering is used for the ‘sub stituent’ ring and lettering for the ‘root’ ring, and all are put in square brackets between substituent and root.

We do not wish to include a great amount of detail, but we shall use purine and pteridine to illustrate the approach and to provide a modest level of familiarity when such names are encountered.

The fused heterocycle is then given its numbering system, starting adjacent to a bridgehead atom to generate the lowest number for the first heteroatom. nucleic acids are bonded to sugar through N-9, the additional potential for tautomerism in the imidazole ring is no longer of concern.

Fused Heterocycles Purines:

Purines, along with pyrimidines, feature as bases in nucleic acids, DNA, and RNA. A purine is the product of fusing a five-membered imidazole ring onto a six-membered pyrimidine ring.

The accepted numbering system unfortunately is non-systematic and treats purine as a pyrimidine derivative, the pyrimidine ring being numbered first and separately from the other ring.

Purine in solution exists as a roughly equimolar mixture of two tautomeric forms, 9H-purine and 7H purine, tautomerism involving the imidazole ring as we have noted earlier.

• The purine systems in nucleic acids, adenine, and guanine, are aminopurines.
• The amino group is on the pyrimidine ring and, as with aminopyrimidines, these compounds exist as their amino tautomers.

Guanine also has an oxygen substituent on the pyrimidine ring, and this adopts the carbonyl form, also following the behavior of oxypyrimidines.

Because adenine and guanine in possible N-protonated forms are produced. This is perhaps unexpected, in that protonation on N-7 would provide a cation that is resonance stabilized in the imidazole ring.

• However, the observed pK_a more closely resembles that of pyrimidine (pK_a 1.3) rather than that of imidazole (pK_a 7.0).
• Amino groups increase basicity (adenine pK_a 4.3), though the oxygen substituent in guanine reduces the effect of the amino group (guanine pK_a 3.3).

In the aminopurines, the position of protonation appears to be N-1 in adenine, whereas it is N-7 in guanine; this presumably reflects the opposing effects provided by amino groups (electron donating) and carbonyl groups (electron withdrawing).

Purine has an acidic pK_a of 8.9, making it somewhat more acidic than phenol (pK_a 10), and a stronger acid than imidazole (pK_a 14.2). The N-9 proton is lost giving an anion with substantial resonance delocalization of charge.

The acidities of adenine (pK_a 9.8) and guanine (pK_a 9.9) are similar, though different protons are removed.

Adenine loses the N-9 proton, but guanine is ionized at N-1. N-1 is part of an amide-like system, and the charge in the conjugate base can be delocalized to the more favorable electronegative oxygen. The effect is most pronounced in uric acid, a metabolite of purines.

Uric Acid, A Purine Metabolite:

Nucleic acid degradation in humans and many other animals leads to the production of uric acid, which is then excreted.

The process initially involves purine nucleotides, adenosine, and guanosine, which are combinations of adenine or guanine with ribose. The purine bases are subsequently modified as shown.

The amino groups are replaced with oxygen. Although there is a biochemical reaction, the same can be achieved under acid-catalyzed hydrolytic conditions and resembles the nucleophilic substitution on pyrimidines.

• The first-formed hydroxy derivative would then tautomerize to the carbonyl structure. In the case of guanine, the product is xanthine, whereas adenine leads to hypoxanthine.
• The latter compound is also converted into xanthine by an oxidizing enzyme, xanthine oxidase. This enzyme also oxidizes xanthine at C-8, giving uric acid.
• Uric acid is not a carboxylic acid but is a relatively strong acid with pK_a 5.8. It has an ‘all-amide’ structure, and there are four potential sites for the loss of a proton.

Deprotonation occurs at N-9. Loss of this proton generates a conjugate base in which the charge can be delocalized to oxygen, giving maximum charge distribution. One resonance form is particularly favorable in having aromaticity in both rings.

Impaired purine metabolism can lead to a build-up of uric acid, and deposition of salts of uric acid as crystals in the joints.

• This causes the painful condition known as gout. One way of treating gout is to reduce uric acid biosynthesis by specific inhibition of the enzyme xanthine oxidase.
• The hypoxanthine analog allopurinol is a drug that is used for this purpose. Allopurinol resembles hypoxanthine, though it contains a pyrazole ring rather than an imidazole ring.

Allopurinol is oxidized by the enzyme to alloxanthine. This product then acts as an inhibitor of the enzyme, binding to the enzyme, but not being modified further and not being released.

Caffeine, Theobromine, And Theophylline:

After the nucleic acid purines adenine and guanine, the next most prominent purine in our everyday lives is probably caffeine.

• Caffeine, in the form of beverages such as tea, coffee, and cola, is one of the most widely consumed and socially accepted natural stimulants.
• Closely related structurally are theobromine and theophylline. Theobromine is a major constituent of cocoa and related chocolate products.

Caffeine is also used medicinally, but theophylline is much more important as a drug compound because of its muscle relaxant properties, utilized in the relief of bronchial asthma.

These compounds competitively inhibit phosphodiesterase, resulting in an increase in cyclic AMP and subsequent release of adrenaline.

• This leads to the major effects: a stimulation of the central nervous system (CNS), a relaxation of bronchial smooth muscle, and induction of diuresis.
• These effects vary in the three compounds. Caffeine is the best CNS stimulant and has weak diuretic action. Theobromine has little stimulant action but has more diuretic activity and also muscle relaxant properties.
• Theophylline also has low stimulant action and is an effective diuretic, but it relaxes smooth muscle better than caffeine or theobromine.
• It has been estimated that beverage consumption may provide the following amounts of caffeine per cup or average measure: coffee, 30–150 mg (average 60–80 mg); instant coffee, 20–100 mg (average 40–60 mg); decaffeinated coffee, 2–4 mg; tea, 10–100 mg (average 40 mg); cocoa, 2–50 mg (average 5 mg); cola drink, 25–60 mg.
• The maximal daily intake should not exceed about 1 g to avoid unpleasant side effects, for example., headaches, and restlessness. An acute lethal dose is about 5–10 g.

Caffeine and theobromine may be obtained in large quantities from natural sources, or they may be obtained by total or partial synthesis. Theophylline is usually produced by total synthesis.

Pteridines:

In pteridines, we have a pyrimidine ring fused to a pyrazine ring. There are, of course, several possible ways of combining these two six-membered ring systems; pteridines are pyrazino[2,3- d]pyrimidines.

• We do not want to consider the chemistry of the pteridine ring system here, but instead, we shall look at the structures of two rather important pteridine-based biochemicals, namely folic acid and riboflavin.
• In the latter case, the pteridine is also fused further to a benzene ring, giving an even more complex ring system, a benzo[g]pteridine.

The accompanying diagram shows the derivation of the fused-ring nomenclature. The oxygenated form of the benzopteridine found in riboflavin is also called an isoalloxazine.

Folic acid (vitamin B₉) is a conjugate of a pteridine unit, p-aminobenzoic acid, and glutamic acid. Deficiency of folic acid leads to anemia, and it is also standard practice to provide supplementation during pregnancy to reduce the incidence of spina bifida.

Folic acid becomes sequentially reduced in the body by the enzyme dihydrofolate reductase to give dihydrofolic acid (FH₂) and then tetrahydrofolic acid (FH₄). Reduction occurs in the pyrazine ring portion.

• Tetrahydrofolic acid then functions as a carrier of one-carbon groups for amino acid and nucleotide metabolism.
• The basic ring system can transfer methyl, methylene, methenyl, or formyl groups, and it utilizes slightly different reagents as appropriate.
• These are shown here; for convenience, we have left out the benzoic acid–glutamic acid portion of the structure.

These compounds are all interrelated, but we are not going to delve any deeper into the actual biochemical relationships.

In any case, you might be able to analyze some of the relationships on a purely chemical basis. For example, tetrahydrofolic acid reacts readily and reversibly with formaldehyde to produce N5 and N10-methylene-FH₄.

• You could consider N-5 of the reduced pteridine ring reacting with formaldehyde (a one-carbon reagent) to give an iminium cation, which could then cyclize via nucleophilic attack of N-10.
• We might also consider reducing the iminium cation with, say, borohydride to give the N-methyl derivative.

These are not necessarily the same as what is occurring in the enzymic reactions, but they should help to make the structures appear rather more familiar.

Now we have seen that the usual reagent for biological methylations is S-adenosylmethionine (SAM).

One occasion where SAM is not employed, for fairly obvious reasons, is the regeneration of methionine from homocysteine, after a SAM methylation. For this, N5-methyl-FH4 is the methyl donor, with vitamin B₁₂ also playing a role as a coenzyme.

Another vitally important methylation reaction involving folic acid derivatives is the production of the nucleic acid base thymine from uracil.

• Uracil is found in RNA, and thymine is a component of DNA; thymine is the methyl derivative of uracil. For continuing DNA synthesis, it is necessary to methylate uracil.
• In practice, it is the nucleotide deoxyuridylate (dUMP) that is methylated to deoxy thymidylate (dTMP). The methylating agent employed here is N⁵, N¹⁰-methylene-FH₄. As a consequence of this reaction, N⁵, N¹⁰– methylene-FH₄ is converted into dihydrofolic acid.
• To keep the reaction flowing, this is reduced to FH₄, and further N⁵, N¹⁰-methylene-FH₄ is produced using a one-carbon reagent.

In this process, the one-carbon reagent comes from the amino acid serine, which is transformed into glycine by the loss of its hydroxymethyl group. The chemistry of the transformations is fairly complex and outside our requirements.

Folic acid derivatives are essential for DNA synthesis, in that they are cofactors for certain reactions in purine and pyrimidine biosynthesis, including the uracil–thymine methylation just described.

• They are also cofactors for several reactions relating to amino acid metabolism.
• The folic acid system thus offers considerable scope for drug action. Mammals must obtain their tetrahydrofolate requirements from their diet, but microorganisms can synthesize this material.
• This offers scope for selective action and led to the use of sulfanilamide and other antibacterial sulfa drugs, compounds that competitively inhibit the biosynthetic enzyme (dihydropteroate synthase) that incorporates p-aminobenzoic acid into the structure.
• Rapidly dividing cells need an abundant supply of dTMP for DNA synthesis, and this creates a need for dihydrofolate reductase activity.

Specific dihydrofolate reductase inhibitors have become especially useful as antibacterials, for example., trimethoprim, and antimalarial drugs, for example., pyrimethamine.

These are pyrimidine derivatives and are effective because of differences in susceptibility between the enzymes in humans and the infective organism.

• Anticancer agents based on folic acid, for example., methotrexate, inhibit dihydrofolate reductase, but they are less selective than the antimicrobial agents and rely on a stronger binding to the enzyme than the natural substrate has.
• They also block pyrimidine biosynthesis. Methotrexate treatment is potentially lethal to the patient and is usually followed by ‘rescue’ with folinic acid (N5-formyl-tetrahydrofolic acid) to counteract the folate-antagonist action.

The rationale is that folinic acid ‘rescues’ normal cells more effectively than it does tumor cells.

Riboflavin:

Riboflavin (vitamin B₂) is a component of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), coenzymes that play a major role in oxidation-reduction reactions.

• Many key enzymes involved in metabolic pathways are covalently bound to riboflavin and are thus termed flavoproteins.
• Riboflavin contains an isoalloxazine ring linked to the reduced sugar ribitol.

The sugar unit in riboflavin is the non-cyclic ribitol so FAD and FMN differ somewhat from the nucleotides we encounter in nucleic acids.

Riboflavin is widely available in foods; dietary deficiency is uncommon, but it manifests itself in skin problems and eye disturbances.

The flavin nucleotides are typically involved in the oxidations creating double bonds from single bonds.

The flavin takes up two hydrogen atoms, represented in the figure as being derived by the transfer of hydride from the substrate and a proton from the medium.

Reductive sequences involving flavoproteins may be represented as the reverse reaction, where hydride is transferred from the coenzyme, and a proton is obtained from the medium.

• The reaction mechanism shown here is in many ways similar to that in NAD⁺ oxidations, i.e. a combination of hydride and a proton; it is less easy to explain adequately why it occurs, and we do not consider any detailed explanation advantageous to our studies.
• We should register only that the reaction involves the N=C–C=N function that spans both rings of the pteridine system.

Some Classic Aromatic Heterocycle Syntheses:

Our study of heterocyclic compounds is directed primarily to an understanding of their reactivity and importance in biochemistry and medicine.

• The synthesis of aromatic heterocycles is not, therefore, a main theme, but it is useful to consider just a few examples to underline the application of reactions in earlier chapters.
• From the beginning, we should appreciate that the synthesis of substituted heterocycles is probably not best achieved by carrying out substitution reactions on the simple heterocycle.
• It is often much easier and more convenient to design the synthesis so that the heterocycle already carries the required substituents, or has easily modified functions.

We can consider two main approaches for heterocycle synthesis, here using pyridine and pyrrole as targets.

We can insert the heteroatom into the rest of the carbon skeleton, or attempt to join two units, one of which contains the heteroatom, using C–C and C–heteroatom linkages.

• To make the new bonds, two reaction types are most frequently encountered.
• Heteroatom–C bond formation is achieved using the heteroatom as a nucleophile to attack an electrophile such as a carbonyl group.
• Aldol-type reactions may be exploited for C–C bond formation, employing enamines and enols/enolate anions.
• We shall now look at some synthetic procedures that merit the descriptor ‘classic’ because of their general application, and their longevity – some have been around for more than 100 years.

Do not worry about remembering the names: these commemorate the originators, and we should instead concentrate on the chemistry, which we shall see is usually a combination of processes we have already met.

Hantzsch Pyridine Synthesis

In its simplest form, this consists of the condensation of a β-ketoester with an aldehyde and ammonia.

• The product is a 1,4-dihydropyridine, which is subsequently transformed into pyridine by oxidation.
• Several separate reactions occur during this synthesis and the precise sequence of events may not be quite as shown below – they may be in a different order.
• The normal Hantzsch synthesis leads to a symmetrical product. The diesters formed may be hydrolyzed and decarboxylated using the base to give pyridines with less substitution.

Note that we are using the ester groups as activating species to facilitate enolate anion chemistry

Skraup Quinoline Synthesis:

The most general method for synthesizing quinolines employs aniline or a substituted aniline, glycerol, sulfuric acid, and an oxidizing agent such as a ferric salt or nitrobenzene.

The first step is acid-catalyzed dehydration of glycerol to the unsaturated aldehyde acrolein. Variations of the Skraup synthesis use different acroleins instead of glycerols.

The initial product is a dihydroquinoline; it is formed via a Michael-like addition, then an electrophilic aromatic substitution that is facilitated by the electron-donating amine function.

• A mild oxidizing agent is required to form the aromatic quinoline.
• The Skraup synthesis can be used with substituted anilines, provided these substituents are not strongly electron-withdrawing and are not acid-sensitive.

Bischler–Napieralski Isoquinoline Synthesis:

Isoquinolines are easily prepared by the reaction of an acyl derivative of a β-phenylethylamine with a dehydrating agent, for example., P₂O₅, then using a catalytic dehydrogenation to aromatize the intermediate 3,4- dihydroisoquinoline.

The crucial cyclization step is represented here as dehydrogenation and an iminium-type system, a resonance form of an electrophilic attack involving the aromatic ring of the amide, suitably coordinated with the phosphorus reagent.

• The cyclizing agent P₂O₅ also dehydrates the intermediate hydroxyamine to a dihydroisoquinoline.
• The isoquinoline is then obtained by heating over a catalyst, effectively reversing a catalytic hydrogenation reaction, facilitated by the generation of aromaticity in the product.
• As in the Skraup synthesis above, electron-withdrawing substituents on the aromatic ring will deactivate it towards electrophilic attack, whereas electron-donating substituents will favor the reaction.

Pictet–Spengler Tetrahydroisoquinoline Synthesis

This approach to the isoquinoline ring, albeit a reduced isoquinoline, is mechanistically similar to the Bischler–Napieralski synthesis, in that it involves an electrophilic attack of an iminium cation onto an aromatic ring. In this case, the imine intermediate is formed by reacting a phenylethylamine with an aldehyde.

We have already met this reaction as an analog of the Mannich reaction, which we then interpreted as a nucleophilic attack of an electron-rich phenolic ring pontoon iminium cation.

• Is it electrophilic or nucleophilic? It matters little; they are the same, though the descriptor used depends upon which species you consider the more important, the nucleophilic phenol or the electrophilic iminium cation.
• For effective cyclization, we need an electron-donating substituent para to the point of ring closure since the Mannich-type electrophile is less reactive than the phosphorus-linked intermediates in the Bis cheer–Napieralski synthesis.
• It is also found that a similar group in the ortho position does not work, though we could still write an acceptable mechanism With a good electron-donating substituent like hydroxyl, the whole process, imine formation, and cyclization can occur under ‘physiological’ conditions pH 6–7 at room temperature.
• In nature, this is precisely how tetrahydroisoquinoline alkaloids are biosynthesized, though the reactions are enzyme-controlled

Knorr Pyrrole Synthesis:

This approach to the five-membered pyrrole ring reacts an α-aminoketone with a β-ketoester.

The mechanism will probably involve imine formation and then cyclization via an aldol-type reaction using the enamine nucleophile.

Dehydration leads to the pyrrole. Only the key parts of this sequence are shown below.

The synthesis works well only with an activated ester like ethyl acetoacetate. Otherwise, self-condensation of the α-aminoketone to a dihydropyrazine occurs more readily than the cyclization.

Paal–Knorr Pyrrole Synthesis:

The other major route to pyrroles is the interaction of a 1,4-dicarbonyl compound with ammonia.

The nucleophilic mechanism below shows successive nucleophilic additions of amino groups onto the carbonyls; but, since no intermediates have been isolated, the precise sequence of steps is speculative.

Note, however, that this synthesis gives furans if no ammonia is included. This would involve a nucleophilic attack of an enol tautomer of the substrate onto another carbonyl to give a hemiketal, followed by dehydration. The heteroatom is thus derived from a carbonyl oxygen.

The procedure works well and is usually carried out with acid catalysts under non-aqueous conditions.

Fischer Indole Synthesis:

The most useful route to indoles is the Fischer indole synthesis, in which an aromatic phenylhydrazone is heated in acid.

• The phenylhydrazone is the condensation product from a phenylhydrazine and an aldehyde or ketone. Ring closure involves a cyclic rearrangement process.
• The hydrazine behaves as an amine towards a carbonyl compound and forms the imine-like product, a hydrazone.
• The cyclic rearrangement involves the enamine tautomer of this hydrazone, and proceeds because the cyclic flow of electrons forms a strong C–C bond whilst cleaving a weak N–N bond.
• This produces what appears to be a di-imine. One of these is involved in rearomatization and creates an aromatic amine.

This then attacks the other imine function, and we get the nitrogen equivalent of a hemiketal. Finally, acid-catalyzed elimination of ammonia gives the aromatic indole system.

Unfortunately, the reaction fails with acetaldehyde and cannot, therefore, be used to synthesize indole itself. It is possible to use the ketoacid pyruvic acid instead and decarboxylate the product to yield indole.

The post Heterocycles appeared first on BDS Notes.

From our discussions of bonding, we have learnt something about the arrangement of bonds around various atoms. These concepts are fundamental to our appreciation of the shape of molecules, i.e. stereochemistry.

Before we delve into these matters, let us recap a little on the disposition of bonds around carbon. Bonding at four-valent carbon is tetrahedral, with four sp³-hybridized orbitals mutually inclined at 109.5°.

Remember that the tetrahedral array is demonstrated by experimental measurements, and that hybridization is the mathematical model put forward to explain this observation.

We can conveniently represent the tetrahedral arrangement in two dimensions by using a wedge–dot convention. In this convention, single bonds written as normal lines are considered to be in the plane of the paper.

Bonds in front of this plane, i.e. coming out from the paper, are then drawn as a wedge, whilst bonds behind the plane, i.e. going into the paper, are drawn as a broken or dotted bond.

As we get more familiar with this representation, we may begin to abbreviate it by showing either the wedge or the dotted bond, rather than both.

Of course, it is important to remember that these abbreviated forms actually represent a tetrahedral array and not something with three bonds planar plus one other.

Drawing Stereostructures:

Bonding at three-valent carbon is trigonal planar with bond angles of 120°, an observation that we account for through sp² hybridization plus the formation of a π bond by the overlap of p orbitals.

Thus, an alkene double bond involves electrons in sp² hybrid orbitals making σ single bonds and the remaining electrons in p orbitals overlapping to produce the π-bond component of the double bond.

We can draw this as a planar representation, all single bonds in the plane of the paper, or show the π bonding in the plane of the paper, so that some bonds now require to be drawn in wedge form and others in dotted form.

Bonding at two-valent carbon is linear, i.e. bond angles are 180°, and the triple bond comprises two π bonds and a σ single bond formed from sp hybrid orbitals.

The two π bonds are at right angles to each other. sp hybrid Although most of the atoms in the framework of an organic molecule tend to be carbon, other atoms, such as oxygen and nitrogen, are routinely encountered.

We can consider the arrangement of bonds around these atoms as approximately the same as the sp³– hybridized tetrahedral array seen with carbon. One (nitrogen) or two (oxygen) of the sp³ orbitals will be occupied by lone pair electrons.

The consequences of this include the fact that the two single bonds to oxygen are not linear, but are inclined at about 109°, and the three bonds to nitrogen are similarly not planar.

When oxygen or nitrogen are linked to another atom, For Example. carbon, by double bonds, the arrangement will be equivalent to the trivalent carbon, i.e. trigonal planar with a π bond perpendicular to the plane.

Lone pair electrons (one lone pair for nitrogen, two in the case of oxygen) will occupy nonbonding sp² orbitals.

A triple bond to nitrogen, as bonding at nitrogen and oxygen approximates to that at carbon via lone pairs; in cyanide, will dictate a linear arrangement, with a nitrogen lone pair occupying a nonbonding sp orbital.

Bond angles depend upon the type of hybridization as just described, but in most molecules, they appear to be very similar.

There can often be a small degree of variation because of the nature of the precise atoms being bonded, and the presence of lone pair electrons but the level of consistency is very high

Similarly, bond lengths are also remarkably consistent, depending mainly on the nature of the atoms bonded and whether bonds are single, double, aromatic, or triple.

With bond lengths and bond angles being sufficiently consistent between molecules, it is possible to predict the shape and size of a molecule using simple molecular models or computer graphics.

Stereoisomers

For a given molecular formula there is often more than one way of joining the atoms together, whilst still satisfying the rules of valency.

Such variants are called structural isomers or constitutional isomers compounds with the same molecular formula but with a different arrangement of atoms.

A simple example is provided by C₄H₁₀, which can be accommodated either by the straight-chained butane or by the branched-chain isobutane (2-methylpropane).

Stereoisomers, on the other hand, are compounds with the same molecular formula, and the same sequence of covalently bonded atoms, but with a different spatial orientation.

Two major classes of stereoisomers are recognized, conformational isomers and configurational isomers.

Conformational isomers, or conformers, interconvert easily by rotation about single bonds. Configurational isomers interconvert only with difficulty and, if they do, usually require bond breaking. We shall study these in turn

Conformational Isomers

Conformations Of Acyclic Compounds

Let us consider first the simple alkane ethane. Since both carbons have a tetrahedral array of bonds, ethane may be drawn in the form of a wedge–dot representation.

Now let us consider the rotation of the right-hand methyl group about the C–C bond, and we eventually get to a different wedge–dot representation as shown.

This is more easily visualized by looking at the molecule from one end down the C–C bond and this gives us what is termed a Newman projection.

The Newman projection shows the hydrogen atoms and their bonds, but the carbons are represented by a circle; since we are looking down the C–C bond, we cannot see the rear carbon.

A further feature is that the C–H bonds of the methyl closest to us are shown drawn to the centre of this circle, whilst those of the rear methyl are partially obscured and drawn only to the edge of this circle.

We can draw a similar Newman projection for the second wedge–dot representation, but the C–H bonds of the front and rear methyls will appear to be on top of each other.

We therefore draw a slightly modified version showing all bonds, but must remember that this really represents a system where the bonds at the rear are obscured by the bonds at the front.

In the sawhorse representation, the molecule is viewed from an oblique angle, and all bonds can be seen.

The two representations shown here are actually two different conformers of ethane; there will be an infinite number of such conformers, depending upon the amount of rotation about the C–C bond.

Although there is fairly free rotation about this bond, there does exist a small energy barrier to rotation of about 12 kJ mol^-1 due to the repulsion of the electrons in the C–H bonds.

By inspecting the Newman projections, it can be predicted that this repulsion will be a minimum when the C–H bonds are positioned as far away from each other as possible.

This is when the dihedral angle between the C–H bonds of the front and rear methyls is 60°, as exists in the left-hand conformer.

This conformation is termed the staggered conformation. On the other hand, electronic repulsion will be greatest when the C–H bonds are aligned, as in the right-hand conformer.

This conformation is termed the eclipsed conformation. In between these two extremes, there will be other conformers of varying energies, depending upon the degree of rotation.

Energies for these will be greater than that of the staggered conformer, but less than that of the eclipsed conformer.

Indeed, if one considers a gradual rotation about the C–C bond, the energy diagram will take the form of a sine wave, because rotations of either 120° or 240° will produce an indistinguishable conformer of identical energy. This is shown in.

It follows that the preferred conformation of ethane is a staggered one; but, since the energy barrier to rotation is relatively small, at room temperature there will be free rotation about the C–C bond.

Let us now consider rotation about the central C–C bond in butane. Rotation about either of the two other C–C bonds will generate similar results as with ethane above.

Wedge–dot, Newman, and sawhorse representations are all shown; use the version that appears most logical to you.

As we rotate the groups, we shall get a series of staggered and eclipsed conformers. The energy barrier to rotation will be larger than the 12 kJ mol^-1 seen with ethane.

This is because, in addition to the similar electronic repulsion in the bonds, there is now a spatial interaction involving the large methyl groups.

It follows that the repulsive energy associated with a methyl–methyl interaction will be larger than a methyl–hydrogen interaction, which in turn will be larger than that arising from hydrogen–hydrogen interactions.

Logically then, we predict that the energy of the eclipsed conformer in which the methyl groups are aligned will be higher than that in which there are methyl–hydrogen alignments and that there will be two equivalent versions of the latter.

Similarly, of the low-energy staggered conformers, there will be two equivalent ones where the carbon–methyl bonds are inclined at 60° to each other, and one in which the carbon–methyl bonds are inclined at 180°.

We can also predict that the latter conformer, which has the methyl groups as far away from each other as possible, will be of lower energy than the alternative staggered conformers, where there must be at least some spatial interaction between the methyl groups.

The staggered conformer with maximum separation of methyl groups is termed the anti-conformer (Greek: ant i = against), whilst the two other ones are termed gauche conformers (French: gauche = left).

The energy diagram observed reflects these predictions, and the energy difference between the low-energy staggered anti-conformer and the highest energy eclipsed conformer is about 18.8 kJ mol^-1.

There will still be free rotation about C–C bonds in butane at room temperature, but the larger energy barrier compared with that for ethane means that the staggered conformers are preferred.

Calculations show that, at room temperature, about 70% of molecules will be in the anti-conformer and about 15% in each gauche conformed.

Conformations Of Cyclic Compounds

Cyclopropane, Cyclobutane, Cyclopentane, Cyclohexane

The practical consequences of conformational isomerism become much more significant when we consider cyclic compounds. The smallest ring system will contain three atoms; in the case of hydrocarbons, this will be cyclopropane.

Now, simple geometry tells us that the inside angle in cyclopropane must be 60°. This is considerably less than the 109.5° of tetrahedral carbon.

The consequences are that the amount of overlap of the sp³ orbitals in forming the C–C bonds must be considerably less than in an acyclic system like ethane. With poorer overlap, we get a potentially weaker bond that can be broken more easily.

We term this ring strain, and although three-membered rings exist and are quite stable, they are frequently subject to ring-opening reactions.

A further feature of three-membered rings is that they must be planar, and a consequence of this is that, in cyclopropane, all C–H bonds are in the high energy eclipsed state.

There can be no conformational mobility to overcome this. In cyclobutane, the internal angle is 90°. Consequently, there is high ring strain, but this is not as great as in cyclopropane.

If cyclobutane were planar, all C–H bonds would be in the high-energy eclipsed state. It transpires that cyclobutane is not planar, since it can adopt a more favourable conformation in which eclipsing is reduced, and the ring appears puckered.

This appears to be achieved by pushing pairs of opposite carbons in different directions; but, in reality, it is only a combination of rotations about C–C bonds as we have seen with the simpler acyclic compounds.

It is not possible to achieve the ideal 60° staggered arrangement, but it does produce a lower energy conformed. Of course, there are two alternative ways of doing this, depending on whether pairs of carbons are ‘pushed’ or ‘pulled’.

Both conformers will be produced equally and can interconvert at room temperature because the energy barrier is fairly small at about 5.8 kJ mol^-1.

The interconversion of the two forms is depicted by the equilibrium arrow, comprised of two half arrows. At equilibrium, both conformers coexist, and in this case, in equal amounts since they have the same energy.

The planar form of cyclobutane will be the energy maximum in the interconversion of conformers.

Compounds With Cyclopropane Or Cyclobutane Rings

A cyclopropane ring has the highest level of ring strain in the carbocycles. This means that they are rather susceptible to ring-opening reactions, but it does not mean that they are unstable and cannot exist.

Indeed, there are many examples of natural products that contain cyclopropane rings, and these are perfectly stable under normal conditions.

One group of natural cyclopropane derivatives of special importance is the pyrethrins, insecticidal components of pyrethrum flowers, and widely used in agriculture and in the home.

These compounds have very high toxicity towards insects without being harmful to animals and man and are rapidly biodegraded in the environment.

The pyrethrins are esters of two acids, chrysanthemum acid and pyrethrin acid, with three alcohols, pyrethrolone, cicerone, and jasmine, giving six major ester structures.

The acids contain the cyclopropane ring, and this appears essential for insecticidal activity.

Many semi-synthetic esters, For Example. bioresmethrin, permethrin, and phenothrin have been produced and these have increased toxicity towards insects and also extended lifetimes.

All such esters retain a high proportion of the natural chrysanthemum acid or pyrethrin acid structure. The drugs naltrexone and nalbuphine are semi-synthetic analogues of the analgesic morphine.

Morphine is a good painkiller, but has some unpleasant side effects, the most serious of which is the likelihood of becoming addicted.

Nalbuphine is a modified structure containing a cyclobutane ring as part of the tertiary amine function.

Extending the size of the nitrogen substituent makes the drug larger and allows it to exploit extra binding sites on the receptor that morphine cannot interact with.

Nalbuphine is found to be a good analgesic with fewer side effects than morphine. Naltrexone incorporates a cyclopropane ring in the nitrogen substituent.

This, together with the other structural modifications, produces a drug that has hardly any analgesic effects but is a morphine antagonist.

Accordingly, it can be used to assist in the detoxification of morphine and heroin addicts.

Let us move on to cyclopentane, where geometry tells us the internal angle is 108°.

This is so close to the tetrahedral angle of 109.5° that cyclopentane can be considered essentially free of ring strain. However, planar cyclopentane would have all its C–H bonds eclipsed, which is obviously not desirable.

Accordingly, it adopts a lower energy conformation in which one of the carbon atoms is out of planarity. ‘Pushing’ this carbon out of the plane is achieved by rotation about C–C bonds, and it reduces eclipsing along all but one of the C–C bonds.

The energy barrier to this conformational change is about 22 kJ mol^-1. There is no reason why any one particular carbon should be out of the plane, and at room temperature there is rapid interconversion of all possible variants.

Again, a planar form would feature as the energy maximum in the interconversions. The conformation with four carbons in the plane and one out of a plane is termed an envelope conformation.

This terminology comes from the similarity to an envelope with the flap open. For cyclohexane, the calculated internal angle is 120° if the molecule were to be planar, but the tetrahedral angle of 109.5° turns out to be perfect if the molecule is non-planar.

It is possible to construct a cyclohexane ring from tetrahedral carbons without introducing any strain whatsoever. The ring shape formed in this way is termed a chair conformation.

There is a considerable resemblance to a folding chair having a backrest and legrest, though the open seat might be regarded as a distinct disadvantage.

Not only is the bond angle perfect, but it also turns out that all C–H bonds are in a staggered relationship with adjacent ones. The chair conformation cannot be improved upon.

The total ring strain in various cycloalkanes compared with their strain-free acrylic counterparts has been estimated, as shown in Table.

Thus, small rings like cyclopropane and cyclobutane have considerable ring strain, and cyclohexane is effectively strain-free.

Larger rings (8–11 atoms) have more ring strain than might be predicted, certainly much more than cyclohexane, but any puckering that reduces ring strain actually creates eclipsing. We shall meet rings containing more than six carbons only infrequently.

How To Draw Chair Conformations Of Cyclohexane

You can only appreciate stereochemical features if you can draw a representation that correctly pictures the molecule.

One of the most challenging is the chair conformation of cyclohexane. Practice makes perfect; so this is how it is done.

Note that the wedges and bold bonds help to show how we are looking at the cyclohexane chair.

In practice, particularly to speed up the drawing of structures, we tend to omit these. Then, by convention, the lower bonds represent the nearest part of the ring.

When one looks at the hydrogens in the chair conformation of cyclohexane, one can see that they are of two types.

Six of them are parallel to the central rotational axis of the molecule, so are termed axial. The other six are positioned around the outside of the molecule and are termed equatorial.

One might imagine, therefore, that these two types of hydrogen would have some different characteristics, and be detectable by an appropriate spectral technique.

Such a technique is NMR spectroscopy; but, at room temperature, only one type of proton is detectable.

At room temperature, all hydrogens of cyclohexane can be considered equivalent; this is a consequence of conformational mobility and the interconversion of two-chair conformation.

This interconversion may be considered as the simultaneous pushing down/pulling up of carbons on opposite sides of the ring, as indicated in the lefthand structure.

As a result, the ring ‘flips’ into an alternative conformation, also a chair, as in the righthand structure. This ring flip is actually achieved by rotation about several of the C–C bonds at the same time.

The ring flip can be demonstrated with suitable molecular models, and it is possible to feel the resistance in the model to this rotation, which represents the energy barrier to the change.

Both conformers have the same energy, but the energy barrier is about 42 kJ mol^-1. The energy barrier looks high compared with those in ethane or butane, but this is because the interconversion involves rotations about several C–C bonds at the same time.

Look at the hydrogen atoms shown labelled in the left-hand structure. Note particularly that, after the ring flip, the axial hydrogen becomes equatorial, whilst the equatorial hydrogen becomes axial. Similar changes occur at all other positions.

With rapidly interconverting conformers, the hydrogens cannot be distinguished by NMR spectroscopy and they all merge to give a single signal. However, as one cools.

In the sample, the energy available to overcome the interconversion energy barrier diminishes, until at a sufficiently low temperature, the interconversion stops, and two types of hydrogen are detectable in the NMR spectrum.

This temperature is −89°C. Measurement of this temperature allows the energy barrier to be calculated.

If we look at the two-dimensional hexagon representation for cyclohexane, we could put in the bonds to hydrogens as wedges (up bonds) or dotted lines (down bonds).

We now know the cyclohexane ring is not planar, but has a chair conformation. We shall frequently want to use the hexagon representation, and it will be necessary to assign hydrogens or other substituents onto the chair representation with the correct stereochemistry.

At this stage, it is salutary to look at both the two-dimensional hexagon and the chair representations of cyclohexane. Note particularly that we must not confuse ‘up’ with axial, and ‘down’ with equatorial.

As the structures show, ‘up’ hydrogens or substituents will alternate axial and equatorial as we go around the ring positions.

The chair is not the only conformation that cyclohexane might adopt. An alternative boat conformation is attained if the ring flip-type process is confined to just one carbon.

The name boat comes from the similarity to boats formed by paper folding; sea-worthiness is rather questionable.

Again, there is no ring strain in this conformation, but it turns out that some of the C–H bonds are eclipsed, as seen in the accompanying Newman projection.

In addition, the hydrogens at the top of the structure are getting rather close to each other, and there is some interaction, termed a flagpole interaction, again from the nautical analogy.

Both the eclipsing and the flagpole interactions can be minimized when the boat conformation undergoes further subtle changes by rotation about C–C bonds to form the twist-boat.

This is a result of twisting the flagpole hydrogens apart. Making a molecular model of the boat conformation immediately shows how easy it is to modify it to the twist-boat variant; the boat conformation is quite floppy compared with the chair, which is very rigid.

An energy diagram linking the chair, boat and twist-boat conformations is shown. The boat conformation is represented by an energy maximum.

In practice, only the chair conformation is important for cyclohexane, since the energy differences between it and the other conformations make them much less favourable.

However, there are plenty of structures where cyclohexane rings are forced into the boat or twist-boat conformation because of other limiting factors.

For example, bornane is a terpene hydrocarbon where opposite carbons in a cyclohexane ring are bridged by a methylene group. This is stereochemically impossible to achieve with a chair – the carbons are too far apart.

However, it is possible with a boat confirmation. In such a structure, there are no further possibilities for conformational mobility – the conformation is now fused in and no further changes are possible, even though there may be unfavourable eclipsing interactions.

In cyclohexene, the double bond and adjacent carbons must all be planar. The remainder of the molecule avoids unfavourable eclipsing interactions by adopting what is termed a half-chair conformation.

This would also be found in a cyclohexane ring fused onto an aromatic ring (tetrahydronaphthalene) or fused to a three-membered ring.

The half-chair conformation in cyclohexane (without the double bond) is thought to be equivalent to the energy maximum in Figure that must be overcome in the chair–twist-boat interconversion.

Substituted cyclohexanes

The ring-flipping conformational mobility in the unsubstituted compound cyclohexane has little practical significance; but, when the ring is substituted, we have to take ring flip into account, because one particular conformation is usually favoured over the other

Let us look at a simple example, namely methylcyclohexane. Ring flip in the case of methylcyclohexane achieves interconversion of one conformer where the methyl group is equatorial into a conformer where this group is axial (compare the hydrogens in cyclohexane).

It turns out that the conformer with the equatorial methyl group is favoured over the conformer where the methyl group is axial. The energy difference of these two conformers is estimated to be about 7.1 kJ mol^-1; this is the energy difference, not the barrier to interconversion.

Because of this energy difference, the equilibrium mixture at room temperature has about 95% conformers with the equatorial methyl and only 5% where the methyl is axial.

We can account for the difference in energy between the two conformers quite easily using the reasoning we applied earlier for the acyclic hydrocarbon butane.

We need to consider a Newman projection looking down the 2,1 bond. When the methyl is axial, it can be seen that there will be a gauche interaction between this methyl and the ring methylene (C-3); a second, similar interaction will be seen if we look down the 6,1 bond.

Now, in the conformer where the methyl is equatorial, the Newman projection shows the most favourable anti-arrangement for the methyl and methylene(s); there will be a similar anti-interaction if we look down the 6,1 bond.

On this basis alone, we can predict that the equatorial conformer is of lower energy and, thus, more favoured.

However, there is a further feature that destabilizes the axial conformer, and that is the spatial interaction between the axial methyl and the axial hydrogens at positions 3 and 5, termed a 1,3-diaxial interaction.

Together, they account for the equilibrium mixture consisting mainly of the equatorial conformer. We can indicate this by using arrows of unequal size in the equilibrium equation.

Note that it is not necessary to consider both forms of cyclohexane, where the methyl is either wedged (up) or dotted (down).

If the cyclohexane ring were planar, the two structures would be the same, since one merely has to turn the structure over to get the other.

Although the cyclohexane ring is not planar, it turns out that the two structures are still identical, because of the ring flip process

This is shown below. One set of conformers is simply the upside-down version of the other.

Now, as the substituent gets bigger, the proportion of axial conformer will diminish even further.

With a substituent as big as a tert-butyl group, the equilibrium is such that essentially all molecules are in the equatorial conformation; in general terms, we can consider that a tert-butyl group will never be axial.

Although analysis of the consequences of ring flip in monosubstituted cyclohexane is pretty straightforward, the presence of two or more substituents requires careful consideration to decide which conformer, if any, is the more favoured.

Let us illustrate the approach using 1,4- dimethylcyclohexane. Now, two configurational isomers of this structure can exist, namely trans and cis.

The terms trans and cis are used to describe the configuration, not conformation, of the isomers; in the trans isomer, the two methyl substituents are on opposite sides (faces) of the ring (Latin: trans = across).

Whereas in the cis isomer, they are on the same side of the ring (Latin: cis = on this side). These concepts will become clear when we reach them.

In the trans isomer, one methyl is written down (dotted bond) whilst the other is written up (wedged bond).

If we transform this to a chair conformation, as shown in the left-hand structure, the down methyl will be equatorial and the up methyl will also be equatorial.

With ring flip, both of these substituents then become axial as in the right-hand conformer. From what we have learned about monosubstituted cyclohexanes, it is now easily predicted that the equatorial conformer will be very much favoured over the diaxial conformed.

In the cis isomer, both methyls are written with wedges, i.e. up. In the left-hand chair conformation, one methyl is therefore axial and the other is equatorial.

With a ring flip, the axial methyl becomes equatorial and the equatorial methyl becomes axial. Both conformers have one equatorial methyl and one axial methyl; they must, therefore, be of the same energy, so form a 50:50 equilibrium mixture.

In fact, it is also easy to see that the rotation of either structure about its central axis produces the other structure, a clear illustration that they must be energetically equivalent.

Note that the cis isomer with both methyls down is actually the same compound viewed from the opposite side. This type of reasoning may be applied to other dimethylcyclohexanes, as indicated in the figure.

There is no easy way to predict the result; it must be deduced in each case. One conformer is of much lower energy in the cases of trans-1,2-, cis-1,3-, and trans-1,4-dimethylcyclohexane; both conformers have equal energy in the cases of cis-1,2-, trans-1,3-, and cis-1,4-dimethylcyclohexane.

Should the two substituents be different, and especially of different sizes, then the simple reasoning used above with two methyl substituents will need adapting; the larger substituent will prefer to be equatorial.

Where we have three or more substituents, the most favoured conformer is going to be the one with the maximum number of equatorial substituents, or perhaps where we have the largest substituents equatorial. This is seen in the following examples.

How To Draw Conformational Isomers And To Flip Cyclohexane Rings

Interpreting a two-dimensional stereochemical structure, converting it into a conformational drawing, and considering the consequences of ring flip can cause difficulties.

The process can be quite straightforward if you approach it systematically. We saw early that, if we draw cyclohexane in typical two-dimensional form.

The bonds to the ring could be described as ‘up’ or ‘down’, according to whether they are wedged or dotted. This is how we would see the molecule if we viewed it from the top.

When we look at the molecule from the side, we now see the chair conformation; the ring is not planar as the two-dimensional form suggests.

Bonds still maintain their ‘up’ and ‘down’ relationship, but this means bonds shown as ‘up’ alternate axial–equatorial around the ring; they are not all axial or all equatorial.

Whilst the ring flip process changes equatorial bonds to axial bonds, and vice versa, it does not change the ‘up’–‘down’ relationship.

Let us consider the trimethylcyclohexane isomer shown below. All three substituents are ‘up’. We need to use one of the carbons as a reference marker; let us choose the top one.

I like to make this the left-hand carbon in the chair; to make the process more obvious, we could turn the structure so that our reference carbon is also on the left.

It is most important to have this reference carbon, so that as we put the various substituents in we put them on the correct carbons.

Now draw the two chair conformations of cyclohexane, both having the reference carbon on the left. The carbons opposite our reference point must be furthest right.

If we draw the structures one above the other, lefthand carbons and right-hand carbons should be aligned.

Draw axial and equatorial bonds at the relevant carbons where we have the substituents and identify them as ‘up’ or ‘down’.

Since we are interpreting the structure as though we are looking down on it from the top, the lower part of the ring represents the near-most part of the conformational drawing.

It can also help to number the carbons. Then fill in the substituents as necessary.

In this example, our three methyl groups are all ‘up’, which means that in one conformer the groups will be axial, equatorial, and axial, whereas in the other they will be equatorial, axial, and equatorial.

The latter conformer, with the most equatorial substituents, will be the favoured one. A word of warning is appropriate here.

As we shall see in due course (see Box 3.11), merely changing a substituent from, say, equatorial to axial without flipping the ring changes the configuration, and can produce a different molecule. It would also destroy the ‘up’ or ‘down’ identifier.

To take this general principle to its extreme, we noted above that tert-butyl groups are sufficiently large that they never occupy an axial position.

It is possible to make di-tert-butylcyclohexanes where conformational mobility would predict that one of these groups would have to be axial, namely cis- 1,2-, trans-1,3- or cis-1,4-derivatives.

As a result, in these cases, we do not see an axial tert-butyl, but instead, the ring system adopts the less favourable twist-boat conformation.

It follows, therefore, that there must be a greater energy difference between chair conformations carrying axial and equatorial tert-butyl substituents than there is between chair and twist-boat conformations.

These conformational changes are shown for trans-1,3-di-tert-butylcyclohexane.

We noted earlier that bonds around nitrogen and oxygen atoms occupied some of the tetrahedral arrays, with lone pairs taking up other orbitals.

This means that we can use essentially the same basic principles for predicting the shape and conformation of heterocycles as we have used for carbocycles.

A substituent on the heteroatom is considered to be larger than the lone pair electrons. Some common examples are shown below.

As we shall see the heteroatom may have other influences, and there are sometimes unexpected effects involving a substituent adjacent to the heteroatom.

Conformationo Flindane

Chlorination of benzene gives an addition product that is a mixture of stereoisomers known collectively as hexachlorocyclohexane (HCH). At one time, this was incorrectly termed benzene hexachloride.

The mixture has insecticidal activity, though activity was found to reside in only one isomer, the so-called gamma isomer, γ-HCH.

γ-HCH, sometimes under its generic name lindane, has been a mainstay insecticide for many years and is about the only example of the chlorinated hydrocarbons that has not been banned and is still available for general use.

Although chlorinated hydrocarbons have proved very effective insecticides, they are not readily degraded in the environment, they accumulate and persist in animal tissues, and have proved toxic to many bird and animal species.

The stereochemistry of the γ-isomer is shown in the diagram, and when converted into a conformational stereo drawing it can be seen that there are three axial chlorines and three equatorial ones.

Ring flip produces an alternative conformation of equal energy, but it can be seen that this is identical to the first structure; rotation through 180◦ produces an identical and, therefore, superimposable structure.

It can be seen that conformational change will not stop the compound from interacting with the insect receptor site.

Configurational Isomers

As we have now seen, conformational isomers interconvert easily by rotation about single bonds. Configurational isomers, on the other hand, are isomers that interconvert only with difficulty, and it usually requires bond breaking if they do interconvert.

Optical Isomers: Chirality And Optical Activity

If tetrahedral carbon has four different groups attached, it is found that they can be arranged in two different ways. These molecules are not superimposable and they have a mirror-image relationship to each other. This is most easily seen with models.

Such an arrangement is called chiral (Greek: cheir = hand), and the carbon atom is termed a chiral centre or stereogenic centre. Look at your two hands.

You will see that they appear identical (allowing for minor blemishes or broken fingernails). However, do what you will, it is not possible to superimpose them, and you should be able to appreciate the mirror image relationship.

The two different arrangements – non-superimposable mirror images – are called enantiomers (Greek: enantiosis = opposite), and we say that enantiomers have different configurations.

The configuration is thus the spatial sequence about a chiral centre. It is also apparent that enantiomers are not going to interconvert readily, and to achieve interconversion we would have to break one of the bonds and then remake it so as to get the other configuration.

Note that the enantiomer of a particular compound can be drawn by reversing two of the substituents; this is actually much easier than drawing the mirror image compound, especially in more complicated structures. As an alternative, the wedge–dot relationship could be reversed.

Molecules that are superimposable on their mirror images are said to be achiral. With tetrahedral carbon, this is typically the case when two or more of the attached groups are the same.

This introduces a plane of symmetry into the molecule; molecules with a plane of symmetry can be superimposed on their mirror images.

Note that chirality is not restricted to tetrahedral carbon; it can also be associated with other tetrahedral systems, such as quaternary nitrogen compounds.

However, non-quaternary nitrogen, although tetrahedral, is not chiral. There is a rapid inversion that converts one enantiomer into the other; effectively, the lone pair does not maintain its position.

The energy barrier to interconversion is about 25 kJ mol^-1, which is sufficiently low that inversion occurs readily at room temperature.

This usually makes it impossible to obtain neutral amines in optically active form; quaternization stops this inversion. We shall later need to introduce a related term, prochiral. The concept of prochirality is discussed.

Manipulating Stereostructures

It is not always easy to look at stereo structures – two-dimensional representations of three-dimensional molecules – and decide whether two separate representations are the same or different.

To compare structures, it is usually necessary to manipulate one or both so that they can be compared directly. Here are a few demonstrations of how to approach the problem on paper.

Of course, constructing models for comparison is the easiest method, but there will always be occasions when we have to figure it out on paper.

Optical activity is the ability of a compound to rotate the plane of polarized light.

This property arises from an interaction of the electromagnetic radiation of polarized light with the unsymmetric electric fields generated by the electrons in a chiral molecule.

The rotation observed will clearly depend on the number of molecules exerting their effect, i.e. it depends upon the concentration.

Observed rotations are thus converted into specific rotations that are characteristic of the compound according to the formula below.

The observed rotation in degrees is divided by the sample concentration (g ml−1 ) and the sample tube length (decimetres). The unusual units used transform the measured small rotations into more manageable numbers.

The specific rotation is then usually in the range 0–1000◦; the degree units are strictly incorrect but are used for convenience.

The polarized light must be monochromatic, and for convenience and consistency, the D line (589 nm) in the sodium spectrum is routinely employed.

Both the temperature and solvent may influence the rotation somewhat, so must be stated.

Enantiomers have equal and opposite rotations. The (+)- or dextrorotatory enantiomer is the one that rotates the plane of polarization clockwise (as determined when facing the beam).

And the (−)- or laevorotatory enantiomer is the one that rotates the plane anticlockwise. In older publications, d and l were used as abbreviations for dextrorotatory and laevorotatory respectively.

But these are not now employed, thus avoiding any possible confusion with D and L.

An equimolar mixture of enantiomers is optically inactive since the individual effects from the two types of molecule are cancelled out.

This mixture is called a racemic mixture or racemate and can be referred to as the (±)-form.

A mixture of enantiomers in unequal proportions has a rotation numerically less than that of either enantiomer; this measurement could be used to determine the proportions of each.

Note that it is not possible to predict the sign or magnitude of the optical activity for a particular enantiomer; it must be measured experimentally.

The presence of more than one chiral centre in a molecule results in an optical rotation that reflects a contribution from each centre, though this is unlikely to be a simple summation.

It must also be appreciated that a positive contribution from one centre may be reduced, countered, or cancelled out by a negative contribution arising from another centre or centre.

Optical Purity And Enantiomeric Excess

A racemic mixture contains equal amounts of the two enantiomeric forms of the compound and has an optical rotation of zero: the optical rotations arising from each of the two types of molecule are cancelled out.

It follows that a mixture of enantiomers in unequal proportions will have a rotation that is numerically less than that of an enantiomer.

Here, we see how to use the measured optical activity to determine the proportions of each enantiomer in the mixture, and therefore its optical purity.

Optical purity is a measure of the excess of one enantiomer over the other in a sample of a compound.

There are a number of occasions when optical purity is of interest. We shall see later that many drugs are chiral compounds, and that biological activity often resides in just one enantiomer.

To minimize potential side effects, it is desirable to supply the drug in a single enantiomeric form.

This might be achieved by devising a synthetic procedure that produces a single enantiomer, an enantiospecific synthesis.

However, syntheses that are enantiospecific can be difficult to achieve, and it is more likely that the procedure is only enantioselective, i.e. it produces both enantiomers but with one predominating.

Alternatively, it is possible to separate the racemic mixture into the two enantiomers. This might not be achieved in a single step.

In both cases, it is usually necessary to monitor just how much of the desired enantiomer is present in the product mixture.

To illustrate the calculation of optical purity, we shall consider another type of reaction of interest, racemization.

This is the conversion of a single enantiomer into a racemic mixture of the two enantiomers. It depends upon the chemical nature of the compound and whether this is easily achievable.

One compound that racemizes readily is hyoscyamine, a natural alkaloid found in deadly nightshade, which is used as an anticholinergic drug.

The natural compound is laevorotatory,\([\alpha]_{\mathrm{D}}^{20}\) − 21° (EtOH), and the enantiomer is almost devoid of biological activity.

Upon heating with a dilute base such as 1% NaOH for about an hour, hyoscyamine racemizes, and the solution becomes optically inactive.

At shorter times, racemization is incomplete and the solution will still be optically active. Consider first a very simple situation in which exactly half of the material has racemized.

Half of the material is now optically inactive, consisting of equal amounts of each enantiomer, whilst the other half is still unchanged.

Since the concentration of the unchanged part is half of the original concentration, the optical rotation will also have dropped to half its original value.

The solution will contain 50% laevorotatory isomer and 50% racemate. However, the racemate is itself a 50 :

50 mixture of the two enantiomers, so the solution actually contains 25% dextrorotatory and 25 + 50% = 75% laevorotatory enantiomers.

Now let us consider when measurements indicate \([\alpha]_{\mathrm{D}}^{20}-9.2^{\circ}\). Calculations now tell us that the sample is 56.2% racemic, and contains 71.9% laevorotatory enantiomer and 28.1% dextrorotatory enantiomer. These figures are derived as follows:

The optical purity (%) = \(\frac{\text { specific rotation of sample }}{\text { specific rotation of pure enantiomer }} \times 100\)

= -9.2/ = 21 x 100 = 43.8%

The sample thus contains 43.8% of laevorotatory enantiomer and 100 − 43.8% = 56.2% of the racemate, the latter contributing no overall optical activity.

The racemate contains equal amounts of laevorotatory and dextrorotatory enantiomers, i.e. it contributes 28.1% of each isomer to the overall mixture.

Therefore, we have 43.8 + 28.1 = 71.9% of laevorotatory enantiomer, and 28.1% of dextrorotatory enantiomer in the partially racemized mixture.

Many workers use the equivalent term percentage enantiomeric excess rather than optical purity:

% Enantiomeric excess = \(\frac{\begin{array}{l}
\text { moles of one enantiomer}-\text { moles of other enantiomer }
\end{array}}{\text { total moles of both enantiomers }} \times 100\)

but this is exactly equivalent to optical purity. From the above calculations, one can see that the laevorotatory enantiomer (71.9%) is in excess of the dextrorotatory enantiomer (28.1%) by 43.8%.

The physical properties of enantiomers and racemates, except for optical rotation and melting points, are usually the same.

The melting points of (+)- and (−)- enantiomers are the same, though that of the racemate is usually different and can be greater or less than the melting point of the enantiomers.

Most spectral properties, For Example. NMR, mass spectrometry, etc., of (+)-, (−)-, and (±)-forms are indistinguishable.

However, pharmacological properties are frequently different, because they may depend upon the overall shape of the compound and its interaction with a receptor.

Pharmacological Properties Of Enantiomers

Although most physical properties of enantiomers are identical, pharmacological properties may be different.

There are examples of compounds where:

• Only one enantiomer is active;
• Both enantiomers show essentially identical activities;
• Both enantiomers have similar activity, but one enantiomer is more active;
• Enantiomers show different pharmacological activities.

These observations may reflect the proximity of the chiral centre to the part of the molecule that binds with the receptor site.

If binding to the receptor involves the chiral centre, then we may see activity in only one enantiomer.

But if binding does not involve the chiral centre, then there may be similar activities for each enantiomer.

Binding close to the chiral centre may cause the same type of activity but of a different magnitude. A different pharmacological activity for each enantiomer almost certainly reflects different receptors.

Further, drug absorption, distribution, and elimination from the body may vary due to differences in protein binding, enzymic modification, etc, since proteins are also chiral entities.

Thus, the anticholinergic activity of the alkaloid hyoscyamine is almost entirely confined to the (−)-isomer, and the (+)-isomer is almost devoid of activity.

The racemic (±)-form, atropine, has approximately half the activity of the laevorotatory enantiomer.

An anticholinergic drug blocks the action of the neurotransmitter acetylcholine and thus occupies the same binding site as acetylcholine.

The major interaction with the receptor involves that part of the molecule that mimics acetylcholine, namely the appropriately positioned ester and amine groups.

The chiral centre is adjacent to the ester and also influences binding to the receptor.

The major constituent of caraway oil is (+)-carvone and the typical caraway odour is mainly due to this component.

On the other hand, the typical minty smell of spearmint oil is due to its major component, (−)- carvone.

These enantiomers are unusual in having quite different smells, i.e. they interact with nasal receptors quite differently. The two enantiomeric forms are shown here in their half-chair conformations.

One of the most notorious and devastating examples of a drug’s side effects occurred in the early 1960s when thalidomide was responsible for many thousands of deformities in newborn children.

Thalidomide was marketed in racemic form as a sedative and antidepressant and was prescribed to pregnant women.

Although one enantiomer, the (R)-form, has useful antidepressant activity, it was not realized at that time that the (S)-form.

Thought to be inactive, actually has mutagenic activity and causes defects in the unborn fetus.

Furthermore, the (S)-isomer also has antiabortive activity, facilitating retention of the damaged fetus in the womb, so that any natural tendency to abort a damaged fetus is suppressed.

It is now general policy in the pharmaceutical industry to release new drugs as optically pure isomers, rather than as racemates.

It is desirable to minimize the amount of foreign chemicals a patient is subjected to since even the inactive portion of a drug has to be metabolized and removed from the body.

Such tragedies as occurred with thalidomide may also be avoided. Where a drug is supplied as a single enantiomer, the optical isomer is often incorporated into the drug name, For Example. dexamfetamine, dexamethasone, levodopa, levomenthol, levothyroxine.

Nevertheless, many racemic compounds are currently used as drugs, including atropine, mentioned above, and the analgesic ibuprofen.

Ibuprofen is an interesting case, in that the (S)-(+)-form is an active analgesic, but the (R)-(−)-enantiomer is inactive.

However, in the body, there is some metabolic conversion of the inactive (R)-isomer into the active (S)-isomer, so that the potential activity from the racemate is considerably more than 50%.

It shows a mechanism to account for this isomerism. There are two approaches to producing drugs as a single enantiomer. If a synthetic route produces a racemic mixture, then it is possible to separate the two enantiomers by a process known as resolution.

This is often a tedious process and, of course, half of the product is then not required.

The alternative approach, and the one now favoured, is to design a synthesis that produces only the required enantiomer, i.e. a chiral synthesis.

Note, the descriptors R and S for enantiomers and RS for racemates are defined.

Cahn–Ingold–Prelog System To Describe Configuration At Chiral Centres

The arrangement of groups around a chiral atom is called its configuration, and enantiomers have different configurations.

Therefore, it is necessary for us to have a means of describing configuration so that we are in no doubt about which enantiomer we are talking about.

Although enantiomers have equal and opposite optical rotations, the sign of the optical rotation does not tell us anything about the configuration.

The system adopted by IUPAC for describing configuration was devised by Cahn, Ingold, and Prelog, and is often referred to as the R, S convention.

The approach used is as follows:

• Assign an order of priority, 1, 2, 3, and 4, to the substituents on the chiral centre.
• View the molecule through the chiral centre towards the group of lowest priority, i.e. priority 4.
• Now consider the remaining groups in order of decreasing priority. If the sense of decreasing priority 1 → 2 → 3 gives a clockwise sequence, then the configuration is described as R (Latin: rectus = right); if the sequence is anticlockwise, then the configuration is described as S (Latin: sinister = left).

The remaining part of the procedure is to assign the priorities. The IUPAC priority rules form a rather long document in order to encompass all possibilities.

Here is a very short version suitable for our requirements. Note that it applies to both acyclic and cyclic compounds.

• A higher atomic number precedes a lower one, for example. Br > Cl > S > O > N > C > H.
• For isotopes, higher atomic mass precedes lower, for example. T > D > H.
• If atoms have the same priority, then secondary groups attached are considered. If necessary, the process is continued to the next atom in the chain.

the first atom is carbon in both cases;
consider the second atom:
the second atom is carbon in both cases;
consider the next atom(s):
carbon directly bonded to two further
carbons have higher priority than carbon
directly bonded to just one further carbon

• Double and triple bonds are treated by assuming each atom is duplicated or triplicated.

As simple examples of the approach, let us consider the amino acid (−)-serine and the Krebs cycle intermediate (+)-malic acid.

It is now possible to incorporate the configuration of the compound into its nomenclature to give more detail. (−)-Serine becomes (−)-(S)-serine, whilst (+)- malic acid becomes (+)-(R)-malic acid.

Because there is no relationship between (+)/(−) and configuration (R)/(S), it is necessary to quote both optical activity and configuration to convey maximum information.

The descriptor (RS ) is used to indicate a (±) racemic mixture.

Note also that the configuration (R) or (S) is defined by the priority rules, and configuration (R) could easily become (S) merely by altering one substituent.

For instance, all the amino acids found in proteins can be represented by the formula

Now all these amino acids that are chiral (glycine, R = H is achiral) have the (S) configuration except for cysteine, which is (R).

Just looking at the structures, one might imagine that they would all have the same configuration.

And indeed one can consider that they have; they differ only in the nature of the R group, but are all arranged around the chiral centre in the same manner.

However since (R) and (S) are only descriptors of configuration, the designation depends upon the nature of the R group.

In most cases, R is an alkyl or substituted alkyl, so it has a lower priority than carboxyl.

In the case of cysteine, R = CH2SH, and since S has a higher atomic number than any of the other atoms under consideration, this group will have a higher priority than the carboxyl. The net result is that cysteine is (R)- cysteine.

Configurations in cyclic compounds are considered in the same way as those in acyclic compounds.

If you cannot get an answer with the first atom, move on to the next, even though this may mean working around the ring system. Consider, for example, the stereoisomer of 3-methylcyclohexanol.

This has two chiral centres, C-1 and C-3. It can readily be deduced that this isomer is actually (1S,2R)- 3-methylcyclohexanol.

At both centres, two of the groups under consideration for priority assignment are part of the ring system.

These are only differentiable when one comes to the ring substituent, the methyl group when one considers C-1 and the hydroxyl when one considers C-3.

In each case, the substituted arm is going to take precedence over the unsubstituted arm. A more interesting example (6-aminopenicillanic acid) containing heterocyclic rings is discussed.

Configurations in 6-aminopenicillanic acid

Let us look at the common substructure of penicillin antibiotics, namely 6-aminopenicillanic acid.

To illustrate some aspects of working out whether a chiral centre is allocated the R or S configuration.

First of all, there are three chiral centres in this molecule, carbons 3, 5 and 6; note that carbon 2 is not chiral, since two of the groups attached are methyls. Only the three carbons indicated have four different groups attached.

The chirality at C-5 is assigned in the usual way. The groups attached have easily assigned priorities, with S > N > C > H. The configuration is thus 5R.

For the chirality at position 3, the priorities are assigned N > C–S > C–O > H.

Now a very useful hint. Since the group of lowest priority is wedged/up, it is rather difficult to imagine the sequence when viewed from the rear.

Accordingly, view the sequence from the front, which is easy, and reverse it.

From the front, the sequence for C-3 looks clockwise, so if viewed from the rear, it must be anticlockwise, and the descriptor is 3S.

Note how we consider substituents in the standard way even if they are part of a ring system. If you cannot get an answer with the first atom, move on to the next around the ring system.

Lastly, suppose one is asked to draw a particular configuration at C-6, namely 6R.

There is no way one can visualize a particular configuration, so the approach is to draw one and see if it is correct; if it is not correct, then change it by reversing wedged/dotted bonds.

And which to try first? Well, always put the group of lowest priority, usually H, away from you, i.e. dotted or down. Then you can see the clockwise/anticlockwise relationship easily from the front.

In this case, the version with H down gave the 6R configuration; but, if it were to be wrong, then the alternative configuration at this centre would be the required one, i.e. a wedged bond to the hydrogen.

Geometric Isomers

Restricted rotation about double bonds or due to the presence of ring systems leads to configurational isomers termed geometric isomers.

Thus, we recognize two isomers of but-2-ene, as shown below, and we term these cis and trans isomers. We have met these terms earlier.

With a double bond, rotation would destroy the π bond that arises from the overlap of p orbitals; consequently, there is a very large barrier to rotation.

It is of the order of 263 kJ mol^-1, which is very much higher than any of the barriers to rotation about single bonds that we have seen for conformational isomerism.

Accordingly, cis and trans isomers do not interconvert under normal conditions. Ring systems can also lead to geometric isomerism.

And cis and trans isomers of cyclopropane-1,2-dicarboxylic acid similarly do not interconvert; interconversion would require the breaking of bonds.

The terms cis and trans are used to describe the configuration, which is considered to be the spatial sequence about the double bond or the spatial sequence relative to a ring system.

The cis isomer has substituents on the same side of the double bond or ring system (Latin: cis = on this side), whereas the trans isomer has substituents on opposite sides (Latin: trans = across).

With simple compounds, like the isomers of but-2- ene, the descriptors cis and trans are quite satisfactory, but a compound such as 3-methylpent-2-ene causes problems.

Do we call the isomer below cis because the methyls are on the same side, or trans because the main chain goes across the bond?

For double bonds, the configuration is now usually described via the non-ambiguous E,Z nomenclature, assigned using the Cahn–Ingold–Prelog priority rules for substituents on each carbon.

First, consider each carbon of the double bond separately, and assign priorities to its two substituents. Then consider the double bond with its four substituents.

If the two substituents of higher priority are on the same side of the double bond, the configuration is Z (German: zusammen = together), whereas if they are on opposite sides, the configuration is E (German: entgegen = across).

Thus, for the 3-methylpent-2-ene isomer we can see that, for C-2, the substituents are methyl and hydrogen with priorities methyl > hydrogen.

For C-3, we have substituents methyl and ethyl, with ethyl having the higher priority.

Higher priority. Thus, the high-priority groups are on opposite sides of the double bond, and this isomer has the E configuration.

The alternative arrangement with high-priority substituents on the same side of the double bond has the Z configuration.

Configurations Of Tamoxifen, Clomifene And Triprolidine

The oestrogen-receptor antagonist tamoxifen is used in the treatment of breast cancer and is highly successful.

Clomifene is also an oestrogen-receptor antagonist but is principally used as a fertility drug, interfering with feedback mechanisms and leading to ova release, though this often leads to multiple pregnancies.

As can be deduced from the application of the Cahn–Ingold–Prelog priority rules, high-priority groups are positioned on the same side of the double bond in each case.

Note that the substituted aromatic ring has higher priority than the unsubstituted ring. Both tamoxifen and clomifene thus have the Z configuration.

The antihistamine drug triprolidine has the E configuration; note that the heterocyclic pyridine ring takes priority over the benzene ring, even though the latter has a substituent.

Priority is deduced by working along the carbon chain towards the first atom that provides a decision, in this case, the nitrogen atom in the pyridine.

Configurational Isomers With Several Chiral Centres

Configurational isomerism involving one chiral centre provides two different structures, the two enantiomers.

If a structure has more than one chiral centre, then there exist two ways of arranging the groups around each chiral centre.

Thus, with n chiral centres in a molecule, there will be a maximum number of 2n configurational isomers. Sometimes, as we shall there are less.

Starting with two chiral centres, there should, therefore, be four stereoisomers.

And this is nicely exemplified by the natural alkaloid (−)-ephedrine, which is employed as a bronchodilator drug and decongestant.

Ephedrine is (1R,2S)-2-methylamino-1-phenylpropan- 1-ol, so has the structure and stereochemistry shown.

Now the other three of the possible four stereoisomers are the (1S,2S), (1R,2R), and (1S,2R) versions. These are also shown, and mirror-image relationships are emphasized.

The (1S,2R) isomer is the mirror image of (−)-ephedrine, which has the (1R,2S) configuration. Therefore, it is the enantiomer of (−)-ephedrine, and can be designated (+)-ephedrine.

Note that the enantiomeric form has the opposite configuration at both chiral centres.

The other two isomers are the (1 S,2S) and (1R,2R) isomers, and these two also share a mirror image relationship, have the opposite configuration at both chiral centres, and are, therefore, a pair of enantiomers.

From a structure with two chiral centres, we thus have four stereoisomers that consist of two pairs of enantiomers.

Stereoisomers that are not enantiomers we term diastereoisomers, or sometimes diastereomers. Thus, the (1S,2S) and (1R,2R) isomers are diastereoisomers of the (1R,2S) isomer.

Other enantiomeric or diastereomeric relationships between the various isomers are indicated in the figure.

We have seen earlier that enantiomers are chemically identical except in optical properties, although biological properties may be different.

On the other hand, diastereoisomers have different physical and chemical properties, and probably different biological properties as well.

As a result, they are considered a completely different chemical entity and are often given a different chemical name.

The (1S,2S) and (1R,2R) isomers are thus known as (+)- pseudoephedrine and (−)-pseudoephedrine respectively.

Interestingly, (+)-pseudoephedrine has similar biological properties to (−)-ephedrine and it is used as a bronchodilator and decongestant drug in the same way as ephedrine.

One more useful piece of terminology can be introduced here. This is the term epimer. An epimer is a diastereoisomer that differs in chirality at only one centre.

Thus, (−)-pseudoephedrine is the 2-epimer of (−)-ephedrine, and (+)-pseudoephedrine is the 1-epimer of (−)-ephedrine.

The epimer terminology is of greater value when there are more than two chiral centres in the molecule.

Suppose we have a compound with three chiral centres, at positions 2, 3, and 4 in some unspecified carbon chain, with configurations 2R,3R,4S. There would thus exist a total of 23 = 8 configurational isomers.

The enantiomer would have the configuration 2S,3S,4R, i.e. changing the configuration at all centres.

The 2S,3R,4S diastereoisomer could then refer to as ‘the 2-epimer’, and the 2R,3S,4S diastereoisomer as ‘the 3-epimer’, since we have changed the stereochemistry at just one centre, keeping other configurations the same.

Drawing Enantiomers And Epimers: 6-aminopenicillanic Acid

The structure of the natural isomer of 6-aminopenicillanic acid is shown. You are asked to draw the structure of its enantiomer and its 6-epimer.

The enantiomer will have the configuration changed at all chiral centres, whereas the 6-epimer retains all configurations except for that at position 6.

Note that it is not necessary to draw the mirror image compound for the enantiomer, just reverse the wedge–dot relationship for the bonds at each chiral centre.

This is much easier and less prone to errors whilst transcribing the structure. Now for a rather important point.

In a compound such as (−)-ephedrine there are going to be many different conformations as a result of rotation about the central C–C bond.

Three of them are shown here, the energetically most favourable staggered conformer with all large groups anti, a less favourable staggered conformer, and a high-energy eclipsed version.

However, note carefully that changing the conformation does not affect the spatial sequence of the chiral centres, i.e. it does not change the configuration at either chiral centre.

This seems a trivial and rather obvious statement, and indeed it probably is in the case of acyclic compounds.

It is when we move on to cyclic compounds that we need to remember this fundamental concept because a common mistake is to confuse conformation and configuration.

The same stereochemical principles are going to apply to both acyclic and cyclic compounds. With simple cyclic compounds that have little or no conformational mobility, it is easier to follow what is going on.

Consider a disubstituted cyclopropane system. As in the acyclic examples, there are four different configurational stereoisomers possible, comprising two pairs of enantiomers. No conformational mobility is possible here.

However, in a cyclohexane system, we also need to consider the conformational mobility that generates two different chair forms of the ring.

Let us consider 3-methylcyclohexanecarboxylic acid. This has two chiral centres, and thus there are four configurational stereoisomers. These are the enantiomeric forms of the trans and cis isomers.

Each isomer can also adopt a different chair conformation as a consequence of ring flip.

We thus can write down eight possible stereoisomers, comprised of two interconvertible conformers for each of the four non-interconvertible configurational isomers.

Put another way, there are four configurational isomers (22 = 4), but each can exist as two possible conformational isomers.

Note that you can also see the mirror image relationship in the conformational isomers.

Of course, in practice, some conformers are not going to be energetically favourable. The cis compound has favoured equatorial and unfavoured diaxial conformers.

The trans compound has one equatorial and one axial substituent; we can assume that the larger carboxylic acid group will prefer to be equatorial.

Do appreciate that cyclohexane rings with 1,2- or 1,3-substitution fit into the above discussions; however, if we have 1,4-substitution there are no chiral centres in the molecule.

Since two of the groups are the same at each possible site! However, cis and trans forms still exist; these are geometric isomers and can still be regarded as diastereoisomers.

We can spot this type of situation by looking for symmetry in the molecule.

Both cis- and trans-4- methylcyclohexanecarboxylic acid isomers have a plane of symmetry, and, as we saw for simple tetrahedral carbons, this symmetry means the molecule is achiral.

Configurations A Nd Conformations: Avoiding Confusion

At this stage, a word of caution: do not confuse conformation with configuration. Different conformations interconvert easily; different configurations do not interconvert without some bond-breaking process.

We commented above that changing the conformation did not affect the spatial sequence of chiral centres and used ephedrine as a rather trivial and obvious example.

Rotation about single bonds did not change the configuration at either chiral centre. To emphasize this point, look at the following relationships for trans-3-methylcyclohexyl bromide.

Don’t confuse conformation with configuration

Ring flip of the upper left structure produces an alternative conformer. Ring flip does not change the configuration.

The axial–equatorial relationship (conformation) is modified, but the up–down relationship (configuration) is still there.

The enantiomer of this structure has the alternative configuration at both chiral centres, but it cannot be produced from the first structure by any simple isomerization process.

However, it is still conformationally mobile. The figure thus shows the conformational isomerism for two different configurational isomers, the enantiomeric pair.

A common mistake that can be made when one is trying to draw the different conformers that arise from ring flip in a cyclohexane compound is to remember vaguely that axial groups become equatorial, and vice versa, and to apply this change without flipping the ring.

Of course, as can be seen from looking at the compounds below, transposing the equatorial bromine to axial and the axial methyl to equatorial changes the configuration at both centres.

So we have produced the enantiomer. This is a configurational isomer and not a conformer.

Meso Compounds

Now for a rather unexpected twist. We have seen that if there are n chiral centres there should be 2n configurational isomers, and we have considered each of these for n = 2 (For example. ephedrine, pseudoephedrine).

It transpires that if the groups around chiral centres are the same, then the number of stereoisomers is less than 2n. Thus, when n = 2, there are only three stereoisomers, not four.

As one of the simplest examples, let us consider in detail tartaric acid, a component of grape juice and many other fruits. This fits the requirement, since each of the two chiral centres has the same substituents.

We can easily draw the four predicted isomers, as we did for the ephedrine–pseudoephedrine group and two of these represent the enantiomeric pair of (−)-tartaric acid and (+)-tartaric acid.

Now let us consider the other pair of isomers, and we shall see the consequences of the substituent groups being the same.

Because these two structures are actually superimposable and, therefore, only represent a single compound.

This is not so easily seen with the staggered conformers drawn, so it is best to rotate these about the 2,3-bond to give an eclipsed conformer.

They can both be rotated to give the same structure, so they represent only a single compound. This is called meso-tartaric acid (Greek: mesos = middle).

Furthermore, since we have superimposable mirror images, there can be no optical activity. We can see why a compound with chiral centres should end up optically inactive by looking again at the eclipsed conformer.

The molecule itself has a plane of symmetry, and because of this symmetry, the optical activity conferred by one chiral centre is equal and opposite to that conferred by the other and, therefore, is cancelled out.

It has the characteristics of a racemic mixture but as an intramolecular phenomenon. A meso compound is defined as one that has chiral centres but is itself achiral.

Note that numbering is a problem in tartaric acid because of the symmetry and that positions 2 and 3 depend on which carboxyl is numbered as C-1.

It can be seen that (2R,3S) could easily have been (3R,2S) if we had numbered from the other end, a warning sign that there is something unusual about this isomer.

The same stereochemical principles apply to both acyclic and cyclic compounds.

With simple cyclic compounds that have little or no conformational mobility, it can even be easier to follow what is going on. Let us first look at cyclopropane-1,2-dicarboxylic acid.

These compounds were considered as examples of geometric isomers, and cis and trans isomers were recognized.

This is essentially the same as the tartaric acid example, without the conformational complication. Thus, there are two chiral centres, and the groups around each centre are the same.

Again, we get only three stereoisomers rather than four, since the cis compound is an optically inactive meso compound.

There is a plane of symmetry in this molecule, and it is easy to see that one chiral centre is mirrored by the other so we lose optical activity.

Conformational mobility, such as we get in cyclohexane rings, makes the analysis more difficult, and manipulating molecular models provides the clearest vision of the relationships.

Let us look at 1,2- dimethylcyclohexane as an example. Again, we have met the cis and trans isomers when we looked at conformational aspects. Here, we need to consider both configuration and confirmation.

In the trans compound, two mirror-image enantiomeric forms can be visualized. These will be the (+)- and (−)-trans isomers.

Note particularly that conformational changes may also be considered, but these do not change configuration, so we are only seeing different conformers of the same compound.

The above scheme thus shows two interconvertible conformers (upper and lower structures) for each of the two non-interconvertible enantiomers (left and right structures).

The cis compound provides the real challenge, however. If we draw version A, together with its mirror image C, they do not look capable of being superimposed.

However, conformer A may be ring flipped to an equal-energy conformer B, and this will have a corresponding mirror image version D.

Now consider a 120° rotation of version A about the central axis; this will give D.A. A similar 120° rotation of version C about the central axis will give B.

It follows, therefore, that if a simple rotation of one structure about its axis gives the mirror image of a conformational isomer, then we cannot have enantiomeric forms but must have the same compound.

These are thus two different conformers of an optically inactive meso compound. It may require manipulation of models to really convince you about this!

Now, although the cyclohexane ring is not planar, the overall consequences for trans- and cisdimethylcyclohexane can be predicted by looking at the two-dimensional representations.

It is clear that this representation of cis-dimethylcyclohexane shows a plane of symmetry, and we can deduce it to be a meso compound.

No such plane of symmetry is present in the representation of trans-methylcyclohexane.

Why does this approach work? Simply because the transformation of planar cyclohexane (with eclipsed bonds) into a non-planar form (with staggered bonds) is a conformational change achieved by rotation about single bonds.

The fact that cyclohexane is non-planar means we may have to invoke the conformational mobility to get the three-dimensional picture.

Our consideration of meso compounds leads us to generalize:

• A molecule with one chiral centre is chiral;
• A molecule with more than one chiral centre may be chiral or achiral.

Now let us extend this generalization with a further statement:

• A molecule may be chiral without having a chiral centre.

This is the subject of the next section.

Chirality Without Chiral Centres

We shall restrict discussions here to three types of compounds. In the first, we get what is termed torsional asymmetry, where chirality arises because of restricted rotation about single bonds.

The commonest examples involve two aromatic rings bonded through a single bond (biphenyls).

If large groups are present in the ortho positions, these prevent rotation about the interring single bond.

The most favourable arrangement to minimize interactions is when the aromatic rings are held at right angles to each other. As a result, two enantiomeric forms of the molecule can exist.

Because of the size of the ortho groups, it is not possible to interconvert these stereoisomers merely by rotation. Even when we only have two different types of substituent, as shown, we get two enantiomeric forms.

The second type of compound is called an allene; these compounds contain two double bonds involving the same carbon. These compounds exist but are often difficult to prepare and are very reactive.

It is the concept of chirality which is more important here than the chemistry of the compounds. If a carbon atom is involved in two double bonds, it follows that the π bonds created must be at right angles to each other.

The consequence of this is that the substituents on the other carbons of the allene are also held at right angles to each other. Again, two enantiomeric forms of the molecule can exist.

The third example of chirality without a chiral centre is provided by s p i r o compounds, which we shall meet later when we consider the stereochemistry of polycyclic systems.

But at this stage, it is worth noting that they provide a third example of chirality without a chiral centre.

Spiro compounds contain two ring systems that have one carbon in common, and it is easy to see this carbon could be chiral if four different groupings are present.

A nice natural example, the antibiotic griseofulvin, is shown here.

However, it is also possible to visualize spiro compounds with groupings that are not all different, where enantiomeric forms exist because mirror image compounds are not superimposable.

The diamine shown is chiral, in that the mirror image forms are not superimposable, even though only two types of substituent are attached to the spiro centre.

Both rings in this compound will have the chair conformation, but it is not easy to draw these because one ring will always be viewed face-on.

The solution is to ensure the spiro centre is not on the left or right tip of either ring.

With biphenyls, allenes, and spiro compounds, groups are held at right angles by a rigid system, and this feature allows the existence of non-superimposable mirror image stereoisomers, i.e. enantiomers.

It is useful to think of this arrangement as analogous to a simple chiral centre, where the tetrahedral array also holds pairs of groups at right angles.

In contrast to tetrahedral carbon, it is not even necessary for all the groups to be different to achieve chirality, as can be seen in the examples above.

Torsionalasymmetry: Gossypol

The concept of torsional asymmetry is not just an interesting abstract idea. Some years ago, fertility in some Chinese rural communities was found to be below normal levels, and this was traced back to the presence of gossypol in dietary cottonseed oil.

Gossypol acts as a male contraceptive, altering sperm maturation, spermatozoid motility, and inactivation of sperm enzymes necessary for fertilization.

Extensive trials in China have shown the antifertility effect is reversible after stopping the treatment, and it has potential, therefore, as a contraceptive for men.

Gossypol is chiral due to restricted rotation, and only the (−)-isomer is pharmacologically active as an infertility agent. The (+)-isomer has been found to be responsible for some toxic symptoms.

Most species of cotton (Gossypium) produce both enantiomers of gossypol in unequal amounts, with the (+)-enantiomer normally predominating over the (−)-isomer.

It has proved possible to separate racemic (±)-gossypol from this type of mixture – the racemate complexes with acetic acid.

Whereas the separate enantiomers do not. The racemic form can then be resolved to give the useful biologically active (−)-isomer.

Prochirality

Enantiotopic Groups

We have defined chirality in terms of ‘handedness’, such that mirror image stereoisomers are not superimposable.

In the case of tetrahedral carbon, chirality is a consequence of having four different groups attached to it.

If two or more groups were the same, then the compound would be termed achiral. Now we introduce another term, prochiral. Achiral molecules that can become chiral by one simple change are called prochiral.

The simplest example we could include under this definition would be an achiral molecule in which two groups are the same.

The two like groups are termed enantiotopic, in that separate replacement of each would generate enantiomers.

This seems an unnecessary complication. Why do we want to call an achiral centre prochiral? What benefits are there?

Well, remember that the Cahn–Ingold–Prelog system allowed us to describe a particular chiral arrangement of groups at a chiral centre; prochirality now allows us to distinguish between the two like groups at an achiral centre.

When might we want to do that? The following example from biochemistry shows the type of occasion when we might need to identify one or other of the like groups.

The enzyme alcohol dehydrogenase oxidizes ethanol to acetaldehyde, passing the hydrogen to the coenzyme nicotinamide adenine dinucleotide NAD+.

This is the enzyme that restores normal service after excessive consumption of alcoholic drinks.

By specifically labelling each hydrogen in turn, and then observing whether the substrate loses or retains the label in the enzymic reaction, it has been determined which hydrogen is lost from the methylene group of ethanol.

How then, in an unambiguous fashion, can we describe which hydrogen is lost? We define the two hydrogens as pro-R and pro-S.

By considering the effect of increasing their effective priorities according to the Cahn–Ingold–Prelog system; this is simply achieved if we consider having deuterium instead of protium (normal hydrogen).

Then, if replacing a particular hydrogen with deuterium produces a chiral centre with the R configuration, that hydrogen is termed the pro-R hydrogen.

Similarly, increasing the priority of the other hydrogen should generate the S configuration, so that that hydrogen is termed the pro-S hydrogen.

We can also label hydrogens in a structure as HR and HS according to this procedure.

We can thus deduce that alcohol dehydrogenase stereospecifically removes the pro-R hydrogen from the prochiral methylene.

This example is from biochemistry. It is a feature of biochemical reactions that enzymes almost always catalyse reactions in a completely stereospecific manner.

They are able to distinguish between enantiotopic hydrogens because of the three-dimensional nature of the binding site.

There are also occasions where chemical reactions are stereospecific; refer to the stereochemistry of E2 eliminations for typical examples.

Citric Acid Has Three Prochiral Centres

The Krebs cycle is a process involved in the metabolic degradation of carbohydrates.

It is also called the citric acid cycle because citric acid was one of the first intermediates identified.

Once formed, citric acid is modified by the enzyme aconitase through the intermediate cis-aconitic acid to give the isomeric isocitric acid.

This is not really an isomerization, but the result of dehydration followed by rehydration. Both steps feature stereospecific anti-processes, i.e. groups are removed or added from opposite sides of the molecule.

First, let us look closely at the structure of citric acid. It has three prochiral centres. Two of these are the methylenes, but note that the central carbon is also prochiral.

It has two groups the same, namely the –CH₂CO₂H groups. The loss of water from citric acid is an anti-elimination, so the hydroxyl is lost together with one of the methylene hydrogens.

The hydrogen lost has been found to be the pro-R hydrogen from the pro-R–CH₂CO₂H group.

This is followed by an anti-addition reaction in which water is added to the new double bond but in the reverse sense. The hydrogen retained throughout the process is shown with an asterisk.

Note that we can only label this hydrogen as pro-S in citric acid; in cis-aconitic acid and isocitric acid, it is no longer attached to a prochiral centre, and we must resort to some other labelling system, namely the asterisk.

This is a nice example of enzymic stereospecificity. It involves the specific removal of one hydrogen atom from a substrate that appears to have four equivalent hydrogens.

Because of the three-dimensional characteristics of both the enzyme and the substrate, the apparently equivalent side chains on the central carbon are going to be positioned quite differently and the enzyme is able to distinguish between them.

Further, it also distinguishes between the two hydrogens of a methylene group.

An interesting consequence of this stereospecificity is that because only one of the citric acid side chains is modified in the aconitase reaction, it takes further turns of the cycle before the material entering the cycle (acetyl-CoA) is actually degraded.

A reaction that gives a mixture of isomeric products with one isomer predominating would be termed stereoselective.

Enantiotopic Faces

We have thus seen that there could be a need to distinguish between two similar groups attached to tetrahedral carbon, and have exploited the Cahn–Ingold–Prelog priorities to label the separate groups.

We also need to consider another way in which a chiral centre might be generated, and that is by the addition of a group to a planar system.

For example, if we reduce a simple ketone that has two different R groups with lithium aluminium hydride we shall produce a racemic alcohol product.

This is because hydride can be delivered to either face of the planar carbonyl group with equal probability.

In marked contrast, nature’s reducing agent, reduced nicotinamide adenine dinucleotide (NADH), delivers hydride in a stereospecific manner because it is a cofactor in an enzyme-catalysed reaction.

For example, reduction of pyruvic acid to lactic acid in vertebrate muscle occurs via attack of hydride to produce just one enantiomer, namely (S)-lactic acid.

We can see from the diagram that hydride must be delivered from the front face as shown, but it makes sense to have a more precise descriptor for faces than front or back.

Once again, the Cahn–Ingold–Prelog system can help us out. We assign priorities to the three groups attached to the planar carbon.

We then consider the descending sequence and decide whether this is clockwise or anticlockwise; the face that provides a clockwise sequence is then labelled Re and the face that provides an anticlockwise sequence is labelled Si.

These are simply variants on R and S, in fact, the first two letters of rectus and sinister. Note that there is no correlation between Re or Si and the chirality R or S of the tetrahedral product formed.

It can now be seen that, in the enzymic reduction of pyruvic acid to lactic acid, hydride is delivered to the Re face of the pyruvic acid.

A molecule such as pyruvic acid is said to have two enantiotopic faces. An attack of a reagent onto the Reface yields one enantiomer, whereas an attack onto the Si face will produce the other enantiomer.

The Re and Si descriptors are similarly applied to the carbon atoms making up C=C bonds.

This gets a little more complex, in that a C=C bond generates four faces to be considered, two at each carbon. It is necessary to systematically deduce the descriptor for each, as shown below.

NADH Delivers Hydride From A Prochiral Centre; NAD+ Has Enantiotopic Faces

NADH (reduced nicotinamide adenine dinucleotide) is utilized in biological reductions to deliver hydride to an aldehyde or ketone carbonyl group.

A proton from water is used to complete the process, and the product is thus an alcohol. The reaction is catalysed by an enzyme called dehydrogenase.

The reverse reaction may also be catalysed by the enzyme, namely the oxidation of an alcohol to an aldehyde or ketone. It is this reverse reaction that provides the dehydrogenase nomenclature.

During the reduction sequence, NADH transfers a hydride from a prochiral centre on the dihydropyridine ring.

And is itself oxidized to NAD + (nicotinamide adenine dinucleotide) that contains a planar pyridinium ring.

In the oxidation sequence, NAD+ is reduced to NADH by acquiring hydride to an enantiotopic face of the planar ring. The reactions are completely stereospecific.

The stereospecificity depends upon the enzyme in question. Let us consider the enzyme alcohol dehydrogenase, which is involved in the ethanol-to-acetaldehyde interconversion.

It has been deduced that the hydrogen transferred from ethanol is directed to the Re face of NAD+, giving NADH with the 4R configuration.

In the reverse reaction, it is the 4-pro-R hydrogen of NADH that is transferred to acetaldehyde.

Note also that the transfer of hydride to the carbonyl compound is also stereospecific, as is the removal of hydrogen from the prochiral centre of ethanol in the reverse reaction.

We should note that prochiral molecules have the potential to become chiral if we make certain changes.

We have used the term enantiotopic to identify the groups at sp³-hybridized carbon or the faces of sp²– 2-hybridised carbon where alternative changes lead to the production of enantiomers.

However, if there is also a chiral centre in the molecule, then the same changes would lead to the formation of diastereoisomers, not enantiomers. Such groups or faces are now correctly termed diastereotopic.

Separation Of Enantiomers: Resolution

We saw that enantiomers have the same physical and chemical properties, except for optical activity, and thus they behave in exactly the same manner.

We also saw, however, that this generalization did not extend to biological properties, and that there were compelling reasons for administering drugs as a single enantiomer rather than a racemate.

At some stage, therefore, it might be necessary to have the means of separating individual enantiomers from a racemic mixture. This is termed resolution.

The traditional method has been to convert enantiomers into diastereoisomers.

Because diastereoisomers have different physical and chemical properties and can, therefore, be separated by various methods.

Provided one can convert the separated diastereoisomers back to the original compound, this offers a means of separating or resolving enantiomers.

The simplest method has been to exploit salt formation by reaction of a racemic acid (or base) with a chiral base (or acid).

For example, treating a racemic acid with a chiral base will give a mixture of two salts that are diastereoisomeric.

Although there is no covalent bonding between the acid and base, the ionic bonding is sufficient that the diastereoisomeric salts can be separated by some means, typically fractional crystallization.

Although fractional crystallization may have to be repeated several times, and, therefore, is tedious, it has generally been an effective means of separating the diastereoisomeric salts.

Finally, the salts can separately be converted back to the acid, completing the resolution

The bases generally employed in such resolutions have been natural alkaloids, such as strychnine, brucine, and ephedrine.

These alkaloids are more complex than the general case shown in the figure, in that they contain several chiral centres.

Tartaric acid has been used as an optically active acid to separate racemic bases.

Of course, not all materials contain acidic or basic groups that would lend themselves to this type of resolution. There are ways of introducing such groups, however, and a rather neat one is shown here.

Racemic alcohol may be converted into a racemic acid by reaction with one molar equivalent of phthalic anhydride; the product is a half ester of a dicarboxylic acid.

This can now be subjected to the resolution process for acids and, in due course, the alcohols can be regenerated by hydrolysis of the ester.

A significant improvement in the fractional crystallization process came with the introduction of chiral phases for column chromatography. This allows simple chromatographic separation of enantiomers.

In practice, it is effectively the same principle, that of forming diastereoisomeric complexes with the chiral material comprising the column.

One enantiomer binds more tightly than the other and, therefore, passes through the column at a different rate. The two enantiomers thus emerge from the column as separate fractions.

It has also proved possible to exploit the enantiospecific properties of enzymes to achieve the resolution of a racemic mixture during chemical synthesis.

Enzymes are proteins that catalyse biochemical reactions with outstanding efficiency and selectivity.

This is a consequence of the size and shape of the enzyme’s binding site, a feature that is determined by the sequence of amino acid residues in the protein.

The selectivity of enzymes means that they carry out reactions on one functional group in the presence of others that might be affected by a chemical reagent.

It also means that they can be stereoselective, either performing reactions in a stereospecific manner or only reacting with substrates with a particular chirality.

As a simple example, racemic ester structures may be resolved by the use of ester hydrolysing enzymes called lipases.

With the appropriate choice of enzyme, it has been found that only one enantiomer of the racemic mixture is hydrolysed, whilst the other remains unreacted.

It is then a simple matter to separate the unreacted ester from the alcohol. The unreacted ester may then be hydrolysed chemically, thus achieving resolution of the enantiomeric alcohols.

Fischer Projections

Fischer projections provide a further approach to the two-dimensional representations of three-dimensional formulae.

They become particularly useful for molecules that contain several chiral centres and are most frequently encountered in discussions of sugars.

To start, though, let us consider just one chiral centre, and choose the amino acid we met earlier, (−)-(S)-serine.

The Fischer projection is drawn with groups on horizontal and vertical lines, but without showing the chiral carbon atom.

Should you put in this carbon atom, it can no longer be considered that you are representing stereochemistry.

The Fischer projection then implies that horizontal bonds are wedged, whilst vertical bonds are dotted, and it thus speeds up the drawing of stereochemical features.

For (−)-(S)-serine, the wedge–dot version is what one would see if one looked down on the right-hand stereostructure as indicated.

Accordingly, we can now transform stereostructures into Fischer projections, and vice versa. The only significant restrictions are

• We should draw the longest carbon chain vertical;
• We should place the carbon of the highest oxidation state at the top.

However, when we come to manipulate Fischer’s projections, we may need to disregard these restrictions in the interests of following the changes.

Manipulations we can do to a Fischer projection may at first glance appear confusing, but by reference to a model of a tetrahedral array, or even a sketch of the representation, they should soon become quite understandable, perhaps even obvious.

The molecular manipulations shown are given to convince you of the reality of the following statements.

• Rotation of the formula by 180° gives the same molecule.

• Rotation of any three groups clockwise or anticlockwise gives the same molecule.

• The exchange of any two groups gives the enantiomer.

• Rotation of the formula by 90◦ gives the enantiomer.

It is also surprisingly easy to assign R or S configurations to chiral carbons in the Fischer projections.

But, because horizontal lines imply wedged bonds (towards you) and vertical lines imply dotted bonds (away from you), there are important guidelines to remember:

• If the group of lowest priority is on the vertical line, a clockwise sequence gives the R configuration;
• if the group of lowest priority is on the horizontal line, a clockwise sequence gives the S configuration.

These do not represent a different set of rules from the clockwise = R, anticlockwise = S conventions we already use It is merely a consequence of the lowest priority group being down (dotted bond) on the vertical line, but up (wedged) on the horizontal line.

We have noted that, if the lowest priority group is wedged, it is easier to look at the sequence from the front, and then reverse it to give us the sequence as viewed from the rear, i.e. towards the group of lowest priority.

Let us apply these principles to tartaric acid. This compound has two chiral centres; but, as we saw previously, only three stereoisomers exist, since there is an optically inactive meso compound involved.

We can draw these three stereoisomers as Fischer projections, reversing the configurations at both centres to get the enantiomeric stereoisomers, whilst the Fischer projection for the third isomer, the meso compound, is characterized immediately by a plane of symmetry.

For (+)-tartaric acid, the configuration is (2R,3R), and for (−)-tartaric acid it is (2S,3S).

For both chiral centres, the group of lowest priority is hydrogen, which is on a horizontal line. In fact, this is the case in almost all Fischer projections, since, by convention, the vertical line is the longest carbon chain.

Thus, we have to reverse our normal configurational thinking: a clockwise sequence of priorities gives S and an anticlockwise sequence gives R.

The configuration of the meso isomer can be deduced by abstracting the appropriate portions from the other two structures and assigning equivalent configurations.

It should be appreciated that a Fischer projection involving more than one chiral centre actually depicts an eclipsed conformer, which is naturally a high-energy state and is normally an unlikely arrangement of atoms.

We need to bear this in mind when we transpose Fischer projections into wedge–dot stereochemical drawings.

Further manipulations are necessary to give lower energy staggered conformers. This is illustrated here with the five-carbon sugar (−)-ribose.

However, as we shall see shortly, Fischer-projection-derived eclipsed conformers are particularly useful in deducing the stereochemistry in cyclic forms of sugars.

D And L configurations

The concept of D and L as configurational descriptors is well established, particularly in amino acids and sugars; frankly, however, we could live without them and save ourselves a lot of confusion.

Since they are so widely used, we need to find out what they mean, but in most cases, the information conveyed is less valuable than sticking with R and S.

D and L sugars

The simplest of the sugars is glyceraldehyde, which has one chiral centre. Long before R and S were adopted as descriptors, the two enantiomers of glyceraldehyde were designated as D and L.

D-(+)-Glyceraldehyde is equivalent to (R)-(+)-glyceraldehyde, the latter configuration being fully systematic.

Configurations in other compounds were then related to the configurations of Dand L-glyceraldehyde by direct comparison of Fischer projections.

For example, (+)-glucose (= dextrose) is represented by a Fischer projection that defines the configuration at all four chiral centres.

Since the configuration at position 5 in (+)-glucose can be directly related to that in D-(+)-glyceraldehyde, (+)-glucose is said to have the D configuration and is thus termed D-(+)-glucose.

By similar reasoning, the enantiomer of glucose has the L configuration and is termed L-(−)-glucose. Now the limitations of this system become obvious when one realizes that D and L refer to the configuration at just one centre.

By convention, the highest numbered chiral centre, and the remaining configurations are not specified, except by the name of the sugar.

Fischer Projections Of Glucose And Stereoisomers

The sugar glucose has four chiral centres; therefore, 24 = 16 different stereoisomers of this structure may be considered. These are shown below as Fischer projections

The 16 stereoisomers are divided into D and L groups, which reflect only the configuration at the highest numbered chiral centre, namely C-5.

The chirality at other centres is defined solely by the name given to the sugar, so we have eight different names for particular configurational combinations.

Note that although D and L strictly refer to the configuration at only one centre, L-glucose is the enantiomer of D-glucose and, therefore, must have the opposite configuration at all chiral centres.

A change in configuration at only one centre produces a diastereoisomer that has different chemical properties and is accordingly given a different name.

Whilst this system of nomenclature has some obvious shortcomings, it is analogous to the ephedrine and pseudoephedrine example where we were considering just two chiral centres.

A more systematic approach (though not one that is used) might give all the above sugars the same name, for example. hexose.

But specify the chirality at each centre, for example. D-(+)-glucose would be (+)-(2R,3S,4R,5R)-hexose and L-(−)-galactose would become (−)-(2S,3R,4R,5S)-hexose.

Instead, we have the eight different names in two configurational classes, D and L.

We can also use the term epimer to describe the relationship between isomers, where the difference is in the configuration at just one centre. This is shown for the four epimers of D-(+)-glucose.

An interesting observation with the 16 stereoisomers is that the optical activity of a particular isomer does not appear to relate to the configuration at any particular chiral centre.

Stereochemistry In Hemiacetal Forms Of Sugars From Fischer Projections

In solution, aldehyde sugars normally exist as cyclic hemiacetals through the reaction of one of the hydroxyls with the aldehyde group, giving a strain-free six- or five-membered ring.

The Fischer projection for the sugar is surprisingly useful in predicting the configuration and conformation of the cyclic form.

The approach is straightforward. Since cyclic hemiacetal formation requires a hydroxyl group as the nucleophile to attack the protonated carbonyl.

We put this hydroxyl group on the vertical, thus getting all the ring atoms onto the vertical. This requires the rotation of three groups attached to the appropriate atom, C-5 in the case of D-(+)-glucose.

Such rotation does not affect the configuration at C-5. Then put in the stereochemistry implied by the Fischer projection, using wedges and dots. This structure should then be turned on its side.

The ring formation is considered by joining up the C-5 hydroxyl and the carbonyl at the rear of the structure.

Note that, as drawn, this eclipsed conformer from the Fischer projection actually has these atoms quite close together, so that ring formation is easily achieved and, most importantly, easily visualized.

The net result is a cyclic system looking like the Haworth representation that is commonly used, especially in biochemistry books.

The Haworth representation nicely reflects the up–down relationships of the various substituent groups, but is uninformative about whether these are equatorial or axial.

The last step, therefore, is to transcribe this representation into a chair conformation, as shown, so that we see the conformational consequences.

The alternative chair conformation, should we draw it instead, would be less favoured than that shown because of the increased number of axial substituents.

The conformation of D-glucose is the easily remembered one, in that all the substituents are equatorial.

A similar procedure is shown for D-(−)-ribose, which, although it is capable of forming a six-membered cyclic form, is found to exist predominantly as a five-membered ring.

D and L amino acids

There is a correlation between D- and L-glyceraldehyde and D- and L-amino acids, in that it is possible to convert one system chemically into another without affecting the integrity of the chiral centre.

The fine detail of the transformations need not concern us here. The net result is that D- and L-amino acids have the general configurations shown.

Note that all the amino acids found in proteins are of the L configuration (except the achiral glycine); D-amino acids are found in some polypeptide antibiotics.

As we pointed out this brings up an apparent anomaly in nomenclature. In all protein L-amino acids, except for cysteine, this represents an S configuration; cysteine, because of its high-priority sulfur atom has the R configuration.

One further point; as mentioned, the now obsolete descriptors d and l are abbreviations for dextrorotatory (+) and laevorotatory (−) respectively. They do not in any way relate to D and L.

Polycyclicsy Stems

Many molecules of biological or pharmaceutical importance contain polycyclic ring systems, and we have already met some examples in other contexts, For Example. penicillins.

There are three main ways in which rings can be joined together, according to whether they share one atom, two atoms, or more than two atoms. These are termed spiro, fused, or bridged systems respectively.

Examples are shown where six-membered rings are joined in various ways, but the concepts apply equally to rings of other sizes.

Spiro Systems

Spiro systems have two rings sharing a single carbon atom, and since this has essentially a tetrahedral array of bonds, the bonds starting the two rings must be arranged perpendicular to each other.

If there is an appropriate substitution on the rings, then this can lead to the spiro centre becoming chiral.

Natural Spirocom Pounds

Spiro compounds are exemplified by several natural product structures. One of these is the antifungal agent griseofulvin produced by cultures of the mould Penicillium griseofulvin.

Griseofulvin is the drug of choice for many fungal infections, but it is ineffective when applied topically, so is administered orally.

Griseofulvin has two chiral centres, one of which is the spiro centre, so there are potentially four configurational isomers for the structure. Natural griseofulvin has the configurations shown.

Solasodine and tomatidine are steroidal alkaloids produced by potatoes (Solanum tuberosum) and tomatoes (Lycopersicon esculente) respectively.

These compounds, as glycosides, are responsible for the toxic properties of the foliage and green fruits of these plants.

They are not present in potato tubers, unless green, or in ripe tomato fruits. Both compounds contain a spiro system, a nitrogen analogue of a ketal.

A spiroketal is present in diosgenin from Dioscorea species, a raw material used for the semi-synthesis of steroidal drugs.

Note that solasodine and tomatidine demonstrate the different configurations at the spiro centre; all natural spiroketals have the same stereochemistry at the spiro centre as in diosgenin.

Fused Ring Systems

Fused ring systems are particularly common. It is logical to suppose that fusing on one or more additional ring systems is going to have stereochemical consequences.

In particular, the conformational changes seen with single-ring systems are likely to be significantly modified. Initially, let us consider two cyclohexane rings fused together, giving a bicyclic system called decalin.

Two configurational isomers exist, trans- and cisdecalin, according to the stereochemistry of ring fusion.

The trans or cis relationship is most easily seen with the hydrogens at the ring fusion carbons, but it also follows that the bonds forming part of the second ring can be considered to share a trans or cis relationship to each other.

It is usual practice to show the stereochemistry in the former way, via the ring fusion substituents.

The situation is in many ways analogous to transand cis-1,2-methylcyclohexane and these afford useful comparisons as we consider conformational changes.

Now, trans-decalin forms a rather rigid system, and it transpires that the only conformational mobility possible is ring flip of chairs to very much less favourable boats.

Since both bonds of the second ring are equatorial with respect to the first ring, any other type of conformational change would require these to become axial.

It is impossible to join the two axial bonds into a ring system as small as six carbons; hence, there is no conformational mobility.

On the other hand, cis-decalin is conformationally mobile, and a simultaneous flipping in both rings produces a new conformer of equal energy.

This is not easy to visualize. In the scheme, the middle conformer has one ring viewed face-on, so we have resorted to the rotation of the structure to get an appreciation of the new conformer with its rings in chair form.

It is best to have models to appreciate this conformational flexibility.

It is quite clear, though, that an axial bond becomes equatorial and an equatorial one becomes axial, just as with substituents in the cis- 1,2-dimethylcyclohexane analogue.

However, it is probably reassuring to appreciate that this conformational flexibility in two cis-fused cyclohexane rings is lost when a third ring is fused, and in many of the fused ring systems of interest to us, it becomes of no further consequence.

Since the second ring in trans-decalin effectively introduces two equatorial substituents to the first ring, whilst cis-decalin.

It provides one equatorial and one axial substituent, it is logical to predict that transdecalin should have a lower energy than cis-decalin.

This is indeed the case, the energy difference being about 12 kJ mol^-1. When we considered trans- and cis-1,2-dimethylcyclohexane.

We found that only three configurational isomers exist, enantiomeric forms of the trans isomer, together with the cis isomer, which is an optically inactive meso compound.

The meso relationship could be deduced from the plane of symmetry in the hexagon representation.

When we look at the structures of trans- and cisdecalin, it is apparent that a further plane of symmetry, through the ring fusion, is present in both structures.

This means that each isomer is superimposable on its mirror image; consequently, there are only two configurational isomers of decalin, one trans and one cis.

The situation in trans- and cis-decalin is complicated by the symmetry elements.

If this symmetry is destroyed, For Example. by introducing dimethyl substituents, we get back to reassuringly familiar territory in which two chiral centres lead to four configurational isomers.

The same is true in the trans- and cis-1,2- dimethylcyclohexane series. Fusing rings of different sizes can produce significant restraints, especially when rings of less than six carbons are involved.

However, the characteristics of these fused systems can be deduced logically by applying our knowledge of single-ring systems.

Fusion of a five-membered ring to a six-membered ring gives a hydride system, and, as with decalins, cis and trans forms are possible.

Because the cyclopentane ring is more planar than a cyclohexane ring this causes deformation and increases strain at the ring fusion.

This deformation is more easily accommodated with the cis-fusion than the transfusion, and, in contrast to the decalins, the cis isomer has a lower energy than the trans isomer (by about 1 kJ mol^-1).

As in the decalins though, the cis form is conformationally mobile, whereas the trans form is fixed.

The fusion of rings of different sizes reduces symmetry in the structures; instead of the rather unusual situation with the decalins, where there are only two configurational isomers.

The hydrindanes exist in the anticipated three isomeric forms, two enantiomeric trans isomers and a meso cis isomer (compare 1,2- dimethylcyclohexane).

Isomerizations Influenced By Ring Fusions

Epimerization Of Cis-Decalone If cis-decalone is treated with a mild base, it is predominantly isomerized to trans-decalone. This can be rationalized by considering stereo drawings of the two isomers.

The ring fusion in cis-decline means that bonds forming the second ring have a relationship to the first ring in which one bond is equatorial and one axial.

In contrast, both such bonds in trans-decline are equatorial to the first ring. We can predict, therefore, that trans-decline has a lower energy than cis-decline.

The isomerization is brought about because the carbonyl group is adjacent (α) to the hydrogen at the ring fusion.

This hydrogen is relatively acidic and may be removed by the base, generating the enolate anion.

The enolate anion must now be planar around the site of ring fusion and, by a reversal of the process, may pick up a proton from either side of the double bond.

However, instead of getting a 1:1 mixture of the two possible isomers, this reaction very much favours the trans isomer because of its lower thermodynamic energy. The equilibrium mixture contains principally trans-decline.

Epimerization Of Etoposide The anticancer agent etoposide contains a five-membered lactone function that is significantly strained because it is trans-fused.

This material is readily converted into a relatively strain-free cis-fused system by treating with very mild alkali, For Example. traces of detergent, and produces an epimer called picroetoposide. This isomer has no significant biological activity.

The epimerization can be formulated as involving an enolate anion, as above. However, in contrast to the decalin example above, cis-hydride is of lower energy than trans-hydride.

In this particular case, on reverting back to a carbonyl compound, the planar enolate anion is presented with the alternatives of receiving.

A proton from one face to form a strained trans-fused system, or from the other face to form a strain-free cis-fused system.

The latter is very much preferred, so much so that the conversion of etoposide into its epimer is almost quantitative.

Although we can rationalize this behaviour simply by considering the hydride-type rings.

The fusion of this system to an aromatic ring causes additional distortion, and the effect becomes even more pronounced in favour of the cis-fused system.

This behaviour contrasts with the racemization of hyoscyamine to atropine, which also involves an enolate anion derived from an ester system.

As the term racemization implies, atropine is a 50:50 mixture of the two enantiomers. It shows how the proportion of each epimer formed can be influenced by other stereochemical factors.

The fusion of a three-membered ring into a six-membered ring has much more serious limitations.

A three-membered ring must be planar, so it will distort the ring it is being fused to, and this restricts stereochemical possibilities.

For example, epoxycyclohexane can, therefore, only be cis-fused, and the six-membered ring is forced to adopt the half-chair conformation we saw with cyclohexene.

There will be conformational mobility in this ring provided that there are no other ring fusions to prevent this.

Note that, in situations where a ring fusion produces chiral centres, we can find the number of configurational isomers possible is less than that predicted from the 2n guidelines.

This may be the consequence of symmetry, in that an isomer is the same as its mirror image, as we have seen above.

However, it can also be the result of restrictions caused by the ring fusion, so that one centre effectively defines the chirality of another, thus reducing the number of combinations.

In epoxycyclohexanes, no trans-fused variants can exist. Note that a cyclohexane system will be forced into a similar half-chair conformation by fusing a planar aromatic ring onto a cyclohexane ring (a tetrahydronaphthalene system).

Shapes Of Steroids

Steroids all contain a tetracyclic ring system comprised of three six-membered rings and one five-membered ring fused together. Cholesterol is the best known of the steroids.

It is an essential structural component of animal cells, though the presence of excess cholesterol in the blood is definitely associated with the incidence of heart disease and heart attacks.

Whilst cholesterol typifies the fundamental structure, further modifications to the side chain and the ring system help to create a wide range of biologically important natural products.

For Example. sterols, steroidal saponins, cardioactive glycosides, bile acids, corticosteroids, and mammalian sex hormones.

Because of the profound biological activities encountered, many natural steroids, together with a considerable number of synthetic and semi-synthetic steroidal compounds, are routinely employed in medicine.

The markedly different biological activities observed emanating from compounds containing a common structural skeleton are, in part.

Ascribed to the functional groups attached to the steroid nucleus and, in part, to the overall shape conferred on this nucleus by the stereochemistry of ring fusions.

Let us start with cholestane, which is the basic hydrocarbon skeleton of cholesterol. This structure has all ring fusions trans.

By logical extension trans-decalin and trans-hydride can be deduced to have approximately the shape illustrated.

Because of the transfusions, there is no conformational mobility except for the unlikely flipping of ring A into a boat form, which we can ignore.

The overall shape of cholestane is a rather rigid and flattish structure. The rings are designated A–D as indicated.

Cholesterol has a double bond in ring B at the A–B ring fusion, so this distorts the rings by demanding that the arrangement around the double bond is planar.

It is not possible to depict this perfectly in a typical two-dimensional representation.

The natural progestogen hormone progesterone also has a double bond at the A–B ring fusion, but this time in ring A, so a similar distortion in ring A is required.

The fungal sterol ergosterol has double bonds at positions 5 and 7, both in the B ring, which consequently should become essentially planar.

The picture shown is a rough approximation. The antifungal effect of polyene antibiotics, such as amphotericin and nystatin.

depends upon their ability to bind strongly to ergosterol in fungal membranes. They do not bind significantly to cholesterol in mammalian cells, so this provides selective toxicity.

The binding to ergosterol is very much influenced by the changes in shape conferred by the extra double bond in ring B.

In oestrogens, such as estradiol, the A ring is aromatic. Consequently, this ring is planar and distorts ring B accordingly; again, it is difficult to draw this perfectly.

The stereochemical outcome makes oestrogens seem rather more flattened than the original all-trans arrangement in cholestane.

More dramatic changes are made to the shape of the steroid skeleton if ring fusions become cis rather than trans.

The most important examples involve the A–B and C–D ring fusions. It is not difficult to work out how the modified skeleton looks after these changes.

The approach is to start from the all-trans system and to delete the appropriate ring, though retaining the bonds to the unchanged part as a guide to putting in the new ring.

This provides us with three of the bonds in the new ring, and it is just necessary to fill in the rest, using earlier decalin or hydrindane templates.

The approach is used to show the shape of cholic acid, one of the bile acids secreted into the gut to emulsify fats and encourage digestion. Cholic acid is characterized by a cis fusion of rings A and B.

Digitoxigenin has cis fusions for both A–B and C–D rings. Glycosides of digitoxigenin are the powerful heart drugs found in the foxglove, Digitalis purpurea.

Note how a cis ring fusion changes the more-or-less flat molecule of cholestane into a molecule with a significant ‘bend’ in its shape; digitoxigenin has two such ‘bends’.

These features are important in the binding of steroids to their receptors and partially explain why we observe quite different biological activities from compounds containing a common structural skeleton.

Most natural steroids have the stereochemical features seen in cholesterol, though, as we have seen, there may be some variations, particularly with respect to ring fusions affecting the A and D rings.

Note that transfusion at the hydride C–D ring junction is energetically less favourable than a cis fusion. but most natural steroid systems actually have this transfusion.

We have met diosgenin as an example of a natural spiro compound. and further examination of the structure shows the Δ⁵ double bond as in cholesterol, a second five-membered ring cis-fused onto the five-membered ring D, as well as the spiro fusion of a six-membered ring.

Before this structure dismays you, take it slowly and logically. It should not be too difficult to end up with the stereo drawing shown here.

The Shape Of Penicillins

Penicillins are the most widely used of the clinical antibiotics. They contain in their structures an unusual fused ring system in which a four-membered β-lactam ring is fused onto a five-membered thiazolidine.

Both rings are heterocyclic, and one of the ring fusion atoms is nitrogen. These heteroatoms do not alter our understanding of molecular shape, since we can consider that they also have an essentially tetrahedral array of bonds or lone pair electrons.

We have seen that, in cyclobutane and cyclopentane, a lower energy conformation is attained if the rings are not planar.

If one fuses a five-membered ring onto a four-membered ring, models demonstrate that it is only possible to have a cis fusion in such a structure.

And that conformational freedom in the four-membered ring disappears if we are to achieve this bonding; the four-membered ring reverts to a more planar shape.

It is still possible to have the five-membered ring non-planar, thereby reducing eclipsed interactions.

The cis fusion in which one of the fusion atoms is nitrogen merely indicates that the nitrogen lone pair electrons occupy the remaining part of the tetrahedral array.

It does, however, mean that inversion at the nitrogen atom is not possible, since that would hypothetically result in the formation of the impossible trans-fused system.

The ring fusion has thus frozen the nitrogen atom into one configuration. Fusion of a four-membered ring onto a six-membered ring is also only possible with a cis fusion.

Cephalosporins provide excellent examples of such compounds, and the comments made above for penicillins are equally valid for these compounds.

Bridged Ring Systems

In bridged ring compounds, rings share more than two atoms, and the bridge can consist of one or more atoms. We have already met an example in bornane.

which we used as an illustration of how a cyclohexane ring can be forced into a boat conformation to achieve the necessary bonding.

If we inspect the ring system of bornane, omitting the methyl groups, we can see that there are actually several bridges of different lengths spanning the bridgehead atoms, depending upon which atoms are considered.

This is used in nomenclature, as illustrated below, including in square brackets all the bridges, listed in decreasing lengths.

Numbering, when necessary, always starts from a bridgehead atom. A closer inspection of the shape of bicyclo[2,2,2]octane (best with a model), which has two-carbon bridges, shows that each ring system has the boat conformation.

Note that the ring systems with small bridges illustrated here can have no conformational mobility, and are quite fixed. Bornane also has no configurational isomers.

If we are going to bridge a cyclohexane ring with a one-carbon bridge, there is only one way to achieve this; in other words, the configuration at the second bridgehead is fixed by that chosen at the first.

A similar situation confronted us with fused rings, in that, in order to achieve the fusion of a small ring, only a cis fusion was feasible.

Furthermore, bornane has a plane of symmetry and can be superimposed on its mirror image, so only one configurational isomer can exist.

We should compare this system with a 1,4- disubstituted cyclohexane such as 4-methylcyclohexanecarboxylic acid.

There is a plane of symmetry in this molecule, so there are no chiral centres; but geometric isomers exist, allowing cis and trans stereoisomers.

The restrictions imposed by bridging have now destroyed any possibility of geometric isomerism.

When we move on to camphor, a ketone derivative of bornane, we find this can exist in two enantiomeric forms because the plane of symmetry has been destroyed.

Nevertheless, there are only two configurational isomers despite the presence of two chiral centres; bridging does not allow the other two variants to exist.

β-Pinene is representative of a bicyclo[3,1,1]heptane system. This natural product has two chiral centres, but can exist only in the (+)- and (−)-enantiomeric forms shown.

Stereochemistry Of Tropane Alkaloids

The tropane alkaloids (−)-hyoscyamine and (−)-hyoscine are found in the toxic plants deadly nightshade (Atropa belladonna) and thornapple (Datura stramonium) and are widely used in medicine.

Hyoscyamine, usually in the form of its racemate atropine, is used to dilate the pupil of the eye, and hyoscine is employed to control motion sickness. Both alkaloids are esters of (−)-tropic acid.

The alcohol portion in hyoscyamine is tropine; in hyoscine, it is the epoxide scopine.

Tropine is an example of an azabicyclo[3,2,1]octane system with a nitrogen bridge, whereas scopine is a tricyclic system with a three-membered epoxide ring fused onto tropine.

Note that systematic nomenclature considers an all-carbon ring system with one carbon replaced by nitrogen; hence, tropane is an azabicyclooctane.

There are several interesting stereochemical features accommodated within these structures.

First, both tropine and scopine are optically inactive meso compounds; despite the chiral centres, two for tropine and four for scopine,

Both compounds have a plane of symmetry so that optical activity conferred by one centre is cancelled out by its mirror image centre.

The optical activities of hyoscyamine and hyoscine are derived entirely from the chiral centre in the tropic acid portion. Atropine, the racemic form of hyoscyamine, is the ester of tropine with (±)-tropic acid.

Note also that, although we normally see rapid inversion at a nitrogen atom, the N-methyl group in hyoscyamine is preferentially in the lower energy equatorial position of the chair-like piperidine ring, as would be predicted.

However, in hyoscine, the N-methyl group has been found to be axial, not the expected equatorial. This seems to arise to minimize interaction with the extra epoxide ring in scopine.

When we look at another tropane alkaloid, cocaine, we get a different scenario. Cocaine is obtained from the coca plant Erythroxylum coca and is a powerful local anaesthetic, but now known primarily as a drug of abuse.

There is no chiral centre in the acid portion, which is benzoic acid, but the optical activity of cocaine comes from the alcohol methylecgonine.

Because of the ester function in methylecgonine, the tropane system is no longer symmetrical, and the four chiral centres all contribute towards optical activity.

Now, you may have noticed that the hydroxyl group in methylecgonine is oriented differently from that in tropine.

In methylecgonine, it is easy to define the position of the hydroxyl, since this is a chiral centre and we can use the R/S nomenclature.

An alternative stereoisomer of tropine exists, and this is called pseudotropine.

How can we define the configuration for the hydroxyl when the plane of symmetry of the molecule goes through this centre and means this centre is not chiral but can exist in two different arrangements?

This is a situation allowed for in the IUPAC nomenclature rules, because if we are faced with two groups which are the same but have opposite chiralities, then the group with R chirality has a higher priority than the group with S chirality.

Applying this rule, tropine would have the S configuration and pseudotropine the R configuration at this centre. Because of the plane of symmetry.

These atoms are not strictly chiral, and this is taken into account by using lower-case letters; tropine is s and pseudotropine is r.

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A fundamental concept in chemistry is associated with proton loss and gain, i.e. acidity and basicity.

Acids produce positively charged hydrogen ions H⁺ (protons) in aqueous solution; the more acidic a compound is, the greater the concentration of protons it produces.

In water, protons do not have an independent existence but become strongly attached to a water molecule to give the stable hydronium ion H₃O⁺. In the Bronsted–Lowry definition

• An acid is a substance that will donate a proton;
• A base is a substance that will accept a proton.

Thus, the acid HCl ionizes in water to produce H₃O⁺ and Cl− ions.

H₃O⁺ is termed the conjugate acid (of the base H₂O) and Cl− is termed the conjugate base (of the acid HCl).

In general terms, cleavage of the H–A bond in an acid HA is brought about by a base, generating the conjugate acid of the base, together with the conjugate base of the acid. You may wish to read that sentence again!

The Lewis definition of acids and bases is rather more general than the Brønsted–Lowry version (which refers to systems involving proton transfer) in that:

An acid is an electron-pair acceptor;

A base is an electron-pair donor.

Thus, Lewis acids include such species as boron trifluoride, which can react with trimethylamine to form a salt.

There is no fundamental difference between trimethylamine acting as a Brønsted base or as a Lewis base, except that in the Bronsted concept, it donates its electrons to a proton electrophile, whereas as a Lewis base, it donates its electrons to a Lewis acid electrophile.

Acidity And pKa Values

For the ionization of the acid HA in water

⇒ \(\mathrm{H}_2 \mathrm{O}+\mathrm{HA} \stackrel{K}{\rightleftharpoons} \mathrm{H}_3 \mathrm{O}^{\oplus}+\mathrm{A}^{\ominus}\) the equilibrium constant K is given by the formula

⇒ \(K=\frac{\left[\mathrm{A}^{-}\right]\left[\mathrm{H}_3 \mathrm{O}^{+}\right]}{[\mathrm{HA}]\left[\mathrm{H}_2 \mathrm{O}\right]}\)

where [HA] signifies the concentration of HA, etc. However, because the concentration of water is essentially constant in aqueous solution, a new equilibrium constant Ka is defined as

⇒ \(K_{\mathrm{a}}=\frac{\left[\mathrm{A}^{-}\right]\left[\mathrm{H}_3 \mathrm{O}^{+}\right]}{[\mathrm{HA}]}\)

K a is termed the acidity constant, and its magnitude allows us to classify acids as strong acids (a large value for Ka and, consequently, a high H₃O⁺ concentration) or weak acids (a small value for Ka and, thus, a low H₃O⁺ concentration). For example, the strong acid HCl has Ka = 107.

However, for weak acids, the amount of ionization is much less and, consequently, the value of Ka is rather small. Thus, acetic acid CH3CO2H has Ka = 1.76 × 10−5. To avoid using such small numbers as these, Ka is usually expressed in the logarithmic form p Ka where pKa = − log10 Ka Accordingly, the pKa for acetic acid is 4.75:

⇒ \(K_{\mathrm{a}}=\frac{\left[\mathrm{A}^{-}\right]\left[\mathrm{H}_3 \mathrm{O}^{+}\right]}{[\mathrm{HA}]}\)

K a is termed the acidity constant, and its magnitude allows us to classify acids as strong acids (a large value for Ka and, consequently, a high H₃O⁺ concentration) or weak acids (a small value for Ka and, thus, a low H₃O⁺ concentration).

For example, the strong acid HCl has Ka = 107. However, for weak acids, the amount of ionization is much less and, consequently, the value of Ka is rather small. Thus, acetic acid CH3CO2H has Ka = 1.76 × 10−5. To avoid using such small numbers as these, Ka is usually expressed in the logarithmic form p Ka where pKa = − log10 Ka

Accordingly, the pKa for acetic acid is 4.75: pKa = − log(1.76 × 10−5) = −(−4.75) = 4.75

The pKa for hydrochloric acid can similarly be calculated to be −7: pKa = − log(107) = −7

This means there is an inverse relationship between the strength of an acid and pKa:

• A strong acid has a large Ka and, thus, a small pKa, i.e. A− is favoured over HA;
• A weak acid has a small K a and, thus, a large pKa, i.e. HA is favoured over A−

Or, put another way:

• The smaller the value of p Ka, the stronger the acid;
• The larger the value of p Ka, the weaker the acid.

We find that pKa values range from about −12 to 52, but it must be appreciated right from the start that a difference of one pKa unit represents a 10-fold difference in K a and, thus, a 10-fold difference in H3O+ concentration.

A twofold difference in acidity would be indicated by a pKa difference of just 0.3 units (log 2 = 0.3).

Accordingly, a difference of n pKa units indicates a 10n-fold difference in acidity, so the range −12 to 52 represents a huge factor of 1064. A compound with pKa < 5 is regarded as a reasonably strong acid, and those with pKa < 0 are very strong acids.

At first glance, negative pKa values seem rather strange, but this only means that the equilibrium lies heavily towards ionization; Ka is large and, therefore, pKa = − log Ka becomes negative.

⇒ \(\begin{gathered}
\mathrm{H}_2 \mathrm{O}+\mathrm{HA} \rightleftharpoons \mathrm{H}_3 \mathrm{O}+\mathrm{A}^{\ominus} \\
K_{\mathrm{a}}=\frac{\left[\mathrm{A}^{-}\right]\left[\mathrm{H}_3 \mathrm{O}^{+}\right]}{[\mathrm{HA}]} \quad \mathrm{p} K_{\mathrm{a}}=-\log _{10} K_{\mathrm{a}} \\
K_{\mathrm{a}}=0.01 \quad K_{\mathrm{a}}=0.1 \quad K_{\mathrm{a}}=1 \quad K_{\mathrm{a}}=10 \quad K_{\mathrm{a}}=100
\end{gathered}\)

As we use pKa values, we shall find that, in most cases, relative, rather than specific, values are all we need to consider to help us predict chemical behaviour and reactivity. Thus, from pKa values, we can see that acetic acid (pKa 4.75) is a weaker acid than hydrochloric acid (pKa − 7).

pKa values for a wide variety of different compounds. Compounds are listed in order of increasing acidity.

Although the pKa values included extend from about 52 to −10, values in the middle of the range are known most accurately.

This is because they can be measured readily in aqueous solution. Outside of the range from about 2 to 12, pKa values have to be determined in other solvents, or even by indirect methods; results are then extrapolated to give the value in water.

Have been intentionally rounded to stress that a high level of accuracy is usually inappropriate. The range of pKa values that can be measured in water is determined by the ionization of water itself, i.e. −1.74 (the pKa of H₃O⁺) to 15.74 (the pKa of H₂O); Acids that are stronger than H3O+ simply protonate water, whereas bases that are stronger than HO− remove protons from water.

However, the fact that they do have to be measured means that as you look in the literature for the pKa of a particular compound you may find slightly different values can be presented.

Do not let this confuse you. As mentioned above, relative, rather than specific, values are our main concern. We have chosen to present the pKa values as a series of tables, rather than in a single one.

This should help you to locate a particular compound according to its functional group, and we hope that this will also emphasize similarities and differences in related structures.

It also means that you may find some examples turning up in more than one table. As we consider different aspects of chemical reactivity in subsequent chapters, we shall see how pKa values can be used to predict whether a reagent is a good or a poor nucleophile, whether it can function as a good leaving group, and how easy it is to generate anionic nucleophiles.

We shall also find that pKa values can tell us how much of a compound or a drug is ionized under particular conditions and, therefore, whether or not it can be produced in a soluble form.

It is now appropriate to consider some of the electronic and structural features that influence pKa so that we can rationalize and predict relative.

Electronic And Structural Features That Influence Acidity

Electronegativity

The more electronegative an element is, the more it helps to stabilize the negative charge of the conjugate base.

For example, the acidities of compounds of second-row elements in the periodic table increase as the atom to which hydrogen is attached becomes more electronegative:

pKa values for CH₄, NH₃, H₂O and HF are about 48, 38, 16 and 3, respectively, i.e. we have increasing acidity left to right as the electronegativity of the atom attached to hydrogen increases.

Bond energies

Within a single column of the periodic table, acidities increase as one descends the column: pKa values for HF, HCl, HBr, and HI are about 3, −7, −9, and −10 respectively, i.e. we have increasing acidity on descending the group.

This is the reverse of what might be expected simply based on electronegativities but relates to the increasing size of the atom and the corresponding improved ability to disperse the negative charge over the atom.

We are seeing a weakening in bond strengths on descending the group. Similarly, although sulfur is less electronegative than oxygen, thiols (RSH) are more acidic than alcohols (ROH). For example, pKa values for methanethiol and methanol are 10.5 and 16 respectively.

Inductive effects

Electron-donating and electron-withdrawing groups influence acidity by respectively destabilizing or stabilizing the conjugate base.

his inductive effect, a charge polarization transmitted through σ bonds, causes a shift in electron density, and its influence may easily be predicted.

Thus, electron-withdrawing groups increase acidity:

pKa values for the simple carboxylic acid acetic acid and its halogenated derivatives chloroacetic acid, dichloroacetic acid, and trichloroacetic acid are about 4.8, 2.9, 1.3, and 0.7 respectively, the inductive effects of the chlorine atoms spreading the charge of the conjugate base and thus stabilizing it.

Increasing the number of halogen atoms increases this effect, with a consequent increase in acidity. Note that the introduction of one chlorine atom increases acidity by a factor of almost 100, and trichloroacetic acid is a strong acid.

Because of the different electronegativities of the various halogens, we can also predict that fluorine will have a greater effect than chlorine, which in turn will increase acidity more than bromine or iodine.

This is reflected in the observed acidities of monohalogenated acetic acids, though the increased acidity of chloroacetic acid (pKa 2.87) over bromoacetic acid (pKa 2.90) is not apparent because of the rounding-up process.

The inductive effect is a rather short-range effect, and its influence decreases rapidly as the substituent in question is located further away from the site of the negative charge because it has to be transmitted through more bonds.

Thus, the effect on the acidity in butanoic acid derivatives can be seen to diminish with distance. 2- Chlorobutanoic acid (pKa 2.9) shows a significant enhancement in acidity over butanoic acid (pKa 4.9), whereas 3-chlorobutanoic acid (pKa 4.1) and 4-chlorobutanoic acid (pKa 4.5) show rather more modest changes.

Other electron-withdrawing groups that increase the acidity of acids include, listed in decreasing order of their effect: –NO₂, –N+R₃, –CN, –CO₂R, –CO–, –OR and –OH.

Electron-donating groups will have the opposite effect, destabilizing the conjugate base by increasing electron density, and thus producing weaker acids. The most common electron-donating groups encountered are going to be alkyl groups, though the effect from alkyl groups is rather small.

Indeed, it is not immediately apparent why there should be any inductive effect at all since the substitution of hydrogen by alkyl should not lead to any bond polarization. At this point, we should merely note that alkyl groups have a weak electron-donating effect – it may not be strictly an inductive effect.

The pKa value for formic acid (pKa 3.7) makes it more acidic than acetic acid. The electron-donating effect of the methyl group is most marked on going from formic acid to acetic acid since the acidity of propionic acid (pKa 4.9) and butanoic acid vary little from that of acetic acid.

The electron-donating effect from alkyl substituents is relatively small, being considerably smaller than inductive effects from most electron-withdrawing groups, and also rapidly diminishes along a carbon chain.

Alcohols are much less acidic than carboxylic acids; but, as one progresses through the sequence of methanol, ethanol, isopropanol, and tert-butanol, pKa values gradually increase from 15.5 to 19, a substantial decrease in acidity.

Although this was originally thought to be caused by the inductive effects of methyl groups, it is now known to be primarily related to solvation effects. In solution, the conjugate base anion is surrounded by polar solvent molecules.

This solvation helps to stabilize the conjugate base and thus increases the acidity of the alcohol. As we get more alkyl groups, the solvation of the anion is diminished because of the increased steric hindrance they cause and observed acidity also decreases.

Hybridization effects

The acidity of a C–H bond is influenced by the hybridization state of the carbon atom attached to the acidic hydrogen. Dissociation of the acid generates an anion whose lone pair of electrons is held in a hybridized orbital.

We can consider sp orbitals to have more s character than sp² orbitals, and similarly, sp² orbitals to have more s character than sp³ orbitals.

Since s orbitals are closer to the nucleus than p orbitals, it follows that electrons in an sp-hybridized orbital are held closer to the nucleus than those in an sp² orbital; those in an sp² orbital are similarly closer to the nucleus than those in an sp3 orbital.

It is more favourable for the electrons to be held close to the positively charged nucleus, and thus a sp-hybridized anion is more stable than a sp²-hydridized anion, which is more stable than a sp3³-hybridized anion. Thus, the acidity of a C–H bond decreases as the s character of the bond decreases.

The pKa of the hydrocarbon ethane is about 50, that of ethylene is about 44, and that of acetylene is about 25. The hybridization of the C–H bond in ethane is sp³ (25% s character), in ethylene it is sp² (33% s character), and in acetylene it is sp (50% s character).

This makes alkynes (acetylenes) relatively acidic for hydrocarbons. It is also a contributing factor in the acidity of HCN (pKa 9.1), where the conjugate base cyanide is a hybridized anion, though additional stabilization comes from the electronegative nitrogen atom.

So far we have considered the hybridization state of the orbital associated with the anionic charge. However, the hybridization state elsewhere in the molecule may also affect acidity.

The more character an orbital has, the closer the electrons are held to the nucleus, and this effectively makes the atom more electronegative.

This may be explained in terms of hybridization modifying inductive effects, such that sp-hybridized carbons are effectively more electronegative than sp²-hybridized carbons, and similarly, sp²-hybridized carbons are more electronegative than sp³-hybridized carbons.

The pKa values for the following acids illustrate that, as the carbon atom adjacent to the carboxylic acid group changes from sp³ to sp³ to sp hybridization, the acidity increases, in accord with the electronegativity explanation above.

Note that benzoic acid (sp² hybridization) has a similar pKa to acrylic acid (propenoic acid), which also has sp² hybridization.

Resonance delocalizes zation effects

Delocalization of charge in the conjugate base anion through resonance is a stabilizing factor and will be reflected by an increase in acidity.

Drawing resonance structures allows us to rationalize that the negative charge is not permanently localized on a particular atom, but may be dispersed to other areas of the structure. We should appreciate that a better interpretation is that the electrons are contained in a molecular orbital that spans several atoms.

However, drawing resonance structures provides a simple and convenient way of predicting stability through delocalization. The pKa of ethanol is 16, and that of acetic acid is 4.8.

The increased acidity of acetic acid relative to ethanol can be rationalized in terms of the delocalization of charge in the acetate anion, whereas in ethoxide the charge is localized on oxygen. Even more delocalization is possible in the methanesulfonate anion, and this is reflected in the increased acidity of methanesulfonic acid (pKa − 1.2)

We have also shown some representations of acetate and methanesulfonate anions that have been devised to emphasize resonance delocalization; these include partial bonds rather than double/single bonds.

Although these representations are valuable, they can lead to some confusion in interpretation. It is important to remember that there is a double bond in these systems. Therefore, we prefer to draw out the contributing resonance structures.

The alkane propane has pKa 50, yet the presence of the double bond in propene means the methyl protons in this alkene have pKa 43; this value is similar to that of ethylene (pKa 44), where increased acidity was rationalized through sp² hybridization effects.

1,3-Pentadiene is yet more acidic, having pKa 33 for the methyl protons. In each case, increased acidity in the unsaturated compounds may be ascribed to the delocalization of charge in the conjugate base. Note that we use the term allyl for the propenyl group.

Resonance stabilization is also responsible for the increased acidity of a C–H group situated adjacent to a carbonyl group.

The anion is stabilized through delocalization of charge, similar to that seen with the allyl anion derived from propene; but this system is even more favourable, in that delocalization allows the charge to be transferred to the electronegative oxygen atom.

As a result, acetone (pKa 19) is significantly more acidic than propene (pKa 43). Anions of this type, termed enolate anions, are some of the most important reactive species used in organic chemistry

The acidity of a C–H is further enhanced if it is adjacent to two carbonyl groups, as in the 1,3- diketone acetylacetone. The enolate anion is stabilized by delocalization, and both carbonyl oxygens can participate in the process.

This is reflected in the pKa 9 for the protons between the two carbonyls, whereas the terminal protons adjacent to just a single carbonyl have pKa 19, similar to acetone above.

Increased delocalization has a profound effect on the acidity. These two values should be compared with that of the hydrocarbon propane (pKa 50)

Aromatic rings are themselves excellent examples of resonance and delocalization of electrons They also influence the acidity of appropriate substituent groups, as seen in benzoic acids. Benzoic acid (pKa 4.2) is a stronger acid than acetic acid (pKa 4.8), and it is also stronger than its saturated analogue cyclohexane carboxylic acid (pKa 4.9).

The phenyl group exerts an electron-withdrawing effect because the hybridization of the ring carbons is sp²; consequently, electrons are held closer to the carbon atom than in a sp³-hybridized orbital. This polarizes the bond between the aromatic ring and the carboxyl. The pKa of phenylacetic acid (pKa 4.3), compared with acetic acid (pKa 4.8), demonstrates the inductive effect of a benzene ring.

However, we might then expect benzoic acid to be a rather stronger acid than it is, since the phenyl group is closer to the carboxyl group than in phenylacetic acid.

We attribute the lower acid strength to an additional resonance effect in the carboxylic acid that is not favourable in the anion, where it would lead to a carboxylate carrying a double negative charge; therefore, the resonance effect weakens the acid strength.

Further inductive effects from other substituents enhance or counter these effects with predictable results. Thus, a halogen such as chlorine, with a strong inductive effect, produces stronger acids, especially in the case of the ortho derivative. Here, the extra inductive effect is correspondingly closer to the carboxyl group, and it will help to stabilize the conjugate base.

The acid-weakening resonance effects are also diminished by the inductive effects of halogens; it is not favourable to have an electron-withdrawing substituent close to a positive charge.

On the other hand, methyl substituents have a weak electron-donating effect opposing that of the aromatic ring. This also favours resonance in the nonionized acid. There is only a modest effect on acidity, except when the methyl is in the ortho position, where the effect is closer to the carboxyl group.

However, ortho substituents add a further dimension that is predominantly steric. Large groups in the ortho position can influence the carboxyl group, forcing it out of the plane of the ring.

The result is that resonance is now inhibited because the orbitals of the carbonyl group are no longer coplanar with the benzene ring. In almost all cases, the ortho-substituted benzoic acid tends to be the strongest acid of the three isomers.

When substituents can also be involved in the resonance effects, changes in acidity become more marked. Consider hydroxy- and methoxy-benzoic acid derivatives. The pKa values are found to be 3.0, 4.1, and 4.6 for the ortho, meta, and para hydroxy derivatives respectively, and 4.1, 4.1, and 4.5 respectively for the corresponding methoxy derivatives.

Let us ignore the figure for ortho-hydroxybenzoic acid for the moment since there is yet another feature affecting acidity. We then see that the para derivatives are rather less acidic than we might predict merely from the inductive effect of the OH or OMe groups.

PKa values show that these compounds are less acidic than benzoic acid, whereas the inductive effect would suggest they should be more acidic. This is because of a large resonance effect emanating from the substituent in which electronic charge is transmitted through the conjugated system of the aromatic ring into the carboxyl group.

The electron-donating effect originates from the lone pair electrons on oxygen, with overlap into the π electron system. This electron donation will stabilize the non-ionized acid via electron delocalization but would destabilize the conjugate base by creating a double charge in the carboxylate system.

The net result is lower acidity. This electron-donating effect from lone pair electrons is simply a resonance effect but is often termed a mesomeric effect. A mesomer is another term for a resonance structure. We shall use the ‘resonance effect’ rather than the ‘mesomeric effect’ to avoid having alternative terminologies.

We can write a similar delocalization picture for the ortho-substituted compounds, but this is countered by the opposing inductive effect close to the carboxyl.

However, the steric effect, as described above, means large groups in the ortho position can force the carboxyl group out of the plane of the ring. This weakens the resonance effect since delocalization is dependent upon coplanarity in the conjugate system.

Resonance stabilization is not as important for the meta derivatives, where it is only possible to donate electrons towards the ring carbons, which are, of course, not as electronegative as oxygen.

Meta-substitution is the least complicated, in that groups placed there exert their influence almost entirely through inductive effects. It should be noted that, where we have opposing resonance and inductive effects, the resonance effect is normally of much greater magnitude than the inductive effect and its contribution predominates (but see below for chlorine).

The relatively high acidity of ortho-hydroxybenzoic acid (salicylic acid), compared with the other derivatives just considered, is ascribed to intramolecular hydrogen bonding, which is not possible in the other compounds, even with orthomethoxybenzoic acid.

Hydrogen bonding involves a favourable six-membered ring and helps to stabilize the conjugate base. Although some hydrogen bonding occurs in the non-ionized acid, the effect is much stronger in the carboxylate anion.

It should be noted that the electron-donating resonance effects just considered are the result of lone pair electrons feeding into the π electron system.

Potentially, any substituent with a lone pair might do the same, yet we did not invoke such a mechanism with chlorine substituents above. As the size of the atom increases, lone pair electrons will be located in orbitals of higher level, e.g. 3p rather than 2p as in carbon.

Consequently, the ability to overlap the lone pair orbital with the π electron system of the aromatic ring will diminish, a simple consequence of how far from the atom the electrons are mostly located. Chlorine thus produces a low resonance effect but a high inductive effect, and the latter predominates.

Resonance can also influence the acidity of hydroxyl groups, as seen in phenols. Cyclohexanol has pKa 16, comparable to that of ethanol. On the other hand, phenol has pKa 10, making it considerably more acidic than a simple alcohol, though less so than a carboxylic acid.

This increased acidity is explained in terms of delocalization of the negative charge into the aromatic ring system, with resonance structures allowing ring carbons ortho and para to the original phenol group to become electron rich.

Although the aromatic ring acts as an acceptor of electrons and may be termed an electron sink, the charge is dispersed towards carbon atoms, which is going to be less favourable than if it can be dispersed towards more electronegative atoms such as oxygen.

A good illustration of this concept is seen in a series of nitrophenols. The nitro group itself has to be drawn with charge separation to accommodate the electrons and our rules of bonding. However, resonance structures suggest that there is electron delocalization within the nitro group.

With substituted phenols, there can be similar delocalization of charge into the aromatic ring as with phenol, but substituents will introduce their effects, be they inductive or resonance-related.

It can be seen that the nitro group allows further delocalization of the negative charge of the phenoxide conjugate base if it is situated in the ortho or para positions. This increases acidity relative to phenol, and both compounds have essentially the same pKa of 7.2

The effect is magnified considerably if there are nitro groups both ortho and para, so that the pKa for 2,4-dinitrophenol is 4.1. A third nitro group, as in 2,4,6-trinitrophenol, confers even more acidity, and this compound has pKa 0.4, making it a strong acid.

This is reflected in its common name, picric acid. Note that m-nitrophenol has pKa 8.4, and is a lot less acidic than o-nitrophenol or p-nitrophenol. We can draw no additional resonance structures here, and the nitro group cannot participate in further electron delocalization.

The increased acidity compared with phenol can be ascribed to the stabilization of resonance structures with the charge on a ring carbon through the nitro group’s inductive effect.

From the above, it should not be difficult to rationalize the effects of other types of substituents on the acidity of phenols. Thus electron-donating groups, e.g. alkyl, reduce acidity, and electron-withdrawing groups, for example, halogens, increase acidity. With strongly electron-withdrawing groups, such as cyano and nitro, the acid-strengthening properties can be quite pronounced.

A summary list of resonance effects emanating from various groups We should also point out that these very same principles will be used to rationalize aromatic substitution. and this is why we have purposely discussed the acidity of aromatic derivatives in some detail.

Basicity

We have already defined a base as a substance that will accept a proton by donating a pair of electrons. Just as we have used pKa to measure the strength of an acid, we need a system to measure the strength of a base. Accordingly, a basicity scale based on pKb was developed in a similar way to pKa. For the ionization of the base B in water.

⇒ \(\mathrm{B}+\mathrm{H}_2 \mathrm{O} \stackrel{K}{\rightleftharpoons} \mathrm{BH}^{\oplus}+\mathrm{HO}^{\ominus}\) the equilibrium constant K is given by the formula.

⇒ \(K=\frac{\left[\mathrm{HO}^{-}\right]\left[\mathrm{BH}^{+}\right]}{[\mathrm{B}]\left[\mathrm{H}_2 \mathrm{O}\right]}\) and since the concentration of water will be essentially constant, the equilibrium constant Kb and the logarithmic pKb may be defined as \(K_{\mathrm{b}}=\frac{\left[\mathrm{HO}^{-}\right]\left[\mathrm{BH}^{+}\right]}{[\mathrm{B}]}\) with \(\mathrm{p} K_{\mathrm{b}}=-\log _{10} K_{\mathrm{b}}\)

This system has been almost completely dropped in favour of using pKa throughout the acidity–basicity scale. To measure the strength of a base, we use the pKa of its conjugate acid, i.e. we consider the equilibrium.

For Which \(K_{\mathrm{a}}=\frac{[\mathrm{B}]\left[\mathrm{H}_3 \mathrm{O}^{+}\right]}{\left[\mathrm{BH}^{+}\right]}\)

It follows that A strong base has a small Ka and thus a large pKa, i.e. BH⁺ is favoured over B; A weak base has a large Ka and thus a small pKa, i.e. B is favoured over BH⁺.

Or, put another way:

The larger the value of p Ka, the stronger the base;

The smaller the value of p Ka, the weaker the base. The relationship between pKa and pKb can be deduced as follows:

⇒ \(\begin{aligned}
K_{\mathrm{b}} & =\frac{\left[\mathrm{HO}^{-}\right]\left[\mathrm{BH}^{+}\right]}{[\mathrm{B}]} \\
K_{\mathrm{a}} & =\frac{[\mathrm{B}]\left[\mathrm{H}_3 \mathrm{O}^{+}\right]}{\left[\mathrm{BH}^{+}\right]} \\
K_{\mathrm{a}} \times K_{\mathrm{b}} & =\frac{[\mathrm{B}]\left[\mathrm{H}_3 \mathrm{O}^{+}\right]}{\left[\mathrm{BH}^{+}\right]} \times \frac{\left[\mathrm{HO}^{-}\right]\left[\mathrm{BH}^{+}\right]}{[\mathrm{B}]} \\
& =\left[\mathrm{H}_3 \mathrm{O}^{+}\right]\left[\mathrm{HO}^{-}\right]
\end{aligned}\)

Thus, Ka × Kb reduces to the ionization constant for water Kw.

In this reaction, one molecule of water acts as a base and accepts a proton from a second water molecule. This second water molecule, therefore, acts as an acid and donates a proton.

The equilibrium constant K for this reaction is given by the formula and because the concentration of water is essentially constant in an aqueous solution, the new equilibrium constant K w is defined as

⇒ \(K=\frac{\left[\mathrm{H}_3 \mathrm{O}^{+}\right]\left[\mathrm{HO}^{-}\right]}{\left[\mathrm{H}_2 \mathrm{O}\right]\left[\mathrm{H}_2 \mathrm{O}\right]}\)

For every hydronium ion produced, a hydroxide anion must also be formed, so that the concentrations of hydronium and hydroxide ions must be equal. In pure water at 25 ◦C, this value is found to be 10−7 M.

⇒ \(K_{\mathrm{w}}=\left[\mathrm{HO}^{-}\right]\left[\mathrm{H}_3 \mathrm{O}^{+}\right]=10^{-7} \times 10^{-7}=10^{-14}\)

We now have the relationship that Ka × Kb = 10−14, or \(\mathrm{p} K_{\mathrm{a}}+\mathrm{p} K_{\mathrm{b}}=14\)

Electronic And Structural Features That Influence Basicity

Basicity relates to the ability of a compound to use its nonbonding electrons to combine with a proton.

We have already seen that features such as inductive or delocalization effects can make an acid stronger. They increase the stability of the conjugate base and consequently favour the loss of a proton from an acid.

It follows that features that stabilize a conjugate base are going to discourage its protonation, i.e. they are going to make it a weaker base. Thus, a compound in which the electrons are delocalized will be less basic than one in which the electrons are localized.

For example, carboxylate anions (delocalized charge) are going to be weaker bases than alkoxide ions (localized charge).

Anionic (charged) bases are naturally going to be more ready to donate electrons to a positively charged proton than a neutral base (uncharged) that uses lone pair electrons.

Most of our organic bases are not anionic, so we need to look at features that affect basicity, just as we have done for acids. Nitrogen compounds are good examples of organic bases and the ones we shall meet most frequently, though oxygen systems will feature prominently in our mechanistic rationalizations.

Electronegativity

The acidity of an acid HX increases as X becomes more electronegative. Conversely, basicity will decrease as an atom becomes more electronegative. Ammonia is a stronger base than water.

These figures relate to the release of a proton from the conjugate acid, namely ammonium ion and hydronium ion respectively.

This is sometimes confusing; we talk about the pKa of a base when we mean the pKa of its conjugate acid. We cannot avoid this, because it becomes too complicated to use the name of the conjugate acid, but we shall endeavour to show the conjugate acid in structures.

Oxygen is more electronegative than nitrogen, so its electrons are less likely to be donated to a proton. Neutral oxygen bases are generally very much weaker than nitrogen bases, but as we shall see later, the protonation of an oxygen atom is important and the first step in many acid-catalysed reactions, especially carbonyl compounds.

Inductive effects

Electron-donating groups on nitrogen are going to increase the likelihood of protonation and help to stabilize the conjugate acid. They thus increase the basic strength.

The pKa values for the amines ammonia, methylamine, dimethylamine, and trimethylamine are 9.2, 10.6, 10.7, and 9.8 respectively.

The electron-donating effect of the methyl substituents increases the basic strength of methylamine over ammonia by about 1.4 pKa units, i.e. by a factor of over 25 (101.4 = 25.1).

However, the introduction of a second methyl substituent has a relatively small effect, and the introduction of a third methyl group, as in trimethylamine, actually reduces the basic strength to nearer that of methylamine.

This apparent anomaly is a consequence of measuring pKa values in an aqueous solution, where there is more than ample opportunity for hydrogen bonding with water molecules.

Hydrogen bonding helps to stabilize a positive charge on nitrogen, and this effect will decrease as the number of alkyl groups increases.

Therefore, the observed pKa values are a combination of increased basicity with increasing alkyl groups (as predicted via electron-donating effects) countered by a stabilization of the cation through hydrogen bonding, which decreases with increasing alkyl groups. Note that we saw solvent molecules influencing the acidity of alcohols by stabilizing the conjugate base.

When pKa values are measured in the gas phase, where there are no hydrogen bonding effects, they are found to follow the predictions based solely on electron-donating effects.

In water, mono-, di-, and tri-alkylated amines all tend to have rather similar pKa values, typically in the range 10–11.

Electron-withdrawing groups will have the opposite effect. They will decrease electron density on the nitrogen, destabilize the conjugate acid, and thus make it less likely to pick up a proton, so producing a weaker base.

Inductive effects from various groups. For example, groups with a strong electron-withdrawing inductive effect, such as trichloromethyl, decrease basicity significantly.

We have already seen that water is a much weaker base than ammonia because oxygen is more electronegative than nitrogen and its electrons are thus less likely to be donated to a proton. Neutral oxygen bases are also generally very much weaker than nitrogen bases.

Nevertheless, the protonation of an oxygen atom is a critical first step in many acid-catalysed reactions.

Oxygen is so electronegative that inductive effects from substituents have rather less influence on basicity than they would in similar nitrogen compounds. Alcohols are somewhat less basic than water, with ethers weaker still.

This is precisely opposite to what would be expected from the inductive effects of alkyl groups, and the observations are likely to be the result primarily of solvation (hydrogen bonding) effects. Note, the cations shown all have negative pKa values. In other words, they are very strong acids and will lose a proton readily. Conversely, the non-protonated compounds are weak bases.

Hybridization effects

We have seen above that acidity is influenced by the hybridization of the atom to which the acidic hydrogen is attached. The acidity of a C–H bond was found to increase as the s character of the bond increased. The more characters in the orbital, the closer the electrons are held to the nucleus.

Similar reasoning may be applied to basicity. If the lone pair is in a sp² or sp orbital, it is held closer to the nucleus and is more difficult to protonate than if it is in a sp³  orbital.

Accordingly, we find that nitrile nitrogen (lone pair in an sp orbital) is not at all basic (pKa about −10), though ethylamine (lone pair in an sp3 orbital) has pKa 10.7. Imines (lone pair in an sp2 orbital) are less basic than amines. Cyclohexanimine (the imine of cyclohexanone) has pKa 9.2 and is less basic than cyclohexylamine (pKa 10.6)

Similarly, alcohols (sp³ hybridization), although they are themselves rather weak bases, are going to be more basic than aldehydes and ketones (sp² hybridization)

Resonance/delocalize zati on effects

Delocalization of charge in the conjugate base anion contributes to the stabilization of the anion, and thus ionization of the acid is enhanced. Delocalization effects in bases are more likely to stabilize the base rather than the conjugate acid and thus tend to reduce the basicity.

For a summary of various groups that may contribute resonance effects. Pre-eminent among examples is the case of amides, which do not show the typical basicity of amines.

Acetamide, for example, has pKa − 1.4, compared with pKa 10.7 in the case of ethylamine. This reluctance to protonate on nitrogen is caused by delocalization in the neutral amide, in which the nitrogen lone pair can overlap into the π system.

This type of resonance stabilization would not be possible with nitrogen protonated since the lone pair is already involved in the protonation process. Indeed, if amides do act as bases, then protonation occurs on oxygen, not on nitrogen.

Resonance stabilization is still possible in the O-protonated amide, whereas it is not possible in the N-protonated amide. Note that resonance stabilization makes the O-protonated amide somewhat less acidic than the hydronium ion (pKa − 1.7); the amide oxygen is more basic than water.

The carbonyl oxygen of aldehydes and ketones is less basic than that of an alcohol by several powers of 10. We have just seen above that this arises because the lone pair electrons of the carbonyl oxygen are in orbitals that are approximately sp² in character and are more tightly held than the alcohol lone pairs in sp³ orbitals.

The neutral carbonyl group is thus favoured, and the conjugate acid is correspondingly more acidic. On the other hand, protonated carboxylic acids and esters are shown with the proton on the carbonyl oxygen, despite this oxygen having sp² hybridization, whereas the alternative oxygen has sp³ hybridization.

This is a consequence of delocalization, with resonance stabilization being possible when the carbonyl oxygen is protonated, but not possible should the OR oxygen become protonated.

This additional resonance stabilization is not pertinent to aldehydes and ketones, which are thus less basic than the carboxylic acid derivatives. However, these oxygen derivatives are still very weak bases and are only protonated in the presence of strong acids.

In the case of the sulfur analogues thioesters and thioacids, this delocalization is much less favourable.

In the oxygen series, delocalization involves an overlap between the oxygen sp3 orbital and the π system of the carbonyl, which is composed of 2p orbitals.

Delocalization in the sulfur series would require overlap between a sulfur 3p orbital and a carbon 2p orbital, which is much less likely because of the size difference between these orbitals resonance of this type is less favourable in the sulfur esters and acids due to the larger S atom, and less orbital overlap Amidines are stronger bases than amines. The pKa for acetamidine is 12.4.

Amidines are essentially amides where the carbonyl oxygen has been replaced with nitrogen, i.e. they are nitrogen analogues of amides. It is the nitrogen replacing the oxygen that becomes protonated. This is easily rationalized, even though the hybridization here is sp², which in theory should be less basic than the sp³-hybridized nitrogen.

Protonation of the imine nitrogen allows resonance stabilization in the cation, which could not happen if the amide nitrogen were protonated.

In addition, the two resonance structures both have a charge on nitrogen and in fact, are identical. We have a similar situation in the carboxylate anion. Amidines, therefore, are quite strong bases, with the potential for electron delocalization being a greater consideration than the hybridization state of the orbital housing the lone pair.

Now let us look at guanidines, which are even stronger bases. Guanidine itself has pKa 13.6. It can be seen that there is delocalization of charge in the conjugate acid, such that in each resonance structure the charge is favourably associated with one of the three nitrogen atoms. No such favourable delocalization is possible in the neutral molecule, so guanidines are readily protonated and, therefore, are strong bases

Should you need further convincing that resonance stabilization is an important criterion in acidity and basicity, it is instructive to consider the bond lengths in the carboxylate anion and the guanidinium and guanidinium cations. Now we would expect double bonds to be shorter than single bonds, and this is true in the corresponding non-ionized systems.

However, bond length measurements for the carboxylate anion tell us that the bonds in question (C–O/C=O) are the same length, being somewhere between the expected single and double bond lengths.

The same is true of the C–N/C=N bonds in guanidinium and guanidinium cations. This fits in nicely with the concept that the actual ion is not a mixture of the various resonance forms that we can draw, but something in between.

Compare this with the fact that the C–C bond lengths in benzene are somewhere between single and double bonds, and thus do not correspond to either of the Kekule resonance structures.´ If we look at the pKa values for the conjugate acids of cyclohexylamine and the aromatic amine aniline, we see that aniline is the weaker base.

Cyclohexylamine has pKa 10.6, whereas aniline has pKa There is an inductive effect in aniline because the phenyl ring is electron withdrawing.

The carbon atoms of the aromatic ring are sp² hybridized, and more electronegative than sp3-hybridized carbons of alkyl groups. We might, therefore, expect some reduction in basicity.

However, a more prominent effect arises from resonance, which can occur in the uncharged amine, but not in the protonated conjugate acid. This makes the unprotonated amine favourable, and aniline is consequently a very weak base.

This effect is increased if there is a suitable electron-withdrawing group in the ortho or para position on the aromatic ring. Thus, p-nitroaniline and o-nitroaniline have pKa 1.0 and −0.3 respectively.

These aromatic amines are thus even weaker bases than aniline, a result of improved delocalization in the free base. The increased basicity of the ortho isomer is a result of the very close inductive effect of the nitro group; the meta isomer has only the inductive effect, and its pKa is about 2.5.

Of course, those groups that can act as electron-donating groups through resonance will produce the opposite effect, and increase the basicity. Through resonance, groups such as hydroxyl and methoxyl can distribute a negative charge towards the amino substituent, facilitating its protonation.

The pKa values for o-methoxyaniline and p-methoxyaniline are about 4.5 and 5.4 respectively, and that for mmethoxyaniline is about 4.2.

The electron-donating resonance effect is countered by the electron-withdrawing inductive effects of these electronegative substituents so that predictions about basicity become a little more complex.

As we pointed out after our considerations of acidity in aromatic derivatives, we wish to emphasize that the very same principles will be used when we consider aromatic substitution reactions. The methods used to understand the basicity of aromatic derivatives will be applied again in a different format.

A word of warning is now needed! Some compounds may have pKa values according to whether they are acting as acids or as bases.

For example, CH₃OH has pKa 15.5 and −2.2; the first figure refers to methanol acting as an acid via the loss of a proton and giving CH₃O−, and the second value refers to methanol acting as a base, i.e. the conjugate acid losing a proton.

Similarly, CH₃NH₂ has pKa values of 35 and 10.6, again referring to acid and base behaviour. It is important to avoid confusion in such cases, and this requires an appreciation of typical pKa values for simple acids and bases.

There is no way we would encourage memorizing pKa values, but two easily remembered figures can be valuable for comparisons. These are pKa around 5 for a typical aliphatic carboxylic acid, and pKa around 10 for a typical aliphatic amine.

These then allow us to consider whether the compound in question is more acidic, more basic, etc. It then becomes fairly easy to decide that methanol is not a strong acid, like nitric acid say, so that the pKa − 2.2 is unlikely to refer to its acid properties.

Methylamine ought to be basic rather like ammonia, so the pKa value of 35 would appear well out of the normal range for bases and must refer to its acidic properties. In such cases, there appear to be very good reasons for continuing to use pKb values for bases; unfortunately, however, this is not now the convention.

Basicity Of Nitrogen Heterocycles

Our discussions of the basicity of organic nitrogen compounds have concentrated predominantly on simple amines in which the nitrogen atom under consideration is part of an acyclic molecule.

Many biologically important compounds, and especially drug molecules, are based upon systems in which nitrogen is part of a heterocycle. We shall consider the properties of heterocyclic compounds; here, we mainly want to show how our rationalizations of basicity can be extended to a few commonly encountered nitrogen heterocycles.

The basicities of the simple heterocycles piperidine and pyrrolidine vary little from that of a secondary amine such as dimethylamine. pKa values for the conjugate bases of these three compounds are 11.1, 11.3, and 10.7 respectively.

However, pyridine and pyrrole are significantly less basic than either of their saturated analogues. The pyridinium cation has pKa 5.2, making pyridine a much weaker base than piperidine, whereas the pyrrolium cation (pKa − 3.8) can be considered a very strong acid, and thus pyrrole is not at all basic.

Although the nitrogen atom in these systems carries a lone pair of electrons, these electrons are not able to accept a proton in the same way as a simple amine. The dramatic differences in basicity are a consequence of the π electron systems, to which the nitrogen contributes.

Pyridine, like benzene, is an aromatic system with six π electrons. The ring is planar, and the lone pair is held in an sp² orbital.

The increased s character of this orbital, compared with the sp³ orbital in piperidine, means that the lone pair electrons are held closer to the nitrogen and, consequently, are less available for protonation.

This hybridization effect explains the lower basicity of pyridine compared with piperidine. Pyrrole is also aromatic, but there is a significant difference, in that both of the lone pair electrons are contributing to the six- π-electron system.

As part of the delocalized π electron system, the lone pairs are consequently not available for bonding to a proton.

Protonation of the nitrogen in pyrrole is very unfavourable: it would destroy the aromaticity.

It is possible to protonate pyrrole using a strong acid; but, interestingly, protonation occurs on the α-carbon and not on the nitrogen.

Although this still destroys aromaticity, there is some favourable resonance stabilization in the conjugate acid. Let us consider just one more nitrogen heterocycle here, and that is imidazole, a component of the amino acid histidine.

The imidazolium cation has pKa 7.0, making imidazole less basic than a simple amine, but more basic than pyridine. Imidazole has two nitrogen atoms in its aromatic ring system.

One of these nitrogens contributes its lone pair to make up the aromatic sextet, but the other has a free lone pair that is available for protonation. As with pyridine, this lone pair is in an sp² orbital, but the increased basicity of imidazole compared with pyridine is a result of additional resonance in the conjugate acid.

The basicity of some other heterocyclic systems

Polyfunctional Acids And Bases

We have so far considered acids and bases with a single ionizable group, and have rationalized the measured pKa values concerning structural features in the molecule.

This additional structural feature could well have its own acidic or basic properties, and we thus expect that such a compound will be characterized by more than one pKa value.

Before we consider polyfunctional organic compounds, we should consider the inorganic acids sulfuric acid and phosphoric acid.

Sulfuric acid is termed a dibasic acid, in that it has two ionizable groups, and phosphoric acid is a tribasic acid with three ionizable hydrogens. Thus, sulfuric acid has two pKa values and phosphoric acid has three.

Both acids give rise to resonance-stabilized conjugate bases (compared to carboxylate) and are strong acids. Sulfuric acid is stronger, owing to improved resonance possibilities provided by the two S=O functions, as against just one P=O in phosphoric acid. Note particularly, though, that the pKa values for the second and third ionizations are higher than the first.

This indicates that the loss of a further proton from an ion is much less favourable than the loss of the first proton from the non-ionized acid.

Nevertheless, the sulfate dianion is sufficiently well resonance stabilized via the two S=O functions that hydrogensulfate (bisulfate) is still a fairly strong acid. We can generalize that it is going to be more difficult to lose a proton from an anion than from an uncharged molecule.

This is also true of polyfunctional acids, such as dicarboxylic acids. However, it is found that this effect diminishes as the negative centres become more separated.

Thus, pKa values for some simple aliphatic dicarboxylic acids are as shown, the loss of the first proton being represented by pKa1 and the loss of the second by pKa2.

It can be seen that the difference between the first and second pKa values diminishes as the number of methylene groups separating the carboxyls increases, i.e. it becomes easier to lose the second proton as the other functional group is located further away.

It can also be seen that since malonic acid is a stronger acid than acetic acid, then the extra carboxyl is an electron-withdrawing substituent that is stabilizing.

The conjugate base. Again, this effect diminishes rapidly as the chain length increases, as anticipated for an inductive effect. Of course, ionization of a carboxylic acid group to a carboxylate anion reverses the inductive effect, in that the carboxylate will be electron donating, and will destabilize the dianion.

This is reflected in the pKa2 values for malonic, succinic and glutaric acids all being larger than the pKa for acetic acid.

Oxalic acid appears anomalous in this respect, and this appears to be a result of the high charge density associated with the dianion and subsequent solvation effects. In the aromatic benzene dicarboxylic acid derivatives, the pattern is not dissimilar, especially since we have no oxalic acid-like anomaly.

Carboxylic acid groups are electron-withdrawing, and all three diacids are stronger acids than benzoic acid. On the other hand, the carboxylate group is electron donating, and this weakens the second ionization. This makes the second acid a weaker acid than benzoic acid. The effects are greatest in the ortho derivative, where there are also going to be steric factors.

Compounds with two basic groups, for example, diamines, can be rationalized similarly. Here, we must appreciate that both amino groups and ammonium cations are electron withdrawing, the positively charged entity having the greater effect. pKa values for a series of aliphatic diamines.

As the distance between the amino groups increases, the effect of the NH2 on the first protonation diminishes, so that pKa1 values for the 1,3- and 1,4-diamino compounds are very similar to that of ethylamine. Only in 1,2-diaminoethane do we see the electron-withdrawing effects of the second amino group decreasing basicity.

However, for the second protonation, it is clear that an ionized amino group has a much larger effect than a nonionized one.

The effects fall off as the separation increases but persist further. Thus, pKa2 values for the 1,3- and 1,4-diamino compounds are now rather different. The aromatic diamines present a much more complex picture, and we do not intend to justify the observed pKa values in detail.

There are going to be several effects here, with some that provide opposing influences. An amino group has an electron-withdrawing inductive effect but has an electron-donating resonance effect that tends to be greater in magnitude than the inductive effect.

A protonated amino group also has an electron-withdrawing inductive effect that is greater than that of an uncharged amino group. On the other hand, it no longer supplies the electron-donating resonance effect.

As with other disubstituted benzenes, the ortho compound also experiences steric effects that may reduce the benefits of resonance. Both the meta and para diamines are stronger bases than aniline, and the protonation of the first amine in all three compounds considerably inhibits the second protonation.

pH

The acidity of an aqueous solution is normally measured in terms of pH. pH is defined as pH = − log₁₀[H₃O⁺] The lower the pH, the more acidic the solution; the higher the pH, the more basic the solution. The pH scale only applies to aqueous solutions and is only a measure of the acidity of the solution.

It does not indicate how strong the acid is (that is a function of pKa) and the pH of an acid will change as we alter its concentration. For instance, dilution will decrease the H3O+ concentration, and thus the pH will increase. In water, the hydronium ion concentration arises by the self-dissociation equilibrium.

⇒ \(\mathrm{H}_2 \mathrm{O}+\mathrm{H}_2 \mathrm{O} \rightleftharpoons \mathrm{H}_3 \mathrm{O}^{\oplus}+\mathrm{HO}^{\ominus}\)

In this reaction, one molecule of water acts as a base, accepting a proton from a second water molecule. The second molecule is acting as an acid and donating a proton.

For every hydronium ion produced, a hydroxide anion must also be formed, so that the concentrations of hydronium and hydroxide ions must be equal. In pure water at 25 ◦C, this value is found to be 10−7 M.

The equilibrium constant K is given by the formula and because the concentration of water is essentially constant in an aqueous solution, the new equilibrium constant K w (the ionization constant for water) is defined as

⇒ \(K=\frac{\left[\mathrm{H}_3 \mathrm{O}^{+}\right]\left[\mathrm{HO}^{-}\right]}{\left[\mathrm{H}_2 \mathrm{O}\right]\left[\mathrm{H}_2 \mathrm{O}\right]}\)

K w = [HO−][H3O+] = 10−7 × 10−7 = 10−14

This means that the pH of pure water at 25 ◦C is therefore

pH = − log 10−7 = 7

pH 7 is regarded as neither acidic, nor basic, but neutral. It follows that acids have a pH less than 7 and bases have a pH greater than 7.

Kw and pH of neutrality at different temperatures We rapidly become accustomed to the idea that the pH of water is 7.0 and that this represents the pH of neutrality.

Unfortunately, this is only true at 25 C; at other temperatures, the amount of ionization varies, so that Kw will consequently be different. We find that the amount of ionization increases with temperature and the pH of neutrality decreases accordingly.

Kw and pH of neutrality at different temperatures

Calculation of pH: strong acids and bases A strong acid is considered to be completely ionized in water so that the hydronium ion concentration is the same as its molarity.

Thus, a 0.1 M solution of HCl in water has [H₃O+] = 0.1, and pH = − log 0.1 = 1. Similarly, a 0.01 M solution has [H₃O+] = 0.01 and pH = − log 0.01 = 2, and a 0.001 M solution has [H₃O+] = 0.001 and pH = − log 0.001 = 3. It follows from this that, because we are using a logarithmic scale, a pH difference of 1 corresponds to a factor of 10 in hydronium ion concentration.

If the pH is known, then we can calculate the hydronium ion concentration. Since pH = − log[H₃O+] the hydronium ion concentration is given by [H₃O+] = 10−pH For example, if the pH = 4, [H₃O+] = 10−4 = 0.0001 M.

When we have a strong base, our calculations need to invoke the ionization constant for water K w = [H3O+][HO−] = 10−14 Thus, the pH of a 0.1 M solution of NaOH in water is calculated from [HO−] = 0.1, and since [H₃O+][HO−] = 10−14, [H₃O+] must be 10−13.

Hence, the pH of a 0.1 M solution of NaOH in water will be − log 10−13 = 13. A 0.01 M solution of NaOH will have [HO−] = 10−2 and pH = − log 10−12 = 12, and a 0.001 M solution has [HO−] = 10−3 and pH = − log 10−11 = 11.

Weak acids are not completely ionized in aqueous solutions, and the amount of ionization, and thus hydronium ion concentration, is governed by the equilibrium.

⇒ \(\mathrm{HA}+\mathrm{H}_2 \mathrm{O} \rightleftharpoons \mathrm{H}_3 \mathrm{O}^{\oplus}+\mathrm{A}^{\ominus}\) and the equilibrium constant Ka we defined above

⇒ \(K_{\mathrm{a}}=\frac{\left[\mathrm{A}^{-}\right]\left[\mathrm{H}_3 \mathrm{O}^{+}\right]}{[\mathrm{HA}]}\)

However, since [H3O+] must be the same as [A−], we can write

⇒ \(K_{\mathrm{a}}=\frac{\left[\mathrm{H}_3 \mathrm{O}^{+}\right]^2}{[\mathrm{HA}]}\) and therefore

⇒ \(\left[\mathrm{H}_3 \mathrm{O}^{+}\right]=\sqrt{K_{\mathrm{a}}[\mathrm{HA}]}\)

If we take negative logarithms of both sides, we get \(-\log \left[\mathrm{H}_3 \mathrm{O}^{+}\right]=-\frac{1}{2} \log K_{\mathrm{a}}-\frac{1}{2} \log [\mathrm{HA}]\) which becomes

⇒ \(\mathrm{pH}=\frac{1}{2} \mathrm{p} K_{\mathrm{a}}-\frac{1}{2} \log [\mathrm{HA}]\)

Note: This is simply a variant of the Henderson–Hasselbalch equation below, when [A−] = [H₃O⁺]. The calculation of the pH of a weak base may be approached in the same way. The equilibrium we need to consider is \(\mathrm{B}+\mathrm{H}_2 \mathrm{O} \rightleftharpoons \mathrm{BH}^{\oplus}+\mathrm{HO}^{\ominus}\) and the equilibrium constant Kb will be defined as

⇒ \(K_{\mathrm{b}}=\frac{\left[\mathrm{HO}^{-}\right]\left[\mathrm{BH}^{+}\right]}{[\mathrm{B}]}\)

However, since [HO−] must be the same as [BH⁺], we can write

⇒ \(K_{\mathrm{b}}=\frac{\left[\mathrm{HO}^{-}\right]^2}{[\mathrm{~B}]}\) and therefore \(\left[\mathrm{HO}^{-}\right]=\sqrt{K_{\mathrm{b}}[\mathrm{B}]}\)

Now we need to remember that K w = [HO−][H₃O⁺] so that we can replace [HO−] with Kw/[H₃O⁺]; this leads to

⇒ \(\frac{K_{\mathrm{w}}}{\left[\mathrm{H}_3 \mathrm{O}^{+}\right]}=\sqrt{K_{\mathrm{b}}[\mathrm{B}]}\) so that we can replace [HO−] with Kw/[H3O+]; this leads to \(\frac{K_{\mathrm{w}}}{\left[\mathrm{H}_3 \mathrm{O}^{+}\right]}=\sqrt{K_{\mathrm{b}}[\mathrm{B}]}\) and hence \(\left[\mathrm{H}_3 \mathrm{O}^{+}\right]=\frac{K_{\mathrm{w}}}{\sqrt{K_{\mathrm{b}}[\mathrm{B}]}}\)

If we now take negative logarithms of both sides, we get \(-\log \left[\mathrm{H}_3 \mathrm{O}^{+}\right]=-\log K_{\mathrm{w}}+\frac{1}{2} \log K_{\mathrm{b}}+\frac{1}{2} \log [\mathrm{B}]\) Which becomes \(\mathrm{pH}=\mathrm{p} K_{\mathrm{w}}-\frac{1}{2} \mathrm{p} K_{\mathrm{b}}+\frac{1}{2} \log [\mathrm{B}]\)

Calculation of pH: weak acids and bases Consider a 0.1 M solution of the weak acid acetic acid (Ka = 1.76 × 10−5; pKa = 4.75). Since the degree of ionization is small, the concentration of undissociated acid may be considered to be approximately the same as the original concentration, i.e. 0.1. The pH can be calculated using the equation \(\mathrm{pH}=\frac{1}{2} \mathrm{p} K_{\mathrm{a}}-\frac{1}{2} \log [\mathrm{HA}]\)

Thus

pH = 2.38 − 0.5 × log 0.1

= 2.38 − (−0.5)

= 2.88

The calculation of the pH of a weak base can be achieved similarly; but again, since we have a base, our calculations need to invoke the ionization constant for water hus

pH = 2.38 − 0.5 × log 0.1

= 2.38 − (−0.5)

= 2.88

The calculation of the pH of a weak base can be achieved similarly; but again, since we have a base, our calculations need to invoke the ionization constant for water Kw = [H3O+][HO−] = 10−14 and pKa + pKb = 14. Thus, for a 0.1 M solution of ammonia (conjugate acid pKa = 9.24

⇒ \(\mathrm{pH}=\mathrm{p} K_{\mathrm{w}}-\frac{1}{2} \mathrm{p} K_{\mathrm{b}}+\frac{1}{2} \log [\mathrm{B}]\)

and pKb is thus 14 − 9.24 = 4.76. This leads to

pH = 14 − 2.38 + 0.5 × log 0.1

= 14 − 2.38 + 0.5(−1)

= 11.12

Alternatively, we could use

⇒ \(\mathrm{pH}=\frac{1}{2} \mathrm{p} K_{\mathrm{w}}+\frac{1}{2} \mathrm{p} K_{\mathrm{a}}+\frac{1}{2} \log [\mathrm{B}]\) To get the same result:

pH = 7 + 4.62 + 0.5 × log 0.1 = 11.12 These calculations are for the pH of weak acids and weak bases. It is well worth comparing the figures we calculated above for strong acids and bases. Thus, a 0.1 M solution of the strong acid HCl had pH 1, and a 0.1 M solution of the strong base NaOH had pH 13.

Although this produces a similar type of expression to that for the pH of a weak acid above, it does employ pKb rather than pKa. To keep to a ‘pKa only’ concept, we need to incorporate the pKa + pKb = pKw expression. Then we get the alternative formula.

⇒ \(\begin{gathered}
\mathrm{pH}=\mathrm{p} K_{\mathrm{w}}-\frac{1}{2}\left(\mathrm{p} K_{\mathrm{w}}-\mathrm{p} K_{\mathrm{a}}\right)+\frac{1}{2} \log [\mathrm{B}] \\
\mathrm{pH}=\frac{1}{2} \mathrm{p} K_{\mathrm{w}}+\frac{1}{2} \mathrm{p} K_{\mathrm{a}}+\frac{1}{2} \log [\mathrm{B}]
\end{gathered}\)

The pH of salt solutions It should be self-evident that solutions comprised of equimolar amounts of a strong acid, for example, HCl, and a strong base, for example, NaOH, will be neutral, i.e. pH 7.0 at 25 ◦C.

We can thus deduce that a solution of the salt NaCl in water will also have a pH of 7.0. However, salts of a weak acid and strong base or a strong acid and weak base dissolved in water will be alkaline or acidic respectively.

Thus, aqueous sodium acetate is basic, whereas aqueous ammonium chloride is acidic. pH values may be calculated from pKa as follows. Consider the ionization of sodium acetate in water; this leads to an equilibrium in which AcO− acts as base \(\Theta_{\mathrm{OAc}}+\mathrm{H}_2 \mathrm{O} \rightleftharpoons \mathrm{HOAc}+\mathrm{HO}^{\ominus}\)

⇒ \(\begin{aligned}
\mathrm{pH} & =\frac{1}{2} \mathrm{p} K_{\mathrm{w}}+\frac{1}{2} \mathrm{p} K_{\mathrm{a}}+\frac{1}{2} \log [\mathrm{B}] \\
& =7+2.38+0.5 \times \log 0.1 \\
& =7+2.38+0.5 \times(-1) \\
& =8.88
\end{aligned}\)

If we now consider a 0.1 M solution of ammonium chloride in water, where pKa for the conjugate acid NH4+ is 9.24, we have the equilibrium.

⇒ \(\mathrm{NH}_4^{\oplus}+\mathrm{H}_2 \mathrm{O} \rightleftharpoons \mathrm{NH}_3+\mathrm{H}_3 \mathrm{O}^{\oplus}\) in which the ammonium ion is acting as an acid. For the ionization of a weak acid, we calculated above that the pH is given by the equation. \(\mathrm{pH}=\frac{1}{2} \mathrm{p} K_{\mathrm{a}}-\frac{1}{2} \log [\mathrm{HA}]\)

Thus \(\begin{aligned}
\mathrm{pH} & =4.62-0.5 \times \log 0.1 \\
& =4.62-0.5(-1) \\
& =5.12
\end{aligned}\).

In both cases, we are assuming that the concentration of the ion (either AcO− or NH4+) is not significantly altered by the equilibrium and can, therefore, be considered to be equivalent to the molar concentration.

The Henderson–Hasselbalch equation

Ka for the ionization of an acid HA has been defined as

⇒ \(K_{\mathrm{a}}=\frac{\left[\mathrm{A}^{-}\right]\left[\mathrm{H}_3 \mathrm{O}^{+}\right]}{[\mathrm{HA}]}\) and this can be rearranged to give \(\left[\mathrm{H}_3 \mathrm{O}^{+}\right]=K_{\mathrm{a}} \times \frac{[\mathrm{HA}]}{\left[\mathrm{A}^{-}\right]}\)

Taking negative logarithms of each side, this becomes \(-\log \left[\mathrm{H}_3 \mathrm{O}^{+}\right]=-\log K_{\mathrm{a}}+\log \frac{\left[\mathrm{A}^{-}\right]}{[\mathrm{HA}]}\)

or \(\mathrm{pH}=\mathrm{p} K_{\mathrm{a}}+\log \frac{\left[\mathrm{A}^{-}\right]}{[\mathrm{HA}]}\)

This is referred to as the Henderson–Hasselbalch equation, and it is sometimes written as

⇒ \(\mathrm{pH}=\mathrm{p} K_{\mathrm{a}}+\log \frac{[\text { base }]}{\text { [acid] }}\)

Using this relationship, it is possible to determine the degree of ionization of an acid at a given pH. An immediate outcome from this expression is that the pKa of an acid is the pH at which it is exactly half dissociated. This follows from \(\mathrm{pH}=\mathrm{p} K_{\mathrm{a}}+\log \frac{\left[\mathrm{A}^{-}\right]}{[\mathrm{HA}]}\)

But when the concentrations of acid HA and conjugate base A− are equal, then

⇒ \(\log \frac{\left[\mathrm{A}^{-}\right]}{[\mathrm{HA}]}=\log 1=0\)

So that pH = pKa

This means we can determine the pKa of an acid by measuring the pH at the point where the acid is half neutralized. As we increase the pH, the acid becomes more ionized; as we lower the pH, the acid becomes less ionized. For a base, Ka is defined as

⇒ \(K_{\mathrm{a}}=\frac{[\mathrm{B}]\left[\mathrm{H}_3 \mathrm{O}^{+}\right]}{\left[\mathrm{BH}^{+}\right]}\)

which can be rearranged to give \(\left[\mathrm{H}_3 \mathrm{O}^{+}\right]=K_{\mathrm{a}} \times \frac{\left[\mathrm{BH}^{+}\right]}{[\mathrm{B}]}\)

so that the Henderson–Hasselbalch equation is written \(\mathrm{pH}=\mathrm{p} K_{\mathrm{a}}+\log \frac{[\mathrm{B}]}{\left[\mathrm{BH}^{+}\right]}\) \(\mathrm{pH}=\mathrm{p} K_{\mathrm{a}}+\log \frac{[\mathrm{B}]}{\left[\mathrm{BH}^{+}\right]}\) or, as previously

⇒ \(\mathrm{pH}=\mathrm{p} K_{\mathrm{a}}+\log \frac{\text { [base] }}{\text { [acid] }}\)

Again, we can see that the pKa of a base is the pH at which it is half ionized. As we increase the pH, the base becomes less ionized; as we lower the pH, the base becomes more ionized.

A further useful generalization can be deduced from the Henderson–Hasselbalch equation. This relates to the ratio of ionized to non-ionized forms as the pH varies.

A shift in pH by one unit to either side of the pKa value must change the ratio of ionized to non-ionized forms by a factor of 10. Every further shift of pH by one unit changes the ratio by a further factor of 10.

Thus, for example, if the pKa of a base is 10, at pH 7 the ratio of free base to protonated base is 1:103. An acid with pKa 2 at pH 7 would produce a ratio of acid to anion of 105:1.

Calculation of percentage ionization

Using the Henderson–Hasselbalch equation, we can easily calculate the amount of ionized form of an acid or base present at a given pH, provided we know the pKa. For example, consider aqueous solutions of acetic acid (pKa 4.75) first at pH 4.0 and then at pH 6.0. Since \(\mathrm{pH}=\mathrm{p} K_{\mathrm{a}}+\log \frac{\left[\mathrm{A}^{-}\right]}{[\mathrm{HA}]}\) at pH 4.0

⇒ \(\log \frac{\left[\mathrm{A}^{-}\right]}{[\mathrm{HA}]}=\mathrm{pH}-\mathrm{p} K_{\mathrm{a}}=4.0-4.75=-0.75\)

Thus

⇒ \(\frac{\left[\mathrm{A}^{-}\right]}{[\mathrm{HA}]}=10^{-0.75}=0.18\)

If we consider [A−] = I, the fraction ionized, then [HA] is the fraction non-ionized, i.e. 1 − I, and I/(1 − I) = 0.18, from which I may be calculated to be about 0.15 or 15%. At pH 6.0, pH – pKa = 1.25, and the calculation yields I/(1 − I) = 101.25 = 17.8, so that I = 0.95, i.e. 95% ionized. With a base such as ammonia (pKa 9.24), the percentages ionized at pH 8.0 and 10.0 are calculated as follows:

⇒ \(\mathrm{pH}=\mathrm{p} K_{\mathrm{a}}+\log \frac{[\mathrm{B}]}{\left[\mathrm{BH}^{+}\right]}\)

At pH 8.0

⇒ \(\log \frac{[\mathrm{B}]}{\left[\mathrm{BH}^{+}\right]}=\mathrm{pH}-\mathrm{p} K_{\mathrm{a}}=8.0-9.24=-1.24\)

Now with bases, [B] is the non-ionized fraction 1 − I and [BH+] is the ionized fraction I, so (1 − I)/I = 0.057, and therefore I = 0.95, i.e. 95% ionized. At pH 10.0, the calculation yields (1 − I)/I = 100.76 = 5.75, so that I = 0.15, i.e. 15% ionized.

The ionization a-aminoino acids a t p H 7

Peptides and proteins are composed of α-amino acids linked by amide bonds. Their properties, for example, the ability of enzymes to catalyse biochemical reactions, are dependent upon the degree of ionization of various acidic and basic side chains at the relevant pH.

This aspect will be discussed, but, here, let us consider a simple amino acid dissolved in water at pH 7.0. An α-amino acid has an acidic carboxylic acid group and a basic amine group. Both of these entities need to be treated separately.

The carboxylic acid groups of amino acids have pKa values in a range from about 1.8 to 2.6 (see Section 13.1). Let us consider a typical carboxylic acid group with pKa 2.0. Using the Henderson–Hasselbalch equation

We can deduce that \(\log \frac{\left[\mathrm{RCO}_2^{-}\right]}{\left[\mathrm{RCO}_2 \mathrm{H}\right]}=\mathrm{pH}-\mathrm{p} K_{\mathrm{a}}=7.0-2.0=5.0\)

Thus \(\frac{\left[\mathrm{RCO}_2{ }^{-}\right]}{\left[\mathrm{RCO}_2 \mathrm{H}\right]}=10^5=10000: 1\)

Therefore, the carboxylic acid group of an amino acid can be considered to be completely ionized in solution at pH 7.0. Now let us consider the amino group in α-amino acids. The pKa values of the conjugate acids are found to range from about 8.8 to 10.8. We shall consider a typical group with pKa 10.0. From

⇒ \(\begin{gathered}
\mathrm{pH}=\mathrm{p} K_{\mathrm{a}}+\log \frac{\left[\mathrm{RNH}_2\right]}{\left[\mathrm{RNH}_3{ }^{+}\right]} \\
\log \frac{\left[\mathrm{RNH}_2\right]}{\left[\mathrm{RNH}_3{ }^{+}\right]}=\mathrm{pH}-\mathrm{p} K_{\mathrm{a}}=7.0-10.0=-3.0 \\
\frac{\left[\mathrm{RNH}_2\right]}{\left[\mathrm{RNH}_3{ }^{+}\right]}=10^{-3}=1: 1000
\end{gathered}\)

Therefore, as with the carboxylic acid group, we find that the amino group of an amino acid is effectively ionized completely, i.e. fully protonated, in solution at pH 7.0. Therefore, we can deduce that α-amino acids in solution at pH 7.0 exist as dipolar ions; these are called zwitterions (German; Twitter = hybrid)

Some amino acids have additional ionizable groups in their side chains. These may be acidic or potentially acidic (aspartic acid, glutamic acid, tyrosine, cysteine), or basic (lysine, arginine, histidine).

We use the term ‘potentially acidic’ to describe the phenol and thiol groups of tyrosine and cysteine respectively; under physiological conditions, these groups are unlikely to be ionized.

It is relatively easy to calculate the amount of ionization at a particular pH and to justify that latter statement.

Similar calculations as above for the basic side-chain groups of arginine (pKa 12.48) and lysine (pKa 10.52), and the acidic side-chains of aspartic acid (pKa 3.65) and glutamic acid (pKa 4.25) show essentially complete ionization at pH 7.0.

However, for cysteine (pKa of the thiol group 10.29) and for tyrosine (pKa of the phenol group 10.06) there will be negligible ionization at pH 7.0. For cysteine at pH 7.0, the Henderson–Hasselbalch equation leads to

⇒ \(\log \frac{\left[\mathrm{A}^{-}\right]}{[\mathrm{HA}]}=\mathrm{pH}-\mathrm{p} K_{\mathrm{u}}=7.0-10.29=-3.29\)

And \(\frac{\left[\mathrm{A}^{-}\right]}{[\mathrm{HA}]}=10^{-3.29}=5.1 \times 10^{-4}\)

Interestingly, the heterocyclic side-chain of histidine is partially ionized at pH 7.0. This follows from

⇒ \(\begin{gathered}
\log \frac{[\mathrm{B}]}{\left[\mathrm{BH}^{+}\right]}=\mathrm{pH}-\mathrm{p} K_{\mathrm{z}}=7.0-6.00=-1.0 \\
\frac{[\mathrm{B}]}{\left[\mathrm{BH}^{+}\right]}=10^{-1.0}=10
\end{gathered}\)

Which translates to approximately 9.1% ionization. We shall see that this modest level of ionization is particularly relevant in some enzymic reactions where histidine residues play an important role.

Note, however, that when histidine is bound in a protein structure, pKa values for the imidazole ring vary somewhat in the range 6–7 depending upon the protein, thus affecting the level of ionization. The ionic states at pH 7.0 of these amino acids with ionizable side chains are shown below.

Buffers

A buffer is a solution that helps to maintain a reasonably constant pH environment by countering the effects of added acids or bases.

They are used extensively for the handling of biochemicals, especially enzymes, as well as in chromatography and drug extractions. The simplest type of buffer is composed of a weak acid–strong base combination or a weak base–strong acid combination.

This may be prepared by combining the weak acid (or base) with its salt. For example, the sodium acetate–acetic acid combination is one of the most common buffer systems.

Although tabulated data are available for the preparation of buffer solutions, a sodium acetate–acetic acid buffer could be prepared simply by adding sodium hydroxide to an acetic acid solution until the required pH is obtained.

For maximum efficiency, this pH needs to be within about 1 pH unit on either side of the pKa of the weak acid or base used.

Since acetic acid is only weakly dissociated, the concentration of acetic acid will be almost the same as the amount put in the mixture.

On the other hand, the sodium acetate component will be almost completely dissociated, so the acetate ion concentration can be considered the same as that of the sodium acetate used for the solution.

The addition of an acid such as HCl to the buffer solution provides H+, which combines with the acetate ion to give acetic acid.

This has a twofold effect: it reduces the amount of acetate ion present and, by so doing, also increases the amount of undissociated acetic acid.

Provided the amount of acid added is small relative to the original concentration of base in the buffer, the alteration in base: acid ratio in the Henderson–Hasselbalch equation is relatively small and has little effect on the pH value.

Similar considerations apply if a base such as NaOH is added to the buffer solution. This will decrease the amount of undissociated acid, and increase the amount of acetate ion present. The Henderson–Hasselbalch equation may be employed in calculations relating to the properties and effects of buffer solutions.

Preparation of a buffer

One litre of 0.1 M sodium acetate buffer with a pH of 4.9 is required. The pKa of acetic acid is 4.75. From the Henderson–Hasselbalch equation \(\mathrm{pH}=\mathrm{p} K_{\mathrm{a}}+\log \frac{\left[\mathrm{A}^{-}\right]}{[\mathrm{HA}]}\)

Therefore \(4.9=4.75+\log \frac{\left[\mathrm{A}^{-}\right]}{[\mathrm{HA}]}\)

So that \(\log \frac{\left[\mathrm{A}^{-}\right]}{[\mathrm{HA}]}=0.15\) and \(\frac{\left[\mathrm{A}^{-}\right]}{[\mathrm{HA}]}=10^{0.15}=\frac{1.41}{1}\)

This means that the buffer solution requires 1.41 parts sodium acetate to 1 part acetic acid. Therefore, this can be prepared by mixing 1.41/2.41 = 0.585 l of 0.1 M sodium acetate with 1/2.41 = 0.415 l of 0.1 M acetic acid. The amount of sodium acetate in 1 l of solution will thus be 0.0585 M, and the amount of acetic acid will be 0.0415 M.

Buffering effect

If 1 ml of 1 M HCl is added to this sodium acetate buffer solution, the pH change may be calculated as follows. Again, we require the Henderson–Hasselbalch equation:

⇒ \(\mathrm{pH}=\mathrm{p} K_{\mathrm{a}}+\log \frac{\left[\mathrm{A}^{-}\right]}{[\mathrm{HA}]}\)

We are adding [H3O+] of 0.001 M, and this reacts

⇒ \(\Theta_{\mathrm{OAc}}+\mathrm{HCl} \longrightarrow \mathrm{HOAc}+\mathrm{Cl}^{\ominus}\)

so effectively reducing the amount of acetate base by 0.001 M and also increasing the amount of acetic acid by 0.001 M. We can ignore the small change in volume arising from the addition of the acid. The Henderson–Hasselbalch equation becomes

⇒ \(\mathrm{pH}=4.75+\log \frac{0.0585-0.001}{0.0415+0.001}\)

So

⇒ \(\begin{aligned}
\mathrm{pH}=4.75+\log \frac{0.0575}{0.0425} & =4.75+\log 1.35 \\
& =4.75+0.13=4.88
\end{aligned}\)

It can be seen, therefore, that the effect of the addition of the acid is to change the pH value from 4.90 to 4.88, i.e. by just 0.02 of a pH unit.

This contrasts with the effect of adding 0.001 M of HCl to 1 litre of water (pH 7). The new [H3O+] of 0.001 M gives pH = − log[H3O+] = − log 0.001 = 3, i.e. a change of four pH units. If 1 ml of 1 M NaOH was added to this buffer solution, the pH change may be calculated similarly.

We are adding [HO−] of 0.001 M, and this reacts effectively increasing the amount of acetate base by 0.001 M and also decreasing the amount of acetic acid by 0.001 M. The Henderson–Hasselbalch equation becomes

⇒ \(\mathrm{HOAc}+\mathrm{HO}^{\ominus} \longrightarrow \mathrm{H}_2 \mathrm{O}+{ }_{\mathrm{OAc}}\)

⇒ \(\mathrm{pH}=4.75+\log \frac{0.0575+0.001}{0.0415-0.001}\)

So

⇒ \(\begin{aligned}
\mathrm{pH}=4.75+\log \frac{0.0585}{0.0405} & =4.75+\log 1.44 \\
& =4.75+0.16=4.91
\end{aligned}\)

Again, the pH change is minimal, which is the whole point of a buffer solution. Note also that the pH of a buffer solution is essentially independent of dilution. An unbuffered solution of an acid or base would suffer a pH change on dilution because pH relates to hydronium ion concentration.

Dilution of a buffered solution does not affect pH because any such changes are accommodated in the log([A−]/[HA]) component and, therefore, cancel out. We have made certain approximations in deriving the equations, and at very high dilutions the pH does begin to deviate.

The sodium acetate–acetic acid combination is one of the most widely used buffers and is usually referred to simply as acetate buffer.

Other buffer combinations commonly employed in chemistry and biochemistry include carbonate–bicarbonate (sodium carbonate–sodium hydrogen carbonate), citrate (citric acid–trisodium citrate), phosphate (sodium dihydrogen phosphate–disodium hydrogen phosphate), and tris [tris(hydroxymethyl)aminomethane–HCl].

The buffering effect of blood plasma

In humans, the pH of blood is held at a remarkably constant value of 7.4 ± 0.05. In severe diabetes, the pH can drop to pH 7.0 or below, leading to death from acidotic coma.

Death may also occur at pH 7.7 or above because the blood is unable to release CO2 into the lungs.

The pH of blood is normally controlled by a buffer system, within rather narrow limits to maintain life and within even narrower limits to maintain health. The buffering system for blood is based on carbonic acid (H2CO3) and its conjugate base bicarbonate (HCO3−)

⇒ \(\mathrm{H}_2 \mathrm{CO}_3 \rightleftharpoons \mathrm{H}^{\oplus}+\mathrm{HCO}_3^{\ominus}\)

From the Henderson–Hasselbalch equation \(\mathrm{pH}=\mathrm{p} K_{\mathrm{a}}+\log \frac{\left[\mathrm{HCO}_3^{-}\right]}{\left[\mathrm{H}_2 \mathrm{CO}_3\right]}\)

we can see that maintaining the pH depends upon the ratio of bicarbonate to carbonic acid concentrations.

Large quantities of acid formed during normal metabolic processes react with bicarbonate to form carbonic acid. This, however, dissociates and rapidly loses water to form CO2 that is removed via the lungs.

⇒ \(\mathrm{H}_2 \mathrm{CO}_3 \rightleftharpoons \mathrm{H}_2 \mathrm{O}+\mathrm{CO}_2\)

The pH is maintained, therefore, in that a reduction in [HCO₃−] is countered by a corresponding decrease in [H₂CO₃]. The increase in metabolic acid is compensated by a corresponding increase in CO₂. If the pH of blood rises, [HCO₃−] temporarily increases.

The pH is rapidly restored when atmospheric CO₂ is absorbed and converted into H₂CO₃. It is a reservoir of CO₂ that enables the blood pH to be maintained so rigidly. This reservoir of CO₂ is large and can be altered quickly via the breathing rate.

Using pKa values

Predicting acid-base interactions

With knowledge of pKa values, or a rough idea of relative values, one can predict the outcome of acid-base interactions.

This may form an essential preliminary to many reactions, or provide us with an understanding of whether a compound is ionized under particular conditions, and whether or not it is in a soluble form.

As a generalization, acid-base interactions result in the formation of the weaker acid and the weaker base, a consequence of the most stable species being favoured at equilibrium. Thus, a carboxylic acid such as acetic acid will react with aqueous sodium hydroxide to form sodium acetate and water.

Consider the pKa values. Acetic acid (pKa 4.8) is a stronger acid than water (pKa 15.7), and hydroxide is a stronger base than acetate. Accordingly, hydroxide will remove a proton from acetic acid to produce acetate and water, the weaker base–weaker acid combination.

Because of the large difference in pKa values, the position of equilibrium will greatly favour the products, and we can indicate this by using a single arrow and considering the reaction to be effectively irreversible.

Even acids that are not particularly soluble in water, for example, benzoic acid, will participate in this reaction because the conjugate base benzoate is a water-soluble anion.

Bases can be considered in the same way. Thus, methylamine will react with aqueous HCl to produce methylammonium chloride and water. Hydronium is a stronger acid than methylammonium, and methylamine is a stronger base than water, so methylamine will become protonated in aqueous acid.

Again, there is a large difference in pKa values, so the position of equilibrium is well over to the righthand side. Bases that are not particularly soluble in water, e.g. aniline, can easily be made soluble by conversion to the salt form.

However, attempts to make an aqueous solution of the base sodium amide would result in the formation of sodium hydroxide and ammonia.

The amide ion is a strong base and abstracts a proton from water, a weak acid. The reverse reaction is not favoured, in that hydroxide is a weaker base than the amide ion, and ammonia is a weaker acid than water.

Take care with the terminology ‘amide’: the amide anion H2N− is quite different from the amide molecule RCONH₂.

In general, in aqueous solutions, water can donate a proton to any base stronger than the hydroxide ion. If we wish to use bases that are stronger than the hydroxide ion, then we must employ a solvent that is a weaker acid than water.

For example, hydrocarbons (pKa about 50), ethers (pKa about 50), or even liquid ammonia (pKa 38). Since these are all extremely weak acids, they will not donate a proton even to a strong base such as an amide ion. Thus, in liquid ammonia, the amide ion may be used to convert an acetylene to its acetylide ion conjugate base.

Similarly, the amide ion could be used to abstract a proton from a ketone to produce an enolate anion in an essentially irreversible reaction, since the difference in acidities of the ketone and ammonia is so marked.

However, if the base chosen were ethoxide, then enolate anion formation would be dependent on an equilibration reaction since the two acids are more comparable in acidity. As we shall see, this latter system may be used, provided the equilibrium can be disturbed in favour of the products.

Sodium ethoxide may be produced by treating ethanol with sodium hydride. Again, hydride is the strong base, the conjugate base of the weak acid hydrogen, so the reaction proceeds readily.

Sodium tert-butoxide (from sodium hydride treatment of tertbutanol) is a stronger base than sodium ethoxide since tert-butanol (pKa 19) is less acidic than ethanol (pKa 16).

Some bases and acids that are commonly used as reagents to initiate reactions are listed in Table 4.10, in decreasing order of basicity or acidity.

Isotopic labelling using basic reagents By definition, an acid will donate a proton to a base, and it is converted into its conjugate base. Conversely, a base will accept a proton from a suitable donor, generating its conjugate acid.

We can utilize these properties to label certain compounds with isotopes of hydrogen, namely deuterium (2H or D) and tritium (3H or T). To differentiate normal hydrogen (1 H) from deuterium and tritium, it is sometimes referred to as protium.

Because these isotopes are easily detectable and measurable by spectroscopic methods, labelling allows us to follow the fate of particular atoms during chemical reactions, or metabolic studies.

In general, the labelling process is one of exchange labelling, removing protium from an acid, and allowing the conjugate base to accept isotopic hydrogen from a suitable donor, most conveniently and cheaply supplied as labelled water. If the labelled compound is going to be of use, say in metabolic studies, the labelling must be achieved at a position that does not easily exchange again in an aqueous environment. This rules out hydrogens attached to oxygen or nitrogen that can be exchanged through simple acid-base equilibria.

Thus, dissolving acetic acid in deuterated water will rapidly give deuterated acetic acid by acid-base equilibria. However, if the deuterated acetic acid were then dissolved in normal water, the reverse process would wash out the label equally rapidly.

Useful labelled compounds containing deuterium or tritium normally require the isotopic hydrogen to be attached to carbon, so the acid-base equilibrium will require cleavage of a C–H bond, where acid strength is usually very weak.

This will necessitate the use of a very strong base to achieve the formation of the conjugate base. A simple example follows from the reactions considered in Section 4.11.1. We saw that we needed to use a strong base such as the amide ion to form the conjugate base of an acetylene. This reaction was favoured, in that the products were the weaker base acetylide and the weaker acid ammonia.

Upon completion of this ionization, we can then add labelled water D2O. Under these conditions, labelling occurs through the abstraction of a deuteron 2H+ from D2O. This is feasible because acetylide is a stronger base than hydroxide and water is a stronger acid than acetylene.

It is also possible to produce deuterium-labelled acetaldehyde by an exchange reaction with D2O and NaOD. This results in an exchange of all three α-hydrogens for deuterium and depends upon the generation of the conjugate base of acetaldehyde under basic conditions.

Unlike the example of the acetylene above, the feature of this process is that it is an equilibrium. This is because acetaldehyde is a weak acid (pKa 17), a weaker acid in fact than water (pKa 15.7).

In other words, the conjugate base of acetaldehyde (the enolate anion) is a stronger base than hydroxide.

Treatment of acetaldehyde with hydroxide thus generates an equilibrium mixture, with only a small amount of enolate anion present. Nevertheless, since we get a small amount of conjugate base, this can abstract a deuteron from the solvent D₂O in the reverse reaction.

Provided excess D₂O is available, equilibration allows the exchange of all three hydrogens in the methyl group of acetaldehyde. The label introduced is unfortunately not stable enough, in that similar treatment with a strong base and H₂O will reverse the process and incorporate 1 H.

Note that the aldehydic hydrogen is not acidic and, therefore, not removed by base. This follows from consideration of the conjugate base, which has no stabilizing features.

The amount of enolate anion present at equilibrium may be calculated from the pKa values:

⇒ \(K=\frac{[\text { enolate }]\left[\mathrm{H}_2 \mathrm{O}\right]}{[\text { acetaldehyde }]\left[\mathrm{HO}^{-}\right]}\)

⇒ \(\begin{aligned}
& =\frac{[\text { enolate }]\left[\mathrm{H}^{+}\right]}{[\text {acetaldehyde }]} \times \frac{\left[\mathrm{H}_2 \mathrm{O}\right]}{\left[\mathrm{HO}^{-}\right]\left[\mathrm{H}^{+}\right]}=10^{-17} \frac{1}{10^{-15.7}} \\
& =10^{-1.3}=0.05
\end{aligned}\)

i.e. the proportion of enolate is about 5%. Despite the unfavourable equilibrium, this type of reaction works surprisingly well under relatively mild conditions.

By using a much stronger base, example sodium hydride or lithium diisopropylamide, the generation of the conjugate base would be essentially complete.

Treating the enolate anion with D²O would give a deuterium-labelled acetaldehyde, but only one atom of deuterium would be introduced. It is the equilibration process that allows the exchange of all three hydrogens.

Amphoteric compounds: ami no acid ds Amphoteric compounds are compounds that may function as either acid or base, depending upon conditions. We have already met this concept in Section 4.5.4, where simple alcohols and amines have two pKa values according to whether the compound loses or gains a proton.

Of course, with alcohols and amines, acidity and basicity involve the same functional group. Other amphoteric compounds may contain separate acidic and basic groups. Particularly important examples of this type are the amino acids that makeup proteins.

The carboxylic acid groups of protein amino acids have pKa values of about 2, ranging from about 1.8 to 2.6, making them significantly more acidic than simple alkanoic acids (pKa about 5).

For the amino groups, the pKa values of the conjugate acids are found to range from about 8.8 to 10.8, with most of them in the region 9–10. These values are thus much closer to those of simple amines (pKa about 10).

Protein amino acids are α-amino acids, the amino and carboxylic acid groups being attached to the same carbon. Thus, the groups are close and will exert maximum inductive effects.

The increased acidity of the carboxylic group, therefore, reflects the electron-withdrawing inductive effect of the amino group, or, more correctly, the ammonium ion. This is because we should not consider the amino acid as the nonionized structure but as the doubly charged form termed a zwitterion (German: Twitter = hybrid)

Consider the two pKa values. The carboxylic acid group (pKa²) is a stronger acid than the protonated NH² group (pKa 9). Thus, the carboxylic acid will protonate the amino group, and, in pure water (pH 7), amino acids having neutral side chains will exist predominantly as the doubly charged zwitterion.

This may be considered an internal salt, and could be compared to ammonium acetate, the salt formed when ammonia (pKa 9.2) reacts with acetic acid (pKa 4.8)

At low pH (acidic solution), an amino acid will exist as the protonated ammonium cation, and at high pH (basic solution) as the amino carboxylate anion. The intermediate zwitterion form will predominate at pHs between these extremes.

The uncharged amino acid has no real existence at any pH. Ironically, we are so familiar with the terminology amino acid, yet such a structure has no real existence! Amino acids are ionic compounds, solids with a high melting point.

We can appreciate that the ionization of the carboxylic acid is affected by the electron-withdrawing inductive effect of the ammonium residue; hence the increased acidity when compared with an alkanoic acid.

Similarly, the loss of a proton from the ammonium cation of the zwitterion is influenced by the electron-donating inductive effect from the carboxylate anion, which should make the amino group more basic than a typical amine. That this is not the case is thought to be a solvation effect (compare simple amines).

The pH at which the concentration of the zwitterion is a maximum is equal to the isoelectric point pI, strictly that pH at which the concentrations of cationic and anionic forms of the amino acid are equal. With a simple amino acid, this is the mean of the two pKa values:

⇒ \(\mathrm{pH}=\frac{\mathrm{p} K_{\mathrm{al}}+\mathrm{p} K_{\mathrm{a} 2}}{2}\)

This is deduced from \(K_{\mathrm{a} 1}=\frac{\left[\mathrm{H}^{+}\right][\mathrm{zw} \text { zwitterion }]}{[\text { cation }]} \text { and } K_{\mathrm{a} 2}=\frac{\left[\mathrm{H}^{+}\right][\text {anion }]}{[\text { zwitterion }]}\)

It follows, therefore, that pH = pKa1 when [cation] = [zwitterion], and that pH = pKa2 when [zwitterion] = [anion]. At the isoelectric point, [cation] = [anion]; thus

⇒ \(\begin{aligned}
{[\text { cation }] } & =\frac{\left[\mathrm{H}^{+}\right][\mathrm{zwitterion}]}{K_{\mathrm{a} 1}} \\
& =[\text { anion }]=\frac{K_{\mathrm{a} 2}[\mathrm{zwitterion}]}{\left[\mathrm{H}^{+}\right]}
\end{aligned}\)

Therefore \(K_{\mathrm{a} 1} \times K_{\mathrm{a} 2}=\left[\mathrm{H}^{+}\right]^2\) and by taking negative logarithms, it follows that \(\mathrm{pH}=\frac{\mathrm{p} K_{\mathrm{a} 1}+\mathrm{p} K_{\mathrm{a} 2}}{2}\)

For alanine, pKa1 = 2.34 and pKa2 = 9.69, so pI = 6.02. Most amino acids have a pI around them.

Note that a number of the protein amino acids also have ionizable functions in the side-chain R group. These may be acidic or potentially acidic (aspartic acid, glutamic acid, tyrosine, cysteine), or basic (lysine, arginine, histidine). These amino acids are thus characterized by three pKa values.

We have used the term ‘potentially acidic’ to describe the phenol and thiol groups of tyrosine and cysteine respectively; under physiological conditions, these groups are unlikely to be ionized. Let us consider lysine, which has a second amino group in its side chain.

The pKa values for lysine are 2.18 (CO2H), 8.95 ( α-NH2), and 10.52 ( ε-NH2). Note that the ε-amino group has a typical amine pKa value, and is a stronger base than the α-amino group. In a strongly acidic solution, lysine will be present as a di-cation, with both amino groups protonated.

As the pH is raised, the most acidic proton will be lost first, and this is the carboxyl proton (pKa 2.18). As the pH increases further, protons will be lost first from the more acidic α-ammonium cation (pKa 8.95), and lastly from the least acidic ε-ammonium cation (pKa 10.52). Again, there is no intermediate with uncharged functional groups.

The isoelectric point for lysine is the pH at which the compound is in an electrically neutral form, and this will be the average of pKa2 (the cation) and pKa3 (the dipolar ion). For lysine, pKa² = 8.95 and pKa3 = 10.52, so pI = 9.74.

Glutamic acid is an example of an amino acid with an acidic side chain. The pKa values are 2.19 (CO2H), 4.25 ( γ-CO2H), and 9.67 (NH2). Here, the γ-carboxyl is more typical of a simple carboxylic acid.

In a strongly acidic solution, glutamic acid will be present as a cation, with the amino group protonated. As the pH is raised, a proton will be lost from the most acidic group, namely the 1- 1-carboxyl (pKa 2.19), followed by the γ-carboxyl (pKa 4.25). Lastly, the proton from the α-ammonium cation (pKa 9.67) will be removed. Yet again, there is no intermediate with uncharged functional groups.

The isoelectric point for glutamic acid is the pH at which the compound is in an electrically neutral form, and this will be the average of pKa1 (the cation) and pKa2 (the dipolar ion). For glutamic acid, pKa1 = 2.19 and pKa2 = 4.25, so pI = 3.22.

Isoelectric points are useful concepts for the separation and purification of amino acids and proteins using electrophoresis. Under the influence of an electric field, compounds migrate according to their overall charge.

As we have just seen for amino acids, this very much depends upon the pH of the solution. At the isoelectric point, there will be no net charge, and, therefore, no migration towards either anode or cathode.

Ionization of morphine: extraction from opium

Morphine is the major alkaloid in opium, the dried latex obtained from the opium poppy, Papaver somniferum. About 25% of the mass of opium is composed of alkaloids, with morphine constituting about 12–15%. Morphine is a powerful analgesic and remains one of the most valuable for relief of severe pain.

However, most of the morphine extracted from opium is processed further to give a range of semi-synthetic drugs, with enhanced or improved properties. A means of extracting morphine from the other alkaloids in opium is thus desirable.

Alkaloids are found mainly in plants and are nitrogenous bases, typically primary, secondary, or tertiary amines. The basic properties facilitate their isolation and purification.

Water-soluble salts are formed in the presence of mineral acids, and this allows the separation of the alkaloids from any other compounds that are neutral or acidic. It is a simple matter to take a plant extract in a water-immiscible organic solvent and extract this solution with aqueous acid.

Salts of the alkaloids are formed, and, being water soluble, these transfer to the aqueous acid phase. On basifying the acid phase, the alkaloids revert to an uncharged form and may be extracted into fresh organic solvent.

Opium contains over 40 different alkaloids, all of which will be extracted from opium by the procedure just described. It then remains to separate morphine from this mixture. Of the main opium alkaloids, only morphine displays some acidic properties as well as basic properties.

Although a tertiary amine, morphine also contains a phenolic group. The acidity of this group can be exploited for the preferential extraction of morphine from an organic solvent by partitioning with an aqueous base.

Thus, if a solution of opium alkaloids in an organic solvent, for example, dichloromethane, is shaken with aqueous NaOH, only morphine will ionize at this pH, and it will form the water-soluble phenolate anion.

The other alkaloids will remain non-ionized and stay in the organic layer, allowing their separation from the aqueous morphine phenolate fraction.

By adding acid to the aqueous fraction, the phenolate will become protonated to give the non-ionized phenol, which may be extracted by shaking with the organic solvent.

Care is needed during the acidification since the addition of too much acid would ionize the amine and create another water-soluble ion, the protonated amine.

This would stay in the aqueous phase and not be extracted by an organic solvent. The optimum pH will be the isoelectric point as described under amino acids.

This is the pH at which the concentrations of cationic and anionic forms of morphine are equal and is the mean of the two pKa values.

Morphine has pKa (phenol) 9.9, and pKa (amine) 8.2, so that pI = 9.05. Note that the protonated amine is a stronger acid than the phenol so the intermediate between the two ionized forms will be the non-ionized alkaloid.

We can then use the Henderson–Hasselbalch equation \(\mathrm{pH}=\mathrm{p} K_{\mathrm{a}}+\log \frac{\text { [base] }}{\text { [acid] }}\) to calculate the relative amounts of the ionic forms at this pH.

For the phenol, pKa 9.9 \(\log \frac{[\text { base }]}{\text { [acid] }}=\mathrm{pH}-\mathrm{p} K_{\mathrm{a}}=9.05-9.9=-0.85\)

Thus [base]/[acid] = 10−0.85 = 0.14 or about 1:7.

For the amine, pKa 8.2

⇒ \(\log \frac{[\text { base ] }}{\text { [acid] }}=\mathrm{pH}-\mathrm{p} K_{\mathrm{a}}=9.05-8.2=0.85\)

Thus [base]/[acid] = 100.85 = 7.08 or about 7:1.

At pH 9.05, the phenol–phenolate equilibrium favours the phenol by a factor of 7: 1, and the amine–ammonium ion equilibrium favours the amine by a factor of 7: 1. In other words, the non-ionized morphine predominates, and this can thus be extracted into the organic phase. What about the amounts in ionized form; are these not extractable?

By solvent extraction of the non-ionized morphine, we shall set up a new equilibrium in the aqueous phase, so that more non-ionized morphine is produced at the expense of the two ionized forms.

A second solvent extraction will remove this, and we shall effectively recover almost all the morphine content. A third extraction would make certain that only traces of morphine were left in ionized forms.

pKa and drug absorption

Cell membranes are structures containing lipids and proteins as their main components. Many drug molecules are weak acids or bases and can, therefore, exist as ionized species, depending upon their pKa values and the pH of the environment.

One of the more important concepts relating to drug absorption is that ionized species have very low lipid solubility, and are unable to permeate through membranes. Only the non-ionized drug is usually able to cross membranes. A range of pKa values covered by some common drugs

If we invoke the Henderson–Hasselbalch equation \(\mathrm{pH}=\mathrm{p} K_{\mathrm{a}}+\log \frac{\text { [base] }}{\text { [acid] }}\)

where pH = pKa for the drug, then, for a weak acid

⇒ \(\log \frac{[\text { base }]}{\text { [acid }]}=\log \frac{\text { [ionized }]}{\text { [non-ionized }]}=0\)

In other words, [ionized] = [non-ionized] = 50%. As we saw in Section 4.9, a shift in pH by one unit to either side of the pKa value must change the ratio of ionized to non-ionized forms by a factor of 10.

For pH=pka+1

⇒ \(\begin{aligned}
\log \frac{[\text { ionized] }}{\text { [non-ionized] }} & =1 \text { and therefore } \frac{\text { [ionized] }}{\text { [non-ionized] }} \\
& =10
\end{aligned}\)

for ph=pKa-1

⇒ \(\begin{aligned}
\log \frac{\text { [ionized] }}{[\text { non-ionized] }} & =-1 \text { and therefore } \frac{\text { [ionized] }}{\text { [non-ionized] }} \\
& =0.1
\end{aligned}\)

For a weak base, we have a similar relationship, though \(\log \frac{[\text { base }]}{[\text { acid }]}=\log \frac{\text { non-ionized] }}{\text { [ionized] }}\)

The human body has many different pH environments. For example, blood plasma has a rigorously controlled pH of 7.4 gastric juice is usually strongly acidic (pH from about 1 to 7), and urine can vary from about 4.8 to 7.5.

It is possible to predict the qualitative effect of pH changes on the distribution of weakly acidic and basic drugs, especially concerning gastric absorption and renal excretion:

Very weak acids with pKa values greater than 7.5 will be essentially non-ionized at all pH values in the range of 1–8 so that absorption will be largely independent of pH.

Acids with pKa values in the range 2.5–7.5 will be characterized by significant changes in the proportion of non-ionized drugs according to the pH. As the pH rises, the percentage of nonionized drugs decreases, and absorption therefore also decreases.

Absorption of stronger acids (pKa less than about 2.5) should also depend upon pH, but the fraction that is non-ionized is going to be very low except under the most acidic conditions in the stomach. Absorption is typically low, even under acidic conditions.

Basic drugs will not be absorbed from the stomach, where the pH is strongly acidic.

The excretion of drugs will be affected by the pH of the urine. If the urine is acidic, weak bases are ionized and there will be poor re-absorption. With basic urine, weak bases are non-ionized and there is more re-absorption.

The pH of the urine can be artificially changed in the range of 5–8.5: oral administration of sodium bicarbonate (NaHCO3) increases pH values, whereas ammonium chloride (NH4Cl) lowers them.

Thus, urinary acidification will accelerate the excretion of weak bases and retard the excretion of weak acids. Making the urine alkaline will facilitate the excretion of weak acids and retard that of weak bases.

The post Acids And Bases appeared first on BDS Notes.

From the beginning of chemistry, scientists have devised means of representing the materials they are discussing and have gradually developed a comprehensive range of shorthand notations.

These cover the elements themselves, the bonding between atoms, the arrangement of atoms in molecules, and, of course, a systematic way of naming compounds that are accepted and understood throughout the scientific world.

The study of carbon compounds provides us with the subdivision ‘organic chemistry’, and a few simple organic compounds can exemplify this shorthand approach to molecular representations.

The primary alcohol propanol (systematically propane-1-ol or 1- propanol, formerly n-propanol, n signifying normal or unbranched) can be represented by a structure showing all atoms, bonds, and lone pair or nonbonding electrons.

Lines are used to show what we call single bonds, indicating the sharing of one pair of electrons.

In writing structures, we have to remember the number of bonds that can be made to a particular atom, i.e. the valency of the atom.

In most structures, carbon is tetravalent, nitrogen trivalent, oxygen divalent, and hydrogen and halogens are univalent. These valencies arise from the number of electrons available for bonding.

More often, we trim this type of representation to one that shows the layout of the carbon skeleton with attached hydrogens or other atoms.

This can be a formula-like structure without bonds, or it can be one showing just the principal bonds, those of the carbon chain.

However, for many complex structures, even these approaches become too tedious, and we usually resort to a shorthand version that omits most, if not all, of the carbon and hydrogen atoms.

Propanol is now shown as a zig-zag chain with an OH group at one end. The other end of the chain, where it stops, is understood to represent a methyl group; three attached hydrogens have to be inferred.

At a point on the chain, two hydrogens are assumed, because two bonds to carbons are already shown. In a structure where three bonds joined, a single additional hydrogen would be assumed (see vinyl chloride, below).

Double bonds, representing the sharing of two pairs of electrons, are inferred by writing a double line. Vinyl chloride (systematically chloroethene) is shown as two different representations according to the conventions we have just seen for propanol.

Note that it is customary always to show the reactive double bond so that CH₂CHCl would not be encountered as an abbreviation for vinyl chloride.

The six-membered cyclic system in aromatic rings is usually drawn with alternating double and single bonds, i.e. the Kekule form´, and it is usually immaterial which of the two possible versions is used.

Aniline (systematically aminobenzene or benzenamine) is shown with and without carbons and hydrogens. It is quite rare to put in any of the ring hydrogens on an aromatic ring.

However, it is sometimes convenient to put some in on the substituent, For Example. on a methyl, as in toluene (methylbenzene), or an aldehyde group, as in benzaldehyde.

Benzene strictly does not have alternating double and single bonds, but the aromatic sextet of electrons is localized in a π orbital system and bond lengths are somewhere in between double and single bonds.

The zig-zag arrangement is convenient so that we see where carbons are located (a long straight line would not tell us how many carbons there are).

But it also mimics the low-energy arrangement (conformation) for such a compound. Note that it is usual to write out the hydroxyl, or some alternative group, in full.

This group, the so-called functional group, tends to be the reactive part of the molecule that we shall be considering in reactions.

When we want an even more concise method of writing the molecule, abbreviations for an alkyl (or aryl) group may be used, in which case propanol becomes PrOH. Some more common abbreviations are given later.

To represent this, a circle may be drawn within the hexagon. Unfortunately, this version of benzene becomes quite useless when we start to draw reaction mechanisms.

And most people continue to draw benzene rings in the Kekule form. In some cases, such as fused rings, it is actually incorrect to show the circles.

Thus, naphthalene has only 10 π electrons, one from each carbon, whereas the incorrect two-circle version suggests it has 12 π electrons.

We find that, in the early stages, students are usually happier to put in all the atoms when drawing structures, following earlier practices.

However, you are urged to adopt the shorthand representations as soon as possible. This saves time and cleans up the structures of larger molecules.

Even a relatively simple molecule such as 2-methylcyclohexane carboxylic acid, a cyclohexane ring carrying two substituents, looks like a mess when all the atoms are put in.

By contrast, the line drawing looks neat and tidy and takes much less time to draw.

Do appreciate that there is no strict convention for how you orientate the structure on paper. In fact, we will turn structures around, as appropriate, to suit our needs.

For example, the amino acid tyrosine has three functional groups, i.e. a carboxylic acid, a primary amine, and a phenol.

How we draw tyrosine will depend upon what modifications we might be considering, and which functional group is being altered.

You will need to be able to reorientate structures without making mistakes, and also to be able to recognize different versions of the same thing.

A simple example is with esters, where students have learned that ethyl acetate (ethyl ethanoate) can be abbreviated to CH₃CO₂C₂H₅.

When written backwards, i.e. C₂H₅OCOCH₃, the ester functionality often seems less recognizable.

Partial Structures

We have just seen that we can save a lot of time and effort by drawing structures without showing all of the atoms. When we come to draw reaction sequences.

We shall find that we have to repeat large chunks of the structure each time, even though no chemical changes are occurring in that part of the molecule.

This is unproductive, so we often end up writing down just that part of the structure that is of interest, i.e. a partial structure.

This will not cause problems when you do it, but it might when you see one and wish to interpret it.

In the representations overleaf, you can see the line drawing and the version with methyls that stresses the bond ends.

Both are satisfactory. When we wish to consider the reactivity of the double bond, and perhaps want to show that reaction occurs irrespective of the alkyl groups attached to the double bond.

We put in the abbreviation R (see below), or usually just omit them. When we omit the attached groups.

It helps to show what we mean by using wavy lines across the bonds, but in our urge to proceed, we tend to omit even these indicators.

This may cause confusion in that we now have what looks like a double bond with four methyls attached, not at all what we intended.

A convenient ploy is to differentiate this from a line drawing by putting in the alkene carbons.

Functional groups

The reactivity of a molecule derives from its functional group or groups.

In most instances, the hydrocarbon part of the molecule is likely to be unreactive, and the reactivity of the functional group is largely independent of the nature of the hydrocarbon part.

In general terms, then, we can regard a molecule as R–Y or Ar–Y, a combination of a functional group Y with an alkyl group R or aryl group Ar that is not participating in the reaction under consideration.

This allows us to discuss reactivity in terms of functional groups, rather than the reactivity of individual compounds.

Of course, most of the molecules of interest to us will have more than one functional group; it is this combination of functionalities that provides the reactions of chemical and biochemical importance.

Most of the functional groups we shall encounter are included, which also contain details for their nomenclature.

It is particularly important that when we look at the structure of a complex molecule we should visualize it in terms of the functional groups it contains.

The properties and reactivity of the molecule can generally be interpreted in terms of these functional groups.

It may sometimes be impossible to consider the reactions of each functional group in complete isolation, but it is valuable to disregard the complexity and perceive the simplicity of the structure.

With a little practice, it should be possible to dissect the functional groups in complex structures such as morphine and amoxicillin.

Systematic nomenclature

Organic compounds are named according to the internationally accepted conventions of the International Union of Pure and Applied Chemistry (IUPAC).

Since these conventions must cover all eventualities, the documentation required spans a book of similar size to this volume. A very much-abbreviated version suitable for our requirements is given here:

• The functional group provides the suffix name;
• With two or more functional groups, the one with the highest priority provides the suffix name;
• The longest carbon chain containing the functional group provides the stem name;
• The carbon chain is numbered, keeping minimum values for the suffix group;
• Side-chain substituents are added as prefixes with appropriate numbering, listing them alphabetically.

The stem names are derived from the names of hydrocarbons. Acyclic and cyclic saturated hydrocarbons (alkanes) in the range C₁ –C₁₂ are listed.

Aromatic systems are named in a similar way, but additional stem names need to be used. Parent aromatic compounds of importance are benzene, naphthalene, anthracene, and phenanthrene.

The last three contain fused rings, and they have a fixed numbering system that includes only those positions at which substitution can take place.

It is anticipated that readers will already be familiar with many of the general principles of nomenclature and will be able to name a range of simple compounds.

It is not the object of this section to provide an exhaustive series of instructions for naming every class of compound.

Instead, the examples chosen here have been selected to illustrate some of the perhaps less familiar aspects that will be commonly encountered and to foster a general understanding of the approach to nomenclature.

Alternative names are shown in some cases; this should emphasize that there is often no unique ‘correct’ name.

Sometimes, it can be advantageous to bend the rules a little so as to provide a neat name rather than a fully systematic one.

Typically, this might mean adopting a lower-priority functional group as the suffix name.

It is important to view nomenclature as a means of conveying an acceptable unambiguous structure rather than a rather meaningless scholastic exercise.

Other examples and specialized aspects will occur in subsequent chapters. For example, heterocyclic nomenclature will be treated in more detail at the appropriate time.

Stereochemical descriptors are omitted here but will be discussed under stereochemistry.

Systematic nomenclature: some example

• alkenes have higher priority than halides; the suffix is -ene
• longest carbon chain is seven carbons: heptane
• numbering is chosen to give the lowest numbers for the double bonds; 2-ene denotes 2,3-double bond, 4-ene denotes 4,5-double bond
• the European system hepta-2,4-diene is less prone to errors than the US system 2,4-heptane
• an additional syllable -a- is used but is not obligatory; heptadiene is easier to say than heptdiene

• Alcohols have higher priority than alkynes; the suffix is -ol
• The longest carbon chain is six carbons: hexane
• Numbering is chosen to give the lowest number for alcohol
• The European system hex-5-yn-2-ol keeps numbers and functionalities together

• Acids have higher priority than amines; remember ‘amino acids’
• The suffix is -oic acid
• One of the methyls is part of the five-carbon chain, the others are substituents
• Note the use of 4,4-, which shows both methyls are attached to the same carbon; 4-dimethyl would not be as precise

• The highest priority group is a ketone; suffix -one
• The longest carbon system is the ring cyclohexane numbering is around the ring starting from ketone as position 1
• 2,5-diene conveys 2,3- and 5,6-double bonds
• Note 2,5-dienone means two double bonds and one ketone; in contrast, endzone which would be one double bond and two ketones

• The highest priority group is aldehyde; suffix -al
• The amino group at 4 is also substituted; together they become ethylamine
• The alternative name invokes a seven-carbon chain with one carbon (C-5) replaced by nitrogen; this is indicated by using the extra syllable -aza-, so the chain becomes 5-azaheptane

• Simple ethers are best named as an alkyl alkyl ether
• The phenylmethyl group is commonly called benzyl
• An acceptable alternative is as an alkoxy alkane: the alternative ethoxy toluene would require an indication of the point of attachment
• The second alternative invokes a three-carbon chain with one carbon replaced by oxygen; this is indicated by using the extra syllable -oxa-, so the chain becomes 2-oxabutane

• Esters are named alkyl alkanoate – two separate words with no hyphen or comma
• Alkyl signifies the alcohol part from which the ester is constructed, whilst alkanoate refers to the carboxylic acid part
• but-2-yl means the ester is constructed from the alcohol butan-2-ol; 3-phenylpropanoate means the acid part is 3-phenylpropanoic acid
• Note the numbers 2 and 3 are in separate words and do not refer to the same part of the molecule

• This is a methyl ester of a substituted benzoic acid; the ring is numbered from the point of attachment of the carboxyl
• The acid portion for the ester is 2-substituted
• The ether group is most easily treated as a methoxy substituent on the benzene ring

• The carboxylic acid takes priority; suffix usually-oic acid
• The carboxylic acid is here treated as a substituent on the cyclohexane ring; the combination is called cyclohexanecarboxylic acid

• This is a secondary amide of butanoic acid; thus the root name is butanamide
• Two methyl substituents are on position 3, and one on the nitrogen, hence N,3,3-trimethyl; the N is given in italics

• A ketone in which the longest chain is two carbons; thus the root name is methanone
• The phenyl substituent is on the carbonyl, therefore at position 1
• Without the 1-substituent, ethanone is actually an aldehyde, and would be ethanal!
• The alternative methyl phenyl ketone is a neat and easy way of conveying the structure
• This structure has a common name, acetophenone, which derives from an acetyl (CH₃CO) group bonded to a phenyl ring

• An amine; suffix usually -amine
• The root name can be phenylamine, as an analog of methylamine, or the systematic benzydamine; in practice, the IUPAC accepted name is aniline
• The ring is numbered from the point of attachment of the amino group
• The prefixes ortho-, meta-, and para- are widely used to denote 1,2-, 1,3-, or 1,4-arrangements respectively on an aromatic ring; these are abbreviated to o-, m-, and p-, all in italics

• This could be named as an alcohol, or as a phenol
• As an alcohol (butanol), there is a substituted phenyl ring attached at position 2
• Note the phenyl and its substituents are bracketed to keep them together and to separate their numbering (shown underlined) from that of the alcohol chain
• As a phenol, the substituted butane side-chain is attached through its 2-position so has a root name but-2-yl to show the position of attachment; again, this is in brackets to separate its numbering from that of the phenol
• di-, tri-, tetra-, etc. are not part of the alphabetical sequence for substituents; dimethoxy comes under m, whereas trihydroxy would come under h, etc.

• This is a thioether, which can be named as a thioether or as a sulfide
• An alternative invokes a four-carbon chain with one carbon replaced by sulfur using the extra syllable -thia-; this chain thus becomes 2-thiabutane
• Note how the (trimethyl)methyl group is most frequently referred to by its long-established name of tertiary-butyl, abbreviated to tert-butyl, or t-butyl

• This contains an amine, an amide, and a carboxylic
• The amide group as a substituent is termed carbamoyl;
• It is rather easier to consider the amide as amino and
• The ketone is indicated by oxo-; do not confuse this
• The common name is glutamic acid; it is an amino acid found in proteins
• There now follow several examples demonstrating how to convert a systematic name into a structure, with appropriate guidance hints.
• For added relevance, these are all selected from routinely used drugs. Again, any stereochemical aspects are not included.

Converting systematic names into structures: selected drug molecules

1-chloro-3-ethylpent-1-en-4-yn-3-ol (ethchlorvynol)

Main chain is pentane (C₅) number it 

Put in unsaturation

• 1-ene (=1,2-ene)
• 4-yne (=4,5-yne)

Put in substituents

• 1-chloro
• 3-ethyl
• 3-hydroxy (3-ol)

4-aminohex-5-enoic acid (vigabatrin)

The main chain is hexane (C₆) number it

Put in unsaturation 5-ene (=5,6-ene)

A main functional group is an acid (-oic acid) this will be carbon-1

Put in substituent 4-amino

2-(2-chlorophenyl)-2-methylaminocyclohexanone (ketamine)

The main chain is cyclohexane (C₆) number it

A main functional group is a ketone (-one) which will be carbon-1

Put in substituents 2-methylamino = 2-amino carrying a methyl (contrast aminomethyl = methyl carrying an amino) 2-(2-chlorophenyl) = 2-chlorophenyl at position 2; the phenyl carries a chloro substituent at its own position 2; note the use of brackets to separate the two types of numbering

5-methyl-2-(2-propyl)-cyclohexanol (menthol)

Main chain is cyclohexane (c6) number it

Main functional group is an alcohol (-ol) this will be carbon-1

put substituents 5-methyl 2-(2-propyl)=2-propyl position 2;
2-propyl is a peopyl group joined via its 2-position

1-(3,4-dihydroxyphenyl)-2-dimethylaminoethanol (adrenaline; epinephrine)

Main chain is ethane (C₂) number it

Main functional group is an alcohol (-ol) this will be carbon-1

Put in substituents 2-methylamino = 2-amino carrying a methyl 1-(3,4-dihydroxyphenyl) = 3,4-dihydroxyphenyl at position 1; the phenyl carries hydroxy substituents at its own positions 3 and 4; note the use of brackets to separate the two types of numbering

1-benzyl-3-dimethylamino-2-methyl-1-phenylpropyl propionate (dextropropoxyphene)

This is an ester (two words, -yl -oate) the -oate part refers to the acid component, the -yl part to the esterifying alcohol

Main chain of acid is propane (C₃) main chain of alcohol is propane (C₃)
these are numbered separately (the ester has two separate words)

No substituents on acid component

Put in substituents on alcohol components 1-phenyl; 1-benzyl; 2-methyl; 3-dimethylamino

Join with acid components via an ester linkage

2-[4-(2-methylpropyl)phenyl]propanoic acid (ibuprofen)

Main chain propane (C₃) number it

A main functional group is an acid (-oic acid) this will be carbon-1

Put in substituents consider brackets; we have square brackets with curved brackets inside initially ignore the contents of the curved brackets and their numbering (4), this reduces to 2-[phenyl] propanoic acid, which indicates phenyl at position 2 on propanoic acid

4-(2-methylpropyl)phenyl = 2-methylpropyl at position 4 of the phenyl;

2-methylpropyl = propyl with methyl at position 2 note the brackets separate different substituents and their individual numbering systems

2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide (lidocaine; lignocaine)

This is an amide; acetamide is the amide of acetic acid (C₂)

number it; the carbonyl carbon is C-1

There are two main substituents, on C-2 and the nitrogen, with brackets to keep the appropriate groups together the substituent at C-2 is diethylamino.

An amino which is itself substituted with two ethyl groups the substituent on the nitrogen is 2,6-dimethylphenyl, a phenyl group substituted at positions 2 and 6 on the phenyl.

Put in substituents

Common Groups And Abbreviations

In drawing structures, we are already using a sophisticated series of abbreviations for atoms and bonding.

Functional groups are also abbreviated further, in that –CO₂H or –CHO convey considerably more information to us than the simple formula does.

Other common abbreviations are used to specify particular alkyl or aryl groups in compounds, to speed up our writing of chemistry.

It is highly likely that some of these are already familiar, such as Me for methyl, and Et for ethyl. Others are included.

Common Non – Systematic Names

Systematic nomenclature was introduced at a relatively late stage in the history of chemistry, and thus common names had already been coined for a wide range of chemicals.

Because these names were in everyday usage, and familiar to most chemists, a number have been adopted by IUPAC as the approved name, even though they are not systematic.

These are thus names that chemists still use, that are used for labeling reagent bottles, and are those under which the chemical is purchased.

Some of these are given in Table 1.4, and it may come as a shock to realize that the systematic names school chemistry courses have provided will probably have to be ‘relearned’.

The use of the old terminology n- (normal) for unbranched hydrocarbon chains, with i- (iso), s- (secondary), and t- (tertiary) for branched chains is still quite common with small molecules and can be acceptable in IUPAC names.

Trivial Names For Complex Structures

Biochemical and natural product structures are usually quite complex, some exceedingly so, and fully systematic nomenclature becomes impracticable.

Names are thus typically based on so-called trivial nomenclature, in which the discoverer of the natural product exerts his or her right to name the compound.

The organism in which the compound has been found is frequently chosen to supply the root name, For Example. hyoscyamine from Hyoscyamus, atropine from Atropa, or penicillin from Penicillium.

Name suffixes might be -in to indicate a constituent of’, -side to show the compound is a sugar derivative, -genin for the aglycone released by hydrolysis of the sugar derivative -toxin for a poisonous constituent, or they may reflect chemical functionality, such as -one or -ol.

Traditionally, -one is always used for alkaloids (amines). Structurally related compounds are then named as derivatives of the original, using standard prefixes, such as hydroxy-, methoxy-, methyl-, dihydro-, homo-, etc. for added substituents, or deoxy-, dimethyl-, dimethoxy-, dehydro-, nor-, etc.

For removed substituents. Homo- is used to indicate one carbon more, whereas nor- means one carbon less. The position of this change is then indicated by the systematic numbering of the carbon chains or rings.

Some groups of compounds, such as steroids and prostaglandins, are named semi-systematically from an accepted root name for the complex hydrocarbon skeleton.

Drug names chosen by pharmaceutical manufacturers are quite random and have no particular relationship to the chemical structure.

Acronyms

Some of the common reagent chemicals and solvents are usually referred to by acronyms, a sequence of letters derived from either the systematic name or a trivial name.

We shall encounter some of these in due course, and both names and acronyms will be introduced when we first meet them. For reference purposes, those we shall meet are also listed.

Far more examples occur with biochemicals. Those indicated cover many, but the list is not comprehensive.

Pronunciation

As you listen to chemists talking about chemicals, you will soon realize that there is no strict protocol for pronunciation.

Even simple words like ethyl produce a variety of sounds. Many chemists say ‘eethyle’.

But the Atlantic divide gives us ‘ethel’ with short ‘e’s, and continental European chemists often revert to the German pronunciation ‘etool’.

There is little to guide us in the words themselves, since methane is pronounced ‘meethayne’ whilst methanol tends to have short ‘e’, ‘a’, and ‘o’, except for occasional cases.

Mainly European, when it may get a long ‘o’. On the other hand, propanol always seems to have the first ‘o’ long, and the second one short.

Vinyl can be ‘vinil’ or ‘vynyl’ according to preference, and amino might be ‘ameeno’ or ‘amyno’. Need we go on? Your various teachers will probably pronounce some common words quite differently.

Try to use the most commonly accepted pronunciations, and don’t worry when a conversation with someone involves differences in pronunciation.

As long as there is mutual understanding, it’s not really important how we say it. By and large, chemists are a very tolerant group of people.

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Intermediary Metabolism

Intermediary metabolism is the all-encompassing name given to the highly integrated network of chemical reactions by which organisms obtain energy from their environment and synthesize those molecules necessary for their continued well-being and existence.

These are the reactions that we consider to comprise ‘biochemistry’, and they are usually studied as part of a biochemistry course. However, if we choose to study these reactions as part of organic chemistry, we shall see some interesting, elaborate, and often quite complex transformations taking place.

Two very important characteristics differentiate these reactions from those we have already encountered. First, they are almost always enzyme-mediated reactions and thus take place readily at near-neutral pH and at ambient temperatures.

Second, they are also highly regulated, and their participation can be switched on or off, or otherwise finely controlled by the organism according to its needs.

As we look at some of the reactions of intermediary metabolism, we shall rationalize them in terms of the chemistry that is taking place.

In general, we shall not consider here the involvement of the enzyme itself, the binding of substrates to the enzyme, or the role played by the enzyme’s amino acid side chains.

We looked at specific examples where we know just how an enzyme is able to catalyze a reaction. Examples such as aldolase and triose phosphate isomerase, enzymes of the glycolytic pathway, and citrate synthase from the Krebs cycle were considered in some detail.

It may be advantageous to look back at these examples in order to underline the participation of an enzyme.

A large proportion of the substrates used in intermediary metabolism are in the form of phosphates. Phosphates are favored in nature since they usually confer water solubility on the compound, and provide a functional group that is able to bind to enzymes through simple electrostatic bonding.

In many cases, the phosphate group may also feature as a chemically reactive functional group – phosphates are good leaving groups. In many structures, the abbreviation P is used to represent the phosphate group and PP the diphosphate (or pyrophosphate) group:

At physiological pH values, these groups will be ionized as shown, but in schemes where structures are given in full, the non-ionized acids are usually depicted.

This is done primarily to simplify structures, eliminate the need for counterions, and avoid mechanistic confusion. Likewise, amino acids are usually shown in non-ionized form, although they will typically exist as zwitterions:

Ionized and non-ionized forms of many com-pounds are regarded as synonymous in the text, thus citrate/citric acid, acetate/acetic acid or others may be used according to the author’s whim and context, and should not be considered as having any special relevance.

Oxidation Reactions And ATP

The currency unit for energy in biochemical reactions is the nucleotide derivative ATP, adenosine triphosphate. We have already discussed this molecule, where we rationalized many of the reactions of phosphates in terms of them being analogs of carbonyl compounds.

Only the triphosphate portion of ATP is involved chemically in the energy processes; the remaining complex part of the molecule is a recognizable feature that allows binding to the enzyme.

The triphosphate portion can be visualized as containing two anhydride functions and one ester function. We have seen that hydrolysis of anhydrides is achieved much more easily than hydrolysis of esters, an observation that can be related to the nature of the leaving group.

Accordingly, hydrolysis of the anhydride bond liberates considerably more energy than does hydrolysis of the ester bond, and it is anhydride hydrolysis that is crucial to ATP’s role in biochemistry. Hydrolysis of ATP to ADP liberates energy, which can be coupled to energy-requiring processes.

Alternatively, energy-releasing processes can be coupled to the synthesis of ATP from ADP.

Hydrolysis of ATP to ADP is rationalized simply as a nucleophilic attack of water onto the terminal P=O double bond, followed by cleavage of the anhydride bond and expulsion of ADP as the leaving group.

There are two anhydride linkages in ATP, but nucleophilic attack in the enzyme-controlled reaction usually occurs on the terminal P=O (hydrolysis of ATP to ADP), and only occasionally do we encounter attack on the central P=O (hydrolysis of ATP to adenosine monophosphate, AMP).

Both reactions yield the same amount of energy, ΔG—34 kJmol^-1. This is not surprising, since in each case the same type of bond is being hydrolyzed.

The further hydrolysis of AMP to adenosine breaks an ester linkage and liberates only a fraction of the energy, ΔG — 9 kJmol^-1, and this reaction is not biochemically important.

Oxidation reactions are the main providers of energy for ATP synthesis. Whilst oxidation usually involves the incorporation of one or more oxygen atoms, in its simplest form it can be thought of as a loss of electrons.

Thus, the transformation of ferrous ion to ferric ion is an oxidation reaction and involves the loss of one electron. Such electrons can be considered as carrying the energy released from the oxidation reactions.

In biochemical reactions, these electrons are eventually passed to oxygen, which becomes reduced to water. Overall, the oxidation of a substrate AH2 could be represented by the equation

⇒ \(\mathrm{AH}_2+1 / 2 \mathrm{O}_2 \rightleftharpoons \mathrm{A}+\mathrm{H}_2 \mathrm{O} \text { large negative } \Delta \mathrm{G}\)

and this reaction has the potential to liberate energy, i.e. it has a large negative ΔG.

Now this reaction is not possible directly. We are not accustomed to seeing our food spontaneously reacting with atmospheric oxygen and igniting because of the energy released!

However, food such as carbohydrates, fat, and protein is oxidized after we have eaten it, and energy is released and utilized by our bodies. The secret is to react AH₂ through the involvement of a suitable coenzyme, not directly with oxygen. This reaction can be considered as

⇒ \(\left.\mathrm{AH}_2+\mathrm{X}(\text { oxidized }) \rightleftharpoons \mathrm{A}+\mathrm{X} \text { (reduced }\right)\)

where X is the coenzyme. The reaction is catalyzed by an enzyme termed dehydrogenase, which removes two hydrogen atoms from the substrate. The coenzyme system involved can generally be related to the functional group being oxidized in the substrate.

If the oxidation process is then a pyridine nucleotide, nicotinamide adenine dinucleotide (NAD⁺) or nicotinamide adenine dinucleotide phosphate (NADP⁺), tends to be utilized as hydrogen acceptor. One hydrogen from the substrate (that bonded to carbon) is transferred as hydride to the coenzyme, and the other, as a proton, is passed to the medium.

NAD⁺ and NADP⁺ may also be used in the oxidations

The reverse reaction, i.e. reduction, is also indicated in the scheme and may be compared with the chemical reduction process using complex metal hydrides, for example, LiAlH₄₁or NaBH₄, namely nucleophilic addition of hydride and subsequent protonation.

The reduced forms NADH and NADPH are conveniently regarded as hydride-donating reducing agents. We also noted that there were stereochemical features associated with these coenzymes.

During a reduction sequence, there is the stereospecific transfer of hydride from a prochiral center on the dihydropyridine ring, and it is delivered to the carbonyl compound also in a stereospecific manner.

In practice, NADPH is generally employed in reductive processes, whereas NAD⁺ is used in oxidations.

Should the oxidative process be the conversion then the coenzyme used as acceptor is usually a flavin nucleotide: flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN).

These entities are bound to the enzyme in the form of a flavoprotein, and take up two hydrogen atoms, represented in the figure as being derived by the addition of a hydride from the substrate and a proton from the medium.

Reductive sequences involving flavoproteins may be represented as the reverse reaction, where hydride is transferred from the coenzyme, and a proton is obtained from the medium.

After the substrates containing either CH-OH or CH₂-CH₂ functional groups have been oxidized by the dehydrogenase enzyme-coenzyme system, energy abstraction from the oxidative transformation now depends upon reoxidation of the reduced coenzyme.

⇒ \(\left.\begin{array}{l}
\mathrm{NAD}(\mathrm{P}) \mathrm{H}+1 / 2 \mathrm{O}_2 \rightarrow \mathrm{NAD}(\mathrm{P})^{+}+\mathrm{H}_2 \mathrm{O} \\
\mathrm{FADH}_2+1 / 2 \mathrm{O}_2 \rightarrow \mathrm{FAD}+\mathrm{H}_2 \mathrm{O} \\
\mathrm{FMNH}_2+1 / 2 \mathrm{O}_2 \rightarrow \mathrm{FMN}+\mathrm{H}_2 \mathrm{O}
\end{array}\right\} \text { large negative } \Delta \mathrm{G}\)

This will be one of the processes shown, all of which have a large negative ΔG and are capable of harnessing this energy via the synthesis of ATP molecules. However, even these are not achievable directly, and the electron transport chain of oxidative phosphorylation is utilized.

Oxidative Phosphorylation And The Electron Transport Chain

The total oxidation of an organic compound using molecular oxygen as the electron acceptor has the potential to yield a very large amount of energy, sufficient for the synthesis of several molecules of ATP from ADP if there could be one efficiently coupled oxidation process.

It is quite unrealistic to achieve this in one step; instead, a multistage process termed oxidative phosphorylation is employed. This process removes packets of energy, more nearly corresponding to the amounts required for the synthesis of single ATP molecules from ADP.

Oxidation of a compound, per atom of oxygen used, can yield up to three molecules of ATP, representing an energy efficiency of about 50%. It takes place in the mitochondria via a sequence of redox (reduction-oxidation) reactions known as the electron transport chain or the respiratory chain and provides the principal source of ATP for an aerobic cell.

The electron transport chain involves a series of compounds acting together, achieving the removal of hydrogen equivalents from organic molecules and eventually reacting them with oxygen to form water.

The first part of the chain involves the transfer of a pair of hydrogen atoms, whereas only electrons are transferred in the final stages of the pathway. The individual molecules involved are NAD⁺, flavoprotein, coenzyme Q (ubiquinone), and a number of cytochromes.

We have already seen the redox reactions of NAD⁺ and flavoproteins containing FAD or FMN. The term coenzyme Q or ubiquinone covers a range of structures (as shown), depending on the length of the hydrocarbon side-chain, which varies according to species. In humans, the redox carrier is coenzyme Q₁₀ (n = 10).

Ubiquinone is readily reduced to ubiquinol, a process requiring two protons and two electrons; similarly, ubiquinol is readily oxidized back to ubiquinone.

This redox process is important in oxidative phosphorylation, in that it links hydrogen transfer to electron transfer. The cytochromes are haem-containing proteins.

As we have seen, haem is an iron-porphyrin complex. Alternate oxidation-reduction of the iron between Fe²⁺ (reduced form) and Fe³⁺ (oxidized form) in the various cytochromes is responsible for the latter part of the electron transport chain.

The individual cytochromes vary structurally, and their classification (a, b, c, etc.) is related to their absorption maxima in the visible spectrum.

Most compounds oxidized by the electron transport chain donate hydrogen to NAD⁺, and then NADH is reoxidized in a reaction coupled with the reduction of a flavoprotein.

During this transformation, sufficient energy is released to enable the synthesis of ATP from ADP. The reduced flavoprotein is reoxidized via the reduction of coenzyme Q; subsequent redox reactions then involve cytochromes and electron transfer processes rather than hydrogen transfer.

In two of these cytochrome redox reactions, there is sufficient energy release to allow ATP synthesis. In due course, electrons are passed to oxygen, which is converted into water in the presence of protons.

The total process whereby hydrogen atoms are passed to NAD⁺ generates three molecules of ATP per pair of hydrogen atoms. However, substrates with the CH₂-CH₂ grouping that are oxidized by flavoproteins effectively bypass the first ATP generation step, so only produce two molecules of ATP per pair of hydrogen atoms.

The electron transport chain is vital to aerobic organisms. Interference with its action may be life-threatening.

Thus, cyanide and carbon monoxide bind to haem groups and inhibit the action of the enzyme cytochrome c oxidase, a protein complex that is effectively responsible for the terminal part of the electron transport sequence and the reduction of oxygen to water.

What has been achieved by the participation of coenzyme systems and the electron transport chain is twofold.

First, there is no need for the substrate AH₂ to react with oxygen. Second, it provides common routes for the oxidation of many different organic compounds, rather than a specific route for every compound, a vast variety of which will be present in the normal diet.

Although it is somewhat simplistic, many of the non-oxidative reactions of intermediary metabolism can be viewed as additional chemical transformations designed to provide substrates containing either CH-OH or CH₂-CH₂ that may then be subjected to dehydrogenation.

The Glycolytic Pathway

The glycolytic pathway, or glycolysis, is a metabolic sequence in which glucose is broken down to pyruvic acid. The subsequent fate of pyruvate then depends upon whether or not the organism is aerobic or anaerobic: Under aerobic conditions, pyruvate is oxidized via oxidative phosphorylation; under anaerobic conditions, pyruvate is converted further into compounds such as lactate or ethanol, depending upon the organism.

The first step in glycolysis is the phosphorylation of glucose to give the ester glucose 6-phosphate. The glucose starting material may well have come from hydrolysis of starch obtained in the diet, or by utilization of glycogen reserves.

This phosphorylation step is achieved by the reaction of the 6-hydroxyl with the anhydride ATP, during which process ATP is converted into ADP.

This process is driven by the energy contained in the anhydride function of ATP, and represents an expenditure of energy to get the metabolic process started, though the overall objective of glycolysis is to acquire energy via the synthesis of ATP molecules.

Glucose 6-phosphate is then isomerized to fructose 6-phosphate. This conversion of an aldose sugar to a ketose sugar is easy to rationalize in terms of keto-enol tautomerism.

We should first consider the open-chain form of glucose 6-phosphate, rather than its pyranose hemiacetal form. The open-chain aldose has the requirements for enolization, namely a hydrogen α to the aldehyde carbonyl group.

Enolization produces in this case an enediol, which can revert to a keto form in two ways, i.e. reforming the open-chain aldose or, alternatively, producing the ketose fructose 6-phosphate. The enediol may be considered a common enol for the two enolization processes.

The open-chain form of fructose 6- phosphate may then form a hemiketal, as shown, generating a furanose ring.

Further phosphorylation, again using ATP as in the first reaction, converts fructose 6-phosphate into fructose 1,6-diphosphate.

Again, there is the expenditure of energy by the use of ATP; we have now used two molecules of ATP, and there has been no net generation of energy. This represents a significant investment before any rewards are forthcoming.

For the subsequent reactions, we need to consider fructose 1,6-diphosphate in its open-chain form rather than the hemiketal originally drawn. Now follows the reverse aldol reaction catalyzed by aldolase, as we have already discussed in some detail elsewhere.

For a simple chemical interpretation, we can write this as involving enolate anions, either as leaving the group in the forward reaction or as a nucleophile in the reverse reaction, but the enzymic reaction is known to utilize enamine derivatives.

It is worth emphasizing again that organisms make use of this reaction both in its forward direction for carbohydrate metabolism and in its reverse direction for carbohydrate synthesis, according to requirements.

The reverse aldol reaction results in the form- is not on the direct pathway, and is converted into a tion of dihydroxyacetone phosphate and glycersecond molecule of glyceraldehyde 3-phosphate by aldehyde 3-phosphate. Dihydroxyacetone phosphate is the enzyme triose phosphate isomerase.

This is achieved by two keto-enol tautomerism reactions and a common enol. Mechanistically, it is identical to the isomerization of glucose 6-phosphate to fructose 6-phosphate seen earlier in the sequence, so we can move on to the next step of the pathway.

In this step, the aldehyde group of glyceraldehyde 3-phosphate appears to be oxidized to an acid, which becomes phosphorylated, and hydrogen is passed to NAD⁺, which becomes reduced to NADH. We shall see shortly that the fate of this NADH is quite significant.

At first glance, this oxidation-phosphorylation reaction seems rather obscure. It becomes much more logical when we see that the enzyme achieves this via a multi-stage process.

Critical to the reaction is the involvement of a thiol group on the enzyme. This reacts with the aldehyde group of the substrate glyceraldehyde 3-phosphate to form a hemithioacetal. It is this intermediate that reacts with NAD⁺ since it contains an oxidizable CH-OH function.

The product is then a thioester. The thioester is attacked by a phosphate nucleophile, and a stepwise sequence involving thiol-containing enzyme since it contains a good leaving group, EnzS-, the reaction product is released from the enzyme as 1,3- diphosphoglycerate.

If we look at the structure of 1,3-diphosphoglycerate, we can see that it is actually an anhydride, albeit a mixed anhydride of carboxylic acid and phosphoric acid. Accordingly, we expect it to be fairly reactive toward nucleophiles, and indeed it is.

It is sufficiently reactive that hydrolysis liberates enough energy to synthesize ATP from ADP.

ATP synthesis is achieved by ADP acting as the nucleophile towards this mixed anhydride, attacking the P=O bond, with the carboxylate being the leaving group.

Note that this reaction is favored, whereas the alternative possibility involving hydrolysis of the phosphate ester does not occur.

This is precisely what we would predict knowing the different reactivities of anhydrides and esters.

This direct synthesis of ATP by a process in which ADP acquires an additional phosphate from a suitable donor molecule is often termed substrate-level phosphorylation, differentiating it from ATP synthe¬sis that is achieved through oxidative phosphorylation.

After donating its phosphate group to ADP, 1,3- diphosphoglycerate is converted into 3-phospho- glycerate. This reaction is followed by enzymic modification to 2-phosphoglycerate.

Although this reaction is catalyzed by a mutase, which perhaps suggests this is a rearrangement reaction, there is no transfer of the phosphate group to the adjacent hydroxyl.

Instead, this reaction proceeds via an intermediate diphosphate, so we are actually seeing a phosphorylation-dephosphorylation or esterification-hydrolysis sequence. There is an unusual aspect of this extra phosphorylation, and that is that no ATP is involved.

Instead, the new phosphate group is derived from the enzyme itself. Although this is unexpected, it does avoid another energy-requiring reaction and the use of precious ATP.

Then follows an elimination reaction, in which water is removed from 2-phosphoglycerate to yield phosphoenolpyruvate.

This reaction is catalyzed by an enzyme called enolase; though this may appear quite straightforward, it is chemically unusual. Eliminations depend upon the presence of a suitable leaving group, and by far the better-leaving group in 2-phosphoglycerate is the phosphate.

We might predict that the product from an elimination reaction on 2-phosphoglycerate would logically be the alternative enol system. That this does not occur indicates and emphasizes the enzyme’s special contribution to the reaction.

The product phosphoenolpyruvate is able to donate its phosphate group directly to ADP, resulting in ATP synthesis.

Substrate-Level Phosphorylation

Although phosphoenolpyruvate is only an enol ester, hydrolysis gives an unfavoured enol; tautomerism to the keto form is the driving force for the reaction and results in a large negative ΔG

This is another example of substrate-level phosphorylation but differs from the earlier example that involved hydrolysis of a mixed anhydride. Here, we have merely the hydrolysis of an ester, and thus a much lower release of energy.

In fact, with 1,3- diphosphoglycerate, we specifically noted the difference in reactivity between the anhydride and ester groups. So how can this reaction lead to ATP synthesis? The answer lies in the stability of the hydrolysis product, enolpyruvic acid.

Once formed, this enol is rapidly isomerized to its keto tautomer, pyruvic acid, with the equilibrium heavily favoring the keto tautomer.

The driving force for the substrate-level phosphorylation reaction is actually the position of equilibrium in the subsequent tautomerization.

This completes the glycolytic pathway; well, almost. To maintain the operation of the pathway, the NAD⁺ used in the conversion of glyceraldehyde 3-phosphate into 1,3-diphosphoglycerate must be regenerated from its reduced form NADH, since only small amounts of the coenzyme will be available to the organism.

If the organism is aerobic, then it is possible to use the oxidative phosphorylation processes to regenerate NAD⁺ from NADH, and in so doing also achieve the synthesis of ATP.

However, for anaerobes, or for aerobes under temporary anaerobic conditions, pyruvate synthesized in the last reaction is modified further to achieve this end.

For example, certain organisms use NADH to reduce pyruvate to lactate and regenerate NAD⁺. This process might occur in actively exercised muscles, when there is a temporary shortage of oxygen, leading to a build-up of lactic acid and ensuing cramp pains.

Other organisms are equipped to produce ethanol, by employing a thiamine diphosphate-dependent decarboxylation of pyruvate to acetaldehyde and NAD⁺ is regenerated by reducing the acetaldehyde to ethanol.

This is a characteristic of baker’s yeast and forms the essential process for both bread making (production of CO₂) and the brewing industry (formation of ethanol).

The glycolytic pathway is crucial to anaerobes for ATP production; this is reflected in the fact that ATP synthesis is achieved via substrate-level phosphorylation, and does not depend on the availability of oxidative phosphorylation.

The energy yield from glycolysis for the anaerobic decomposition of glucose to 2 mol of lactic acid may be calculated as follows:

1. 2 mol of ATP are used up in phosphorylations;
   2 mol of ATP are gained per half molecule of glucose, i.e. a total of 4 mol ATP;
2. Net yield from glucose  → 2 mol lactic acid = 2 mol ATP.

The Krebs Cycle

The Krebs cycle is sometimes still referred to as the citric acid cycle, citric acid being one of the intermediates involved, and even the tricarboxylic acid cycle, in that several of the intermediates are triads.

As the name suggests, the process is a cycle, so that there is a reasonably constant pool of intermediates functioning in an organism, and material for degradation is processed via this pool of intermediates.

Overall, though, the material processed does not increase the size of the pool. The compound that enters the cycle is the thioester acetyl-coenzyme A (acetyl-CoA).

We have just seen that anaerobic organisms metabolize pyruvate from the glycolytic pathway by various means, but that the prime objective is to reoxidize NADH to NAD⁺.

In aerobic organisms, reoxidation of NADH is achieved via oxidative phosphorylation, generating ATP in the process, and there is no longer any need to sacrifice pyruvate for this purpose.

Accordingly, pyruvate from glycolysis is converted into acetyl-CoA by a process known as oxidative decarboxylation. but, for the moment, it can be represented by the equation.

The whole process is multi-step, and catalyzed by the pyruvate dehydrogenase enzyme complex, which has three separate enzyme activities.

During the transformation, an acetyl group is effectively removed from pyruvate, and passed via carriers of thiamine diphosphate (TPP) and lipoic acid eventually to coenzyme A, a complex material whose principal functional group involved in metabolic reactions is a thiol.

The requirement for NAD⁺ is to reoxidize the lipoic acid carrier. It is worth mentioning that the pyruvate → acetaldehyde conversion we considered at the end of the glycolytic pathway involves the same initial sequence, and pyruvate decarboxylase is another thiamine diphosphate-dependent enzyme.

Acetyl-CoA is a thioester of acetic acid with coenzyme A. It is a remarkably common intermediate in many metabolic degradative and synthetic pathways, for which the reactivity of the thioester function plays a critical role.

There are two major sources of the acetyl-CoA entering the Krebs cycle: glycolysis via the oxidative decarboxylation of pyruvate and fatty acid degradation. There are other minor sources of acetyl-CoA, including the metabolism of amino acids from protein.

The Krebs cycle intermediate that reacts with acetyl-CoA is oxaloacetate, and this reacts via an aldol reaction, giving citryl-CoA. However, the enzyme citrate synthase also carries out hydrolysis of the thioester linkage, so that the product is citrate; hence the terminology ‘citric acid cycle’.

The hydrolysis of the thioester is actually responsible for disturbing the equilibrium and driving the reaction to completion.

The aldol reaction is easily rationalized, with acetyl-CoA providing an enolate anion nucleophile that adds to the carbonyl of oxaloacetate – easily rationalized, but surprising.

Oxaloacetate is more acidic than acetyl-CoA, in that there are two carbonyl groups flanking the methylene. If one were to consider a potential base-catalyzed reaction between these two substrates, then logic suggests that oxaloacetate would be preferentially converted into an enolate anion nucleophile.

This could then attack the carbonyl of acetyl-CoA, but via a Claisen reaction, since there is a thiolate leaving group.

That citrate synthase achieves an aldol reaction (as shown) reflects that the enzyme active site must have a basic residue appropriately positioned to abstract a proton from acetyl-CoA allowing it to act as the nucleophile.

Citrate is subsequently isomerized to isocitrate; this involves dehydration and rehydration via the intermediate cis-aconitate. Both reactions are catalyzed by the single enzyme aconitase. They may be considered simply as acid-catalyzed elimination followed by acid-catalyzed addition reactions.

Worthy of note in this reaction is that citrate displays prochirality. The methylene carbons may be considered prochiral, in that enzymic elimination of a proton is likely to be entirely stereospecific.

In addition, the apparently equivalent side chains on the central carbon are also prochiral and going to be positioned quite differently on the enzyme.

This means that only one of these side chains is involved in the dehydration-rehydration sequence, and it can be shown from labeling studies that the side chain modified is not the one that was recently derived from acetyl-CoA as a nucleophile.

In isocitrate, there is a CHOH group that is available for oxidation via the coenzyme NAD⁺ and the enzyme isocitrate dehydrogenase. NADH will then be reoxidized via oxidative phosphorylation, and lead to ATP synthesis.

The oxidation product from isocitrate is oxalosuccinate, β-ketoacid that easily decarboxylates through an intramolecular hydrogen-bonded system.

Although thermal (non-enzymic) decarboxylation would probably occur readily, it turns out that the enzyme isocitrate dehydrogenase also catalyzes this reaction.

The product is 2-oxoglutarate, sometimes referred to as α-oxoglutarate or α-ketoglutarate. Note specifically that we have just lost one of the carbon atoms. Oxaloacetate (a C₄ compound) reacted with acetyl-CoA (C₂) to give citrate (C₆), and this reaction now gives us a C₅ compound; to complete the cycle and get back to C₄, we shall need to lose another carbon atom.

This is achieved in the next reaction catalyzed by 2-oxoglutarate dehydrogenase.

Now this reaction is effectively a repeat of the pyruvate → acetyl-CoA oxidative decarboxylation we saw at the beginning of the Krebs cycle. It similarly requires thiamine diphosphate, lipoic acid, coenzyme A, and NAD⁺.

A further feature in common with that reaction is that 2-oxoglutarate dehydrogenase is also an enzyme complex comprised of three separate enzyme activities.

2-Oxoglutarate is thus transformed into succinyl-CoA, with the loss of a further carbon as CO₂, and producing NADH that can be exploited in ATP synthesis via oxidative phosphorylation.

Note that, because of the prochirality in citric acid and subsequent enzymic selectivity, neither of the carbon atoms lost in the two decarboxylations originates from the acetyl-CoA molecule added in the first reaction, the aldol addition.

These carbons are not lost until further cycles of the pathway have been completed.

The product succinyl-CoA is able to participate in ATP synthesis as an example of substrate-level phosphorylation – we met some other examples in the glycolytic pathway.

Essentially, hydrolysis of succinyl-CoA liberates sufficient energy that it can be coupled to the synthesis of ATP from ADP.

However, guanosine triphosphate (GTP) is the nucleoside phosphate produced, rather than ATP. ATP is then produced indirectly from GTP.

There appears to be no obvious reason why this reaction should be coupled to the synthesis of GTP, rather than to the direct synthesis of ATP; the other product of the reaction is succinate.

When we investigate this substrate-level phosphorylation reaction in detail, we find it also involves a molecule of phosphate. Phosphate reacts initially with succinyl-CoA, converting the thioester into an acyl phosphate, which is, of course, a mixed anhydride.

It is actually hydrolysis of this mixed anhydride that can be coupled to nucleoside triphosphate synthesis, and it is fitting to compare this with the formation and hydrolysis of 1,3-diphosphoglycerate in the glycolytic pathway.

As we move on in the Krebs cycle, the next reaction is the oxidation of the CH₂-CH₂ grouping in succinate to give the unsaturated diacid fumarate. We have already looked at this type of oxidation and seen that it involves a dehydrogenase enzyme coupled to a flavin nucleotide coenzyme.

For this reaction, the coenzyme is FAD. The reduced form of FAD can then be reoxidized to FAD via oxidative phosphorylation, generating energy in the form of ATP in the process.

The sequence continues with hydration, and addition and results in the formation of oxaloacetate, which of water, to produce malate, which completes the cycle and regenerates the substrate to an oxidizable CHOH group. Oxidation involves NAD⁺, which reacts with further acetyl-CoA.

This sequence of reactions, namely oxidation of CH₂-CH₂ to CH=CH, then hydration to CH₂-CHOH, followed by oxidation to CH₂-CO, is a sequence we shall meet again in the P-oxidation of fatty acids.

The first oxidation utilizes FAD as a coenzyme and the second NAD⁺. In both cases, participation in the oxidative phosphorylation system allows the regeneration of the oxidized coenzyme and the subsequent generation of energy in the form of ATP.

The energy yield from the Krebs cycle by the aerobic breakdown of pyruvate may be calculated as follows. overall:

CH₃COCO₂H + 3 H₂O → 3 CO₂ + 5 x 2H

• five pairs of hydrogen atoms are available for oxidation;
• four pairs are passed to NAD⁺ and via the respiratory chain yield 4 x 3 = 12 mol ATP;
• one pair is passed to FAD and via the respiratory chain yields 2 mol ATP;
• there is also the gain of one ATP via GTP;
• therefore, there will be a total yield of 15 mol ATP.

By combining the glycolytic pathway, the Krebs cycle, and oxidative phosphorylation, the energy yield from the aerobic degradation of glucose will be

Total = 38 mols ATP

The total yield of 38 mol ATP by aerobic degradation of glucose may not be achieved under all circumstances, but it is, nevertheless, considerably more efficient than that from the anaerobic breakdown, namely 2 mol ATP.

Oxidation Of Fatty Acids

Fat degradation provides a major source of energy for most organisms. Fats are esters of glycerol with long-chain fatty acids and are hydrolyzed by the action of enzymes called lipases. This gives the alcohol portion, glycerol, together with a range of fatty acids, such as stearic acid.

Most fats taken in the diet provide a range of fatty acids of varying chain length and different levels of unsaturation, according to the source. Because of their biosynthetic origin, the vast majority of fatty acids have an even number of carbon atoms.

Glycerol provides a minor source of energy, in that it can be modified readily to glyceraldehyde 3- phosphate, one of the intermediates in the glycolytic pathway.

The fatty acids are metabolized by a process termed β-oxidation, which involves the sequential removal of two-carbon units via oxidation at the β- position. The process for saturated fatty acids will now be described.

Metabolism Of Saturated Fatty Acids

The free fatty acid needs activating before it can be metabolized. This is achieved by conversion into its thioester by esterification with coenzyme A.

We have already seen that thioesters are reactive entities, and it is reasonable, therefore, to suppose that such activation will cost energy. It is achieved in a two-stage reaction catalyzed by a single enzyme, an acyl-CoA synthetase. Energy is supplied in the form of ATP.

The fatty acid is initially converted into an acyl- AMP derivative by attack of the carboxylate as a nucleophile onto the P=O system of ATP, with loss of diphosphate as a leaving group.

This reaction is far from favorable, and the equilibrium is disturbed by subsequent pyrophosphatase-catalyzed hydrolysis of diphosphate into two molecules of phosphate.

This means that the energy demands (ATP →  AMP) are equivalent to two ATP → ADP transformations. However, the product fatty acyl-AMP is actually a reactive mixed anhydride and may be attacked by the thiol group of coenzyme A, giving the required thioester. We have met an analogous series of reactions in non-ribosomal peptide biosynthesis.

Oxidation at the β-position is then achieved by the same sequence of dehydrogenation, hydration, and dehydrogenation reactions that we have seen earlier in the succinate → fumarate → malate → oxaloacetate transformations in the Krebs cycle.

Because of the enzyme specificity in the hydration step, the new carbonyl group is introduced P to the original thioester carbonyl.

The sequence includes two dehydrogenation reactions and involves both FAD and NAD⁺ as coenzymes. The reduced forms of these coenzymes can be reoxidized by means of oxidative phosphorylation, and can, therefore, yield ATP.

Although the reactions just described form the basis of β-oxidation, the terminology β-oxidation when applied to fatty acid metabolism is usually understood to include the next step, the sequential chain shortening.

There follows cleavage of acetyl-CoA from the end of the chain via a reverse Claisen reaction. This requires the use of a molecule of coenzyme A as a nucleophile, with the loss of the enolate anion of acetyl-CoA as the leaving group.

The net result is the production of a new fatty acyl-CoA that is two carbons shorter than the original and a molecule of acetyl-CoA that can be metabolized via the Krebs cycle.

Thus, a fatty acid such as stearic acid (C₁₈), after activation, can undergo the P-oxidation and chain-shortening process eight times, producing nine molecules of acetyl-CoA for further metabolism.

stearic acid can undergo P-oxidation / chain shortening 8x

CH₃(CH₂)₁₆CO₂H —► 9 CH₃CO-SCoA ⇒ Krebs cycle

The overall energy yield from β-oxidation may thus be calculated as follows:

• Each sequence of β-oxidation involves the passage of one pair of hydrogen atoms to FAD (which yields 2 mol ATP via the respiratory chain) and one pair to NAD⁺ (which yields 3 mol ATP via the respiratory chain).
• Stearoyl-CoA thus produces 8 x 5 = 40 mol ATP from eight β-oxidations.
• The nine acetyl-CoA moles generated will yield 9 x 12 = 108 mol ATP via the Krebs cycle.
• However, the activation of stearic acid to stearoyl- CoA is achieved by the reaction ATP → AMP, which is the effective loss of 2 mol ATP; nevertheless, only one activation step is necessary per fatty acid.
• The total yield is thus 40 + 108 — 2 = 146 mol ATP.

Comparison Of Fat And Carbohydrate As Energy Stores

It is instructive to compare the energy yield from three molecules of glucose (C₁₈) with that from one molecule of stearic acid (also C₁₈).

• We saw that aerobic degradation of each molecule of glucose via glycolysis and the Krebs cycle gave 38 mol ATP; three molecules would thus give 3 x 38 = 114 mol ATP.
• Stearic acid gives 146 mol ATP. The higher energy yield per carbon atom from fatty acid compared with carbohydrates reflects its higher level of reduction, which consequently allows more oxidation.
• Thus, fat is logically the preferred storage molecule for carbohydrates. This is borne out in practice.
• A 70 kg man would typically have fat reserves of about 7 kg, equivalent to his energy needs for 1 month, and carbohydrate reserves of about 0.35 kg, equivalent to his energy needs for only about 1 day.
• This is undoubtedly why low-carbohydrate diets have proved so effective for rapid weight loss. As soon as the reserves of carbohydrates are used up, the body resorts to metabolizing fat for its energy needs. This continues whenever carbohydrate intake is limited.

It should also be appreciated that although carbohydrates can readily be converted into fat, fat is not readily converted into carbohydrates in animals. Fat metabolism produces acetyl-CoA, which is then usually metabolized completely via the Krebs cycle.

Metabolism Of Unsaturated Fatty Acids

Much of the fat taken in via the diet will contain unsaturated fatty acids, particularly that portion that originates from plant material. For example, the fats in olive oil contain up to 85% oleic acid (C₁₈ unsaturated), and only relatively small amounts of saturated fatty acids.

Animal fats have a much higher proportion of saturated fatty acid derivatives, but they still contain a substantial level of unsaturated fatty acids.

The fatty acid analysis of butterfat, for example, shows it contains about 28% oleic acid, and most of the remainder is composed of saturated fatty acids: 13% stearic acid (C₁₈), 29% palmitic acid (C₁₆), 12% myristic acid (C₁₄), and other shorter- chain saturated fatty acids.

The vast majority of natural unsaturated fatty acids have one or more double bonds with the Z or cis configuration.

The metabolism of unsaturated fatty acids is similar to that of the saturated compounds just described, but additional enzymic reactions are necessary.

Thus, oleoyl-CoA, the CoA ester of oleic acid, will undergo β-oxidation three times, until the C₁₂ derivative is reached. The 3,4-Z-double bond in this compound now prevents the normal dehydrogenation step that should introduce a 2,3-double bond.

As a result, the normal degradative process stops until this compound is isomerized to the normal intermediate with a 2,3-E-double bond by the action of an isomerase enzyme.

By means of this additional step, the P-oxidation process can then continue as normal. The energy yield will be only slightly less than that for stearoyl-CoA since there is the omission of transfer of one pair of hydrogen atoms to FAD, and consequently loss of 2 mol ATP.

Of course, the double bond in the starting ester may end up in the correct position for the P-oxidation processes, but it turns out that the usual Z or cis configuration of this double bond is wrong for normal enzymes.

Although hydration of the Z double bond occurs, the configuration of the hydroxy derivative is wrong for the subsequent dehydrogenase, so an inversion to the required configuration is achieved by the action of an epimerase enzyme. β-Oxidation processes can then continue normally.

These processes are shown for the CoA ester of linoleic acid, the most common of the polyunsaturated acids.

Synthesis Of Fatty Acids

Fatty acid synthesis provides an organism with a means of storing energy in the form of an organic reverse Claisen reaction molecule that can be degraded by oxidative reactions whenever necessary.

In principle, fatty acid synthesis is the reverse of fatty acid metabolism, though there are some fundamental differences, which are quite logical when we consider the chemical reactivity of the intermediate reagents.

Fatty acid degradation involves a reverse Claisen reaction Therefore, we could consider using the Claisen reaction in fatty acid synthesis.

However, a more favorable pathway is used, employing a more reactive nucleophile. Rather than using the enolate anion derived from acetyl-CoA, nature uses the enolate anion derived from malonyl-CoA. Malonyl-CoA is obtained from acetyl-CoA by means of an enzymic carboxylation reaction, incorporating CO₂ (usually from the soluble form bicarbonate).

Now CO₂ is a particularly unreactive material, so this reaction requires the input of energy (from ATP) and the presence of a suitable coenzyme, biotin, as the carrier of CO₂.

The conversion of acetyl-CoA into malonyl-CoA increases the acidity of the α-hydrogens since the acidic protons are flanked by two carbonyl groups, and thus it is easier to generate a nucleophile for the Claisen condensation. We should relate this to the use of diethyl malonate rather than ethyl acetate as a nucleophile.

The Claisen reaction can now proceed smoothly, but nature introduces another little twist. The carboxyl group introduced into malonyl-CoA is simultaneously lost by a decarboxylation reaction during the Claisen condensation.

Accordingly, we now see that the carboxylation step helps to activate the α- carbon and facilitate Claisen condensation, and the carboxyl is immediately removed on completion of this task.

An alternative rationalization is that decarboxylation of the malonyl ester is used to generate the acetyl enolate anion without any requirement for a strong base.

The processes of fatty acid biosynthesis are catalyzed by the enzyme fatty acid synthase. In animals, this is a multifunctional protein containing all of the catalytic activities required, whereas in bacteria and plants, it is an assembly of enzymes that can be separated.

Acetyl-CoA and malonyl-CoA themselves are not involved in the condensation step: they are converted into enzyme-bound thioesters.

The Claisen reaction follows, giving the acetoacetyl thioester (β-ketoacyl-SEnz; R=H), which is reduced stereospecifically to the corresponding P- hydroxy ester, consuming NADPH in the reaction. Then follows the elimination of water, giving the E (trans) α,β-unsaturated ester.

Reduction of the double bond again utilizes NADPH and generates a saturated acyl-SEnz (fatty acyl-SEnz; R=H) that is two carbons longer than the starting material.

This can feed back into the system, condensing again with malonyl thioester, and going through successive reduction, dehydration, and reduction steps, gradually increasing the chain length by two carbons for each cycle, until the required chain length is obtained.

At that point, the fatty acyl chain can be released as a fatty acyl-CoA or as a free acid. The chain length actually elaborated is probably controlled by the specificity of the thioesterase enzymes that subsequently catalyze release from the enzyme.

Note that the reduction, dehydration, and reduction steps are essentially the reverse of the oxidation, hydration, and oxidation steps in fatty acid metabolism, though the enzymes and coenzymes involved are different.

Fatty Acid Synthase

Fatty acid synthesis is catalyzed in animals by the enzyme fatty acid synthase, which is a multifunctional protein containing all of the catalytic activities required.

Bearing in mind the necessity to provide a specific binding site for the various substrates involved, and then the fairly complex sequence of reactions carried out, it raises the question of just how it is possible for this process to be achieved at the enzymic level.

Nature has devised an elaborate but satisfyingly simple answer to this problem.

The fatty acid synthase protein is known to contain an acyl carrier protein (ACP) binding site, and also an active-site cysteine residue in the β-ketoacyl synthase domain.

Acetyl and malonyl groups are successively transferred from coenzyme A esters and attached to the thiol groups of Cys and ACP.

The Claisen condensation occurs, and the processes of reduction, dehydration, and reduction then occur whilst the growing chain is attached to ACP. The ACP carries a phosphopantetheine group exactly analogous to that in coenzyme A (pantothenic acid: vitamin B₅).

This phosphopantetheine group provides a long flexible arm, enabling the growing fatty acid chain to reach the active site of each enzyme in the complex, and allowing the different chemical reactions to be performed without releasing intermediates from the enzyme.

The chain is then transferred to the thiol of Cys, and the process can continue.

Making the process even more efficient, animal fatty acid synthase is a dimeric protein containing two catalytic centers, and it is able to generate two growing chains at the same time.

The monomeric subunits are also arranged head to tail so that the acyl group of one unit actually picks up a malonyl extender from the other unit.

It is interesting that the sequence of enzyme activities along the protein chain of the enzyme complex does not correspond with the order in which they are employed.

A similar approach is employed in the formation of peptides such as peptide antibiotics.

In marked contrast to the ribosomal biosynthesis of proteins, where a biological production line interprets the genetic code, many natural peptides are known to be synthesized by a more individualistic sequence of enzyme-controlled processes, in which each amino acid is added as a result of the specificity of the enzyme involved.

The many stages of the whole process appear to be carried out by a multi-functional enzyme termed a non-ribosomal peptide synthase.

Pantothenic acid bound to the enzyme as pantothenic is used to carry the growing peptide chain through its thiol group.

The long ‘pantothenic arm’ allows different active sites on the enzyme to be reached in the chain assembly process, a process remarkably analogous to the fatty acid synthase mechanism.

Amino Acids And Transamination

The synthesis of amino acids depends upon the ami- nation of the Krebs cycle intermediate 2-oxoglutarate to glutamate, a process of reductive amination. This can occur when a high concentration of ammonium ions is available and involves NADH or NADPH as a reducing agent.

The reaction involves the formation of an imine through the reaction of ammonia with the ketone, followed by the reduction of this imine. As we noted earlier, nicotinamide coenzymes may also participate in imine reductions as well as aldehyde/ketone reductions, further emphasizing the imine-carbonyl analogy.

The reverse reaction, removal of ammonia from glutamate, is also of importance in amino acid catabolism.

Glutamate can then participate in the formation of other amino acids via the process called transamination. Transamination is the exchange of the amino group from an amino acid to a keto acid and provides the most common process for the introduction of nitrogen into amino acids and for the removal of nitrogen from them.

The reaction is catalyzed by a transaminase enzyme, and the coenzyme pyridoxal phosphate (PLP) is required.

The process initially features the formation of an imine intermediate (aldimine) using the amine group of the amino acid with the aldehyde group of PLP. The imine function formed is conjugated with the aromatic pyridine ring.

Accordingly, protonation of the pyridine nitrogen (as would occur at physiological pH) makes the α-hydrogen of the original amino acid considerably more acidic.

This can be removed, in a process rather like that seen with conjugated carbonyl compounds. This generates a dihydropyridine ring system; the process is effectively imine-enamine tautomerization, though in an extended conjugated system.

Deprotonation then produces a new imine (ketamine) and also restores aromaticity in the pyridine ring. However, because of the conjugation, it allows protonation at a position that is different from where the proton was originally lost.

The net result is that the imine double bond has effectively moved to a position adjacent to its original position. Hydrolysis of this new imine group generates a keto acid and pyridoxamine phosphate.

The remainder of the sequence is now a reversal of this process. This now transfers the amine function from pyridoxamine phosphate to another keto acid.

The glutamic acid-2-oxoglutaric acid couple features as the usual donor-acceptor molecules for the amino group, and glutamate transaminase is thus the most important of the transaminases.

Transamination allows the amino group to be transferred from glutamic acid to a suitable keto acid. In the reverse mode, the amino group can be transferred from an amino acid to 2-oxoglutaric acid.

Equilibrium constants for the reactions catalyzed by transaminases are close to unity, so the reactions proceed readily in either direction.

It now becomes possible, as shown, to transfer the amino group of one amino acid, which may be readily available, to provide another amino acid, which could be in short supply.

This has obvious advantages over the process seen for glutamate synthesis via the reductive amination of 2-oxoglutarate, in that it no longer requires the intervention of free ammonia.

We thus have the situation that some organisms are able to carry out the fixation of ammonia via reductive amination, whereas others manipulate via transamination the amino acid structures obtained from protein in the diet.

PLP-dependent Reactions

We have just noted the role that pyridoxal phosphate plays as a coenzyme (cofactor) in transamination reactions. Pyridoxal 5′-phosphate (PLP) is crucial to a number of biochemical reactions.

PLP, together with a number of closely related materials that are readily converted into PLP, for example, pyridoxal, pyridoxine, and pyridoxamine, are collectively known as vitamin B₆, which is essential for good health.

At neutral pH, PLP is considerably ionized, so that the phenol group loses a proton and the pyridine nitrogen is protonated. Of course, the phosphate will also be ionized.

These ionic centers facilitate binding to the enzyme, but, for clarity, they are omitted from the mechanisms shown. However, the positively charged nitrogen is essential for the cofactor’s chemical reactivity, and we need to invoke it in the mechanisms.

In transamination, we have seen the formation of an imine, in which the protonated nitrogen acts as an electron sink, making the α-hydrogen of the original amino acid acidic and facilitating its removal.

The reversal of this process could potentially occur with deprotonation from either face of the C=N double bond, and a mixture of aldimines would result, leading to the generation of a racemic amino acid. This accounts for the mode of action of PLP-dependent amino acid racemase enzymes.

Of course, the enzyme controls the removal and supply of protons; this is not a random event. One important example of this reaction is the alanine racemase, employed by bacteria to convert L-alanine into D-alanine for cell wall synthesis.

In transamination and racemization reactions, we have seen the loss of a proton from the aldimine, i.e. breaking of bond a. Let us now consider the two alternatives, namely the breaking of bonds b or c, to explore further the scope for PLP-dependent reactions.

Breaking of bond b accounts for PLP-dependent decarboxylations. Decarboxylation of the intermediate aldimine is facilitated in the same way as the loss of a proton in the transamination sequence.

The protonated nitrogen acts as an electron sink, and the conjugated system allows the loss of the carboxyl proton, with subsequent bond breaking and loss of CO₂.

The resultant imine may subsequently be hydrolyzed, releasing an amine (the decarboxylated amino acid) and regenerating PLP. There are many examples of decarboxylation of amino acids.

PLP- Dependent Amino Acid Decarboxylations

An important example of PLP-dependent amino acid decarboxylation is the conversion of histidine into histamine. Histamine is often involved in human allergic responses, for example, to insect bites or pollens.

Stress stimulates the action of the enzyme histidine decarboxylase and histamine is released from mast cells. Topical antihistamine creams are valuable for pain relief, and oral antihistamines are widely prescribed for nasal allergies such as hay fever.

Major effects of histamine include dilation of blood vessels, inflammation and swelling of tissues, and narrowing of airways. In serious cases, life-threatening anaphylactic shock may occur, caused by a dramatic fall in blood pressure.

The catecholamines noradrenaline (norepinephrine) and adrenaline (epinephrine) are amines derived via decarboxylation of amino acids. Noradrenaline is a mammalian neurotransmitter and adrenaline, the ‘fight or flight’ hormone, is released in animals from the adrenal gland as a result of stress.

These compounds are synthesized by successive hydroxylation and N-methylation reactions on dopamine.

Dopamine is the decarboxylation product of DOPA, dihydroxyphenylalanine, and is formed in a reaction catalyzed by DOPA decarboxylase. This enzyme is sometimes referred to as aromatic amino acid decarboxylase, since it is relatively non-specific in its action and can catalyze the decarboxylation of other aromatic amino acids, for example, tryptophan and histidine.

DOPA is itself derived by aromatic hydroxylation of tyrosine, using tetrahydrobiopterin as a cofactor.

The neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) is formed from tryptophan by hydroxylation and then decarboxylation, paralleling the tyrosine → dopamine pathway.

The non-specific enzyme aromatic amino acid decarboxylase again catalyzes the decarboxylation.

5-HT is a neurotransmitter found in cardiovascular tissue, the peripheral nervous system, blood cells, and the CNS. It mediates many central and peripheral physiological functions, including contraction of smooth muscle, vasoconstriction, food intake, sleep, pain perception, and memory, a consequence of it acting on several distinct receptor types.

Although 5-HT may be metabolized by monoamine oxidase, platelets, and neurons possess a high-affinity 5-HT re-uptake mechanism. This mechanism may be inhibited by the widely prescribed antidepressant drugs termed selective serotonin reuptake inhibitors (SSRI), for example, fluoxetine (Prozac®), thereby increasing levels of 5-HT in the CNS.

Yet another neurotransmitter, γ-aminobutyric acid or GABA, is formed by PLP-dependent decarboxylation of an amino acid, in this case, glutamic acid.

GABA acts as an inhibitory transmitter in many different CNS pathways. It is subsequently destroyed by a transamination reaction in which the amino group is transferred to 2-oxoglutaric acid, giving glutaric acid and succinic semialdehyde.

This also requires PLP as a cofactor. Oxidation of the aldehyde group produces succinic acid, a Krebs cycle intermediate.

The breaking of bond c is going to be less common than deprotonation or decarboxylation. In most amino acids, R is an alkyl group, so there is little chance of losing R as a cation. Indeed, the only occasions on which we can break bond c are when R is hydroxymethyl (as in serine) or a similar grouping (as in threonine).

In both cases, bond breaking is facilitated by the hydroxyl, in that the lone pair can feed into the conjugated system. The result in the case of serine is the loss of formaldehyde, whereas, in the case of threonine, it is the loss of acetaldehyde.

The fragment attached to pyridoxal will be the same in both cases and after hydrolysis, it is released as the amino acid glycine.

In case this seems a bit complicated, consider the reverse reaction, which would be an attack of an electron-rich system onto the carbonyl group of an aldehyde, i.e. an aldol reaction. Therefore, what we are seeing here is merely a reverse aldol-type reaction.

It is interesting to see how different products are formed according to which of the three different bonds is cleaved in the aldimine derived from an amino acid and PLP.

There is one other point to ponder though. What determines the type of cleavage that occurs?

The answer must lie in the enzyme and how it binds the substrate, and it merely becomes a consideration of stereochemistry. By considering the shape of the aldimine, we see that the pyridine ring and the adjacent C=N double bond must be planar to achieve maximum orbital overlap and conjugation.

Electrons from the bond that is broken should feed smoothly into this planar conjugated system. This requires the bond to be positioned at right angles to the plane and, therefore, parallel to the p orbitals.

As shown in the accompanying diagrams, rotation about the N-C bond positions the vulnerable group towards the exterior face of the enzyme so that it can be attacked.

Do appreciate that one enzyme will not catalyze all three types of reaction. We need different enzymes to accomplish a particular reaction on a particular substrate.

Rotation about the N-C bond positions the vulnerable group for reaction; it also positions the R and/or CO₂^– groups so that they can interact and bind to the enzyme, thus providing specificity.

One further point for the sake of accuracy; we have omitted it to simplify the mechanistic features.

PLP does not exist as the free aldehyde when it is bound to the enzyme, but actually uses the aldehyde group in its binding.

An imine linkage is formed between the aldehyde and the primary amine group of a lysine residue in the enzyme active site. When the substrate RNH₂ binds to the enzyme to produce the intermediates shown above, it achieves this by a transamination reaction.

A similar transamination, in the reverse sense, takes place at the end of the reaction sequence to displace the product but still retains PLP bound to the enzyme via the lysine group.

Should the chemistry seem a little complicated, remind yourself of the mechanism for hemiacetal and acetal formation; that featured an oxygen analogy for the transimination sequence.

If we had included this additional series of reactions in the above descriptions, it would have obscured the simple principles of the PLP-dependent reactions.

TPP-Dependent Reactions

Thiamine diphosphate (TPP) is the coenzyme for the pyruvate dehydrogenase complex that catalyzes the oxidative decarboxylation of pyruvate to acetyl- CoA and thus links the glycolytic pathway to the Krebs cycle. Later in the Krebs cycle, TPP is the cofactor for the 2-oxoglutarate dehydrogenase complex, which catalyzes a similar reaction on 2-oxoglutarate.

In the glycolytic pathway, the pyruvic acid → acetaldehyde conversion also features TPP. A further enzyme, transketolase, has the unexpected property of transferring a two-carbon fragment between carbohydrates in the pentose phosphate pathway, and TPP is again involved.

The biosynthetic pathways to two amino acids, valine, and isoleucine, also involve TPP-dependent enzymes. All of these reactions employ TPP as a carrier of an acyl anion equivalent.

TPP thus plays a very important role in carbohydrate metabolism. The parent alcohol thiamine is one of the B group vitamins, namely vitamin B₁; dietary deficiency leads to the condition beriberi, characterized by neurological disorders, loss of appetite, fatigue, and muscular weakness.

The conversion of pyruvic acid into acetyl-CoA is conveniently written according to the equation

This simple equation conceals a quite complex reaction sequence involving not just TPP, but other coenzymes, including lipoic acid. Let us first inspect the nature of TPP.

With its pyrimidine ring and diphosphate grouping, TPP looks rather like a nucleotide, but the central ring system is a thiazole rather than a sugar.

This heterocyclic ring is alkylated on nitrogen and is thus a thiazolium salt. This plays a key role in the reactivity of TPP.

The proton in the thiazolium ring is relatively acidic (pK_a about 18) and can be removed by even weak bases to generate the carbanion or ylid. A ylid (also ylide) is a species with positive and negative charges on adjacent atoms; this ylid is an ammonium ylid with extra stabilization from the sulfur atom.

This ylid can act as a nucleophile and is also a reasonable leaving group. In addition, the carbonyl group of pyruvic acid is followed by decarboxylation, the positive nitrogen in the ring acting as an electron sink.

The resulting molecule is an enamine, but because of the neighboring heteroatoms, it is extremely electron-rich. It accepts a proton, and this achieves tautomerism of the enamine to the iminium ion.

This is followed by a reverse aldol reaction, which also regenerates the ylid as the leaving group. This sequence would thus accommodate the pyruvic acid → acetaldehyde conversion catalyzed by pyruvate decarboxylase in the glycolytic pathway.

In oxidative decarboxylation of pyruvate to acetyl- CoA, the enzyme-bound disulfide-containing coen¬zyme lipoic acid is also involved. The electron-rich enamine intermediate, instead of accepting a proton, is used to attack sulfur in the lipoic acid moiety.

This leads to fission of the S-S bond, and thereby effectively reduces the lipoic acid fragment. Regen¬eration of the TPP ylid via the reverse aldol-type reaction leaves the acetyl group bound to the dihydrolipoic acid as a thioester.

This acetyl group is then released as acetyl-coA by displacement with the thiol coenzyme A. The bound dihydrolipoic acid frag¬ment must then be reoxidized to restore its function.

An exactly equivalent reaction is encountered in the Krebs cycle in the conversion of 2-oxoglutaric acid into succinyl-coA.

Each of the complexes pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase actually contains three enzyme activities. We can now readily appreciate what the separate activities might be.

In the case of pyruvate dehydrogenase, the individual activities are pyruvate dehydrogenase (requires cofactor TPP), dihydrolipoamide acyltransferase (requires cofactors lipoic acid and coenzyme A), and dihydrolipoamide dehydrogenase (requires cofactors FAD and NAD⁺).

The requirement for NAD⁺ is to reoxidize FADH₂ after regeneration of lipoic acid. The ‘lipoamide’ terminology indicates that the lipoic acid is enzyme bound through its carboxylic acid group to an amino group of lysine via an amide linkage.

At the end of the first paragraph in this section, we stated ‘All these reactions employ TPP as a carrier of an acyl anion equivalent’. An acyl anion is an unlikely species since we would consider locating a negative charge on the carbon of a carbonyl group as definitely unfavorable.

However, the following scheme should emphasize how we can consider that TPP is effectively removing and transferring an acetyl anion equivalent in the above reactions.

TPP And Acyl Anion Equivalents

A slightly different acyl anion equivalent is transferred in transketolase reactions, and this anion is then used in a subsequent aldol reaction.

Transketolase removes a two-carbon fragment from keto sugars such as xylulose 5-phosphate (alternatively fructose 6-phosphate or sedoheptulose 7-phosphate) through the participation of the thiamine diphosphate ylid.

The nucleophilic attack of this ylid onto the ketone carbonyl results in an addition product that then fragments by a reverse aldol reaction. This generates a chain-shortened aldose, for example, glyceraldehyde 3-phosphate from xylulose 5-phosphate, and the two-carbon acyl anion equivalent attached to TPP.

Then, in what is formally a reverse of this reaction, this carbanion equivalent can attack another aldose, such as erythrose 4-phosphate, extending its chain length by two carbons.

Transketolase is crucial to metabolism in creating a link between the pentose phosphate pathway and glycolysis.

Reactions Catalysed By Transketolase Enzymes:

An additional enzyme that transfers C₃ rather than C₂ units is called transaldolase, but, in common with aldolase, this enzyme utilizes an imine-enamine mechanism through an imine link with lysine, and does not involve TPP.

Biotin-Dependent Carboxylations

We have briefly noted the role of biotin when we considered the biosynthesis of fatty acids. Biotin is a carrier of carbon dioxide and is involved in carboxylation reactions.

In fatty acid biosynthesis, we noted how acetyl-CoA was transformed by carboxylation into the more effective nucleophilic agent malonyl-CoA, thus facilitating the Claisen reaction.

Structurally, biotin (vitamin H) is composed of two fused five-membered heterocycles, a cyclic urea, and a cyclic sulfide (tetrahydrothiophene).

Carbon dioxide is a normally unreactive material, and in combination with biotin requires the input of energy (from ATP). Carbon dioxide is usually present as the soluble form of bicarbonate, and this reacts with ATP to form a mixed anhydride, as part of the reaction catalyzed by the carboxylase.

This mixed anhydride carboxylates the coenzyme in a biotin-enzyme complex. Biotin is bound to a lysine residue in the enzyme as an amide. The carboxylation reaction is effectively a nucleophilic attack of the cyclic urea on the mixed anhydride.

In what can be considered a reversal of this sequence, the acetyl-CoA acts as the nucleophile and is carboxylated to malonyl-CoA with displacement of the biotin–enzyme system.

Fixation of carbon dioxide by biotin–enzyme complexes is not unique to acetyl-CoA, and another important example occurs in the generation of
oxaloacetate from pyruvate in the synthesis of glucose from non-carbohydrate sources (gluconeogenesis).

This reaction also allows replenishment of Krebs cycle intermediates when compounds are drawn off for biosynthetic purposes, for example, amino acid synthesis.

The post The Organic Chemistry Of Intermediary Metabolism appeared first on BDS Notes.

The nucleic acids DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are the molecules that play a fundamental role in storing genetic information, and the subsequent manipulation of this information. They are polymers whose building blocks are nucleotides, which are themselves combinations of three parts: a heterocyclic base, a sugar, and phosphate.

The most significant difference in the nucleotides comprising DNA and RNA is the sugar unit, which is deoxyribose in DNA and ribose in RNA. The term nucleoside is used to represent a nucleotide lacking the phosphate group, i.e. the base–sugar combination.

The general structure of nucleotides and nucleosides is shown below:

Before we analyze nucleotide structure in detail, it is perhaps best that we consider the nature of the various parts. In nucleic acid structures, there are five different bases and two different sugars.

The bases are monocyclic pyrimidines (see or bicyclic purines and all are aromatic. The two purine bases are adenine (A) and guanine (G), and the three pyrimidines are cytosine (C), thymine (T) and uracil (U). Uracil is found only in RNA, and thymine is found only in DNA. The other three bases are common to both DNA and RNA. The heterocyclic bases are capable of existing in more than one tautomeric form. The forms shown here are found to predominate in nucleic acids. Thus, the oxygen substituents are in keto form, and the nitrogen substituents exist as amino groups.

Sugars:

The two sugars are pentoses, D-ribose in RNA and 2-deoxy-D-ribose in DNA. In all cases, the sugar is present in a five-membered acetal ring form, i.e. a furanoside. The base is combined with the sugar through an N-glycoside linkage at C-1, and this linkage is always β.

Purine bases are linked through N-9, and pyrimidines through N-1. When numbering nucleosides and nucleotides, we use primed numbers for the sugar, since non-primed numbers are already employed in the base part. There are thus four different nucleosides for each type of nucleic acid, as shown.

Nucleosides in RNA:

Nucleosides in DNA:

The phosphate group of nucleotides is attached via a phosphate ester linkage, and may be attached to either C-5′ or C-3′. As we shall see, nucleosides in nucleic acids are joined together through a phosphate linkage between the 3′-hydroxyl of one sugar and the 5′-hydroxyl of another. As a result, hydrolysis of nucleic acid could give us nucleotides containing either 5′- or 3′-phosphate groups. It is usual, however, to consider nucleic acids as composed of nucleotides containing a 5′-phosphate group.

The accepted nomenclature for the various components in RNA and DNA

Nomenclature of bases, nucleosides, and nucleotides:

Nucleic Aids

1. DNA

The nucleic acids comprise a long unbranched chain of nucleotide monomeric units. The nucleotides are linked together via the phosphate group, which joins the sugar units through ester linkages, usually referred to as phosphodiester bonds.

• The phosphodiester bond links the 5′ position of one sugar with the 3′ position of the next. A short portion of a DNA molecule
• The nucleic acid chain is thus composed of alternating units of sugar and phosphate, with the bases appearing as side chains from the sugar components. The nucleotide chain has ends, referred to as the 5′- and 3′-ends, according to the sugar hydroxyl that is available for further bonding.
• Though we shall not be considering this aspect further, in some organisms, especially bacteria and some viruses, the two ends of the DNA chain are joined together so that we encounter a circular form of DNA.
• Nucleic acid structures generally need to be written in a much-abbreviated form. The sugar-phosphate backbone is taken for granted; it can be indicated by a line, with the attached bases defined.
• Even this is tedious. It is thus reduced further to the sequence of attached bases. The base sequence of the nucleic acid is the standard way of defining its structure; strictly, the structure is a sequence of nucleotides.
• By convention, the base sequence is written from the 5′-end to the 3′-end, so that the short strand of DNA would be given as–ACGT–

Perhaps the most far-reaching feature of nucleic acids is the ability of the bases to hydrogen bond to other bases. This property is fundamental to the double helix arrangement of the DNA molecule, and the translation and transcription via RNA of the genetic information present in the DNA molecule.

The polymeric strand of DNA coils into a helix, and it is bonded to a second helical strand by hydrogen bonds between appropriate base pairs. In DNA, the base pairs are adenine–thymine and guanine-cytosine. It should be appreciated that each of these bases is planar and that the hydrogen-bonded base pair is also planar. The hydrogen-bonded N–H–N and N–H–O interatomic distances are in the range 2.8–3.0 A. By comparison, N–H and O–H˚ bonds are typically about  1.0 Å.

Thus, each purine is specifically linked to a pyrimidine by either two or three hydrogen bonds. The result of these interactions is that each nucleotide recognizes and bonds with its complementary partner.

This specific base pairing means that the two strands in the DNA double helix are complementary. Wherever adenine appears in one strand, thymine appears opposite it in the other; wherever cytosine appears in one strand, guanine appears opposite it in the other. We shall see later the significance of base pairing between adenine and uracil. The latter base is found in RNA instead of thymine.

The DNA double helix has both chains twisting on a common axis. The bases are directed inwards to allow hydrogen bonding, and the sugar and phosphodiester parts of the main chain form the outside portion.

The planes of the base pairs are perpendicular to the helix axis so that the molecule looks like a spiral staircase with the base-pair combinations forming the treads.

The helix makes A complete turn every 10 base pairs along the chain. The two strands are complementary:

If you know the sequence along one chain, you can write down the sequence along the other via the base pairing relationship. Note, however, that the chains are antiparallel, i.e. they run in opposite directions.

• This is indicated in the schematic diagram in
• One further point arises because the glycoside bonds between the sugars and bases of a particular base pair are not directly opposite each other.
• This is easily appreciated from the illustrations of hydrogen-bonded base pairings. The consequence of this is that the grooves along the outside of the double helix array are of unequal width, and are termed the major groove and the minor groove.
• These grooves contain many water molecules through interaction with amino and carbonyl groups of the bases, and are distinguishable to agents that bind to DNA,

Example: Some anticancer drugs.

2. Replication of DNA

During cell division, the DNA molecule is replicated so that each daughter cell will carry its DNA molecule. During the process, the two strands of DNA unwind, and each strand then acts as the template for the synthesis of a new strand; in each case, the new strand is complementary to the original because of the base-pairing restrictions.

Each new double helix is comprised of one strand that was part of the original molecule and one strand that is newly synthesized. Not surprisingly, this is a very simplistic description of a quite complex process, catalyzed by enzymes known as DNA polymerases.

The precursors for the synthesis of the new chain are the nucleoside triphosphates, dATP, dGTP, dTTP, and dCTP. We have already met ATP when we considered anhydrides of phosphoric acid; these compounds are analogs of ATP, though the sugar is deoxyribose rather than ribose.

These triphosphate anhydrides are susceptible to nucleophilic attack by hydroxyl groups. Chain extension is simply an esterification reaction utilizing the 3′-hydroxyl of the sugar in the growing chain, with diphosphate as a good leaving group.

• The correct nucleoside triphosphate is selected because of the hydrogen bonding properties of base pairs. This also provides the correct alignment so that the reaction can occur.
• In the illustration, the next base in the original DNA strand is thymine, which dictates that only an adenine nucleotide can hydrogen bond and form the complementary base pair.
• The esterification occurs, with the loss of diphosphate as the leaving group, and the new daughter strand is extended by one nucleotide.
• The process repeats as the enzyme moves on to the next position on the original DNA strand. Hydrolysis of diphosphate to two molecules of phosphate provides some of the driving force to facilitate the reaction.

3. RNA

RNA differs structurally from DNA in three important ways. First, as indicated above, the sugar in RNA is ribose, not 2-deoxyribose.

• Second, thymine is replaced by uracil, so that the four bases are adenine, uracil, guanine, and cytosine.
• The third difference is that RNA is usually single-stranded. Although an RNA molecule may be single-stranded, it does not exclude the possibility of partial double-stranded sequences being present. In such cases, the molecule doubles back on itself and coils up with a complementary base sequence elsewhere.
• Remember that complementary sequences now involve A–U rather than A–T hydrogen-bonding interactions.
• DNA stores the genetic information for a cell, but it is RNA that participates in the processes by which this information is used. RNA molecules are classified according to their function or cellular location.

Three major forms are found in prokaryotic cells:

1. Messenger RNA (mRNA) carries genetic information from DNA to ribosomes, the organelles responsible for protein synthesis;
2. Ribosomal RNA (rRNA) is an integral part of the ribosomes
3. Transfer RNA (tRNA) carries the amino acid residues that are added to the growing peptide chain during protein synthesis.

4. The genetic code

It is the sequence of bases along one of the strands of the DNA molecule, the coding strand, that provides the information for the synthesis of proteins, especially enzymes, in an organism.

• A complementary sequence exists along the second strand, and this is termed the template strand. A gene is a segment of DNA that contains the information necessary for the synthesis of one protein.
• Each amino acid in a protein is specified by a sequence of three nucleotides, termed a codon.
• A codon is usually designated in terms of the base sequence, however, just as we saw with nucleic acid sequences above. With four different bases, there are 43 = 64 different combinations of three bases (codons) available, more than enough for the 20 different amino acids found in proteins.
• Most amino acids can be specified by two or more different codons, and three particular codons are known to carry the signal for stop, i.e. chain termination.
• The signal for start is the same as for methionine (unusual in having only one codon rather than several) and means that all proteins should begin with a methionine residue.
• Since this is not the case, the inference is that many proteins are subsequently modified by cleaving off a fragment that contains this starter amino acid residue.

The codon combinations A codon can be the DNA sequence in the coding strand or the related sequence found in mRNA.The table shows the mRNA sequences since we shall be using these during consideration of protein synthesis. The DNA sequences merely have thymine (T) in place of uracil (U), as appropriate. The sequence is always listed from the 5′-end to the 3′-end.

The genetic code: mRNA sequences:

Transcription of DNA to mRNA:

5. Messenger RNA synthesis transcription

Although the amino acid sequence of a protein is defined by the sequence of codons in DNA, it is RNA that participates in the interpretation of this sequence and the subsequent joining together of amino acids. The process starts with the synthesis of mRNA, in a process called transcription.

• Part of the DNA double helix, corresponding to the gene in question, is unwound. Rather like in the replication of DNA, the base sequence is used to synthesize a new nucleic acid strand.
• However, this time only one strand is interpreted, the template strand, and ribonucleotides (ATP, GTP, CTP, and UTP) are used in the new chain assembly instead of deoxyribonucleotides.
• The sequence of ribonucleotides incorporated is dictated by the sequence of nucleotides in DNA and depends on hydrogen bonding between pairs of bases.
• In RNA synthesis, uracil nucleotides are employed rather than thymine nucleotides. The result is a synthesis of a single strand of RNA with a sequence analogous to the coding strand of DNA, except that U replaces
• T. Coupling of the ribonucleotide units is catalyzed by the enzyme RNA polymerase and is mechanistically the same as with DNA replication above, i.e. esterification of a hydroxyl via a phosphoric anhydride.

6. Transfer RNA and translation

Although messenger RNA is synthesized in the cell nucleus, it then moves to the cytoplasm and to the ribosomes, where protein biosynthesis occurs.

• These particles are composed of two subunits, termed 50S and 30S, and are combinations of rRNA and protein.
• The ribosomes are responsible for binding the two other types of RNA, mRNA (which contains the genetic code) and tRNA (which carries the individual amino acids).
• tRNA molecules are very small compared with the other forms of RNA, being less than 100 nucleotides.
• The size of mRNA reflects the number of amino acid residues in the protein being synthesized but could be a thousand or more nucleotides. rRNA is the most abundant of the three types of RNA, and in size covers a range from about 75 to 3700 nucleotides.
• A tRNA molecule is specific for a particular amino acid, though there may be several different forms for each amino acid. Although relatively small, the polynucleotide chain may show several loops or arms because of base pairing along the chain.
• One arm always ends in the sequence cytosine–cytosine–adenosine. The 3’hydroxyl of this terminal adenosine unit is used to attach the amino acid via an ester linkage.

However, it is now a section of the nucleotide sequence that identifies the tRNA–amino acid combination, and not the amino acid itself.

• A loop in the RNA molecule contains a specific sequence of bases, termed an anticodon, and this sequence allows the tRNA to bind to a complementary sequence of bases, a codon, on mRNA.
• The synthesis of a protein from the message carried in mRNA is called translation, and a simplified representation of the process as characterized in the bacterium.
• Escherichia coli is shown below. Initially, the amino acid is activated by an ATP-dependent process, producing an aminoacyl-AMP.
• A hydroxyl group in ribose, part of a terminal adenosine group of tRNA, then reacts with this mixed anhydride. In this way, the amino acid is bound to tRNA via an ester linkage as an aminoacyl-tRNA.

The tRNA involved will be specific for the particular amino acid. A detailed mechanism for this process has been considered in

The mRNA is bound to the smaller 30S subunit of the bacterial ribosome. The mRNA is a transcription of one of the genes of DNA and carries the information as a series of three-base codons. The message is read (translated) in the 5’to 3’direction along the mRNA molecule.

• The aminoacyl-tRNA anticodon (UAC) allows binding via hydrogen bonding to the appropriate codon (AUG) on mRNA. In prokaryotes, the first amino acid encoded in the sequence is N-formylmethionine (fMet).
• Although the codon for initiation ( N-formylmethionine) is the same as that for methionine, the initiator tRNA used is different from that employed for the incorporation of methionine elsewhere in the peptide chain.
• The initiator aminoacyl-tRNA is thus bound and positioned at the P (for peptidyl) site on the ribosome. The next aminoacyl-tRNA a tRNA specific for alanine) is also bound via a codon (GCG)–anticodon (CGC) interaction and is positioned at an adjacent A (for aminoacyl) site on the ribosome.

This allows peptide bond formation to occur, with the amino group of the amino acid in the A site attacking the activated ester in the P site. The peptide chain is thus initiated and has become attached to the tRNA located in the A site.

The tRNA at the P site is no longer required and is released from the ribosome. Then the peptidyl-tRNA at the A site is translocated to the P site by the ribosome moving along the mRNA a codon at a time, exposing the A site for a new aminoacyl-tRNA appropriate for the particular codon, and a repeat of the elongation process occurs. The cycles of elongation and translocation continue until a termination codon is reached, and the peptide or protein is then hydrolyzed and released from the ribosome.

Note that the protein is synthesized from the N-terminus towards the C-terminus. Some special features of proteins are elaborated by secondary transformations that are not part of the translation process. The N-formylmethionine initiator may be hydrolyzed to methionine, or, as we have already indicated, the methionine unit may be removed altogether. Other post-translational changes to individual amino acids may be seen,

Example:

The hydroxylation of proline to hydroxyproline or the generation of disulfide bridges between cysteine residues

Antibiotics that interface with ribosomal peptide biosynthesis

Many of the antibiotics used clinically are active because of their ability to inhibit protein biosynthesis in bacteria. The individual steps of protein biosynthesis all seem susceptible to disruption by specific agents.

Some specific examples are listed below:

•  Inhibitors of transcription
  • Rifampicin(inhibits RNA polymerase)
• Inhibitors of aminoacyl-tRNA binding to ribosome
  • Tetracyclines (bind to 30S subunit of the ribosome and prevent attachment of aminoacyl-tRNA)
• Inhibitors of translation
  • Streptomycin (binds to 30S subunit of the ribosome, causes mRNA to be misread)
  • Erythromycin (binds to 50S subunit of the ribosome, inhibits translocation)
  • Chloramphenicol (binds to 50S subunit, inhibits peptidyltransferase activity)

Naturally, if such materials are going to be useful as antibiotic drugs, we require a selective action. We need to be able to inhibit protein biosynthesis in bacteria, whilst producing no untoward effects in man or animals. Although the mechanisms for protein biosynthesis are essentially the same in prokaryotes and eukaryotes, there are some subtle differences, e.g. the ribosome and how the process is initiated. Without such differences, the agent would be toxic to man as well as to bacteria.

Nucleosides as antiviral agents

Viruses are responsible for many human and animal diseases, with a variety of symptoms and levels of severity.

• Common viral illnesses include colds, influenza, cold sores (herpes), and childhood infections such as chickenpox, measles, and mumps.
• More serious conditions include meningitis, poliomyelitis, and human immunodeficiency virus (HIV), the latter potentially leading to acquired immune deficiency syndrome (AIDS).
• Viruses are simpler than bacteria and consist essentially of nucleic acid (either DNA or RNA) enclosed in a protein coat.
• Those causing chickenpox, smallpox, and herpes belong to the DNA virus group, whereas those responsible for influenza, measles, mumps, meningitis, poliomyelitis, and HIV are classified as RNA viruses. Viruses have no metabolic machinery of their own, and for their very existence are intracellular parasites of other organisms.
• To survive and reproduce, they have to tap into the metabolic processes of the host organism.
• For this reason, it is difficult to find drugs that are selective towards viruses without damaging the host.
• Most antiviral agents are only effective whilst the virus is replicating, and viral replication is very far advanced by the time the infection is detectable. There are relatively few effective antiviral drugs, and most of these are nucleoside derivatives.

Aciclovir

Aciclovir (acyclovir) was one of the first effective selective antiviral agents. It is a guanine derivative of value in treating herpes viruses, though it does not eradicate them, and is only useful if drug treatment is started at the onset of infection.

Aciclovir is a member of a group of nucleoside derivatives termed acyclonucleosides, in that there is an incomplete sugar ring.

• The structural relationship to 2′-deoxyguanosine should be very clear.
• Aciclovir is converted into its monophosphate by the viral enzyme thymidine kinase – some viruses also possess enzymes that facilitate their replication in the host cell.
• The viral enzyme turns out to be much more effective than that of the host cell, and conversion is, therefore, mainly in infected cells.
• The monophosphate is subsequently converted into triphosphate by the host cell enzymes.
• Aciclovir triphosphate inhibits viral DNA polymerase, much more so than it does the host enzyme, and so terminates DNA replication.

Zidovudine:

Zidovudine is 3′-azido-3′-deoxythymidine and is a derivative of deoxythymidine in which an azide group replaces the 3′-hydroxyl.

It is better nown as the anti-AIDS drug AZT.

• The AIDS virus is an RNA retrovirus. In retroviruses, an enzyme reverse transcriptase makes a DNA copy of viral RNA (contrast transcription: making an RNA copy of DNA).
• This DNA copy is then integrated into the host genome and gets transcribed into both new viral RNA and mRNA for translation into viral proteins. AZT is an inhibitor of reverse transcriptase.
• AZT is phosphorylated by cellular enzymes to the triphosphate, which competes with normal substrates for the formation of DNA by reverse transcriptase and blocks viral DNA synthesis.
• Mammalian DNA polymerase is relatively unaffected, but there can be some toxic effects. AZT is used in AIDS treatment along with other antiretroviral drugs.

Some Other Important Nucleosides And Nucleotides ATP SAm Coenzyme A, NAD, FAD

The terminology nucleotide or nucleoside immediately directs our thoughts toward nucleic acids.

• Remarkably, nucleosides and nucleotides play other roles in biochemical reactions that are no less important than their function as part of nucleic acids.
• We also encounter more structural diversity.
• It is rare that the chemical and biochemical reactivities of these derivatives relate specifically to the base plus sugar part of the structure, and usually reside elsewhere in the molecule.

Almost certainly, it is this base plus sugar part of the structure that provides a recognition feature for the necessary enzymes that utilize these compounds.

ATP:

ATP, adenosine triphosphate, provides the currency unit for energy in biochemical reactions (see and is simply a triphosphate variant of a standard RNA nucleotide. It is, of course, the biosynthetic precursor for adenine-based units in RNA. As we have already seen, the functions of ATP can be related to hydrolytic reactions in the triphosphate (anhydride) part of the molecule.

SAM:

SAM, S-adenosylmethionine, has been encountered as a biological methylating agent, carrying out its function via a simple S_N2 reaction.

This material is a nucleoside derivative formed by nucleophilic attack of the thiol group of methionine onto ATP. It provides in its structure an excellent leaving group, the neutral Sadenosylhomocysteine.

Coenzyme A

Coenzyme A is another adenine nucleotide derivative, with its primary functional group, a thiol, some distance away from the nucleotide end of the molecule. This thiol plays an important role in biochemistry via its ability to form thioesters with suitable acyl compounds.

We have seen how thioesters are considerably more reactive than oxygen esters, with particular attention being paid to their improved ability to form enolate anions, coupled with thiolates being excellent leaving groups.

Nature’s oxidizing agents NAD⁺ and NADP⁺, and the corresponding reducing agents NADH and ⁺, are all dinucleotide derivatives).

• Indeed, the full names betray this: NAD is nicotinamide adenine dinucleotide. From the structures of nucleic acids, one interprets a dinucleotide as a repeated nucleotide. This would have two bases attached to a chain that reads.
• Phosphate–sugar–phosphate–sugar: A phosphodiester linkage. Note that these NAD derivatives have a sugar-phosphate–phosphate–sugar sequence, a broader interpretation of dinucleotide terminology.

The reactive center in these compounds relates to the pyridine ring in nicotinamide, which is capable of accepting or donating hydride equivalents according to its oxidation state.

• We have seen that, in biochemical reactions, NADH and NADPH may be considered analogs of complex metal hydride reagents.
• Here is our first example, then, of a nucleotide where the base, nicotinamide, is different from those in nucleic acids.
• FAD shares a lot of features with NAD⁺ and NADP⁺, but contains two new variants:
• A sugar that is neither ribose nor deoxyribose and a fairly complex heterocyclic base flavin. The new sugar is ribitol, non-cyclic because it contains no carbonyl group.

The chemistry of FAD is concentrated in the flavin part, and features oxidation/reduction processes. FMN, flavin mononucleotide, is simply the flavin-containing

Cyclic AMP

The nucleotide cyclic AMP (3′,5′-cyclic adenosine monophosphate, cAMP) is a cyclic phosphate ester of particular biochemical significance. It is formed from the triester ATP by the action of the enzyme adenylate cyclase, via nucleophilic attack of the ribose 3′-hydroxyl onto the nearest P = O group, displacing diphosphate as the leaving group. It is subsequently inactivated by hydrolysis to 5′-AMP through the action of a phosphodiesterase enzyme

cAMP functions in cells as a second messenger, a mediator molecule that transmits the signal from a hormone. Other second messengers identified include Ca²⁺, prostaglandins, diacylglycerol, and the equivalent cyclic phosphate derivative of guanosine, cyclic GMP.

• cAMP is the mediator for a variety of drugs, hormones, and neurotransmitters, including adrenaline, glucagon, calcitonin, and vasopressin. Such compounds produce their effects by increasing or decreasing the catalytic activity of adenylate cyclase, thus raising or lowering the cAMP concentration in a cell.
• A pyrophosphatase activity rapidly removes the other reaction product, disturbing the equilibrium, and making the reaction unidirectional cAMP, in turn, is responsible for the activation of various protein kinases that regulate the activity of cellular proteins by phosphorylation of serine and threonine residues using ATP.

The phosphorylated and nonphosphorylated forms of the enzymes catalyze the same reaction, but at quite different rates; often, one of the forms is essentially inactive. This means the activity of enzymes may be switched on or off by addition or removal of phosphate groups, and can thus be controlled by hormones.

Caffeine in tea and coffee inhibits the phosphodiesterase that degrades cAMP. The resultant increase in cAMP levels, therefore, mimics the action of mediators such as the catecholamines that modulate adenylate cyclase. Caffeine and the related theophylline (both purine alkaloids are thus effective stimulants of the CNS.

Nucleotide Biosynthesis

Nucleic acids are synthesized in nature from nucleoside triphosphates, which are coupled by a chain extension process.

• We have seen that coupling is simply an esterification reaction utilizing the 3′-hydroxyl of the sugar of the growing chain, with diphosphate as a good leaving group.
• Nucleoside triphosphates, especially ATP, have other major biochemical roles. A full discussion of the origins of these compounds is outside our requirements, but there are some features of particular interest pertinent to our understanding of these compounds.
• One of these is that several of the biosynthetic reactions require the involvement of ATP, demonstrating that nucleotide production requires input from other nucleotides.
• Another interesting aspect is the quite different approach nature adopts for the synthesis of pyrimidine or purine nucleotides.
• Pyrimidine nucleotides are made by adding a preformed pyrimidine ring to the sugar-phosphate.

On the other hand, the purine ring of purine nucleotides is built up gradually, and assembly occurs with the growing ring attached to the sugar-phosphate. A common intermediate for all the nucleotides is 5-phosphoribosyl-1-diphosphate (PRPP), produced by successive ATP-dependent phosphorylations of ribose. This has an α-diphosphate leaving group that can be displaced in S_N2 reactions.

Similar S_N2 reactions have been seen in glycoside synthesis and biosynthesis and for the synthesis of aminosugars. For pyrimidine nucleotide biosynthesis, the nucleophile is the 1-nitrogen of uracil- 6-carboxylic acid, usually called orotic acid. The product is the nucleotide orotidylic acid, which is subsequently decarboxylated to the now-recognizable uridylic acid (UMP).

Formation of UTP requires successive phosphorylations using ATP. CTP is, in turn, formed from UTP by an amination reaction in the pyrimidine ring, with the amino acid glutamine supplying the nitrogen; this is also an ATP-dependent reaction.

Glutamine also supplies an amino function to start purine nucleotide biosynthesis. This complex little reaction is again an S_N2 reaction on PRPP, but only an amino group from the amide of glutamine is transferred. The product of the enzymic reaction is thus 5-phosphoribosylamine.

The amino group now provides the nucleus for purine ring formation, an extended series of reactions we shall not describe.

• The first-formed purine product is inosine 5′-phosphate (IMP), which leads to either AMP or GMP; these require amination at alternative sites and utilize either GTP- or ATP-dependent reactions for amination.
• GTP or ATP (as appropriate) will also be required for further phosphorylations to produce the nucleotide triphosphates.
• 2 ‘Deoxyribonucleotides are generally formed by reduction of ribonucleoside diphosphates
• . This involves a series of redox reactions in which NADP+ and FAD play a role, with a subsequent electron transport chain. DNA contains thymine rather than uracil, so thymidine triphosphate (dTTP) is a requirement.
• Methylation of dUMP to dTMP is a major route to thymine nucleotides and is dependent upon N5, N10-methylenetetrahydrofolate as the source of the methyl group.

Determination Of Nucleotide Sequence

Restrictions on endonucleases

Natural DNA molecules are extremely large, and for sequence determination, it is necessary to cleave them into manageable fragments. This may be accomplished by using enzymes, called restriction endonucleases, which are obtained mainly from bacterial sources.

• These enzymes appear to have developed so that a cell can destroy foreign, particularly viral, DNA.
• The enzymes, of which several hundred are available, cleave the DNA at specific points in the chain, dictated by a series of nucleotides, typically three to six nucleotides.
• For example, the enzyme EcoRI (from Escherichia coli ) cleaves a GAATTC sequence between G and A.
• Also important is the property that most restriction endonucleases cleave both strands of DNA because the recognition sequence reads the same both ways: the complementary strand to GAATTC (in 5’→ 3’direction) is CTTAAG (in 3’→ 5’direction).
• The restriction endonuclease recognizes a specific sequence, but the probability of these sequences occurring in a given DNA molecule is usually quite low; therefore, cleavage produces only a few fragments.
• The use of a different enzyme on the same DNA will produce different fragments, but there then will be an overlap of sequences.
• Hence, sequencing of both sets of fragments should allow the full sequence to be deduced. This deductive approach is thus similar to that used in amino acid sequencing.

Chemical sequencing

Before separation, double-stranded restriction fragments are labeled chemically, by attaching a radioactive or fluorescent marker to the 5′-end of the chain.

For example: Radioactive 32P-labelled phosphate may be added using labeled ATP in an enzymic reaction.

• The labeled fragments are then separated chromatographically using conditions that are known to cause strand separation into single-stranded DNA molecules.
• The separated fragments are then split into four portions, and each portion is treated chemically with a suitable reagent.
• The reagent needs to induce cleavage reactions, but it shows selectivity for the different nucleotides.
• Now this could potentially lead to almost total cleavage, but the trick is to use reagents at concentrations so low that, statistically, only one cleavage occurs per chain. The reagents are dimethyl sulfate and hydrazine (only two reagents, but read on), and though we shall not consider the full mechanisms of the reactions here.

They may be summarized as follows

• Me₂SO4, then aqueous piperidine; cleavage at G;
• Me₂SO4 and aqueous formic acid, then aqueous piperidine; cleavage at A and G;
• Aqueous hydrazine (H₂NNH₂), then aqueous piperidine; cleavage at C and T;
• Aqueous hydrazine (H₂NNH₂) and NaCl, then aqueous piperidine; cleavage at C

Dimethyl sulfate

Dimethyl sulfate is an effective methylating agent Methylation of the purine rings in guanine and adenine makes them susceptible to hydrolysis and subsequent rupture. This, in turn, makes the glycosidic bond vulnerable to attack, and the heterocycle is displaced from the phosphodiester. The phosphodiester bond can then be cleaved by basic hydrolysis (aqueous piperidine).

• Guanine is methylated on the imidazole ring at N-7, whereas adenine undergoes methylation at N-3.
• Under the conditions used, guanine is methylated more readily than adenine; therefore, cleavage of the DNA occurs predominantly where there a guanine residues.
• However, by treating the methylated DNA with acid, cleavage at the methylated adenine sites becomes enhanced, and the chain is broken at sites that originally contained either adenine or guanine.
• The pyrimidines cytosine and thymine both react with hydrazine, which initially attacks the unsaturated carbonyl system and then leads to ring opening.

Again, base treatment is used to hydrolyze the phosphodiester bond.

This reaction becomes selective for cytosine in the presence of NaCl, which suppresses the reaction with thymine.

• The reaction products from the four reactions are then separated by gel electrophoresis in parallel lanes.
• This procedure will separate the components according to their charge (mainly from phosphate groups) and their size.
• The smallest species will migrate furthest. After chromatography, the gel is visualized by autoradiography, detecting bands via the radioactive tracer used. The base sequence can be read directly from the gel by the pattern of bands produced using the following reasoning

Consider a short sequence as shown (by convention written from 5′-end to 3′-end):

AGTCGGAACGTA

This is labelled at the 5’end with 32P to give

³²P–AGTCGGAACGTA

Cleavage at the 5′-side of G residues using the first reagent (Me₂SO₄, then aqueous piperidine) leads to fragments

³²P – A

³²P – AGTC
³²
P – AGTCG

³²P – AGTCGGAAC

Of course, there will be other fragments that do not contain the 5′-end with its ³²P label, but we shall not detect any of these since they contain no radioactive label.

Corresponding fragments will be produced when we use the other three types of cleavage reactions. The resultant chromatogram with the four reaction mixtures will then look something like though the bands will be much closer together in practice.

Bands that occur in the left-hand lane represent guanine, and bands that occur in the second lane but not the first lane represent adenine. Similarly, bands in the third lane but not the fourth lane represent thymine, and bands that occur in the fourth lane represent cytosine. By reading up the chromatogram, the sequence AGTCGGAAC may be deduced. It is possible to distinguish about 200 bands on a single gel.

The process is so reliable that automated equipment is available to perform routine analyses. As an alternative to using radioactive labeling, a modification uses differently colored fluorescent dyes, one for each base-selective reaction. All samples are then applied in one lane, and the base sequence can then be read automatically from the color of the bands along the gel. Similar sequencing methodology can be applied to RNA samples.

Oligonucleotide Synthesis: The phosphoramidite Method

The ability to synthesize chemically short sequences of single-stranded DNA (oligonucleotides) is an essential part of many aspects of genetic engineering.

The method most frequently employed is that of solid-phase synthesis, where the basic philosophy is the same as that in solid-phase peptide synthesis.

• In other words, the growing nucleic acid is attached to a suitable solid support, protected nucleotides are supplied in the appropriate sequence, and each addition is followed by repeated coupling and deprotection cycles.
• As with peptide synthesis, similar considerations must be incorporated into the methodology. Vulnerable functional groups in the base, the sugar, and the phosphates will need to be protected.
• The groups to be coupled may need suitable activation, and after the coupling reaction, the protecting groups must be removed under mild conditions.
• In addition, we need to attach the starting material to the support, and eventually, the product will need to be released from the support.

Nevertheless, the procedure is efficient and has allowed the development of automatic DNA synthesizers capable of preparing oligonucleotides of up to about 150 residues.

In solid-phase syntheses, oligonucleotides are usually synthesized in the 5′-direction from an immobilized 3′-terminus

• The solid phase is generally silica or controlled pore glass (CPG), which has been derivatized to provide a spacer molecule carrying a primary amino group. This spacer group is used to bring the nucleotide away from the support and allow the reagents free access.
• The first residue, as a nucleoside (i.e. without phosphate), is affixed to the support via its 3′-hydroxyl, using a succinic acid residue to achieve bonding, and also extend the spacer further.

The succinic acid residue thus has an amide link at one end and an ester link to the sugar of the nucleoside. In practice, the ester linkage is performed first.

Protection

Protection  of the 5′-hydroxyl of the sugar unit is usually as a dimethoxytrityl ether (trityl: triphenyl methyl), by reaction with dimethoxytrityl chloride

. The dimethoxytrityl group is bulky, and the reaction only occurs at the primary 5′-hydroxyl of the sugar group, the secondary 3′-hydroxyl being too hindered to react.

• This protecting group is easily removed by treatment with acid, even more easily than trityl groups, since the electron-donating methoxy groups stabilize the triaryl methyl carbocation that is an intermediate in the deprotection reaction
• The bases adenine, guanine, and cytosine all contain exocyclic amino substituents that require protection since these are potential nucleophiles
• . They are converted into amides that are stable to the other reagents used in the process, yet can be removed readily by basic hydrolysis.

The most effective protecting groups are isobutyryl for the amino group of guanine and benzoyl for adenine and cytosine. Thymine has no exocyclic nitrogen and does not need protection.

Protection and activation of the phosphate moiety is achieved by employing a phosphoramidite derivative, –P(OR)NR₂. This reagent has phosphorus in its PIII oxidation state; the phosphate that we finally require contains PV. Favoured R groups in the phosphoramidite are 2-cyanoethyl for OR and 2-propyl (isopropyl) for NR₂. The reagent used to attach this to the 3′-hydroxyl is the phosphorodiamidate shown, the hydroxyl displacing an NR₂ group in the presence of tetrazole as a mild acidic catalyst.

In what is essentially a repeat of this reaction, the 5’hydroxyl of a second nucleoside can couple to this intermediate; this is the crucial coupling reaction in the sequence shown below.

Of course, the product does not have a phosphate linker between the two nucleosides, and phosphorus is still in the wrong oxidation state.

• This is remedied by oxidation of the dinucleotide phosphite to a phosphotriester using iodine. We now have the required phosphate linker, though it is still protected with the cyanoethyl group.
• This is retained at this stage. The dimethoxytrityl ester-protecting group is now removed by treatment with a mild acid (CCl₃CO₂H), which is insufficiently reactive to hydrolyze the amide protection of bases or the cyanoethyl protection of the phosphate.
• The coupling cycle can now be repeated using a phosphoramidite derivative of the next appropriate nucleoside. The sequences will be continued as necessary until the desired oligonucleotide is obtained.
• It then remains to remove protecting groups and release the product from the support.
• All of these tasks, except for the removal of the dimethoxytrityl group, are achieved by the use of a single deprotection reagent, an aqueous base (ammonia).
• The cyanoethyl groups are lost from the phosphates by base-catalyzed elimination, and amide protection of the bases is removed by base-catalyzed hydrolysis. The latter process also achieves hydrolysis of the succinate ester link to the support.

Copying DNA: The Polymerase Chain Reaction

The polymerase chain reaction (PCR), developed by Mullis, is a simple and most effective way of amplifying, i.e. producing multiple copies of, a DNA sequence. It finds applications in all sorts of areas not immediately associated with nucleic acid biochemistry,

Examples:

Genetic screening, medical diagnostics, forensic science, and evolutionary biology.

The general public is now well aware of the importance of some of these topics.

Example:

The ability to identify a person by DNA analysis, but perhaps does not realize that tiny samples of DNA must be copied millions of times to provide a sample large enough for chromatographic analysis.

• PCR makes use of the heat-stable enzyme DNA polymerase from the bacterium Thermus aquaticus and its ability to synthesize complementary strands of DNA when supplied with the necessary deoxyribonucleoside triphosphates. We have already looked at the chemistry of DNA replication, and this process is the same, though it is carried out in the laboratory and has been automated.
• Although knowledge of the whole nucleotide sequence of the target area of DNA is not required, one must know the sequence of some small stretch on either side of the target area. These data may be known from other sequencing studies; or, surprisingly, it can even be predictable from knowledge of related genes.
• Two single-stranded oligonucleotides, one for each sequence, are then synthesized to act as primers.
• Typically, the primers should contain about 20 nucleotides, and they must be complementary to the DNA sequences of opposite strands.
•  In the schematic illustration of the process, the central target area is indicated, and the primers are depicted as short complementary sequences.

Initially, the double-stranded DNA is heated to separate the strands. The primers are then added and the temperature is lowered so that the primers anneal to the complementary sequences of each strand. In the presence of nucleoside triphosphates, the DNA polymerase enzyme will replicate a length of DNA starting from the 3′-end of a nucleotide, extending the chain towards the 5′-end.

It will thus start chain extension from the 3′-ends of the primers and continue to the end of the DNA strands. This will lead to two double-stranded DNA molecules, composed of initial strands, and primer plus newly synthesized DNA, as shown.

The process is repeated. Heating causes the separation of strands, and cooling allows the primer to attach to the appropriate nucleotide sequence.

• Enzymic chain extension then produces four double-stranded DNA molecules.
• The number of DNA molecules doubles in each cycle of the process, so that after 30 cycles, say, we have 230 molecules (approximately 109 copies).
• However, there is another, less obvious feature that makes the PCR even more useful. In the second cycle, two of the newly synthesized single-stranded chains will be of defined length.
• They will consist of the target area plus two primers; the 5’ends of the primers define the length of DNA.
• Other molecules will be much longer because replication goes on to the end of the template.
• Should you wish to follow this through, you will find that, after the third cycle, there will be eight single-stranded DNA molecules of defined length and eight that are longer.
• After each cycle, the number of defined-length DNA molecules increases geometrically, whereas the number of DNA strands containing sequences outside of the primers only increases arithmetically. This means that, after about 20 cycles, the DNA synthesized is almost entirely composed of molecules whose length is defined by the primers, i.e. the target area plus a short extra length defined by the primers

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