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.

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

The post Nucleosides Nucleotides And Nucleic Acids appeared first on BDS Notes.

Question. Describe clinical features, diagnosis, investigations, and management of rheumatic mitral stenosis.

Answer.

Mitral stenosis is a valvular heart disease. Rheumatic mitral stenosis occurs in elderly people and is most common in females.

Clinical Manifestations of Rheumatic Mitral Stenosis.

Symptoms of Rheumatic Mitral Stenosis.

1. The patient complains of breathlessness and fatigue on exertion.
2. Progression of stenosis leads to dyspnea on rest and even has orthopnea and paroxysmal nocturnal dyspneal.
3. Acute pulmonary edema can also occur.
4. Hemoptysis can be present due to rupture of pulmonary congestion and pulmonary embolism and cough due to pulmonary congestion.
5. Chest pain is present due to pulmonary venous hypertension.

Signs of Rheumatic Mitral Stenosis.

1. Atrial firillation is present.
2. Auscultation: Presence of loud first heart sound, opening snap, and mid-diastolic low-pitched rumbling murmur best heard at the apex.
3. Signs of raised pulmonary capillary pressure: Pleural effusion, crepitation, pulmonary edema.
4. Signs of pulmonary hypertension: RV heave, loud P2
5. Others: Basal crackers, ascites, and pleural effusion

Investigations of Rheumatic Mitral Stenosis.

1. ECG:

1. Right ventricular hypertrophy
2. Left atrial hypertrophy

2. X-ray chest:

1. A prominent left atrial appendage may be seen in the left border of the heart between the pulmonary artery and left ventricle. It indicates left atrial enlargement.
2. Double shadow of enlarged left atrium on the right side of the spine.
3. Signs of pulmonary venous congestion

Mitral Valve Stenosis Causes, Symptoms, Diagnosis, and Treatment

3. Echocardiogram:

1. Show thick immobile mitral cusp
2. Decreased diastolic filling of the left ventricle
3. Decreased valve orifice area
4. Left atrial thrombus, if it is present.

4. Cardiac catheterization is used to assess valvular lesions
and to detect coronary artery disease.

5. Doppler:

1. Pressure gradient across the mitral valve
2. Pulmonary artery pressure
3. Left ventricular function

Diagnosis: Rheumatic Mitral Stenosis.
It is based on physical signs and investigations.

Management of Rheumatic Mitral Stenosis.

Medicinal: Rheumatic Mitral Stenosis.

1. Salt restriction should be done in the diet, or a very low salt diet should be given.
2. Digitalis therapy is given. In the patient with congestive heart failure, Tab. Digoxin 0.25 mg BD is given.
3. Diuretics can be given to control heart failure
4. Anticoagulants such as heparin can be given to prevent embolism
5. Prophylactic oral penicillin V 250 mg BD is given to prevent rheumatic fever. If the patient is allergic of penicillin, erythromycin 250 mg daily orally is given.

Surgical: Rheumatic Mitral Stenosis.
When the patient remains symptomatic despite medical treatment or when mitral stenosis is severe, surgical intervention is needed:

1. Mitral valvotomy:

1. Percutaneous balloon valvotomy is indicated when the mitral valve is noncalcified and without regurgitation.
   The procedure involves the passing of a catheter across the valve and inflation of the balloon to dilate the orifice.
2. Open valvotomy is carried out in patients where balloon valvotomy is not possible or in cases with rest enosis.
   In this procedure, the fusion of the valve is loosened,d and calcium deposits and thrombi are removed.

Causes of Mitral Valve Stenosis

2. Mitral valve replacement: The mitral valve is replaced when there is critical mitral stenosis and/or there is associated mitral regurgitation. Replacement is also done when the mitral valve is severely distorted and calcified.

The post Mitral Valve Stenosis: Causes, Symptoms, Diagnosis, and Treatment Explained appeared first on BDS Notes.

Question 1. Enumerate the causes of Jones criteria of acute rheumatic fever.

Answer. The causes of Jones criteria are:

1. Previous streptococcal infection
2. Recent scarlet fever
3. Positive throat culture from streptococcal A
4. Increased-antistreptolysin O titer.

Question 2. Describe briefly clinical features and management of aortic regurgitation.

Answer. Aortic regurgitation is produced due to acute rheumatic carditis which is associated with other valve involvement and infective endocarditis.

Clinical Features Rheumatic Mitral Stenosis.

Symptoms Rheumatic Mitral Stenosis.

1. In mild to moderate aortic regurgitation:

1. Often asymptomatic
2. On palpitation — pounding of heart is a common symptom
3. Symptoms of left heart failure appear but late

2. In severe aortic regurgitation:

1. Symptoms of heart failure, i.e., dyspnea, orthopnea are present at onset.
2. Angina pectoris is frequent complaint.
3. Arrhythmias are uncommon.

Understanding Rheumatic Fever: Jones Criteria and Aortic Regurgitation

Signs Rheumatic Mitral Stenosis.

1. Collapsing or good volume pulse (wide pulse pressure)
2. Bounding peripheral pulses
3. Dancing carotids (Corrigan’s sign)
4. Capillary pulsation in nail beds (Quincke’s sign)
5. Pistol shots sound and Duroziez’s sign/murmur
6. Head nodding with carotid pulse — de Musset’s sign
7. Cyanosis (peripheral, central or both) may be present
8. Pittng ankle edema may be present.
9. Tender hepatomegaly if right heart failure present.

Jones Criteria for Rheumatic Fever and Aortic Regurgitation

Management Rheumatic Mitral Stenosis.

1. Treatment of underlying causes like endocarditis and syphilis.
2. Surgical: Replacement of aortic valve should be performed before heart failure can develop.
   Serial evaluation of end systolic dimensions should be made and surgery considered when this exceeds 5 mm.
3. Medical:

1. Prophylaxis against bacterial endocarditis before and after surgery
2. Therapy of heart failure if develops

The post Understanding Rheumatic Fever: Jones Criteria and Aortic Regurgitation Explained appeared first on BDS Notes.

Question. Enumerate the causes of cervical lymphadenopathy.
Or
Enumerate the causes of generalized lymphadenopathy. 
Or
Enumerate the causes of lymphadenopathy.

Answer.

1. Infectious Diseases:

1. Viral infections:

• Infectious hepatitis
• Infectious mononucleosis
• AIDS
• Rubella
• Varicella
• Herpes zoster.

2. Bacterial infections:

• Streptococci
• Staphylococci
• Salmonella
• Brucella
• Listeria monocytogenes.

3. Fungal infections:

• Coccidioidomycosis
• Histoplasmosis
• Chlamydial Infections
• Lymphogranuloma venereum
• Trachoma.

4. Mycobacterial infections:

• Tuberculosis
• Leprosy
• Parasitic infestations
• Microfiariasis
• Toxoplasmosis.

5. Spirochetal diseases

• Syphilis
• Yaws
• Leptospirosis.

Lymphadenopathy and Malignancy: Causes and Diagnosis

2. Immunologic Diseases:

• Rheumatoid arthritis
• Systemic lupus erythematosus
• Dermatomyositis
• Serum sickness
• Drug reactions: Phenytoin, hydralazine
• Primary biliary cirrhosis
• Chronic active hepatitis.

3. Malignant Disorders:

1. Haematologic disorders:

• Hodgkin’s lymphoma
• Myeloid leukaemia—blastic crisis
• Chronic lymphatic leukaemia

2. Metastatic tumours:

• Melanoma
• Kaposi’s sarcoma
• Tumours
• Lung
• Breast
• Prostate
• Kidney
• Head and neck
• Gastrointestinal tract.

4. Endocrine disease:

• Hyperthyroidism.

5. Lipid Storage disease:

• Gaucher’s disease
• Niemann­Pick disease.

Lymphadenopathy as a Sign of Cancer

6. Miscellaneous disorders:

• Sarcoidosis
• Amyloidosis
• Sinus histiocytosis.

The post Lymphadenopathy And Malignancy appeared first on BDS Notes.

Question. Outline the management of acute rheumatic fever.
Or
Discuss the management of acute rheumatic fever.

Answer.

Management of Acute Rheumatic Fever.

1. Treatment of acute attack: Rheumatic Fever

1. Bed rest is important to reduce joint pain and cardiac workload.
   Duration of bed rest is guided by markers of inflammation, like temperature,
   WBC count and ESR.
2. Benzathine penicillin 1.2 mu IM 4 hourly. If the patient is allergic to penicillin, erythromycin 40–50 mg/kg for ten days is given.
3. Aspirin usually relieves symptoms of arthritis rapidly.
   A 60 mg/kg body weight starting dose per day is given, divided into 6 doses.
   The dose may be increased to 120 mg/kg body weight.
   This dose may produce severe symptoms like vomiting, tachypnea, and acidosis. Aspirin is given till ESR comes to normal.
4. Corticosteroids like prednisolone produce rapid symptomatic relief than aspirin and are indicated in cases with severe arthritis or carditis.
   Prednisolone is given in doses of 1.2 mg/kg body weight till ESR comes to normal

Rheumatic Fever and Its Management

2. Secondary prevention: Rheumatic Fever

Long-term prophylaxis is needed to prevent further attacks of rheumatic fever.

Management of Rheumatic Fever

1. Benzathine penicillin 1.2 mu IM is injected at an interval of 21 days.
   Further attacks are unusual after the age of 21 years, and treatment can be stopped.
2. To prevent the chances of endocarditis, prophylactic antibiotic therapy should be given.

The post Rheumatic Fever And Its Management appeared first on BDS Notes.

Question.  Describe briefly, diagnosis of rheumatic fever.
Or
Write short notes on Jones’ criteria for rheumatic fever.
Or
Write a short note on Duke Jones’ criteria in acute rheumatic fever.

Answer.

Clinical Rheumatic Fever

1. Fever
2. Arthralgia
3. Previous history of rheumatic fever or rheumatic heart disease.

Laboratory Rheumatic Fever

1. Acute phase reactants (leucocytosis, raised ESR, C­ C-reactive protein)
2. Prolonged PR interval in ECG.

Jones Criteria Explained: Acute Rheumatic Fever Diagnosis

Essential Criteria of Rheumatic Fever

Evidence for recent streptococcal infection, as evidenced by:

1. Increase in ASO titer

1. > 333 Todd units (in children).
2. > 250 Todd units (in adults).
3. Positive throat culture for streptococcal infection
4. Recent history of scarlet fever.

Confirmation of Diagnosis of Rheumatic Fever

The result is based on the Presence of two or more major criteria or one major and two minor criteria; in the presence of essential criteria, it is required to diagnose acute rheumatic fever.

ones Criteria for Acute Rheumatic Fever Diagnosis

The post Jones Criteria Explained: Diagnosing Acute Rheumatic Fever Made Simple appeared first on BDS Notes.

Question. How will you diagnose and manage a case of rheumatic fever? Outline the complications of rheumatic fever.

Answer.

Diagnosis of rheumatic fever is made by the ‘Jones criteria’, which is as follows:

Major criteria for Rheumatic Fever

1. Carditis: Rheumatic Fever

1. It is pancarditis involving the endocardium, myocardium, and pericardium.
2. It manifests as breathlessness, palpitation, and chest pain.
3. Tachycardia, cardiomegaly, and new or changed murmurs
4. Aortic regurgitation in 50% of cases.
5. Pericarditis produces frictional rub and pericardial tenderness.
6. Cardiac failure due to myocardial infarction.

Acute Rheumatic Fever Diagnosis and Management

2. Sydenham’s chorea: Rheumatic Fever

1. Late neurological manifestations that occurs at least three months after the episode of acute rheumatic fever when all signs disappear.
2. More common in females.
3. It is characterized by involuntary dancing movements of hands, feet or face.

3. Polyarthritis: Rheumatic Fever

1. The early feature of illness is non-specific.
2. It is characterized by acute, painful, symmetric, and migratory inflammation of large joints.
3. Classical presentation is acute migratory polyarthritis.
   Pain and swelling in the involved joints subside or disappear as newer joints get affected.

4. Erythema marginatum: Rheumatic Fever

Red macules which fade in the centre, but remain red at the edges, and occur mainly on the trunk and proximal extremities on the face.

Diagnosis of Acute Rheumatic Fever and Treatment

5. Subcutaneous nodules: Rheumatic Fever

They are small, dense, firm, painless, and are best felt over tendons and bones.

Nodules appear more than 3 weeks after the onset of other manifestations.

Clinical Rheumatic Fever

1. Fever
2. Arthralgia
3. Previous history of rheumatic fever or rheumatic heart disease.

Laboratory Rheumatic Fever

1. Acute phase reactants (leucocytosis, raised ESR, C­ C-reactive protein)
2. Prolonged PR interval in ECG.

Essential criteria for Rheumatic Fever

Evidence for recent streptococcal infection as evidenced by:

1. Increase in ASO titer

1. > 333 Todd units (in children).
2. > 250 Todd units (in adults).
3. Positive throat culture for streptococcal infection
4. Recent history of scarlet fever.

Confirmation of Diagnosis: Rheumatic Fever

The result is based on the Presence of two or more major criteria or one major and two minor criteria; in the presence of essential criteria, is required to diagnose acute rheumatic fever.

Management of Rheumatic Fever

1. Treatment of acute attack:

1. Bed rest is important to reduce joint pain and cardiac workload.
   Duration of bed rest is guided by markers of inflammation like temperature,
   WBC count and ESR.
2. Benzathine penicillin 1.2 mu IM 4 hourly. If the patient is allergic to penicillin, erythromycin 40–50 mg/kg for ten days is given.
3. Aspirin usually relieves symptoms of arthritis rapidly.
   A starting dose of 60 mg/kg body weight per day is given, divided into 6 doses.
   The dose may be increased to 120 mg/kg body weight.
   This dose may produce severe symptoms like vomiting, tachypnea, and acidosis. Aspirin is given till ESR comes to normal.
4. Corticosteroids like prednisolone produce rapid symptomatic relief than aspirin and is indicated in cases with severe arthritis or carditis.
   Prednisolone is given in doses of 1.2 mg/kg body weight till ESR comes to normal

2. Secondary prevention: Long-term prophylaxis is needed to prevent further attacks of rheumatic fever.

1. Benzathine penicillin 1.2 mu IM is injected at an interval of 21 days.
   Further attacks is unusual after the age of 21 years, and treatment can be stopped.
2. To prevent the chances of endocarditis, prophylactic antibiotic therapy should be given.

Complications Of Rheumatic Fever

1. Myocardial infarction
2. Mitral stenosis
3. Tricuspid regurgitation
4. Aortic regurgitation
5. Aortic stenosis is rare
6. Mitral regurgitation.

The post Acute Rheumatic Fever Diagnosis And Management appeared first on BDS Notes.

Question. Write a short note on Hepatic Amoebiasis.
Answer.

It is the most common complication of amoebiasis.

• Pathophysiology of Hepatic Amoebiasis
• Amoeba after reaching the liver multiply and block small intrahepatic portal radicles, producing thrombosis and infarction resulting in necrosed areas surrounded by areas of congestion.
• The necrotic area consists of degenerated liver cells, leukocytes, connective tissue strands, and is enmeshed with Entamoeba histolytica.
• Cytolytic enzymes liberated from amoebae destroy the liver parenchyma and fusion of these small necrosed areas results in abscess formation.

Amoebiasis Symptoms 

• An abscess is generally single, but may be multiple. Its walls are lined by a shaggy necrotic zone in whose centre there is thick reddish brown pus containing fragments of liver tissue, necrotic material, and erythrocytes.
• The pus is typically called “Anchovy sauce” and is sterile on culture.

Clinical Features Hepatic Amoebiasis

• The onset of Amoebic hepatitis is insidious, and the patient may present with irregular or intermittent fever
• There is a stretching sensation in the liver area.
• Gradually, with the progression of the disease, anorexia,a hepatic pain and epigastric discomfort appear
• Examination shows a uniform tender hepatomegaly
• There are signs of toxemia

Hepatic Amoebiasis: Symptoms, Diagnosis, and Management

Amoebiasis Treatment

• Jaundice is not very common
• When hepatitis progresses to a liver abscess, pain in the liver area becomes a constant feature
• Intermittent fever, loss of weight, lassitude, peculiar sallowness of skin, irritability, and sleeplessness are common features.

Investigations of Hepatic Amoebiasis

• TLC and DLC show leukocytosis with an increase in polymorphs.
• A stool examination is done, and cysts and trophozoites of
• The amoebic fluorescent antibody titer is positive.

Hepatic Amoebiasis Symptoms and Treatment

Management of amoebic liver abscess/Hepatic Amoebiasis

• Early cases are responding well with metronidazole 800 mg TID for 5 days or tinidazole 2 gm daily for three days.
• Luminal amoebicide: Diloxamide furoate 500 mg 8 hourly for 10 days should be given to determine the luminal cyst.
• If the abscess is large and does not respond to chemotherapy, repeated aspiration under ultrasonic guidance is required.
• Rupture of an abscess into the peritoneal cavity requires immediate aspiration or surgical drainage.

The post Understanding Hepatic Amoebiasis: Symptoms, Diagnosis, and Management appeared first on BDS Notes.