Heterocycles:

Cyclic compounds in which one or more of the ring atoms are not carbon are termed heterocycles; the noncarbon atoms are referred to as heteroatoms.

• We shall limit our discussions to compounds in which the heteroatoms are nitrogen, oxygen, or sulfur.
• To study and understand their properties, heterocycles are conveniently grouped into two classes, i.e. non-aromatic and aromatic.

Non-Aromatic Heterocycles:

We have already met many examples of nonaromatic heterocycles in earlier chapters, for example., cyclic ethers, including epoxides, and cyclic amines, as well as lactones, lactams, and cyclic acetals and ketals.

• From the familiar examples shown below, it should be clear that the standard approach to generating heterocyclic systems requires a difunctional compound containing a leaving group or elec aerophilic center, together with a nucleophilic species that provides the heteroatom.
• The nomenclature of simple heterocyclic ring systems containing one heteroatom is indicated overleaf.

These form a useful reference, but there is little to be gained in committing them to memory.

Note, however, that the most important of these structures tend to have a trivial rather than systematic name, a consequence of long-standing common usage.

• Some of these, for example., tetrahydrofuran and tetrahydropyran, are derived from the name of the corresponding aromatic heterocycle by the concept of reduction.

A few examples of commonly encountered heterocycles with two heteroatoms are also shown.

Numbering always begins at the heteroatom; in the case of morpholine, numbering starts at oxygen, the heteroatom of a higher atomic number.

• Remember that an accepted alternative in nomenclature is to indicate a heteroatom by the prefix aza-, oxo, or thia in the appropriate carbocycle.
• Thus, we could name piperidine as azacyclohexane, and ethylene oxide tetrahydrofuran as oxacyclopentane.

The chemistry of these non-aromatic heterocycles differs little from the chemistry of their acyclic counterparts and we emphasize only the relative reactivity of the three-membered ring systems towards ring opening, thus achieving relief of ring strain.

We have already noted the ring opening of epoxides, and similar reactivity is found with aziridines and iranes.

• Four-membered systems are also considerably strained and reactive towards nucleophiles, though not as readily as the three-membered compounds.
• Some of these heterocycles provide us with valuable laboratory solvents, for example., the ethers tetrahy drofuran and dioxane (1,4-dioxane). Others are useful as organic bases, for example., piperidine, pyrrolidine, and morpholine.

The basicities of the nitrogen derivatives are comparable to those of similar acyclic amines, but physical properties, for example., higher boiling point, make them more versatile than the simple amines.

From the pK_a values shown, there is relatively little difference in basicities for diethylamine, pyrrolidine, or piperidine. Note, however, that morpholine and piperazine are weaker bases than piperidine.

• This is the result of an electron-withdrawing inductive effect from the second heteroatom, making the nitrogen atom both less basic and also less nucleophilic. This makes morpholine a useful base with basicity between that of piperidine and pyridine (pK_a 5.2).
• The second pK_a value for the diamine piperazine is substantially lower than the first since the inductive effect from the protonated amine will withdraw electrons away from the unprotonated amine.

Aromaticity And Heteroaromaticity:

Pyridine is structurally related to benzene: one CH unit has been replaced by N. If we consider the constitutions of the two compounds in more detail, we shall see even closer similarity.

• Thus, we have seen that the ring atoms in benzene are sp² hybridized. The remaining singly occupied p orbitals are oriented at right angles to the plane of the ring, and overlap to form a delocalized π system, extending to form a closed loop above and below the ring.
• Compared with what we might expect for the hypothetical cyclohexatriene, this results in a considerable stabilization, with significantly modified structure and reactivity in benzene. We termed this aromaticity.
• Benzene conforms to Huckel’s rule, which predicts that planar cyclic polyenes containing 4n + 2 π electrons show enhanced stability associated with aromaticity.
• Pyridine is also aromatic: nitrogen contributes one electron in a p orbital to the π electron system, and its lone pair is located in an sp² orbital that is in the plane of the ring and perpendicular to the π electron system.

It also conforms to Huckel’s rule, in that we still have an aromatic ¨ sextet of π electrons.

One of the structural features of benzene that derives from aromaticity is the equal length of the C–C bonds (1.40 A), which lies between the conformal single (1.54 A) and double (1.34˚A) bonds.

• Nevertheless, we continue to draw benzene with single and double bonds because this allows us to represent reaction mechanisms in terms of electron movements.

Pyridine does not have a perfect hexagon shape; the symmetry is distorted because the C–N bonds are slightly shorter (1.34 A) than the C–C bonds (1.39–1.40 A).

Nitrogen is more electronegative than carbon, and this influences the electron distribution in the π electron system in pyridine through inductive effects, such that nitrogen is electron-rich.

• In addition, the nitrogen will also become electron-rich through a resonance effect: several resonance forms may be drawn that have a negative charge on nitrogen. These effects thus reinforce each other.
• The heteroatom thus distorts the π electron cloud of the aromatic ring system, drawing electrons towards the nitrogen and away from the carbons.

The consequences of this are that we can predict that the pyridine nitrogen will react readily with electrophiles, whereas the remainder of the ring system will be resistant to electrophilic attack.

An experimental probe for aromaticity is the chemical shift of the hydrogen signals in NMR spectroscopy.

• The substantially greater δ values for benzene protons (δ 7.27 ppm) compared with those in alkenes (δ 5–6 ppm) have been ascribed to the presence of a ring current that creates its magnetic field opposing the applied magnetic field.
• This ring current is the result of circulating electrons in the π system of the aromatic ring.
• The hydrogen NMR signals for pyridine also appear at relatively large δ values, in the range of 7.1–8.5 ppm, typical of aromatic systems.

The signals do not all appear at the same chemical shift; the heteroatom distorts the π electron distribution and affects the 2/6, 3/4, and 5 positions to different extents.

• Now let us consider pyrrole, where we have a five-membered ring containing nitrogen. Pyrrole is also aromatic.
• This is somewhat unexpected: how can we get six π electrons from just five atoms? The answer is that each carbon contributes one electron as before, but nitrogen now contributes two electrons, its lone pair, to the π electron system.
• We can draw Frost circles to show the relative energies of the molecular orbitals for pyridine and pyrrole.

The picture for pyridine is essentially the same as for benzene, with six π electrons forming an energetically favorable closed shell.

• For pyrrole, we also get a closed shell, and there is considerable aromatic stabilization over electrons in the six atomic orbitals.
• However, the contribution of the nitrogen lone pair to the aromatic sextet in pyrrole makes the nitrogen atom relatively electron deficient. The nitrogen atom should create an inductive effect, as in pyridine, drawing electrons towards the heteroatom.
• However, a consideration of the resonance structures leads to several resonance forms with a positive charge on nitrogen. The resonance effect is opposite to the inductive effect and of greater magnitude.

Overall, the heteroatom distorts the π electron cloud of the aromatic ring system by pushing electrons away from the nitrogen and towards the carbons.

The difference in electron distribution in pyridine and pyrrole manifests itself via the measured dipole moments. More importantly, we shall see that this electron distribution influences the chemical reactivity of the two systems.

• In broad terms, ring systems where the carbons are electron deficient because of the electron-withdrawing effect of the heteroatom, for example., pyridine, are more reactive towards nucleophiles than benzene.
• On the other hand, ring systems where the carbons are electron-rich because of the electron-donating heteroatom, for example., pyrrole, are more reactive towards electrophiles than benzene.
• Note the deliberate choice of terminology here: ring systems where the carbons are electron deficient or electron rich.
• You may meet the older terminology of π-deficient heterocycles and π-excessive heterocycles, but these can give a false impression.
• Each heterocycle contains six π electrons, so it is not the heterocycle that is electron deficient or electron-rich, but the carbons that receive less or more than their equal share because of the effect of the heteroatom.
• Though we shall return to this again, one critical difference between pyridine and pyrrole to note here relates to basicity. Pyridine is a base because its nitrogen still carries a lone pair able to accept a proton.

Pyrrole is not basic: it has already used up its lone pair in contributing to the aromatic sextet.

Six-Membered Aromatic Heterocycles Pyridine:

From our discussions in the last section, we might expect pyridine to display properties associated with the nitrogen function and the aromatic ring.

Not surprisingly, it turns out that the aromatic ring affects the properties of the amine; but, more significantly, the aromatic properties are greatly influenced by the presence of the heteroatom.

Based on our earlier knowledge, and from a simple inspection of its structure, we might expect to observe three types of general reactivity in pyridine. We might expect to see:

Reaction at the heteroatom – the non-bonding electrons on the nitrogen might coordinate to H+ or another suitable electrophile.

Reaction of the aromatic π system – typical elec trophilic substitution as seen for benzene might be expected.

Reaction of the C=N ‘imine’ function – though this is not an isolated imine function but is part of the aromatic ring, its polarization might make it susceptible to nucleophilic attack.

Reassuringly, our predictions turn out to be well-founded.

Pyridine is a base (pK_a pyridinium cation 5.2), but it is a considerably weaker base than a typical non-aromatic heterocyclic amine such as piperidine (pK_a piperidinium cation 11.2).

• This is because the lone pair electrons in pyridine are held in an sp² orbital.
• The increased s character of this orbital, compared with the sp3 orbital in piperidine, means that the lone pair electrons are held closer to the nitrogen, and are consequently less available for protonation.
• The lower basicity of pyridine compared with piperidine is thus a hybridization effect.

Although pyridine is a weak base, it can form salts with acids and is widely used in chemical reactions as an acid scavenger and as a very good polar solvent.

Just as pyridine is a weaker base than piperidine, it is also a poorer nucleophile. Nevertheless, it reacts with electrophiles to form stable pyridinium salts.

• In the examples shown, primary alkyl halides form N-alkylpyridinium salts, whereas acyl halides and anhydrides react to give N-acylpyridinium salts.
• We have already seen the latter compounds involved in esterification reactions, and see the value of pyridine in removing acidic by-products, for example., HCl.

Of course, N-acyl pyridinium salts will easily be hydrolyzed under aqueous conditions.

Even better than pyridine in such reactions is the derivative 4-N, N-dimethylamino pyridine (DMAP), where a resonance effect from the dimethylamino substituent reinforces the nucleophilicity of the pyridine nitrogen.

It then also promotes the acylation step by improving the nature of the leaving group. The example shows its function in a typical esterification process, i.e. acylation of an alcohol.

There are other gains, as well. Pyridine as a solvent is difficult to remove from the products, and it smells quite awful.

• In this reaction, a catalytic amount of DMAP is all that is necessary, and a more acceptable solvent can be employed.
• The pre-eminent reactivity associated with aromatic compounds is the ease of electrophilic substitution.
• As we have already predicted, the pyridine ring is rather unreactive towards electrophilic reagents, and these tend to be attacked by nitrogen instead, making the ring even less reactive.

It is readily seen from the intermediate addition cations and their resonance structures that attack at C-2 or C-4 will be unfavorable, in that one of the resonance forms features an unstable electron-deficient nitrogen cation.

Attack at C-3 is the more likely, simply based on an inspection of resonance structures for the addition cation.

• However, elec trophilic attack still tends to be unfavourable, because many electrophilic reagents, for example., HNO₃ –H₂SO₄, are strongly acidic, and the first effect is protonation on nitrogen.
• The attack of E⁺ on a positively charged pyridinium cation is even less favorable. Under acidic conditions, we require an attack on free pyridine, the concentration of which will be very small.
• Thus, under equivalent conditions, pyridine undergoes electrophilic substitution very much more slowly than benzene, by a factor of about 106.

Even Friedel–Crafts acylations are inhibited, because the nitrogen complexes with the Lewis acid, again leading to a cationic nitrogen.

A striking demonstration of the reduced activity towards electrophiles for the pyridine ring compared with the benzene ring will be seen later when we consider the fused heterocycles quinoline and isoquinoline.

• These contain a benzene ring fused to a pyridine ring; electrophilic substitution occurs exclusively in the benzene ring.
• To facilitate electrophilic substitution, it is possible to first convert pyridine into pyridine N-oxide by the action of a peracid such as peracetic acid or m-chloroperbenzoic acid (MCPBA;).
• N-oxide formation is not peculiar to pyridine, but it is a general property of tertiary amines. There is no overall charge in the molecule, but it is not possible to draw the structure without charge separation.

Although the introduced oxygen atom causes electron withdrawal through an inductive effect, there is a greater and opposing resonance effect that donates electrons into the ring system.

This improves reactivity towards electrophiles. Consideration of resonance structures shows positions 2, 4, and 6 are now electron-rich.

Nitration of pyridine N-oxide occurs at C-4; very little 2-nitration is observed. The pyridine compound can then be regenerated by deoxygenation with triphenylphosphine.

Pyridine, on the other hand, is more reactive than benzene towards nucleophilic aromatic substitution

• This is effectively a reaction towards the C=N ‘imine’ function, as described above.
• The attack is attacked at principally at positions 2 and 4, as predictable from resonance structures of reaction intermediates.

Attack at the 3 position does not allow the nitrogen to help stabilize the negative charge.

However, for an unsubstituted pyridine, the leaving group to finish off this reaction is hydride, which is a strong base and thus a poor leaving group.

• It may be necessary to use an oxidizing agent to function as a hydride acceptor to the Chichibabin reaction to facilitate this type of hydride transfer.

Nevertheless, there is a classic example of this process, known as the Chichibabin reaction, in which pyridine is converted into 2-aminopyridine through heating with sodium amide.

The hydride released appears to abstract a proton from the product since the other product of the reaction is gaseous hydrogen. The aminopyridine anion is finally quenched with water.

• The product is mainly 2-aminopyridine, probably the result of the enhanced inductive effect on carbons immediately adjacent to the electronegative nitrogen.
• It is much more effective to have a better-leaving group in the pyridine system. Thus 2- or 4-chloropyridines react with several nucleophiles to generate substituted products.
• Note that one can predict from the resonance structures that 3-chloropyridine, despite having a satisfactory leaving group, would not be susceptible to nucleophilic substitution at position 3.

It is not possible in the addition of anion to share the charge with nitrogen.

Methylpyridines are called picolines. 2-picoline and 4-picoline may be deprotonated by treatment with a strong base, giving useful anions.

• The methyl acidity results because of resonance stabilization in the conjugate base, providing an enolate anion analog.

However, pK_a values for 2-picoline (32) and 4-picoline (34) show that they are somewhat less acidic than ketones.

These anions can now be used as nucleophiles in several familiar reactions, for example., S_N2 reactions with alkyl halides, or aldol reactions with carbonyl compounds.

It is worthwhile here to relate the behavior of 2-chloropyridine and 2-methylpyridine to carbonyl chemistry.

• If we consider the pyridine ring as an imine, and therefore a carbonyl analog, then with 2-chloropyridine we are seeing reactions that parallel nucleophilic substitution of an acyl halide through an addition–elimination mechanism.
• With 2-methylpyridine we are seeing typical aldol reactions with activated methyl derivatives.

Nicotine, Nicotinic Acid, And Nicotinamide:

Nicotine is an oily, volatile liquid and is the principal alkaloid found in tobacco (Nicotiana tabacum). It can be seen to be a combination of two types of heterocycle, i.e. the aromatic pyridine and the non-aromatic N methylpyrrolidine.

In small doses, nicotine can act as a respiratory stimulant, though in larger doses it causes respiratory depression. Nicotine is the only pharmacologically active component in tobacco, and it is highly addictive.

• On the other hand, tobacco smoke contains several highly carcinogenic chemicals formed by incomplete combustion.
• Tobacco smoking also contributes to atherosclerosis, chronic bronchitis, and emphysema, and is regarded as the single most preventable cause of death in modern society.
• Nicotine, in the form of chewing gum, nasal sprays, or trans-dermal patches, is available for use by smokers who wish to stop the habit.
• Nicotine affects the nervous system, interacting with the nicotinic acetylcholine receptors, and the tight binding is partially accounted for by the structural similarity between acetylcholine and nicotine.
• Curare-like antagonists also block nicotinic acetylcholine receptors. There are other acetylcholine receptors, termed muscarinic, that are triggered by the alkaloid muscarine.

The tropane alkaloid hyoscyamine (see Box 10.9) binds to muscarinic acetylcholine receptors.

Oxidation of nicotine with chromic acid led to the isolation of pyridine-3-carboxylic acid, which was given the trivial name nicotinic acid.

• We now find that nicotinic acid derivatives, especially nicotinamide, are biochemically important.
• Nicotinic acid (niacin) is termed vitamin B3, though nicotinamide is also included under the umbrella term vitamin B3 and is the preferred material for dietary supplements.
• It is common practice to enrich many foodstuffs, including bread, flour, corn, and rice products.

Deficiency in nicotinamide leads to pellagra, which manifests itself in diarrhea, dermatitis, and dementia.

Nicotinic acid and nicotinamide are precursors of the coenzymes NAD⁺ and NADP⁺, which play a vital role in oxidation-reduction reactions and are the most important electron carriers in intermediary metabolism.

We shall look further at the chemistry of NAD⁺ and NADP⁺ shortly, but note that, in these compounds, nicotinamide is bound to the rest of the molecule as an N-pyridinium salt.

An intriguing feature of nicotinic acid formation in animals is that it is a metabolite produced from the amino acid tryptophan.

This means the pyridine ring is formed by biochemical modification of the indole fused-ring system, and, as you might imagine, it involves a substantial sequence of transformations.

Nucleophilic Addition To Pyridinium Salts:

The reaction of nucleophiles with pyridinium salts leads to addition, giving dihydropyridines.

Attack is normally easier at positions 2 or 6, where the inductive effect from the positively charged nitrogen is greatest; however, if these sites are blocked, attack occurs at position 4. This is easily predicted from a consideration of resonance structures.

Thus, treatment of N-methyl pyridinium salts with cyanide produces a mixture of 2- and 4-cyanide hydropyridines, with the 2-isomer predominating.

It is quite difficult to reduce benzene or pyridine because these are aromatic structures. However, partial reduction of the pyridine ring is possible by using complex metal hydrides on pyridinium salts.

• Hydride transfer from lithium aluminum hydride gives the 1,2-dihydro derivative, as predictable from the above comments.
• Sodium borohydride under aqueous conditions achieves a double reduction, giving the 1,2,5,6-tetrahydro derivative, because protonation through the unsaturated system is possible.
• The final reduction step requires catalytic hydrogenation. The reduction of pyridinium salts is of considerable biological importance.

Note the way we can refer to the unsaturated heterocycle by considering it as a reduced pyridine, for example., a dihydropyridine (two double bonds) or tetrahydropyridine (one double bond).

Nicotinamide Adenine Dinucleotide: Reduction Of A Pyridinium Salt:

Nicotinamide adenine dinucleotide (NAD⁺) is a complex molecule in which a pyridinium salt provides the reactive functional group, hence the superscript + in its abbreviation.

• NAD⁺ acts as a biological oxidizing agent, and in so doing is reduced to NADH (reduced nicotinamide adenine dinucleotide).
• An enzyme, a dehydrogenase, catalyzes the process and NAD⁺ is the cofactor for the enzyme. The reaction can be regarded as directly analogous to the hydride reduction of a pyridinium system to a dihydropyridine, as described above.
• We have already seen that NADH can act as a reducing agent, delivering the equivalent of hydride to a carbonyl compound.

In the oxidizing mode, the enzyme can extract hydride from the substrate and use it to reduce the pyridinium salt NAD⁺, producing the dihydropyridine NADH.

The substrate in most reactions of this type is alcohol, which becomes oxidized to an aldehyde or ketone, for example., ethanol is oxidized to acetaldehyde.

• Some reactions employ the alternative phosphorylated cofactor NADP⁺; the phosphate does not function in the oxidation step but is merely a recognition feature helping to bind the compound to the enzyme. The full structures of NAD⁺ and NADP⁺.
• Note that the attack of hydride is at position 4 of the dihydropyridine ring. This is controlled by the enzyme, but it is also probably the only site accessible since the rest of the complex molecule hinders the approach to positions 2 and 6.

Tautomerism: Pyridones

The pyridine ring system may carry substituents, just as we have seen with benzene rings.

• We have encountered several such derivatives in the previous section. Hydroxy or amino heterocycles, however, may sometimes exist in tautomeric forms.
• We have met the concept of tautomerism primarily with carbonyl compounds, and have seen the isomerization of keto and enol tautomers.
• In certain cases, for example., 1,3-dicarbonyl compounds, the enol form is a major component of the equilibrium mixture. amides.
• This was used to explain why amides are fragile bases. Note that such resonance forms of pyridones are favorable, having a positive charge on the nitrogen and a negative charge on the more electronegative oxygen.
• In addition, the structure gains further stabilization from the carbonyl group. The pyridone forms are very much favored over the phenol forms, and typical C=O peaks are seen in the infrared (IR) spectra.

In the example shown, liquid acetylacetone contains about 76% of the enol tautomer.

2-Hydroxy- and 4-hydroxy-pyridines are in equilibrium with their tautomeric ‘amide’ structures containing a carbonyl. These tautomers are called 2- pyridone and 4-pyridone respectively.

This type of tautomerism does not occur with the corresponding benzene derivative phenol, since it would destroy the stabilization conferred by aromaticity.

So why can tautomerism occur with a hydroxypyridiet? It is because 2-pyridone and 4-pyridone still retain aromaticity, with the nitrogen atom donating its lone pair electrons to the aromatic sextet.

• This is more easily seen in the resonance structures and should remind us of the resonance stabilization in amides. This was used to explain why amides are fragile bases.
• Note that such resonance forms of pyridones are favorable, having a positive charge on the nitrogen and a negative charge on the more electronegative oxygen. In addition, the structure gains further stabilization from the carbonyl group.

The pyridone forms are very much favored over the phenol forms, and typical C=O peaks are seen in the infrared (IR) spectra.

Note, however, that we cannot get the same type of tautomerism with 3-hydroxypyridine. In polar solvents, 3-hydroxypyridine may adopt a dipolar zwitterionic form.

• This may look analogous to the previous structure, but appreciate that there is a difference. With 3-hydroxyproline, the zwitterion is a major contributor and arises simply from acid-base properties.
• The hydroxyl group acts as an acid, losing a proton, and the nitrogen acts as a base, gaining a proton.

The structure from 2-pyridone is a minor resonance form that helps to explain charge distribution; the compound is almost entirely 2-pyridone.

Like amides, 2- and 4-pyridones are also very weak bases, much weaker than amines.

• Like amides, they protonate on oxygen rather than nitrogen. This further emphasizes that the nitrogen lone pair is already in use and not available for protonation.
• On the other hand, the N–H can readily be deprotonated; pyridones are appreciably acidic (pK_aabout 11).
• The conjugate base benefits from considerable resonance stabilization, both via the carbonyl group and also via the ring.

The main structures in which charge is associated with the electronegative N or O atoms.

It is thus possible to N-alkylate a pyridone by exploiting its acidity. As with enolate anions, there is the possibility for O-alkylation and N-alkylation.

Although it depends upon the conditions and the nature of the electrophile, carbon electrophiles tend to react with nitrogen rather than oxygen.

A useful reaction of pyridones is conversion into chloropyridines by the use of phosphorus oxychloride POCl₃ in the presence of PCl₅. This appears to react initially on oxygen, forming a good leaving group, which is subsequently displaced by chloride.

Aminopyridines are also potentially tautomeric with corresponding imino forms.

However, 2-aminopyridine and 4-aminopyridine exist almost entirely as amino tautomers – indeed, we have just seen 2-aminopyridine as a product of the Chichibabin reaction.

• Which tautomer is preferred for hydroxy and amino heterocycles is not always easily explained; but, as a generalization, we find that the oxygen derivatives exist as carbonyl tautomers and amino heterocycles favor the amino tautomers.
• At this stage, we should just register the potential for tautomerism in aminopyridines; we shall see important examples with other heterocycles.

Aminopyridines protonate on the ring nitrogen, and 4-aminopyridine is a stronger base than 2-aminopyridine.

This may be rationalized by a consideration of resonance in the conjugate acids.

• The conjugate acids from ring protonation benefit from charge delocalization, which is greater in 4-amino pyridinium than in 2-amino pyridinium.
• This type of delocalization is not possible in 3-aminopyridinium; 3-aminopyridine (pK_a 6.0) is the weakest base of the three aminopy ridines and has basicity more comparable to that of pyridine (pK_a 5.2).

Pyrylium Cation And Pyrones

Pyrones are oxygen analogs of pyridones and are potentially aromatic. However, there is little evidence that the dipolar resonance forms of either 2-pyrone or 4-pyrone make any significant contribution.

• Their chemical behavior suggests they should be viewed more as conjugated lactones (2-pyrones) or vinylogous lactones (4-pyrones) rather than aromatic systems since many reactions lead to ring opening.

The pyrylium cation is isoelectronic with pyridine: it has the same number of electrons and, therefore, we also have aromaticity. Oxygen is normally divalent and carries two lone pairs.

If we insert oxygen into the benzene ring structure, then it follows that, by having one electron in a p orbital contributing to the aromatic sextet, there is a lone pair in an sp² orbital, and the remaining electron needs to be removed, hence the pyrylium cation.

However, oxygen tolerates a positive charge less readily than nitrogen and aromatic stabilization is less than with pyridine.

Flavonoids are natural phenolic systems containing pyrylium and pyrone rings and provide the most prominent examples.

We have met some of these systems under antioxidants. Coumarins contain a 2-pyrone system. Note that all of these compounds are fused to a benzene ring and are strictly benzopyran or benzopyrylium systems.

Dicoumarol And Warfarin:

Warfarin provides us with a slightly incongruous state of affairs: it is used as a drug and also as a rat poison.

• It was developed from a natural product, dicoumarol, and provides us with a nice example of how pyrone chemistry resembles that of conjugated lactones rather than aromatic systems.
• Many plants produce coumarins; coumarin itself is found in sweet clover and contributes to the smell of new-mown hay.
• However, if sweet clover is allowed to ferment, oxidative processes initiated by the microorganisms lead to the formation of 4-hydroxycoumarin rather than coumarin.

4-Hydroxycoumarin then reacts with formaldehyde, also produced via the microbial degradative reactions, and provides dicoumarol.

4-Hydroxycoumarin can be considered an enol tautomer of a 1,3-dicarbonyl compound; conjugation with the aromatic ring favors the enol tautomer. This now exposes its potential as a nucleophile.

• Whilst we may begin to consider enolate anion chemistry, no strong base is required and we may formulate a mechanism in which the enol acts as the nucleophile, in a simple aldol reaction with formaldehyde.

Dehydration follows and produces an unsaturated ketone, which then becomes the electrophile in a Michael reaction. The nucleophile is a second molecule of 4-hydroxycoumarin.

Animals fed spoiled sweet clover were prone to fatal hemorrhages. The cause was traced to the presence of dicoumarol.

• This compound interferes with the effects of vitamin K in blood coagulation, the blood loses its ability to clot, and minor injuries can lead to severe internal bleeding.
• Synthetic dicoumarol has been used as an oral blood anticoagulant in the treatment of thrombosis, where the risk of blood clots becomes life-threatening.

It has since been superseded by warfarin, a synthetic development based on the natural product.

Warfarin was initially developed as a rodenticide and has been widely employed for many years as the first-choice agent, particularly for the destruction of rats.

• After consumption of warfarin-treated bait, rats die from internal hemorrhage.
• Warfarin is synthesized from 4-hydroxycoumarin by a Michael reaction on benzalacetone, again exploiting the nucleophilicity of the hydroxypyrone.

Benzalacetone is the product of an aldol reaction between benzaldehyde and acetone.

Five-Membered Aromatic Heterocycles: Pyrrole

Pyrrole (azacyclopentadiene) is the ring system obtained if we replace the CH₂ group of cyclopenta diene with NH.

• Although cyclopentadiene is certainly not aromatic, pyrrole has an aromatic character because nitrogen contributes two electrons, its lone pair, to the π-electron system.
• We have also noted from resonance forms that nitrogen carries a partial positive charge, and the carbons are electron-rich.

This is stronger than the opposing inductive effect.

This is reflected in the basicity of pyrrole. Pyrrole is a particularly weak base, with pK_a of the conjugate acid −3.8.

First, we should realize that protonation of pyrrole will not occur on nitrogen: nitrogen has already used up its lone pair by contributing to the aromatic sextet, so protonation would necessarily destroy aromaticity.

It is possible to protonate pyrrole using a strong acid, but even then the protonation occurs on C-2 and not on the nitrogen.

• Although this still destroys aromaticity, there is some favorable resonance stabilization in the conjugate acid.
• Protonation on C-3 is not as favorable, in that there is less resonance stabilization in the conjugate acid.
• It turns out that, as opposed to acting as a base, pyrrole is potentially an acid (pKa 17.5); it is not a particularly strong acid, but stronger than we might expect for a secondary amine system (pK_a about 36).
• This is because the anion formed by losing the proton from nitrogen has a negative charge on the relatively electronegative nitrogen, but maintains its aromaticity.

Unlike in pyrrole, the anion resonance structures do not involve charge separation.

It is appropriate here to compare the acidity of cyclopentadiene, which has pK_a 16, is considerably more acidic than most hydrocarbon systems and comparable to water and alcohols.

• Removal of one of the CH₂ protons from the non-aromatic cyclopentadiene generates the cyclopentadienyl anion.
• This anion has an aromatic sextet of electrons, two electrons being contributed by the negatively charged carbon.
• The charge distribution in pyrrole leads us to predict that it will react readily with electrophiles; or, put another way, pyrrole will behave as a nucleophile.
• This is indeed the case, and the ease of elec trophilic substitution contrasts with the behavior of pyridine above, where charge distribution favored nucleophilic attack onto the heterocycle.

Although our resonance description of pyrrole shows negative charge can be dispersed to any ring carbon, pyrrole reacts with electrophiles preferentially at C-2 rather than C-3, unless the 2-position is already substituted.

This may reflect that there is more charge dispersion in the addition cation from attack at C-2 than there is from attack at C-3.

• This is, of course, the same argument as used above for C-protonation; protonation (pyrrole acting as a base) also occurs at C-2.
• As with protonation, electrophiles do not react at the nitrogen center. Pyrrole is very reactive towards electrophiles.

For example, treatment with bromine leads to the substitution of all four positions.

Indeed, it is often difficult to control electrophilic attack so that monosubstitution occurs.

A further problem is that pyrrole polymerizes in the presence of strong acids and Lewis acids, so that typical electrophilic reagents, for example., HNO₃ –H₂SO₄ and RCOCl–AlCl₃, cannot be used. Polymerization involves the conjugate acid functioning as the elec trophile.

To achieve useful monosubstitution it is necessary to employ relatively mild conditions, often without a catalyst.

• Nitration may be accomplished with the reagent acetyl nitrate, giving mainly 2-nitropyrrole.
• Acetyl nitrate is formed by reacting acetic anhydride with fuming nitric acid. Since the other product is acetic acid, there is no strong mineral acid present to cause polymerization.

It is also possible to synthesize 2-acetyl pyrrole simply by using acetic anhydride, and pyrrole can act as the nucleophile in the Mannich reaction.

Although pyrrole is a weak acid, it can be deprotonated by using a strong base, for example., sodium hydride, and the anion can be used in typical nucleophilic reactions.

This allows simple transformations such as N-alkylation, N-acylation, and N-sulfonation. Note particularly that whereas pyrrole reacts with elec trophiles at carbon, usually C-2, the pyrrole anion reacts at the nitrogen atom.

Porphyrins And Corrins:

Pyrrole reacts with aldehydes and ketones under acidic conditions to form polymeric compounds. In many cases these are intractable resin-like materials; however, with appropriate carbonyl compounds, interesting cyclic tetramers can be formed in very good yields.

Thus, pyrrole and acetone react as shown above. This involves pyrrole acting as the nucleophile to attack the protonated ketone in an aldol-like reaction.

• This is followed by the elimination of water, facilitated by the acidic conditions. This gives an intermediate alkylidene pyrrolium cation, a highly reactive electrophile that reacts with another molecule of nucleophilic pyrrole.
• We then have a repeat sequence of reactions, in which further acetone and pyrrole molecules are incorporated.
• The presence of the two methyl substituents from acetone forces the growing polymer to adopt a planar array, and this eventually leads to a cyclic tetramer, the terminal pyrrole attacking the alkylidene pyrrolium cation at the other end of the chain.
• The cyclic tetramer shown is structurally related to the porphyrins. The basic ring system in porphyrins is porphin, which is more oxidized than the tetramer from the pyrrole–acetone reaction, and has four pyrrole rings linked together by methine (–CH=) bridges.
• One of the features of porphin is that it is aromatic. It contains an aromatic 18 π-electron system, which conforms to Huckel’s rule, 4n + 2 with n = 4.
• The aromatic ring weaves around the porphin structure, and is composed entirely of double-bond electrons; it does not incorporate any nitrogen lone pairs.

Note that we can draw Kekule-like resonance structures for porphin. Do not be confused by seeing alternative structures with the double bonds arranged differently.

Porphyrin rings are formed in nature by a process that is remarkably similar to that shown above. Though the sequence contains some rather unusual features, the coupling process also involves nucleophilic attack onto an alkylidene pyrrolium cation. This may be generated from the precursor porphobilinogen by the elimination of ammonia.

One of the important properties of porphyrins is that they are complex with divalent metals, the pyrrole nitrogens being ideally spaced to allow this.

Of vital importance to life processes are the porphyrin derivatives chlorophyll and haem. Chlorophyll (a mixture of structurally similar porphyrins; chlorophyll a is shown) contains magnesium, and is, of course, the light-gathering pigment in plants that permits photosynthesis.

Plants and a few microorganisms use photosynthesis to produce organic compounds from inorganic materials found in the environment, whereas other organisms, such as animals and most microorganisms, rely on obtaining their raw materials in their diet, for example., by consuming plants.

• Haemoglobin, the red pigment in blood, serves to carry oxygen from the lungs to other parts of the body tissue.
• This material is made up of the porphyrin haem and the water-soluble protein globin.
• The haem component shares many structural features with chlorophyll, one of the main differences being the use of Fe²⁺ as the metal rather than Mg²⁺ as in chlorophyll.
• The oxygen-carrying ability of hemoglobin involves a six-coordinate iron, with an imidazole ring from the protein (a histidine residue) occupying the sixth position.
• Porphyrin rings containing iron are also a feature of the cytochromes. Several cytochromes are responsible for the latter part of the electron transport chain of oxidative phosphorylation that provides the principal source of ATP for an aerobic cell.
• Their function involves alternate oxidation-reduction of the iron between Fe²⁺ (reduced form) and Fe³⁺ (oxidized form).

The individual cytochromes vary structurally, and their classification (a, b, c, etc.) is related to their absorption maxima in the visible spectrum. They contain a haem system that is covalently bound to protein through thiol groups.

An especially important example is cytochrome P-450, a coenzyme of the so-called cytochrome P-450-dependent mono-oxygenases.

• These enzymes are frequently involved in biological hydroxylations, either in biosynthesis or in the mammalian detoxification and metabolism of foreign compounds such as drugs.
• Cytochrome P-450 is named after its intense absorption band at 450 nm when exposed to CO, which is a powerful inhibitor of these enzymes.
• A redox change involving the Fe atom allows binding and the cleavage of molecular oxygen to oxygen atoms, with subsequent transfer of one atom to the substrate.
• In most cases, NADPH features as a hydrogen donor, reducing the other oxygen atom to water. Many such systems have been identified, capable of hydroxylating aliphatic or aromatic systems, as well as producing epoxides from alkenes.
• A related ring system containing four pyrroles is seen in vitamin B₁₂, but this has two pyrroles directly bonded and is termed a corrin ring.
• Vitamin B₁₂ is extremely complex and features six-coordinate Co²⁺ as the metal component. Four of the six coordination are provided by the corrin ring nitrogens and a fifth by a dimethyl benzimidazole moiety.
• The sixth is variable, being cyano in cyanocobalamin (vitamin B₁₂), but other anions may feature in vitamin B₁₂ analogs.

Vitamin B₁₂ appears to be entirely of microbial origin, with intestinal flora contributing to human dietary needs. Insufficient vitamin B₁₂ leads to pernicious anemia, a disease that results in nervous disturbances and low production of red blood cells.

Furan And Thiophene:

Furan and thiophene are the oxygen and sulfur analogs respectively of pyrrole. Oxygen and sulfur contribute two electrons to the aromatic sextet but still retain lone pair electrons.

• There is one significant difference, however, in that oxygen uses electrons from a 2p orbital, whereas the electrons that sulfur contributes originate from a 3p orbital.
• In the case of furan H of thiophene, this reduces orbital overlap with the carbon 2p orbitals.
• Both compounds are thus aromatic, and their chemical reactivity reflects what we have learned about pyrrole.

• The most typical reaction is electrophilic substitution. However, we find that pyrrole is more reactive than furan towards electrophiles and thiophene is the least reactive; all are more reactive than benzene
• This relates to the relative stability of positive charges located on nitrogen, oxygen, and sulfur. We used similar electronegativity reasoning to explain the relative basic strengths of nitrogen, oxygen, and sulfur derivatives.

Furan is also the ‘least aromatic’ of the three, i.e. it has the least resonance stabilization, and undergoes many reactions in which the aromatic character is lost, for example., addition reactions or ring opening.

Note that the dipoles of furan and thiophene are opposite in direction to those in pyrrole.

• In furan and thiophene, there is a greater inductive effect opposing the resonance effect, whereas in pyrrole the resonance contribution was greater.

In the non-aromatic analogs, the heteroatom is at the negative end of the dipole in all cases.

Nevertheless, we can interpret the reactions of furan and thiophene by logical consideration as we did for pyrrole.

• In electrophilic substitutions, there is again a preference for 2- rather than 3-substitution, and typical electrophilic reactions carried out under acidic conditions are difficult to control.
• However, because of lower reactivity compared with pyrrole, it is possible to exploit Friedel-Crafts acylations, though using less-reactive anhydrides rather than acyl chlorides, and weaker Lewis acids than AlCl₃.
• Nitration can be achieved with acetyl nitrate rather than nitric acid. In the case of furan, this is slightly anomalous, in that it involves an addition intermediate by combination of the carbocation with acetate.

This subsequently aromatizes by loss of acetic acid. The less-reactive thiophene can even be nitrated with concentrated nitric acid when it yields a mixture of 2- and 3-nitrothiophene.

Six-membered rings with two heteroatoms

Diazines:

A diazabenzene, i.e. a benzene ring in which two of the CH functions have been replaced with nitrogen, is termed a diazine.

• Three isomeric variants are possible; these are called pyridazine, pyrimidine, and pyrazine.
• These structures are all aromatic, the nitrogen atoms functioning in the same way as the pyridine nitrogen, each contributing one p electron to the aromatic sextet, with a lone pair in an sp² orbital.

The diazines are much weaker bases than pyridine (pK_a 5.2).

If we consider the inductive and resonance effects in pyridine, we have seen that these both draw electrons toward the nitrogen.

• Therefore, a second nitrogen will have destabilizing effects on the conjugate acid formed by the protonation of the first nitrogen.
• The order of basicity in pyridazine, pyrimidine, and pyrazine is influenced by secondary effects, which will not be considered here.
• Deprotonation is very difficult and would require extremely strong acids; in the case of pyridazine, it is essentially impossible because of the need to establish positive charges on adjacent atoms.
• In general, we can consider that the extra nitrogen, through its combined effects, makes the other ring atoms more electron deficient than they would be in pyridine; as a result, the diazines are more susceptible to nucleophilic attack than pyridine.
• In pyrazines and pyridazines, the second nitrogen helps by withdrawing electrons from atoms that would carry a negative charge in the addition anion.

In pyrimidines, the two nitrogens share the negative charge of the addition anion, and pyrimidines are the more reactive towards nucleophiles.

Halodiazines react readily with nucleophiles with a displacement of the halide-leaving group.

• This follows what we have seen with halopyridines, but the halodiazines are more reactive because of the influence of the extra nitrogen.
• Thus, 2-chloropyrazine and 3-chloropyridazine easily yield the corresponding amino derivatives on heating with ammonia in an alcohol solution.
• The 2- and 4-halo pyrimidines are even more reactive and substitute at room temperature. This is because of the improved delocalization of negative charge in the addition anion.

5-Halopyrimidines are the least susceptible to nucleophilic displacement: the halogen is neither α nor γ to nitrogen, and cannot benefit from any favorable charge localization on nitrogen.

Diazines are generally resistant to electrophilic attack on carbon, and, as for pyridine, addition to nitrogen is observed.

• Alkyl halides give mono-quaternary salts; di-quaternary salts are not formed under normal conditions.
• Of course, if the diazine ring carries a substituent that makes the starting material non-symmetric, then the product will almost always be a mixture of two isomeric quaternary salts.

Steric and inductive effects rather than resonance effects appear to influence the reaction and formation of the major product.

Tautomerism In Hydroxy- And Amino-Diazines:

We have seen that 2- and 4-hydroxypyridines exist primarily in their tautomeric ‘amide-like’ pyridone forms.

• This preference over the ‘phenolic’ tautomer was related to these compounds still retaining their aromatic character, with further stabilization from the carbonyl group.
• 3- Hydroxypyridine cannot benefit from this additional stabilization. In contrast, 2-aminopyridine and 4-aminopyridine exist almost entirely as amino tautomers, although they are potentially tautomeric with imino forms.
• We also encounter tautomerism in hydroxy- and amino-diazines, and the preference for one tautomeric form over the other follows what we have seen with the pyridine derivatives.
• Thus, except for 5-hydroxy pyrimidine, all the mono-oxygenated diazines exist predominantly in the carbonyl tautomeric form.

We term these ‘amide-like’ tautomers diazinones. 5-Hydroxypyrimidine is analogous to 3- hydroxypyridine, in that the hydroxyl is wrongly positioned for tautomerism.

The diazinon tautomers are identified by using terminology such as 3(2H)-pyridazine for the car bonyl tautomer of 3-hydroxypyrazine.

• The 3(2H) prefix signifies the position of the oxygen (3- pyridazine) and specifies the NH is at position 2.
• Note that, in addition to the diazine–diazinone tau H tomerism, when the nitrogens have a 1,3-relationship there is further tautomerism possible, for example., 4(1H)- pyrimidone 4(3H)-pyrimidone.

Diazinones may be converted into chlorodiazines by the use of phosphorus oxychloride, just as pyridones yield chloropyridines.

Aminodiazines exist in the amino form. These compounds contain two-ring nitrogens and a primary amino group.

Interestingly, they are more basic than the unsubstituted diazine and always protonate on a ring of nitrogen.

• This allows resonance stabilization of the conjugate acid utilizing the lone pair of the amino substituent.
• It has been found that one can predict which nitrogen is protonated from the ring nitrogen–amino substituent relationship, which follows the preference sequence γ > α > β, as in the examples shown.

This can be related to achieving maximum charge distribution over the molecule.

Pyrimidines And Nucleic Acids:

The storage of genetic information and the transcription and translation of this information are functions of the nucleic acids deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

• They are polymers whose building blocks are nucleotides, which are themselves combinations of three parts, i.e. a heterocyclic base, a sugar, and phosphate.
• The bases are either monocyclic pyrimidines or bicyclic purines (see Section 14.1). Three pyrimidine bases are encountered in DNA and RNA, cytosine (C), thymine (T), and uracil (U).
• Cytosine is common to both DNA and RNA, but uracil is found only in RNA and thymine is found only in DNA.

In nucleic acid, the bases are linked through an N-glycoside bond to a sugar, either ribose or deoxyribose; the combination base plus sugar is termed a nucleoside. The nitrogen bonded to the sugar is shown.

We should note particularly that uracil and thymine are dioxypyrimidines, whereas cytosine is an amino oxypyrimidine. All three pyrimidines are thus capable of existing in several tautomeric forms.

The number of possible forms is reduced somewhat by the fact that one of the nitrogens is bonded to the sugar in the nucleic acid; it no longer carries hydrogen to participate in tautomerism.

• The tautomeric forms indicated are found to predominate in nucleic acids. The oxygen substituents exist almost entirely as carbonyl groups, whereas
• the amino group is preferred over possible imino forms. Although we are accustomed to thinking of nucleic acids containing ‘pyrimidine’ bases, this is not strictly correct. Cytosine exists as an aminopyrimidone, and thymine and uracil are pyrimidines. Further, they are not particularly basic.
• Cytosine is the most basic of the three (pK_a 4.6), in that the amino group by a resonance effect can stabilize the conjugate acid (compare 4-aminopyrimidine pK_a 5.7 above). Thymine and uracil are very weak bases, in that they are ‘amide-like’.
• The most far-reaching feature of nucleic acids is the ability of the bases to hydrogen bond to other bases.
• This property is fundamental to the double helix arrangement of the DNA molecule, and the translation and transcription via RNA of the genetic information present in the DNA molecule.
• Hydrogen bonding occurs between complementary purine and pyrimidine bases and involves either two or three hydrogen bonds.
• In DNA, the base pairs are adenine–thymine and guanine-cytosine. In RNA, base pairing involves guanine-cytosine and adenine–uracil.
• This property will be discussed in detail in Section 14.2, but it is worth noting at this stage that hydrogen bonding is achieved between amino substituents (N–H) and the oxygen of carbonyl groups.
• These functions in the pyrimidine bases arise directly from the tautomeric preferences.

Five-Membered Rings With Two Heteroatoms:

We have looked at the five-membered aromatic heterocycles pyrrole, furan, and thiophene. The introduction of a second heteroatom creates azoles. This name immediately suggests that nitrogen is one of the heteroatoms.

• As soon as we consider valencies, we discover that to draw a five-membered aromatic heterocycle with two heteroatoms, it must contain nitrogen.
• A neutral oxygen or sulfur atom can have only two bonds, and we cannot, therefore, have more than one of these atoms in any aromatic heterocycle. On the other hand, there is potential for having as many nitrogens as we like in an aromatic ring.
• Thus, in five-membered aromatic heterocycles with two heteroatoms, we can have two nitrogens, one nitrogen plus one oxygen, or one nitrogen plus one sulfur.

The heteroatoms can be positioned only 1,2 or 1,3. The numbering of the ring system starts from the heteroatom with the higher atomic number; nitrogen will always be the higher of the two numbers in the oxazole and thiazole systems. In imidazole, numbering begins at the NH.

We can visualize these heterocycles as similar to the simpler aromatic systems pyrrole, furan, and thiophene.

• For example, in imidazole, each carbon and nitrogen will be sp² hybridized, with p orbitals contributing to the aromatic π system. The carbon atoms will each donate one electron to the π system.
• Then, as in pyrrole, the NH nitrogen supplies two electrons, and, as in pyridine, the =N– supplies one electron and retains a lone pair.

Oxygen or sulfur would also supply two electrons, as we saw in furan and thiophene.

It also follows that a compound like imidazole has one pyridine-like nitrogen and one pyrrolelike nitrogen.

• We may thus expect to see imidazole having properties resembling a combination of either pyridine- or pyrrole-like reactivity.
• The availability and location of lone pair electrons are crucial to our understanding of imidazole chemistry, and it often helps to include these in the structure.

1,3-Azoles: Imi Dazole, Oxazole, And Thiazole:

Imidazole (pK_a 7.0) is a stronger base than either pyridine (pK_a 5.2) or pyrrole (pK_a − 3.8).

• When we compared the basicity of pyridine with that of the aliphatic amine piperidine (pK_a 11.1), we implicated the higher s character of the pyridine lone pair (sp²) compared with that in piperidine (sp³) to account for pyridine’s lower basicity.
• Even so, imidazole seems abnormally basic for a compound with sp²-hybridized nitrogen.
• Therefore, when we meet structures for the imidazole-containing amino acid histidine, we may encounter either of the tautomeric forms shown.
• Though there imidazole appears to stem from the symmetry of the conjugate acid, and the resonance stability conferred by this.

The 1,3-relationship allows the two nitrogen atoms to share the charge equally. Note that pK_a 7.0 means imidazole is 50% protonated in water.

Imidazole (pK_a 14.2) is also more acidic than pyrrole (pK_a 17.5); this, again, is a feature conferred by symmetry and enhanced resonance stabilization in the conjugate base.

Oxazole (pK_a 0.8) and thiazole (pK_a 2.5) are weak bases. The basicity of the nitrogen is reduced by the presence of the other heteroatom.

Oxygen and sulfur provide a stronger electron-withdrawing inductive effect, compared with nitrogen, but a much weaker electron-releasing resonance effect.

Tautomerism In Imidazoles:

A complicating factor in imidazoles is tautomerism. Imidazole tautomerizes rapidly in solution and consists of two identical tautomers.

• This becomes a problem, though, in an unsymmetrically substituted imidazole, and tautomerism means 4-methylimidazole is in equilibrium with 5-methylimidazole.
• Depending upon substituents, one tautomer may predominate. Tautomerism of this kind cannot occur with N-substituted imidazoles; it is dependent upon the presence of an N–H group.

Tautomerism is also not possible with oxazoles or thiazoles.

Therefore, when we meet structures for the imidazole-containing amino acid histidine, we may encounter either of the tautomeric forms shown. Though there will usually be no indication that tautomers exist, do not think there is a discrepancy in structures.

The Imidazole Ring Of Histidine: Acid-Base Properties

The amino acid histidine contains an imidazole ring. We have just seen that unsubstituted imidazole as a base has pK_a 7.0. From the Henderson–Hasselbalch equation

⇒ \(\mathrm{pH}=\mathrm{p} K_{\mathrm{a}}+\log \frac{\text { [base }]}{\text { [acid }]}\)

we can deduce that in water, at pH 7, the concentrations of acid and conjugate base are equal, i.e. imidazole is 50% protonated.

• The imidazole side-chain of histidine has a pK_a value of 6.0, making it a weaker base than the unsubstituted imidazole.
• This reflects the electron-withdrawing inductive effect of the amino group, or, more correctly the ammonium ion, since amino acids at pH values around neutrality exist as doubly charged zwitterionic forms.
• Using the Henderson–Hasselbalch equation, this translates to approximately 9% ionization of the heterocyclic side-chain of histidine at pH 7.

In proteins, pK_a values for histidine side chains are estimated to be in the range 6–7, so the level of ionization will, therefore, be somewhere between 9 and 50%, depending upon the protein.

This level of ionization is particularly relevant in some enzymic reactions where histidine residues play an important role.

• This means that the imidazole ring of a histidine residue can act as a base, assisting in the removal of protons, or that the imidazolium cation can act as an acid, donating protons as required.
• The terminology used for such donors and acceptors of protons is a general acid catalyst and general base catalyst respectively.
• A typical role for the histidine imidazole ring is shown below, in the enzyme mechanism for a general base-catalyzed hydrolysis of an ester.
• The imidazole nitrogen acts as a base to remove a proton from water, generating hydroxide that attacks the carbonyl. Subsequently, the alkoxide-leaving group is deprotonated by the imidazolium ion.

General Base-Catalysed Ester Hydrolysis:

The beauty of this is that we effectively have the same mechanism as in the hydrolysis of an ester using aqueous sodium hydroxide. However, with the enzyme catalyst, this is all taking place at pH 7 or thereabouts.

• Implicit in the above mechanism, though not emphasized, is the pronounced ability of imidazole rings to hydrogen bond.
• Imidazole resembles water, in that it is both a very good donor and a very good acceptor for hydrogen bonding.

Imidazole (and also pyrazole) has a higher-than-expected boiling point, ascribed to intermolecular hydrogen bonding. This leads to polymeric-like structures for imidazole and dimers for pyrazole.

In enzymic mechanisms, we are not usually going to get imidazole–imidazole hydrogen bonding, but the ability of imidazole to hydrogen bond to water, to other small molecules, and carboxylic acid side chains facilitates the enzyme reaction by correctly positioning the reagents.

Histamine And Histamine Receptors:

Most people have heard of antihistamines, even if they have little concept of the nature of histamine.

Histamine is the decarboxylation product from histidine and is formed from the amino acid by the action of the enzyme histidine decarboxylase.

The mechanism of this pyridoxal phosphate-dependent reaction will be studied in more detail later.

Histamine is released from mast cells during inflammatory or allergic reactions. It then produces its typical response by interaction with specific histamine receptors, of which there are several types.

• H₁ receptors are associated with inflammatory and allergic reactions, and H₂ receptors are found in acid-secreting cells in the stomach. Drugs to target both of these types of receptors are widely used.
• The term antihistamine usually relates to H₁ receptor antagonists. These drugs are valuable for pain relief from insect stings or for the treatment and prevention of allergies such as hay fever.
• Major effects of histamine include dilation of blood vessels, inflammation and swelling of tissues, and narrowing of airways.
• In serious cases, life-threatening anaphylactic shock may occur, caused by a dramatic fall in blood pressure. Remarkably, current H₁ receptor antagonists, for example., diphenhydramine, bear little if any structural similarity to histamine.
• The main clinical use of H₂ receptor antagonists is to inhibit gastric secretion in the treatment of stomach ulcers.

These agents all contain features that relate to the histamine structure, in particular the heterocyclic ring. Cimetidine and ranitidine are the most widely used in this class.

Cimetidine contains an imidazole ring comparable to histamine, a sulfur atom (thioether group) in the side chain, and a terminal functional group based upon a guanidine.

• Ranitidine bears considerable similarity to cimetidine, but there are some important differences.
• The heterocycle is now furan rather than imidazole, and the guanidine has been modified to an amidine. A newer drug, nizatidine, is a variant of ranitidine with a thiazole heterocyclic ring system.

Reactivity Of 1,3-Azoles:

Electrophiles can add to N-3, the azomethine =N–, of 1,3-azoles as they can to the pyridine nitrogen.

N Alkylation is complicated in the case of imidazole by the possibility of forming a dialkyl imidazolium salt; the first-formed protonated N-alkyl imidazole can be deprotonated by imidazole, then alkylated further.

N-acylation is mechanistically similar, and mono-acylation can be accomplished by using two molar equivalents of imidazole to one of the acylating agents, the second mole serving to deprotonate the first-formed N-3-acyl imidazolium salt.

• Note that in Ac dimethylimidazolium salt alkylation and acylation, it is the =N– that acts as the nucleophile; this carries the only lone pair.

However, proton loss occurs from the other nitrogen, giving the impression that the N–H has been alkylated or acylated.

The 1,3-diazoles are much less susceptible to electrophilic substitution than pyrrole, furan, and thiophene, but are more reactive than pyridine. Imidazole is the most reactive and may be nitrated readily.

• Substitution occurs at C-5, but tautomerism then leads to the 4(5) mixture.
• The position of substitution may be predicted from a consideration of resonance structures: attack at C-5 provides maximum delocalization with no particularly unfavorable resonance forms.

There is less delocalization after the attack at C-2; one of the resonance forms has an unfavorable electron-deficient nitrogen.

In general, the 1,3-diazoles do not react by nucleophilic substitution, although imidazole can participate in the Chichibabin reaction with substitution at C-2; the position of substitution is equivalent to that noted with pyridine.

• Nucleophilic species that are strong bases, like sodium amide, are more likely to remove the NH proton (pK_a 14).

However, oxazole and thiazole do not have any NH, and the most acidic proton is that at C-2. The electronegative oxygen and sulfur can support an adjacent negative charge.

It is found that quaternary salts of 1,3-azoles are deprotonated at C-2 in the same way.

• Rates of deprotonation are considerably faster because of the influence of the quaternary center that provides a favorable inductive effect.
• The conjugate base bearing opposite charges on adjacent atoms is termed a ylid (or ylide; pronounced il-ide). This ylid, with neg negative charge on carbon, is potentially a nucleophilic species.
• Thus, it is found that both oxazoline and thiazolium salts undergo H–D exchange at C-2 remarkably quickly under basic conditions, illustrating very simply this nucleophilic behavior.

The Thiazolium Ring In Thiamine:

Thiamine (vitamin B₁), in the form of thiamine diphosphate (TPP), is a coenzyme of considerable importance in carbohydrate metabolism.

• Dietary deficiency leads to the condition beriberi, characterized by neurological disorders, loss of appetite, fatigue, and muscular weakness.
• We shall study several TPP-dependent reactions. At this stage, we should merely examine the structure of thiamine, and correlate its properties with our knowledge of heterocycles.
• Thiamine contains two heterocyclic rings, a pyrimidine, and a thiazole, the latter present as a thiazolium salt.

The pyrimidine portion is unimportant for our understanding of the chemistry of TPP, though it may play a role in some of the enzymic reactions.

The proton in the thiazolium ring is relatively acidic (pK_a about 18) and can be removed by even weak bases to generate the carbanion or ylid; a ylid is a species with positive and negative charges on adjacent atoms.

• This ylid is an ammonium ylid with extra stabilization provided by the sulfur atom. The lid can act as a nucleophile and is also a reasonable leaving group.
• Prominent among TPP-dependent reactions is the oxidative decarboxylation of pyruvic acid to acetyl-CoA; this reaction links the glycolytic pathway to the Krebs cycle.
• The addition of the thiazolium ylid to the carbonyl group of pyruvic acid is the first reaction of this sequence, and this allows the necessary decarboxylation, the positive nitrogen in the ring acting as an electron sink.

In due course, the thiazolium ylid is regenerated as a leaving group. We shall look at this sequence in more detail.

1,2-Azoles: Pyrazole, Isoxazole, And Isothiazole

As in the 1,3-azoles, the =N–nitrogen carries a lone pair of electrons and 1,2-azoles are thus potentially basic.

• However, the direct linking of the two heteroatoms has a base-weakening effect.
• Thus, pyrazole has pK_a 2.5 and isoxazole pK_a − 3.0. The higher basicity in pyrazole is probably related to the symmetry of the contributing resonance structures.

The greater electron-withdrawing effect of oxygen compared with sulfur is reflected in the basicity of isothiazole (pK_a−0.5).

Heterocycles Fused To A Benzene Ring:

Many interesting and important heterocyclic compounds contain fused ring systems.

• Some of the common ones are the result of fusing a heterocycle to equivalent resonance structures in a benzene ring, and these have long-established trivial names, for example., indole, quinoline, and isoquinoline.
• Systematic names can be derived by relating to the parent heterocycle and using the prefix benzo to indicate its fusion with benzene.

It is necessary to define which of the bonds in the heterocycle is fused to benzene, and this is accomplished through the use of a bond descriptor, a lowercase italic letter in square brackets.

Thus, indole is benzo[b]pyrrole, quinoline is benzo[b]pyridine, and isoquinoline, an isomer of quinoline with a different type of fusion, becomes benzo[c]pyridine. A few other examples are shown below.

• Note that the bonds of the heterocycle are lettered starting from the heteroatom.
• Where we have two similar heteroatoms, lettering is chosen to produce the lower alternative. Thus, quinazoline is benzo[d]pyrimidine, not benzo[e]pyrimidine.

Where the heteroatoms are different, just as we number from the atom of higher atomic number, we also letter from the same atom. Hence, benzo[d]isoxazole is quite different from benzo[c]isoxazole.

The final fused ring system is then given a completely new numbering system, different from that of the heterocycle.

• Typically, this starts adjacent to the bridgehead atom and then proceeds around the fused ring. The major criterion is to generate the lowest number for the first heteroatom.
• Note that, in most cases, we have little regard for Kekulé forms of pyridine rings in which the Kekule form of a benzene or pyridine ring is drawn. The three versions of quinoline shown are simply contributing resonance forms.

However, some structures, such as isoindole, benzo[c]furan, or benzo[c]isoxazole above, can only be drawn in one way without invoking charge separation.

Quinoline And Isoquinoline

Quinoline and isoquinoline are benzopyridines. They behave by showing the reactivity associated with either the benzene or the pyridine rings.

• Quinoline is basic with a pK_a of 4.9, similar to that of pyridine (pK_a 5.2).
• As with pyridine, the nitrogen carries a lone pair in an sp² orbital. Alkyl halides and acyl halides also react with nitrogen to give N-alkyl- and N-acyl quinolinium salts.

The N-alkyl salts are stable, but the N-acyl salts hydrolyze rapidly in the presence of water.

Quinoline is much more reactive towards elec trophilic substitution than pyridine, but this is because substitution occurs on the benzene ring, not on the pyridine.

• We have already seen that pyridine carbons are unreactive towards electrophilic reagents, with strongly acidic systems protonating the nitrogen first, further inhibiting the reaction.
• This is again true in quinoline, so that the protonated system is involved in the reaction, and the benzene ring undergoes substitution.

With a nitrating mixture of HNO₃ –H₂SO₄, the products are 5- and 8-nitroquinoline in roughly equivalent amounts.

This may be rationalized by considering the stability of intermediate addition cations.

• When the electrophile attacks at C-5 or C-8, the intermediate cation is stabilized by resonance, each having two favorable forms that do not perturb the aromaticity of the pyridinium system.
• In contrast, for attack at C-6 or C-7, there is only one such resonance form. We used similar reasoning to explain why naphthalene undergoes preferential electrophilic substitution at the α-positions.

Whilst we may be a little unhappy about the protonation of a quinolinium cation to an intermediate that carries two positive charges, we find that N-methylquinolinium salts also undergo nitration at a similar rate to quinoline; so this mechanism appears correct.

Nucleophilic substitution occurs at C-2, and to a lesser extent C-4, as might be predicted from similar reactions with pyridine.

• Chichibabin amination occurs rather more readily than with pyridine, ggiving2-aminoquinoline.
• A typical hydride abstraction process occurs when quinoline is heated with sodium amide.

However, better yields have been achieved by performing the reaction at low temperatures in liquid ammonia solvent and then oxidizing the intermediate dihydroquinoline salt using potassium permanganate.

Quinolines carrying 2- or 4-halo substituents undergo nucleophilic substitution readily, in the same manner as 2- and 4-halo pyridines.

• Hydroxyquinolines with the hydroxyl at positions 2 or 4 exist mainly in the carbonyl form, i.e. 2-quinolone and 4-quinolone.

Note, however, that hydroxyls on the benzene ring would be typical phenols. Again, aminoquinolines follow the pyridine precedent, and the tautomeric imino forms are not observed.

Both 2-aminoquinoline and 4-aminoquinoline pro tonate first on the ring nitrogen, with 4-aminoquin oline being the more basic, the conjugate acid benefiting from increased charge distribution through H⁺ resonance.

No such resonance structures can be drawn for 3-aminoquinoline, which is much less basic (pK_a 4.9).

Quinolone Antibiotics:

The quinolone antibiotics feature as the one main group of antibacterial agents that is synthetic, and not derived from or based upon natural products, as are penicillins, cephalosporins, macrolides, tetracyclines, and aminoglycosides.

The first of these compounds to be employed clinically was nalidixic acid; more recent drugs in current use include ciprofloxacin, norfloxacin, and ofloxacin

‘Quinolone’ as a descriptor is an oversimplification, since nalidixic acid contains two fused pyridine rings rather than a benzopyridine, and ofloxacin has a morpholine ring fused to the quinolone.

• Nevertheless, the quinolone substructure is generally used when referring to this group of antibiotics.

The most important structural features for good antibacterial activity are a carboxylic acid at position 3, a small alkyl group at position 1, a 6-fluorine substituent, and a nitrogen heterocycle, often piperazine, at position 7.

Quinolones are good general antibiotics for systemic infections, and they are particularly useful for urinary tract infections because high concentrations are excreted into the urine.

• The mode of action involves interference with DNA replication by inhibiting DNA gyrase, a bacterial enzyme related to mammalian topoisomerases that breaks and reseals double-stranded DNA during replication.
• Isoquinoline (pK_a 5.4) has a similar basicity to quinoline and pyridine and also undergoes N-alkylation and N-acylation.

Nitration occurs smoothly to give predominantly 5-nitroisoquinoline; the isoquinolinium cation reacts more readily than the quinolinium cation.

Nucleophilic substitution occurs exclusively at position 1 in isoquinoline; the alternative position C-3 is quite unreactive.

This is explained by the loss of benzene resonance in the intermediate anion. Thus, Chichibabin amination gives 1-aminoisoquinoline.

Substitution with displacement of halide occurs readily at C-1 and much less readily at C-3 for the same reasons, i.e. the loss of benzene resonance if C-3 is attacked.

1-Isoquinolone exists completely in the carbonyl form, whereas 1-amino isoquinoline is the normal tautomer.

The basicity of 1-amino isoquinoline (pK_a 7.6) is similar to that of 2-aminoquinoline (pK_a 7.3).

Indole

Indole is the fusion of a benzene ring with a pyrrole. Like quinoline and isoquinoline, indole behaves as an aromatic compound.

• However, unlike quinoline and isoquinoline, where the reactivity was effectively part benzene and part pyridine, the reactivity in indole is modified by each component of the fusion.
• The closest similarity is between the chemistry of pyrroles and indoles. Indoles, like pyrroles, are very weak bases. The conjugate acid of indole has pK_a − 3.5; that of pyr role has pK_a − 3.8.

As in the case of pyrrole, nitrogen has already contributed its lone pair to the aromatic sextet, so N-protonation would necessarily destroy aromaticity in the five-membered ring.

Nevertheless, an equilibrium involving the N-protonated cation is undoubtedly set up, since acid-catalyzed deuterium exchange of the N hydrogen occurs rapidly, even under very mild acidic conditions.

• Protonation eventually occurs preferentially on carbon, as with pyrrole; but there is a difference, in that this occurs on C-3 rather than on C-2. This is the influence of the benzene ring.
• It can be seen that protonation on C-3 allows resonance in the five-membered ring and charge localization on nitrogen.

In contrast, any resonance structure from protonation at C-2 destroys the benzene ring aromaticity.

Indole is very reactive towards electrophiles, and it is usually necessary to employ reagents of low reactivity. Nitration with HNO₃ –H₂SO₄ is unsuccessful (compare pyrrole), but can be achieved using benzoyl nitrate.

It is also possible to brominate and methylate at C-3; however, conditions must be controlled carefully, since further electrophilic reactions may then occur.

Treatment with acetic anhydride leads to 1,3-diacetyl indole. Indole reacts readily as the nucleophile in Mannich reactions. This provides convenient access to other derivatives, as shown below.

Thus, quaternization at the side-chain nitrogen allows the ready elimination of trimethylamine. This is facilitated by the electron-releasing ability of the indole nitrogen and can be brought about by a mild base.

• By choosing KCN as the mild base, the transient 3-methyleneindoleninium salt can be trapped by cyanide nucleophiles, leading to indole acetonitrile.
• Reduction of the nitrile group with LAH provides a route to tryptamine. A simple addition to carbonyl compounds occurs under mild acidic conditions.
• Examples are given illus trate reaction with acetone, an aldol-like reaction, and conjugate addition to methyl vinyl ketone, a Michael-like reaction.

The first-formed alcohol products in aldol-like reactions usually dehydrate to give a 3- 3-alkylidene-3H-indolium cation.

We noted above that pyrrole, though a very weak base, is potentially acidic (pK_a 17.5).

• This was because the anion formed by losing the proton from nitrogen has a negative charge on the relatively electronegative nitrogen, but maintains its aromaticity.

The indole anion is also formed by the loss of the N–H proton (pK_a 16.2) using sodium amide sodium hydride, or even a Grignard reagent as a base.

The indole anion is resonance stabilized, with a negative charge localized mainly on nitrogen and C-3.

• It can now participate as a nucleophile, for example., in alkylation reactions. However, this can lead to N-alkylation or C-alkylation at C-3.
• Which is the predominant product depends upon several variables; but, as a general rule, if the associated metal cation is sodium, then the anion is attacked at the site of highest electron density, i.e. the nitrogen.
• Where the cation is magnesium, i.e. the Grignard reagent, then the partial covalent bonding to nitrogen prevents attack there, and the reaction occurs at C-3.

Indoles In Biochemistry:

Some rather important indole derivatives influence our everyday lives. One of the most common ones is tryptophan, an indole-containing amino acid found in proteins.

• Only three of the protein amino acids are aromatic, the other two, phenylalanine and tyrosine are simple benzene systems.
• None of these aromatic amino acids is synthesized by animals and they must be obtained in the diet. Despite this, tryptophan is surprisingly central to animal metabolism.

It is modified in the body by decarboxylation (see Box 15.3) and then hydroxylation to 5-hydroxytryptamine (5-HT, serotonin), which acts as a neurotransmitter in the central nervous system.

Serotonin mediates many central and peripheral physiological functions, including contraction of smooth muscle, vasoconstriction, food intake, sleep, pain perception, and memory, a consequence of it acting on several distinct receptor types.

• Although 5-HT may be metabolized by monoamine oxidase, platelets and neurons possess a high-affinity mechanism for reuptake of 5-HT.
• This mechanism may be inhibited by the widely prescribed antidepressant drugs termed selective serotonin reuptake inhibitors (SSRI), for example., fluoxetine (Prozac), thereby increasing levels of 5-HT in the central nervous system.
• Migraine headaches that do not respond to analgesics may be relieved by the use of an agonist of the 5-HT1 receptor since these receptors are known to mediate vasoconstriction.
• Though the causes of migraine are not clear, they are characterized by dilation of cerebral blood vessels.
• 5-HT1 agonists based on the 5-HT structure in current use include the sulfonamide derivative sumatriptan, and the more recent agents naratriptan, rizatriptan, and zolmitriptan.

These are of considerable value in treating acute attacks.

Several of the ergot alkaloids also interact with 5-HT receptors. Some are used medicinally, but the most notorious is the semi-synthetic derivative lysergic acid diethylamide (LSD).

• This is itself an indole derivative, though the indole is part of a more complex fused-ring system.
• Nevertheless, from the structural similarities, it is not difficult to see why LSD might trigger 5-HT receptors.
• It has the additional ability to interact with noradrenaline and dopamine receptors, thus generating a complex pharmacological response.
• LSD is probably the most powerful psychotomimetics known, intensifying and distorting perceptions.

Experiences can vary from beautiful visions to living nightmares, and no two ‘trips’ are alike.

Also known to be hallucinogenic are the indole derivatives psilocin and psilocybin found in the so-called magic mushrooms, Psilocybe species.

• Ingestion of these small fungi causes visual hallucinations with rapidly changing shapes and colors.
• Psilocybin is the phosphate of psilocin; although based on 4-hydroxytryptamine, it also acts on 5-HT receptors.

Melatonin is N-acetyl-5-methoxytryptamine, a simple derivative of serotonin. It is a natural hormone secreted by the pineal gland in the brain during the hours of darkness.

• It is involved in controlling the body’s day-night rhythm, the ability to sleep during the night, and to stay awake during the day.
• When given as a drug, melatonin induces sleep and adjusts the internal body clock. It is now used as a means of reducing the effects of jet lag
• Plants also require hormones to trigger their growth patterns. One of these is indole-3-acetic acid, which controls cell elongation and is produced in the growing shoot tips.
• One of the major subdivisions of plant alkaloids is termed the indole alkaloid group. All contain the basic indole heterocycle, and many have valuable pharmacological activity that can be exploited in drug materials.
• The indole portion is very often fused to another heterocycle; we shall see some typical structures, where we shall consider them under fused heterocycles.

Fused Heterocycles

There is ample scope for increasing structural complexity by fusing two or more heterocycles.

• Shown below are a few of the ring systems encountered in natural compounds, many of which have interesting, and potentially useful, biological properties.
• Note that in some cases the rings are fused so that the heteroatom can be at the ring junction and is thus common to both rings.

This gives us even more combinations.

We do not wish to consider these further, but instead, we shall concentrate on just two groups of fused heterocycles of particular importance, the purines and pteridines. Both purine and pteridine are parent heterocycles for nomenclature purposes.

• The systematic procedure for naming fused heterocycles is an extension of what we saw when we considered a benzene ring fused to a heterocycle. The main difference is that we have to identify bonds in two different rings to indicate the fusion.
• We use lettering for bonds in one heterocycle and numbers for bonds in the other. Numbering is used for the ‘sub stituent’ ring and lettering for the ‘root’ ring, and all are put in square brackets between substituent and root.

We do not wish to include a great amount of detail, but we shall use purine and pteridine to illustrate the approach and to provide a modest level of familiarity when such names are encountered.

The fused heterocycle is then given its numbering system, starting adjacent to a bridgehead atom to generate the lowest number for the first heteroatom. nucleic acids are bonded to sugar through N-9, the additional potential for tautomerism in the imidazole ring is no longer of concern.

Fused Heterocycles Purines:

Purines, along with pyrimidines, feature as bases in nucleic acids, DNA, and RNA. A purine is the product of fusing a five-membered imidazole ring onto a six-membered pyrimidine ring.

The accepted numbering system unfortunately is non-systematic and treats purine as a pyrimidine derivative, the pyrimidine ring being numbered first and separately from the other ring.

Purine in solution exists as a roughly equimolar mixture of two tautomeric forms, 9H-purine and 7H purine, tautomerism involving the imidazole ring as we have noted earlier.

• The purine systems in nucleic acids, adenine, and guanine, are aminopurines.
• The amino group is on the pyrimidine ring and, as with aminopyrimidines, these compounds exist as their amino tautomers.

Guanine also has an oxygen substituent on the pyrimidine ring, and this adopts the carbonyl form, also following the behavior of oxypyrimidines.

Because adenine and guanine in possible N-protonated forms are produced. This is perhaps unexpected, in that protonation on N-7 would provide a cation that is resonance stabilized in the imidazole ring.

• However, the observed pK_a more closely resembles that of pyrimidine (pK_a 1.3) rather than that of imidazole (pK_a 7.0).
• Amino groups increase basicity (adenine pK_a 4.3), though the oxygen substituent in guanine reduces the effect of the amino group (guanine pK_a 3.3).

In the aminopurines, the position of protonation appears to be N-1 in adenine, whereas it is N-7 in guanine; this presumably reflects the opposing effects provided by amino groups (electron donating) and carbonyl groups (electron withdrawing).

Purine has an acidic pK_a of 8.9, making it somewhat more acidic than phenol (pK_a 10), and a stronger acid than imidazole (pK_a 14.2). The N-9 proton is lost giving an anion with substantial resonance delocalization of charge.

The acidities of adenine (pK_a 9.8) and guanine (pK_a 9.9) are similar, though different protons are removed.

Adenine loses the N-9 proton, but guanine is ionized at N-1. N-1 is part of an amide-like system, and the charge in the conjugate base can be delocalized to the more favorable electronegative oxygen. The effect is most pronounced in uric acid, a metabolite of purines.

Uric Acid, A Purine Metabolite:

Nucleic acid degradation in humans and many other animals leads to the production of uric acid, which is then excreted.

The process initially involves purine nucleotides, adenosine, and guanosine, which are combinations of adenine or guanine with ribose. The purine bases are subsequently modified as shown.

The amino groups are replaced with oxygen. Although there is a biochemical reaction, the same can be achieved under acid-catalyzed hydrolytic conditions and resembles the nucleophilic substitution on pyrimidines.

• The first-formed hydroxy derivative would then tautomerize to the carbonyl structure. In the case of guanine, the product is xanthine, whereas adenine leads to hypoxanthine.
• The latter compound is also converted into xanthine by an oxidizing enzyme, xanthine oxidase. This enzyme also oxidizes xanthine at C-8, giving uric acid.
• Uric acid is not a carboxylic acid but is a relatively strong acid with pK_a 5.8. It has an ‘all-amide’ structure, and there are four potential sites for the loss of a proton.

Deprotonation occurs at N-9. Loss of this proton generates a conjugate base in which the charge can be delocalized to oxygen, giving maximum charge distribution. One resonance form is particularly favorable in having aromaticity in both rings.

Impaired purine metabolism can lead to a build-up of uric acid, and deposition of salts of uric acid as crystals in the joints.

• This causes the painful condition known as gout. One way of treating gout is to reduce uric acid biosynthesis by specific inhibition of the enzyme xanthine oxidase.
• The hypoxanthine analog allopurinol is a drug that is used for this purpose. Allopurinol resembles hypoxanthine, though it contains a pyrazole ring rather than an imidazole ring.

Allopurinol is oxidized by the enzyme to alloxanthine. This product then acts as an inhibitor of the enzyme, binding to the enzyme, but not being modified further and not being released.

Caffeine, Theobromine, And Theophylline:

After the nucleic acid purines adenine and guanine, the next most prominent purine in our everyday lives is probably caffeine.

• Caffeine, in the form of beverages such as tea, coffee, and cola, is one of the most widely consumed and socially accepted natural stimulants.
• Closely related structurally are theobromine and theophylline. Theobromine is a major constituent of cocoa and related chocolate products.

Caffeine is also used medicinally, but theophylline is much more important as a drug compound because of its muscle relaxant properties, utilized in the relief of bronchial asthma.

These compounds competitively inhibit phosphodiesterase, resulting in an increase in cyclic AMP and subsequent release of adrenaline.

• This leads to the major effects: a stimulation of the central nervous system (CNS), a relaxation of bronchial smooth muscle, and induction of diuresis.
• These effects vary in the three compounds. Caffeine is the best CNS stimulant and has weak diuretic action. Theobromine has little stimulant action but has more diuretic activity and also muscle relaxant properties.
• Theophylline also has low stimulant action and is an effective diuretic, but it relaxes smooth muscle better than caffeine or theobromine.
• It has been estimated that beverage consumption may provide the following amounts of caffeine per cup or average measure: coffee, 30–150 mg (average 60–80 mg); instant coffee, 20–100 mg (average 40–60 mg); decaffeinated coffee, 2–4 mg; tea, 10–100 mg (average 40 mg); cocoa, 2–50 mg (average 5 mg); cola drink, 25–60 mg.
• The maximal daily intake should not exceed about 1 g to avoid unpleasant side effects, for example., headaches, and restlessness. An acute lethal dose is about 5–10 g.

Caffeine and theobromine may be obtained in large quantities from natural sources, or they may be obtained by total or partial synthesis. Theophylline is usually produced by total synthesis.

Pteridines:

In pteridines, we have a pyrimidine ring fused to a pyrazine ring. There are, of course, several possible ways of combining these two six-membered ring systems; pteridines are pyrazino[2,3- d]pyrimidines.

• We do not want to consider the chemistry of the pteridine ring system here, but instead, we shall look at the structures of two rather important pteridine-based biochemicals, namely folic acid and riboflavin.
• In the latter case, the pteridine is also fused further to a benzene ring, giving an even more complex ring system, a benzo[g]pteridine.

The accompanying diagram shows the derivation of the fused-ring nomenclature. The oxygenated form of the benzopteridine found in riboflavin is also called an isoalloxazine.

Folic acid (vitamin B₉) is a conjugate of a pteridine unit, p-aminobenzoic acid, and glutamic acid. Deficiency of folic acid leads to anemia, and it is also standard practice to provide supplementation during pregnancy to reduce the incidence of spina bifida.

Folic acid becomes sequentially reduced in the body by the enzyme dihydrofolate reductase to give dihydrofolic acid (FH₂) and then tetrahydrofolic acid (FH₄). Reduction occurs in the pyrazine ring portion.

• Tetrahydrofolic acid then functions as a carrier of one-carbon groups for amino acid and nucleotide metabolism.
• The basic ring system can transfer methyl, methylene, methenyl, or formyl groups, and it utilizes slightly different reagents as appropriate.
• These are shown here; for convenience, we have left out the benzoic acid–glutamic acid portion of the structure.

These compounds are all interrelated, but we are not going to delve any deeper into the actual biochemical relationships.

In any case, you might be able to analyze some of the relationships on a purely chemical basis. For example, tetrahydrofolic acid reacts readily and reversibly with formaldehyde to produce N5 and N10-methylene-FH₄.

• You could consider N-5 of the reduced pteridine ring reacting with formaldehyde (a one-carbon reagent) to give an iminium cation, which could then cyclize via nucleophilic attack of N-10.
• We might also consider reducing the iminium cation with, say, borohydride to give the N-methyl derivative.

These are not necessarily the same as what is occurring in the enzymic reactions, but they should help to make the structures appear rather more familiar.

Now we have seen that the usual reagent for biological methylations is S-adenosylmethionine (SAM).

One occasion where SAM is not employed, for fairly obvious reasons, is the regeneration of methionine from homocysteine, after a SAM methylation. For this, N5-methyl-FH4 is the methyl donor, with vitamin B₁₂ also playing a role as a coenzyme.

Another vitally important methylation reaction involving folic acid derivatives is the production of the nucleic acid base thymine from uracil.

• Uracil is found in RNA, and thymine is a component of DNA; thymine is the methyl derivative of uracil. For continuing DNA synthesis, it is necessary to methylate uracil.
• In practice, it is the nucleotide deoxyuridylate (dUMP) that is methylated to deoxy thymidylate (dTMP). The methylating agent employed here is N⁵, N¹⁰-methylene-FH₄. As a consequence of this reaction, N⁵, N¹⁰– methylene-FH₄ is converted into dihydrofolic acid.
• To keep the reaction flowing, this is reduced to FH₄, and further N⁵, N¹⁰-methylene-FH₄ is produced using a one-carbon reagent.

In this process, the one-carbon reagent comes from the amino acid serine, which is transformed into glycine by the loss of its hydroxymethyl group. The chemistry of the transformations is fairly complex and outside our requirements.

Folic acid derivatives are essential for DNA synthesis, in that they are cofactors for certain reactions in purine and pyrimidine biosynthesis, including the uracil–thymine methylation just described.

• They are also cofactors for several reactions relating to amino acid metabolism.
• The folic acid system thus offers considerable scope for drug action. Mammals must obtain their tetrahydrofolate requirements from their diet, but microorganisms can synthesize this material.
• This offers scope for selective action and led to the use of sulfanilamide and other antibacterial sulfa drugs, compounds that competitively inhibit the biosynthetic enzyme (dihydropteroate synthase) that incorporates p-aminobenzoic acid into the structure.
• Rapidly dividing cells need an abundant supply of dTMP for DNA synthesis, and this creates a need for dihydrofolate reductase activity.

Specific dihydrofolate reductase inhibitors have become especially useful as antibacterials, for example., trimethoprim, and antimalarial drugs, for example., pyrimethamine.

These are pyrimidine derivatives and are effective because of differences in susceptibility between the enzymes in humans and the infective organism.

• Anticancer agents based on folic acid, for example., methotrexate, inhibit dihydrofolate reductase, but they are less selective than the antimicrobial agents and rely on a stronger binding to the enzyme than the natural substrate has.
• They also block pyrimidine biosynthesis. Methotrexate treatment is potentially lethal to the patient and is usually followed by ‘rescue’ with folinic acid (N5-formyl-tetrahydrofolic acid) to counteract the folate-antagonist action.

The rationale is that folinic acid ‘rescues’ normal cells more effectively than it does tumor cells.

Riboflavin:

Riboflavin (vitamin B₂) is a component of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), coenzymes that play a major role in oxidation-reduction reactions.

• Many key enzymes involved in metabolic pathways are covalently bound to riboflavin and are thus termed flavoproteins.
• Riboflavin contains an isoalloxazine ring linked to the reduced sugar ribitol.

The sugar unit in riboflavin is the non-cyclic ribitol so FAD and FMN differ somewhat from the nucleotides we encounter in nucleic acids.

Riboflavin is widely available in foods; dietary deficiency is uncommon, but it manifests itself in skin problems and eye disturbances.

The flavin nucleotides are typically involved in the oxidations creating double bonds from single bonds.

The flavin takes up two hydrogen atoms, represented in the figure as being derived by the transfer of hydride from the substrate and a proton from the medium.

Reductive sequences involving flavoproteins may be represented as the reverse reaction, where hydride is transferred from the coenzyme, and a proton is obtained from the medium.

• The reaction mechanism shown here is in many ways similar to that in NAD⁺ oxidations, i.e. a combination of hydride and a proton; it is less easy to explain adequately why it occurs, and we do not consider any detailed explanation advantageous to our studies.
• We should register only that the reaction involves the N=C–C=N function that spans both rings of the pteridine system.

Some Classic Aromatic Heterocycle Syntheses:

Our study of heterocyclic compounds is directed primarily to an understanding of their reactivity and importance in biochemistry and medicine.

• The synthesis of aromatic heterocycles is not, therefore, a main theme, but it is useful to consider just a few examples to underline the application of reactions in earlier chapters.
• From the beginning, we should appreciate that the synthesis of substituted heterocycles is probably not best achieved by carrying out substitution reactions on the simple heterocycle.
• It is often much easier and more convenient to design the synthesis so that the heterocycle already carries the required substituents, or has easily modified functions.

We can consider two main approaches for heterocycle synthesis, here using pyridine and pyrrole as targets.

We can insert the heteroatom into the rest of the carbon skeleton, or attempt to join two units, one of which contains the heteroatom, using C–C and C–heteroatom linkages.

• To make the new bonds, two reaction types are most frequently encountered.
• Heteroatom–C bond formation is achieved using the heteroatom as a nucleophile to attack an electrophile such as a carbonyl group.
• Aldol-type reactions may be exploited for C–C bond formation, employing enamines and enols/enolate anions.
• We shall now look at some synthetic procedures that merit the descriptor ‘classic’ because of their general application, and their longevity – some have been around for more than 100 years.

Do not worry about remembering the names: these commemorate the originators, and we should instead concentrate on the chemistry, which we shall see is usually a combination of processes we have already met.

Hantzsch Pyridine Synthesis

In its simplest form, this consists of the condensation of a β-ketoester with an aldehyde and ammonia.

• The product is a 1,4-dihydropyridine, which is subsequently transformed into pyridine by oxidation.
• Several separate reactions occur during this synthesis and the precise sequence of events may not be quite as shown below – they may be in a different order.
• The normal Hantzsch synthesis leads to a symmetrical product. The diesters formed may be hydrolyzed and decarboxylated using the base to give pyridines with less substitution.

Note that we are using the ester groups as activating species to facilitate enolate anion chemistry

Skraup Quinoline Synthesis:

The most general method for synthesizing quinolines employs aniline or a substituted aniline, glycerol, sulfuric acid, and an oxidizing agent such as a ferric salt or nitrobenzene.

The first step is acid-catalyzed dehydration of glycerol to the unsaturated aldehyde acrolein. Variations of the Skraup synthesis use different acroleins instead of glycerols.

The initial product is a dihydroquinoline; it is formed via a Michael-like addition, then an electrophilic aromatic substitution that is facilitated by the electron-donating amine function.

• A mild oxidizing agent is required to form the aromatic quinoline.
• The Skraup synthesis can be used with substituted anilines, provided these substituents are not strongly electron-withdrawing and are not acid-sensitive.

Bischler–Napieralski Isoquinoline Synthesis:

Isoquinolines are easily prepared by the reaction of an acyl derivative of a β-phenylethylamine with a dehydrating agent, for example., P₂O₅, then using a catalytic dehydrogenation to aromatize the intermediate 3,4- dihydroisoquinoline.

The crucial cyclization step is represented here as dehydrogenation and an iminium-type system, a resonance form of an electrophilic attack involving the aromatic ring of the amide, suitably coordinated with the phosphorus reagent.

• The cyclizing agent P₂O₅ also dehydrates the intermediate hydroxyamine to a dihydroisoquinoline.
• The isoquinoline is then obtained by heating over a catalyst, effectively reversing a catalytic hydrogenation reaction, facilitated by the generation of aromaticity in the product.
• As in the Skraup synthesis above, electron-withdrawing substituents on the aromatic ring will deactivate it towards electrophilic attack, whereas electron-donating substituents will favor the reaction.

Pictet–Spengler Tetrahydroisoquinoline Synthesis

This approach to the isoquinoline ring, albeit a reduced isoquinoline, is mechanistically similar to the Bischler–Napieralski synthesis, in that it involves an electrophilic attack of an iminium cation onto an aromatic ring. In this case, the imine intermediate is formed by reacting a phenylethylamine with an aldehyde.

We have already met this reaction as an analog of the Mannich reaction, which we then interpreted as a nucleophilic attack of an electron-rich phenolic ring pontoon iminium cation.

• Is it electrophilic or nucleophilic? It matters little; they are the same, though the descriptor used depends upon which species you consider the more important, the nucleophilic phenol or the electrophilic iminium cation.
• For effective cyclization, we need an electron-donating substituent para to the point of ring closure since the Mannich-type electrophile is less reactive than the phosphorus-linked intermediates in the Bis cheer–Napieralski synthesis.
• It is also found that a similar group in the ortho position does not work, though we could still write an acceptable mechanism With a good electron-donating substituent like hydroxyl, the whole process, imine formation, and cyclization can occur under ‘physiological’ conditions pH 6–7 at room temperature.
• In nature, this is precisely how tetrahydroisoquinoline alkaloids are biosynthesized, though the reactions are enzyme-controlled

Knorr Pyrrole Synthesis:

This approach to the five-membered pyrrole ring reacts an α-aminoketone with a β-ketoester.

The mechanism will probably involve imine formation and then cyclization via an aldol-type reaction using the enamine nucleophile.

Dehydration leads to the pyrrole. Only the key parts of this sequence are shown below.

The synthesis works well only with an activated ester like ethyl acetoacetate. Otherwise, self-condensation of the α-aminoketone to a dihydropyrazine occurs more readily than the cyclization.

Paal–Knorr Pyrrole Synthesis:

The other major route to pyrroles is the interaction of a 1,4-dicarbonyl compound with ammonia.

The nucleophilic mechanism below shows successive nucleophilic additions of amino groups onto the carbonyls; but, since no intermediates have been isolated, the precise sequence of steps is speculative.

Note, however, that this synthesis gives furans if no ammonia is included. This would involve a nucleophilic attack of an enol tautomer of the substrate onto another carbonyl to give a hemiketal, followed by dehydration. The heteroatom is thus derived from a carbonyl oxygen.

The procedure works well and is usually carried out with acid catalysts under non-aqueous conditions.

Fischer Indole Synthesis:

The most useful route to indoles is the Fischer indole synthesis, in which an aromatic phenylhydrazone is heated in acid.

• The phenylhydrazone is the condensation product from a phenylhydrazine and an aldehyde or ketone. Ring closure involves a cyclic rearrangement process.
• The hydrazine behaves as an amine towards a carbonyl compound and forms the imine-like product, a hydrazone.
• The cyclic rearrangement involves the enamine tautomer of this hydrazone, and proceeds because the cyclic flow of electrons forms a strong C–C bond whilst cleaving a weak N–N bond.
• This produces what appears to be a di-imine. One of these is involved in rearomatization and creates an aromatic amine.

This then attacks the other imine function, and we get the nitrogen equivalent of a hemiketal. Finally, acid-catalyzed elimination of ammonia gives the aromatic indole system.

Unfortunately, the reaction fails with acetaldehyde and cannot, therefore, be used to synthesize indole itself. It is possible to use the ketoacid pyruvic acid instead and decarboxylate the product to yield indole.

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A gene mutation is an abrupt inheritable qualitative or quantitative change in the genetic material of an organism.

• Since in most organisms, genes are segments of DNA molecules, a mutation can be regarded as a change in the DNA sequence which is reflected in the change of sequence of corresponding RNA or protein molecules.
• Such a change may involve only one base/base pair or more than one base pair of DNA. Mutations occur randomly, Le., they are not directed according to the requirements of the organism.
• Most mutations occur spontaneously by the environmental effect, however, they can be induced in the laboratory either by radiation, physical factors or chemicals (called mutagens).
• A unicellular organism is more subjected to environmental attacks since it is at the same time a somatic or germ cell.
• In multicellular organisms, the germ cells are distinct and are relatively protected from the environment. Mutation has a significant role to play in the origin of species or evolution.

Historical Background

The earliest record of point mutations dates back to 1791 when Seth Wright noticed a lamb with exceptionally short legs in his flock of sheep.

• Visualising the economic significance of this short-legged sheep. i.e., short-legged sheep could not cross the low stone fence and damage the crop fields in the vicinity, he produced a flock of sheep, each of which had short legs by employing artificial breeding techniques.
• The short-legged breed of sheep was known as the Ancon breed. Later on, the trait of short legs was found to have resulted from a recessive mutation and the short-legged individuals were found to be homozygous recessive.

• Hugo de Vries was the first hybridist who used the term “mutation” to describe the heritable phenotypic changes of the evening primrose.
• Oenothera lamarckiana. Many mutations described by de Vries in O. lamarckiana, are now known to be due to variation in chromosome number or ploidy and chromosomal aberrations (viz. gross mutations).
• The first scientific study of mutation was started in 1910, when Morgan started his work on fruitfly, Drosophila melanogaster and reported white-eyed male individuals among red-eyed male individuals.
• The discovery of white-eyed mutants in Drosophila was followed by an extensive search of other mutants of Drosophila by Morgan and his co-workers and other geneticists.
• Consequently, about 500 mutants of Drosophila have been reported by geneticists all over the world.
• Later on, several cases of mutations have been reported in a variety of microorganisms (for example., bacteriophages, bacteria(Escherichia coli), Neurospora, etc., plants (i.e., pea, snapdragon, maize, etc.) and animals, (i.e., rodents, fowls, human beings, etc.).

Occurrence

Mutations occur frequently in nature and have been reported in many organisms, e.g., Drosophila, mice and other rodents, rats, rabbits, guinea pigs and human beings.

• In Drosophila, the mutation causes white and pink eyes, black and yellow body colours, and vestigial wings. In rodents, the mutations are responsible for black, white and brown coats.
• In human beings, the mutations cause variations in hair colour, eye colour, skin pigmentation and several somatic malformations. Various genetic diseases of human beings such as haemophila, colour blindness, phenylketonuria, etc., form other examples of mutation in human beings.
• How does a mutation act? Any change in the sequence of nucleotides in the DNA will result in the corresponding change in the nucleotide sequence of mRNA.
• This may result in the alignment of different tRNA molecules on mRNA (during protein synthesis).
• Thus, the amino acid sequence, and, hence, the structure and properties of the enzyme formed will be changed. This defective enzyme or structural protein may adversely affect the trait controlled by the protein. In consequence, a mutant phenotype makes its expression.

Kinds Of Mutations

There exists a lot of controversy among geneticists about the possible kinds of mutations. Mutations have been classified variously according to different criteria as follows:

Classification Of Mutation According To Type Of Cells: According to their occurrence in somatic and germinal cells, the following types of mutations have been classified:

Somatic Mutations: The mutations occurring in non-reproductive body cells are known as somatic mutations. The genetical and evolutionary consequences of somatic mutations are insignificant since only single cells and their daughter cells are involved.

• If, however, a somatic mutation occurs early during embryonic life, the mutant cells may constitute a large proportion of body cells and the animal body may be a mosaic of different types of cells.
• Somatic mutations have often been related to malignant(cancerous) growth.
• Examples of somatic mutation have been reported in Oenothera lamarckiana (by Hugo de Vries) and several other cases including human beings.
• In human beings, the somatic mutation causes several fatal diseases such as paroxysmal nocturnal haemoglobinuria, circumscribed neurofibroma, unilateral retinoblastoma and heterochromia of the iris.

Gametic Mutations: The mutations occurring in gamete cells(e.g., sperms and ova) are called gametic mutations. Such mutations are heritable and of great genetic significance. The gametic mutations only form the raw material for natural selection.

Classification of Mutations According to the Size and Quality: According to size, the following two types of mutations have been recognised:

Point Mutation: Wien heritable alterations occur in a very small segment of DNA molecule. a single nucleotide or nucleotide pair, then these types of mutations are called “point mutations”. The point mutations may occur due to the following types of subnucleotidechange in the DNA and RNA.

1. Deletion Mutations: The point mutation which is caused by to loss or deletion of some portion (single nucleotide pair) in a triplet codon of cistron or gene is called deletion mutation. Deletion mutations have been frequently reported in some bacteriophages(Phage T4).
2. Insertion Or Addition Mutation: The point mutations which occur due to the addition of one or more extra nucleotides to a gene or cistron are called insertion mutations. The insertion mutations can be artificially induced by certain chemical substances called mutagens such as acridine dye and proflavin.

A proflavin molecule, it is believed, inserts between two successive bases of a DNA strand, thereby stretching the strand lengthwise.

• At replication, this situation would allow the insertion of an extra nucleotide in the complementary chain at the position occupied by the proflavin molecule.
• The mutations which arise from the insertion or deletion of individual nucleotides and cause the rest of the message downstream of the mutation to be read out of phase are called frameshift mutations.
• They result in the production of an incorrect, hence, inactive protein, due to which death of the cell may occur.

Substitution Mutation: A point mutation in which a nucleotide of a triplet is replaced by another nucleotide is called substitution mutation.

• The substitution mutation affects only a particular triplet codon.
• Such an altered code word (triplet codon) may designate a different amino acid and may result in the production of a protein with a single amino acid substitution.
• The substitution mutations alter the phenotype of an organism variously and are of great genetic significance. They may be of the following types:

Transition: When a purine(for example., adenine) base of a triplet codon of a cistron is substituted by another purine base (for example., guanine) or a pyrimidine (for example., thymine) is substituted by another pyrimidine base,(for example., cytosine) then such kind of substitution is called transition. Transitional substitution mutations occur due to tautomerization.

Tautomerization: In a DNA molecule, normally, the purine, adenine(A) is linked to the pyrimidine, thymine (T), by two hydrogen bonds, while the purine guanine(G) is linked to the pyrimidine, cytosine(C) by three hydrogen bonds.

• Besides the common molecular configurations, each DNA base may have some altered uncommon molecular configuration.
• Such uncommon forms of DNA bases arc generated by single proton shifts and are called rare states or tautomers. A tautomeric shift is believed to occur when the amino(NH,) form of adenine is changed to an imino(NH) form.
• Similarly, a tautomeric shift may occur in thymine changing it from the keto(C – O) form to the rare enol(COH) form. When a base occurs in its rare or tautomeric state. it cannot be linked to its normal partner.
• However, a purine, such as adenine in its rare state can form a bond with cytosine(besides thymine), provided the cytosine is in its normal state.
• Watson and Crick (1953) hypothesised that the occurrence of the bases in their rare states provides a mechanism for mutation during DNA replication.

If for example, adenine in an old chain is in its rare state at the moment that the complementary new chain reaches it, cytosine can pair with it (adenine) and be added to the growing end of the new chain The result of this type of pairing is the formation of a DNA molecule that contains an exceptional base pair.

• This situation is not stable and at the next replication, adenine is expected to return to its common state and to pair with thymine. Cytosine introduced into the complementary strand due to tautomeric .shill in adenine, would then pair with guanine.
• Thus, there would be formed two kinds of DNA molecules, one that is identical to the original DNA and another that has undergone a base pair substitution of(i-C for A-T.
• This transitionally substituted DNA molecule has altered coding at a point and results in recognisable mutation. Such mutations which form during DNA replication are called copy error mutations. Such copy error mutations.
• The abnormal pairing due to transitional substitution may also occur due to the Ionisation of a base at the time of DNA replication. Ionisation involves the loss of hydrogen from the number one nitrogen of a base.
• For example, in its ionised state, thymine can pair with guanine, if the guanine is in its common form. Similarly, guanine in us ionised state can pair with thymine in its common form.
• From any such unstable base pair, a transition will result following the steps outlined for A-T to G-C and C i-C’ to A-T.

Effect Of Chemical Mutagens On Nucleotide Sequence

Alteration In Resting Nucleic Acid

Deamination: Some chemical substances such as nitrous acid cause transitional mutation due to oxidative deamination of DNA bases.

• In the process of oxidative deamination, the amino group(NH₂) of a DNA base is replaced by the hydroxyl(OH) group by the chemical mutagen.
• Thus, adenine is deaminated into hypoxanthine by nitrous acid as shown in the following figure:

By tautomeric shift, the hypoxanthine(HX) is converted into a more common keto-tautomer which pairs with cytosine. The A: T pair, thus, can be converted to a G: C pair. Similarly, deamination converts cytosine to uracil, which has pairing properties similar to thymine and in such a case G: C pair would be changed into an A: T pair.

Hydroxylainine,(HA =NH₂OH) and hydrazine (HZ =NH₂NH₂). When DNA is treated with hydroxylamine (HA), its cytosine base is the strongest reacting base.

• Hydroxylamine probably causes hydroxylation of cytosine at the amino group giving rise to hydroxylcytosinc, which then subsequently pairs with adenine. Thus, hydroxylamine(HA) induces in DNA a GC → AT base pair transition.
• The hydrazine affects DNA by breaking off rings of uracil and cytosine giving rise to pyrazolone and 3-amino pyrazole, respectively. The treatment of RNA or DNA with anhydrous hydrazine results in the destruction of their pyrimidines.

Alkylating agents: Some alkylating agents carry one, two, or more alkyl groups in a reactive form and act as strong mutagens.

Examples of some most extensively studied alkylating agents include diethyl sulphate(DES), dimethyl sulphate (DMS), methyl methane sulphonate (MMS), ethyl ethane sulphonate(EES) and ethyl methane sulphonate(EMS). These mutagens produce mutations in the following ways:

1. They add ethyl or methyl groups to guanine. This makes guanine the base analogue to adenine.
2. They remove the alkylated guanine. This is known as depurination. The loss of the base produces gaps in the DNA chain which may be filled with a wrong base, thus, producing mutation.
3. The gap may also produce a deletion, causing mutation.

Alteration during Replication of Nucleic Acid

Base Analogues: Certain chemical substances have molecular structures similar to the usual DNA bases that, if they are available, such analogues may be incorporated into a replicating DNA strand.

• For example, 5-bromouracil(5BU) or its nucleoside 5-bromodeoxyuridine (5-BUdR) in its usual (keto) form is a structural analogue of thymine (5-methyluracil) and it will substitute for thymine.
• Thus, an A-T pair becomes and remains A-BU. There is some in vitro evidence to indicate the BU immediately adjacent to adenine in one of the DNA strands causes the latter to pair with guanine.
• But, in its rare(enol) state, 5BU behaves similarly to the tautomer of thymine and pairs with guanine.
• This converts A: T to G: C as shown in Figure 1z6.7.

2-Aminopurine(2-AP) is another base analogue which is a relatively undifferentiated purine that apparently can pair with cytosine and thymine.

• It is thought that 2-AP acts by “switching” pyrimidines: for example, it may be incorporated opposite thymine during one round of replication and then pair with a cytosine at the next round to produce an AT → GC transition(see Goodenough and Levine,1974).

Inhibition Of Precursors Of Nucleic Acids: There are some mutagens which interfere with the synthesis of nitrogen bases of nucleic acids such as purines or pyrimidines.

• Often lack of one base either causes breaks or pairing mistakes.
• For example, azaserine(a potent alkylating agent) inhibits purine synthesis and urethane(a mild alkylating agent) is an inhibitor of pyrimidine synthesis.
• However, urethane-induced chromosome breaks are inhibited by thymine.

Transversion: The substitution mutation when involves the substitution or replacement of a purine with a pyrimidine or vice versa then that type of substitution mutation is called transversion mutation.

• The existence of transversion mutation was first of all postulated by E. Freese in 1959. We still have poor information about the mechanism of induction, identification and characterisation of transversion mutations.
• Moreover, it is extremely difficult to recognise transversion mutations genetically. However, they can be recognized only by analysis of amino acid substitutions in proteins.

Effects Of Physical Conditions On Nucleotide Sequence

• High temperature and low pH values are known to affect depurination or loss of purine bases.
• The removal of a purine from a strand of DNA leaves a gap at that point. At the time of replication, it would be possible for any of the four bases to be inserted in the complementary newly formed strand.
• If the inserted nucleotide contained a purine, the complementary strand would contain a transversion.

Multiple Mutations Or Gross Mutations: When changes involve more than one nucleotide
or entire gene, then such mutations are called gross mutations. The gross mutations occur due to rearrangements of genes within the genome and may be of the following types:

1. The rearrangement of genes may occur within a gene. Two mutations within the same functional gene can produce different effects depending on the gene whether they occur in the cis or trans position.
2. The rearrangement of genes may occur in many genes per chromosome.
3. If the number of gene replicas is non-equivalent on the homologous chromosomes, they may cause different types of phenotypic effects on the organisms.
4. Due to the movement of a gene locus, new types of phenotypes may be created, especially when the gene is relocated near heterochromatin. The movement of gene loci may take place due to the following method:

1. Translocation: Movement of a gene may take place to a non-homologous chromosome and this is known as translocation.
2. Inversion: The movement of a gene within the same chromosome is called inversion.

Classification of Mutation According to the Origin

According to the mode of origin, the following two kinds; of mutations have been recognised:

1. Spontaneous Mutations: Spontaneous mutations occur suddenly in nature and their origin is unknown. They are also called “background mutations” and have been reported in many organisms such as Oenothera, maize, bread moulds, microorganisms (bacteria and viruses), Drosophila, mice, human beings, etc.
2. Induced Mutations: Besides naturally occurring spontaneous mutations, the mutations can be induced artificially in living organisms by exposing them to abnormal environments such as radiation, certain physical conditions (i.e., temperature) and chemicals.

The substances or agents which induce artificial mutations are called mutagens or mutagenic agents. Mutagenic agents. The mutagenic agents are of the following kinds:

Radiations: The radiations which are important in mutagenesis are of two categories: one type is ionising radiations such as X-rays and gamma rays; alpha and beta rays; electrons, neutrons, protons and other fast-moving particles.

The second type is non-ionising radiation such as ultraviolet and visible light. Both types of radiation induce mutations by following methods:

Ionising Radiations As Mutagens: Relatively little is known about the mechanism by which ionising radiations cause mutation.

As, we are already familiar that matter is composed of atoms and atoms, in turn, are made up of a positively charged atomic nucleus (with neutrons, and protons) and a surrounding constellation of negatively charged electrons.

• The charges of atomic particles remain so balanced that normal atoms are electrically neutral.
• When ionising radiations pass through matter, they dissipate their energy in part through the ejection of electrons from the outer shell of atoms and the loss of these balancing, negative particles(electrons) leaves atoms which are no longer neutral but are positively charged.
• The positively charged atom is called an ion. The ejected electrons move at high speed; knock other electrons free from their respective atoms and when their energy is dissipated, become attached to other atoms and convert the atoms into negatively charged ions.
• To achieve their stable configuration (i.e., neutral charge), ions undergo many chemical reactions and during these chemical reactions ionising radiation is thought to cause mutation.
• Further, ionizing radiations cause breaks in the poly-sugar phosphate backbone of DNA and, thus, cause chromosomal mutations such as break, deletion, addition, inversion and translocation.
• During the breakage of the DNA molecule due to ionising radiation, the active role of oxygen is predicted. Oxygen is important in the formation of H₂O₂ and H₂O in irradiated water and these products may induce breaks in DNA molecules.

Non-Ionising Radiations As Mutagens: The ultraviolet(UV) light is a non-ionizing radiation which may cause mutation. The most effective wavelength of ultraviolet light-inducing mutations is about 2,600 A°.

• This is a wavelength that is best absorbed by DNA and a wavelength at which proteins absorb little energy.
• When a substance absorbs sufficient energy from the ultraviolet light, some of its electrons are raised to higher energy levels, a slate called excitation.
• The excited molecule becomes reactive and mutated and is called a photoproduct. Dimerization. The ultraviolet radiation produces several effects on DNA, one being the formation of chemical bonds between two adjacent pyrimidine molecules in a polynucleotide and particularly, between adjacent thymine residues.
• As the two thymine residues associate, or dimerize to form a dimer, their position in the DNA helix becomes so displaced that they can no longer form hydrogen bonds with the opposing purines and thus regularity of the helix becomes distorted.
• Thus, dimerization interferes with the proper base pairing of thymine with adenine and results in thymine pairing with guanine. This will produce a T-A to C-G transition.

Temperature As Mutagen: The rate of all chemical reactions is influenced by temperature. It is not surprising that temperature can be mutagenic.

• It is reported that the rate of mutation is increased due to an increase in temperature. For example, an increase of 10°C temperature increases the mutation rate two or three-fold.
• Temperature probably affects both the thermal stability of DNA and the rate of reaction of other substances with DNA.
• A study by Swedish nudists indicated that the scrotal temperature of human males in ordinary clothing is about 3°C higher than that of nude males.
• The higher temperature could well increase the mutation rate nearly two-fold, leading the investigators to suggest that the wearing of pants has possibly been much more unhygienic than fall out from testing of nuclear devices threatens to be.
• They suggested the wearing of kilts as one solution.

Chemical Mutagens: Many chemical substances have been responsible for increasing (the mutability of genes.

• The ability of chemicals to induce mutation was first demonstrated by Auerbach and Robson in 1947 using mustard gas, and related compounds as nitrogen and sulphur mustards, mustard oil and chloracctonc in experiments with male Drosophila melanogaster.
• Since then many chemical compounds which are ordinarily considered to be non-toxic are mutagenic in certain specific situations.
• Any chemical substance that affects the chemical environment of chromosomes is likely to influence, at least indirectly, the stability of DNA and its ability to replicate without error.
• A chemical mutagen can cause mutation only when it enters the nucleus of the cell.
• It can affect the chromosomal DNA in the following two ways:

Direct Gene Change: Certain chemical mutagens affect DNA directly. They affect the constituents of DNA only when DNA is not replicating.

• For example, nitrous acid converts adenine into hypoxanthine and cytosine to uracil by deamination.
• Like nitrous acid, nitrogen mustard, formaldehyde, epoxides, dimethyl and diethyl sulphate, methyl and ethyl methanesulphonate (MMS and EMS) and nitroguanidine (NG) also have direct mutagenic effects on the DNA molecule.

Copy Error: Certain chemical compounds, called base analogues(for example., 5-bromouracil, 2-aminopyrine, etc.) closely resemble certain DNA bases and therefore, act as mutagens.

• During DNA replication, they are incorporated by DNA in place of the normal DNA bases.
• Certain other base analogues such as urethane triazine, caffeine (in coffee, tea and soft drinks), phenol and carcinogens, and acridines (proflavin, etc.), have mutagenic effects.
• Certain inorganic substances such as manganese chloride are mutagenic for many organisms, as, they are the compounds which bind calcium and. thus, interfere with the integrity of the chromosome structure.

Classification Of Mutation According To The Direction

According to their mode of direction, the following types of mutations have been recognised:

• Forward Mutations: In an organism when mutations create a change from wild-type to abnormal phenotype, then that type of mutations are known as forward mutations. Most mutations are forward-type.
• Reverse Or Back Mutations: The forward mutations are often corrected by an error-correcting mechanism so that an abnormal phenotype changes into a wild-type phenotype. They may be of the following types:
• Single-Site Mutations: Some reverse mutations change only one nucleotide in the gene and are called single-site mutations. For example, due to forward mutation the adenine is changed into guanine and backward mutation changes guanine into adenine:

• Mutation Suppressor: When a mutation occurs at a different site from the site where already primary mutation occurred and that mutated gene reverses the effects of the primarily mutated gene, then such(secondary) mutations are called mutation suppressors. They may be of the following types:
• Extragenic suppressor: The extragenic suppressor mutation occurs in a different gene from that of the mutant gene.
  • In E. coli, a gene mutation suppressor gene called rec A(rec for recombination) is known which is necessary for recombination and is found to repair ultraviolet-induced thymine dimers of a gene by a process called postreplication recombinational repair (see Goodenough and Levine, 1974).
• Intragenic Suppressor. The intragenic suppressor mutation occurs in a different nucleotide within the same gene and shifts the reading frame back into the register.
• Photoreactivation: In photoreactivation type reverse mutation, the reversal of ultraviolet-induced thymine dimers takes place by specific enzymes in the presence of visible light waves.
  • During ultraviolet radiation, a particular enzyme is selectively bound to the bacterial DNA.
  • During photoreactivation, the enzyme is activated by visible light and that cleaves the pyrimidine or purine dimers into monomers and restores their original forms.
• Excision Repair Or Dark Reactivation. In an ultraviolet(UV) induced mutation, the reverse mutation may also occur in the absence of light.

According to Howard Flanders and Boyce (1964), dark reactivation includes the following stages:

1. An enzyme possibly endonuclease makes a cut in the polynucleotide strand on either side of the dimer which may be formed due to ultraviolet radiation and excises a short, single-strand segment of the DNA.
2. Another enzyme, possibly exonuclease widens the gap produced by the action of the endonuclease.
3. DNA polymerase synthesises the missing segment, using the remaining opposite strand as a template; and
4. The final gap is closed by some enzymatic rejoining process, (i.e., DNA ligase).

Classification Of Mutation According To Magnitude Of Phenotypic Effect

According to their phenotypic effects following kinds of mutations may occur:

1. Dominant Mutations: The mutations which have dominant phenotypic expression are called dominant mutations. For example, in human beings, the mutation disease aniridia (absence of iris of eyes) occurs due to a dominant mutant gene.
2. Recessive Mutations: Most types of mutations are recessive so they are not expressed phenotypically immediately. The phenotypic effects of mutations of a recessive gene are seen only after one or more generations when the mutant gene can recombine with another similar recessive gene.
3. Isoalleles: Some mutations alter the phenotype of an organism so slightly that they can be detected only by special techniques. Mutant genes that give slightly modified phenotypes are called isoalleles. They produce identical phenotypes in homozygous or heterozygous combinations.
4. Lethal Mutations: According to their effects on the phenotype, mutations may be classified as lethals, subvitals and supervisors.

Lethal mutations result in the death of the cells or organisms in which they occur. Subvital mutations reduces the chances of survival of the organism in which they occur. Supervital mutations, in contrast, cause the improvement of biological fitness under certain conditions:

Classification Of Mutation According To Consequent Change In Amino Acid Sequence

1. Missense Mutations: They change the meaning ofa codon, changing one amino acid into
   another.
2. Temperature-Sensitive Mutations Or T₈ Mutations: If the substitution produces a protein that is active at one temperature (typically 30°C) and inactive at a higher temperature(usually 40- 42°C).
3. Nonsense Or Chain Termination Mutations: They arise when a codon for an amino acid is mutated into a termination codon(UAG, UAA or UGA), resulting in the production of a shorter protein.
   1. Since temperature-sensitive and chain termination mutations exhibit the mutant phenotype only under certain conditions, they are called conditional mutations; they are the most versatile and useful mutations.
4. Silent Mutations: They change a nucleotide but not the amino acid sequence because they affect the third position of the codon, which is usually less important in coding. This is a silent mutation because it leaves the protein sequence unchanged.

Classification Of Mutation According To The Types Of Chromosomes

According To The Types Of Chromosomes, The Mutations May Be Of Following Two Kinds:

1. Autosomal Mutations: This type of mutation occurs in autosomal chromosomes.
2. Sex Chromosomal Mutations: This type of mutation occurs in sex chromosomes.

Mutation Rate

The frequency with which genes mutate spontaneously is called mutation rate. Most genes are relatively stable and mutation is a rare event.

• The great majority ofgenes have mutation rate of 1 × 10^-5, viz., one gamete in 100,000 to one gamete in a million would contain a mutation at a given locus.
• Mutations occur much more frequently in certain regions of the gene than in others. The favoured regions are called hot spots.

The Mutation Rate Is Influenced By Various Factors Which Are As Follows:

1. Genetic Control Of Mutation Rate: There is ample evidence showing that mutation rate is under genetic control, viz., certain genes called mutator genes may increase the mutation rate in Drosophila(Demere, 1937), maize(Rhoades, 1938) and£. coli(Goldstein, 1955).
   • However, certain suppressor genes may decrease the rate of mutation. In bacteria, as well as in eukaryotes, spontaneous mutations most frequently are caused by transposons which are segments of DNA that tend to jump around the genome.
2. Viral Control Of Mutation Rate: The virus reportedly affects the mutability of the host’s genes. Sprague (1963) experimented with maize and suggested that the virus may cause mutation.
   • Familiar (1967) reported that viruses increase the mutation rate in Drosophila melanogaster. But, still, we do not know how viruses increase the mutability of host genes.
3. Environmental Control Of Mutation Rate: Three major environmental factors affect mutation rates, viz, temperature, certain radiations and chemicals.

Method Of Detection Of Sex-Linked Lethal Mutation

H.J. Muller devised an easy method for detecting lethal mutations in the sex chromosomes of Drosophila. This is called a CIB method in which a special type of female fly is employed which carries a normal X chromosome and an abnormal X chromosome.

• The abnormal X chromosome contains an inversion mutation C (which prevents the chromosome to do crossing over with the normal X chromosome, therefore, called crossover suppressor), a recessive lethal mutational gene, I and a dominant gene B for bar-eye.
• In Muller’s CIB technique, these CIB female flies are mated with males which were previously treated with some mutagenic agent (such as Xrays) to cause mutation in some of their sperms.
• The resulting zygotes are of four types and one of these, the CIB male, fails to survive because such embryos contain a recessive lethal which expresses itself when hemizygous.
• Thus, only one class of male(with CIB X chromosome) male remains to fertilize the F₁ females. Each heterozygous CIB female results from the fertilization of a CIB egg and an irradiated X-bearing sperm and some of these sperms will contain mutated X-chromosomes.
• Mated F( heterozygous CIB females are distributed individually into culture tubes in which each lays fertile eggs and so produces a single F₂ culture. The culture produced by females bearing an induced lethal mutation contain only females; whereas females bearing irradiated X-chromosomes in which no recessive lethal has been induced yield cultures containing some wild-type males.
• Thus, if in a population of 1000 cultures, 990 contained some males and 10 contained only females, the induced rate of sex-linked, recessive lethal mutations would be 1 per cent.

Besides the CIB method, there are many more methods such as the Muller-5 method (for the detection of sex-linked lethal mutations); the attached X-method (for the detection of sex-linked visible mutations); balanced lethal systems(for the detection of autosomal mutations); and Stadler’s method and Singleton’s method (for the detection of specific loci in plants).

Practical Applications Of Mutations

Mutations are generally deleterious and recessive for the organisms, therefore, the majority of them are of no practical value.

• A. Gustafsson has estimated that less than one in 1000 mutants produced may be useful in plant I breeding. In India, several useful mutations of various cereals and other crop plants have been developed.
• Wheat. In bread wheat, many useful mutations have been obtained and utilised in plant breeding, e.g., branched ears, lodging resistance, high protein and lysine content, amber seed colour and awned spikelets.
• Dr M.S. Swaminathan, one of the most distinguished and legendary in the field of cytogenetics and plant breeding in the Indian subcontinent, had utilised amber mutation of Mexican wheat variety to develop a new variety of wheat, called Sharbati Sonora while working at Indian Agriculture Research for Institute(IARI), New Delhi.
• According to Dr. N.E. Borlaug(Nobel Laureate), this variety of wheat paved the way for the Green Revolution in India(see Gupta, 1994).
• Rice. In rice, one of the high-yielding varieties Reimei was developed through mutations isolated after gamma irradiation. Certain developed mutants of rice are found to contain increased contents of proteins and lysine. In certain other mutant rice, the duration of the crop was reduced by as many as 60 days.
• Barley. In barley, mutations called erectoides and eceriferum have been induced. These mutants had high yields including several useful characters.

Significance Of Mutation

The vast majority of mutations are deleterious to the organism and are kept at low frequency in the population by the action of natural selection.

• Mutant types are generally unable to compete equally with wild-type individuals.
• Even under optimal environmental conditions, many mutants appear less frequently than expected.

List Of Varieties Of Crop Plants Released By The Use Of Induced Mutations:

Alteration In The Genetic Material Questions And Answers

Question 1. Why are most mutations in structural genes recessive to their wild-type alleles?
Answer:

• Wild-type alleles usually code for complete, functional enzymes or other proteins.
• One active wild-type allele can often cause enough enzyme to be produced so that normal or nearly normal phenotypes result (dominance).
• Mutations of normally functioning genes are more likely to destroy the biological activities of proteins.
• Only in the complete absence of the wild-type gene product would the mutant phenotype be expressed recessiveness.

Question 2. If the mutation rate of a certain gene is directly proportional to the radiation dosage and the mutation rate of Drosophila is observed to increase from 3% at 1000 R to 6% at 2000 R. What percentage of mutations would be expected at 3500 R?
Answer: 10.5%.

Question 3. The X-linked recessive mutations are more easily studied in appropriate organisms than are autosomal ones. Why?
Answer: Recessive mutations are more easily detected in hemizygous males.

Question 4. Some individuals have a patch of blonde hair in a head of brown hair. What types of mutation would this be?
Answer: Somatic mutation.

Question 5. If a drastic alteration occurred in the structure of one of the genes for 28S rRNA, do you think that the translation of mRNA into protein would cease? If not, why not?
Answer:

Translation would not cease since numerous genes for rRNA are present in the genome. The mutation ofone of these would probably not interfere with protein synthesis.

Question 6. What possible explanations can you offer for the reversion of a mutant to the wild-type phenotype?
Answer:

Intragenic mutation within thesame codon, either restoring the original amino acid or resulting in the presence of a compatible amino acid; intragenic mutation within the same cistron, such as one that restores the normal reading frame; intergenic direct suppression, such as alteration in some component directly involved in protein synthesis, for example, tRNA; intergenic indirect suppression by an alteration in the cellular milieu.

Question 7. How many base pairs would have to be deleted in a mutational event to eliminate a single amino acid from a protein and not change the rest of the protein?
Answer: Three, as any other number of deletions (or additions) would cause a frameshift and other amino acid changes.

Question 8. The “dotted” gene in maize(Dt) is a “mutator” gene influencing the rate at which the gene for colourless aleurone(a) mutates to its dominant allele(A) for coloured aleurone. An average of 7.2 coloured dots (mutations) per kernel was observed when the seed parent was dt/dt, a/ a and the pollen parent was Dt/Dt, a/a. An average of 22.2 dots per kernel was observed in the reciprocal cross, How can these results be explained?
Answer:

The seed parent contributes two sets of chromosomes to the triploid endosperm; one Dt gene gives 7.2 mutations/kernel, and two Dt genes increase mutations to 22.2/kernel.

Question 9. What is the difference between substrate and a template transition mutation?
Answer:

In replicating DNA, a transition mutation can occur by tautomerization of a base in the template strand (template transition) or entering the progeny strand (substrate transition).

Question 10. 5-bromouracil, 2-aminopurine, proflavin, ethyl ethane sulphonate and nitrous acid are chemical mutagens. What does each do?
Answer:

• 5-bromouracil(pyrimidine analogue) and 2-aminopurine(purine analogue) are incorporated into DNA as thymine and adenine, respectively.
• However, each undergoes tautomeric shifts more frequently than the normal base. Both cause transitions.
• Nitrous acid also promotes transitions by converting cytosine into uracil, which acts like thymine, and adenine into hypoxanthine, which acts like guanine.
• Proflavin induces insertions and deletions by intercalating and buckling DNA. Ethyl ethane sulphonate removes purine rings and thus promotes transitions and transversions.

Alteration In The Genetic Material Multiple Choice Questions And Answers

Question 1. Mutation is

1. Change that is inherited
2. Change in a parent not inherited
3. Plant growth controlling factor
4. Change which affects the offspring of F₂ generation

Answer: 1. Change that is inherited

Question 2. Gene mutation is

1. Mutation in the genes of DNA
2. Mutation in phosphodiester linkage
3. Mutation in chromosomes
4. Change in the sequence of nitrogenous bases

Answer: 4. Change in the sequence of nitrogenous bases

Question 3. Mutations which normally happen randomly are considered one of the raw materials for evolution because they

1. Are stable
2. Contribute new variation in an organism
3. Cause the death of the organism
4. None of these

Answer: 2. Contribute new variation in an organism

Question 4. Proflavin and acridine orange induce

1. Transitions
2. Transversions
3. Inversions
4. Frameshift mutations

Answer: 4. Frameshift mutations

Question 5. Induction of mutation by X-rays was discovered by

1. Morgan
2. Hugu devices
3. Muller
4. Luria

Answer: 3. Muller

Question 6. Who is associated with the “Green Revolution” in India?

1. B.P. Pal
2. M.S. Swaminathan
3. R.S. Paroda
4. EJ. Butler

Answer: 2. M.S. Swaminathan

Question 7. Low temperature is mutagenic in

1. Wheat
2. Maize
3. Rice
4. Mustard

Answer: 3. Rice

Question 8. Frequency of mutation

1. Varies with characters and organisms
2. Can be increased by X-rays
3. Is greatly affected by environmental factors
4. All of the above

Answer: 4. All of the above

Question 9. Which of the following mutagens can be best used in inducing mutation in microorganisms?

1. X-rays
2. β-rays
3. UV-rays
4. ϒ-rays

Answer: 3. UV-rays

Question 10. Why are haploids superior to diploids in the study of mutations?

1. They have a shorter lifetime
2. Smaller number of chromosomes
3. They allow the expression of recessive mutation immediately
4. Obtained in large numbers

Answer: 3. They allow the expression of recessive mutation immediately

The post Alteration In The Genetic Material Point Mutation appeared first on BDS Notes.

Enols And Enolization

“What are nucleophilic reactions involving enolate anions? A detailed notes and Q&A guide”

Aldehydes and ketones, and other carbonyl compounds having hydrogen atoms on the α-carbon, exist in solution as equilibrium mixtures of two or more isomeric forms.

These isomers are termed the keto form, which is how we normally represent a carbonyl compound, and the enol form, which takes its name from the combination of double bonds and alcohol.

The interconversion of keto and enol forms is termed enolization, or keto-enol tautomerism.

• The two isomeric structures are not resonance forms but are termed tautomers. Resonance forms have the same arrangement of atoms, but the electrons are distributed differently.
• Tau tomers have the atoms arranged differently, and tau tomerism is an equilibrium reaction between the isomeric forms.
• Thus, in the general case shown, the α-hydrogen in the keto tautomer disappears and the oxygen atom gains hydrogen to produce the hydroxyl of the enol system.

To indicate the importance of enolization, equilibrium constants for several substrates. These equilibrium constants are only approximate, and they depend very much on the solvents employed.

• Nevertheless, we can see that the equilibrium constant K = [enol]/[keto] is very small for substrates like acetaldehyde, acetone, and cyclohexanone, with only a few molecules in every million existing in the enol form.
• However, in ethyl acetoac estate, enol concentrations are measured in per cent ages, and in acetylacetone, the equilibrium constant indicates the enol form can be distinctly favoured over the normal keto form.

In hexane solution, only 8% of acetylacetone molecules remain in the keto form.

• Normally then, the keto form we have traditionally written for carbonyl compounds is very much favoured over the enol tautomer.
• The high contribution of enol forms in equilibrium mixtures of the 1,3- dicarbonyl compounds such as ethyl acetoacetate and acetylacetone are ascribed principally to additional stability conferred by the formation of a conjugated enone system, with further stabilization coming from the establishment of the hydrogen bonding in a favourable six-membered ring.

At the other extreme, as in the case of cyclohexadiene, the enol tautomer is the only contributing tautomer, since the enol form (phenol) benefits from the stabilization conferred by the aromatic ring system.

Keto Enol Equilibria:

“Understanding enolate anions through FAQs: Nucleophilic reactions explained”

“How do enolate anions act as nucleophiles in organic chemistry? FAQ answered”

It is important to note that, in 1,3-dicarbonyl compounds such as acetylacetone, enolization involves loss of the α-hydrogen between the two carbonyl groups and not the terminal α-hydrogens.

• Enolization involving the latter α-hydrogens would not generate conjugation stabilization; and despite the possibility of hydrogen bonding, this enol form is not favoured relative to the alternatives.
• Conjugation acid-catalysed tautomerism can only be achieved if the central α-hydrogens, those sandwiched between the two carbonyls, are involved.
• The interconversion of keto and enol forms may be catalysed by both acids and by a base.

In acid, this may be rationalized by a mechanism in which protonation of the carbonyl to give the conjugate acid is followed by loss of the α-proton.

It is important to appreciate the role of the solvent in this transformation, removing and supplying protons, and to understand that tautomerism is not merely a transfer of a proton from the α-carbon to the carbonyl oxygen.

• The rate-determining step in tautomerism will be the removal of the α-hydrogen; protonation of the carbonyl (formation of the conjugate acid) can be considered rapid.
• In base, slow abstraction of the α-hydrogen by the base will be the first step, followed by rapid protonation of the conjugate base, again making use of the solvent for the removal and supply of protons.

This process is thus exploiting the acidity associated with the α-hydrogens (pK_a 19), which is considerably greater than that of the corresponding alkane (pK_a 50).

• The effect of the adjacent carbonyl is to increase the acidity of the α-hydrogens.
• This is a direct consequence of the polarization of the carbonyl arising from the electronegativity of the oxygen atom.
• The conjugate base in this process is called an enolate anion and is stabilized by resonance.
• Of the two resonance forms of the enolate anion, that with the charge on the electronegative oxygen will be preferred over that with a charge on the carbon.
• Note the distinct difference between resonance as shown here, a redistribution of electrons, and tautomerism, as described above.

Tautomers are isomers in equilibrium and have the atoms arranged differently.

“Importance of studying nucleophilic reactions with enolate anions for chemistry students: Questions explained”

In 1,3-dicarbonyl compounds such as acetylace tone, the protons between the two carbonyls will be even more acidic (pK_a 9), since there are now two carbonyl groups exerting their combined influence.

• It can also be seen that resonance in the enolate anion is even more favourable with two carbonyl atoms arranged differently groups.
• This increased stability is not achieved by removal of the terminal α-hydrogens, and in acety lacetone, these have pK_a 20, comparable to that in acetone. Put another way, the treatment of acetylacetone with base preferentially removes a proton from the central methylene.

Enols And Enolization In The Glycolytic Pathway:

Enols and enolization feature prominently in some of the basic biochemical pathways.

• Biochemists will be familiar with the terminology enol as part of the name phosphoenolpyruvate, a metabolite of the glycolytic pathway. We shall here consider it in non-ionized form, i.e. phosphoenolpyruvic acid.
• As we have already noted, in the enolization between pyruvic acid and enol pyruvic acid, the equilibrium is likely to favour the keto form pyruvic acid very much.
• However, in phosphoenolpyruvic acid the enol hydroxyl is esterified with phosphoric acid, effectively freezing the enol form and preventing tautomerism back to the keto form.

• Once the phosphate ester is hydrolysed, there is immediate rapid tautomerism to the keto form, which becomes the driving force for the metabolic transformation of phosphoenolpyruvic acid into pyruvic acid and explains the large negative free energy change in the transformation. This energy release is coupled to ATP formation.
• Tautomerism occurs elsewhere in the glycolytic pathway. The transformation of glyceraldehyde 3-phosphate into dihydroxyacetone phosphate involves two such keto–enol tautomerisms and proceeds through an enediol.

“Common challenges in understanding enolate anion mechanisms effectively: FAQs provided”

• This enediol can be regarded as a common enol tautomer for two different keto structures.
• In other words, there are two ways in which this enediol can tautomerize back to a keto form, and the reaction thus appears to shift the position of the carbonyl group.
• The reaction is enzyme-catalysed, which allows the normal equilibrium processes to be disturbed.
• It is nice to see this series of reactions being repeated in the glycolytic pathway, this time accounting for the transformation of glucose 6-phosphate into fructose 6-phosphate.
• Although the substrates are different, the reacting portion of the molecules is the same as that in the glyceraldehyde 3-phosphate to dihydroxyacetone phosphate transformation. Again, this is an enzyme-catalysed reaction.

Hydrogen Exchange:

The intermediacy of enols or enolate anions may be demonstrated by hydrogen exchange reactions.

• Both acid-catalysed and base-catalysed tautomerism mechanisms involve the removal of a proton from the α-carbon and the supply of a proton from the solvent to the carbonyl oxygen.
• Accordingly, this removal/supply of protons can be observed using isotopes of hydrogen, either radioactive tritium or the stable deuterium, which can be detected easily via NMR techniques.
• Thus, pentane-3-one can be deuterated using a large excess of D₂O, with either acid (DCl) or base (NaOD) catalyst; the acid or base catalyst should also be deuterated to minimize dilution of the label.

After suitable equilibration, usually requiring prolonged heating, the α-positions will become completely labelled with deuterium.

“Why is early learning of enolate anion chemistry critical for organic synthesis? Answered”

Two mechanisms are shown above. The base-catalysed mechanism proceeds through the enolate anion. The acid-catalysed process would be formulated as involving an enol intermediate.

• Note that the terminal hydrogens in pentane-3-one are not exchanged, since they do not participate in the enolization process.
• Of course, it is also possible to re-exchange the labelled hydrogens by a similar process using an excess of ordinary water, a process that might be exploited to determine or confirm the position of labelling in a deuterium-labelled substrate.
• Although this section has been termed hydrogen exchange, it is important to realize that we could also visualize this simply as an enolate anion acting as a base.

This is also true of the next section, and in some of the following sections we shall encounter enolate anions acting as nucleophiles.

Nucleophilic Reactions Involving Enolate Anions Racemization:

The process of hydrogen exchange shown above has implications if the α-carbon is chiral and has hydrogen attached.

• Removal of the proton will generate a planar enol or enolate anion, and regeneration of the keto form may then involve a supply of protons from either face of the double bond, so changing a particular enantiomer into its racemic form.
• Reacquiring a proton in the same stereochemical manner that it was lost will generate the original substrate, but if it is acquired from the other face of the double bond it will give the enantiomer, i.e. together making a racemate.

Note that removal and replacement of protons at the other α-carbon, i.e. the methyl, will occur, but has no stereochemical consequences.

“Factors influencing success with nucleophilic reactions involving enolate anions: Q&A”

The chiral centre must be α to the carbonyl and must contain a hydrogen substituent.

If there is more than one chiral centre in the molecule with only one centre α to the carbonyl, then the other centres will not be affected by enolization, so the product will be a mixture of diastereoisomers of the original compound rather than the racemate.

Sometimes, other features in the molecule may facilitate the formation of the enol or enolate.

Thus, in the ketone shown below, conjugation of the enol double bond with the aromatic ring system helps to stabilize the enol tautomer; therefore, enolization and racemization occur more readily.

It should be noted that the rate of racemization (or the rate of hydrogen exchange) is the same as the rate of enolization since the protonation reaction is fast.

Hence, the rate is typical of a bimolecular process and depends upon two variables, the concentration of carbonyl compound and the concentration of acid (or base).

Rate = k[C=O][acid]

or

Rate = k[C=O][base]

where C=O is the carbonyl substrate and k is the rate constant.

Interconversion Of Monoterpene Stereoisomers Through Enolization:

On heating with either acid or base, the monoterpene ketone isodihydrocarvone is largely converted into one product only, its stereoisomer dihydrocarvone.

“Steps to explain types of nucleophilic reactions with enolate anions: Aldol vs Michael addition: Notes guide”

There are two chiral centres in isodihydrocarvone, but only one of these is adjacent to the carbonyl group and can participate in enolization.

• Under normal circumstances, we might expect to generate an equimolar mixture of two diastereoisomers.
• This is because two possible configurations could result from the chiral centre α to the carbonyl, whereas the other centre is going to stay unchanged.
• We might thus anticipate the formation of a 50:50 mixture of isodihydrocarvone and dihydrocarvone.
• That the product mixture is not composed of equal amounts of isodihydrocarvone and dihydrocarvone can be rationalized by considering stereochemical factors, particularly the conformations adopted by the two compounds, which turn out to favour the product over the starting material.

The favoured conformation of isodihydrocarvone has the large isopropenyl substituent equatorial.

• On forming the enol (or enolate anion), it will adopt the conformation in which both substituents are equatorial (or equatorial-like).
• To revert to a keto tautomer might then involve acquiring a proton from either side of the planar enol/enolate.
• However, there is going to be a distinct preference for forming the more favoured product that has two equatorial substituents.
• This is dihydrocarvone. The equilibrium mixture set up thus contains predominantly dihydrocarvone, rather than an equal mixture of two diastereoisomers.

The second chiral centre contains a large group, and its stereochemical preference effectively dictates the chirality at the second centre, and thus the nature of the product.

Nucleophilic Reactions Involving Enolate Anions Conjugation:

When an enol tautomer reverts to a keto tau timer, it must acquire a proton, and we have already seen that it may be acquired from different faces of the double bond, giving two types of stereochemistry.

• In the example described, the stereochemistry of the product was effectively dictated by the existing chirality at a second centre.
• Now we can see a further variant, in that the stability of the product dictates that an alternative carbon in the enol tautomer receives the proton. This relates to conjugation in the product.

A β,γ-unsaturated carbonyl compound exposed to acid or base is usually converted rapidly into an α,β-unsaturated carbonyl derivative. This isomerization is easily interpreted by considering enolization.

“Role of enolate formation in base-catalyzed reactions: Questions answered”

Removal of an α-proton from a β,γ-unsaturated ketone generates an enolate anion, and this might be transformed back to the β,γ-unsaturated compound by deprotonation at the α-position.

• However, this does not occur because the enolate anion now has conjugated double bonds, and we can propose an alternative mechanism for deprotonation, invoking the conjugation and protonating at the γ-position.
• This protonation is preferred, in that the product is now a conjugated ketone and, therefore, energetically favoured over the non-conjugated ketone. Since all the reactions are equilibria, eventually a more stable product will result.

Conversion Of Pregnenolone Into Progesterone:

An important transformation in steroid biochemistry is the conversion of pregnenolone into progesterone.

• Progesterone is a female sex hormone, a progestogen, but this reaction is also involved in the production of corticosteroids such as hydrocortisone and aldosterone.
• The reaction also occurs in plants, and features in the formation of cardioactive glycosides, such as digitoxin in foxglove.

This enzymic conversion involves two enzymes, a dehydrogenase and an isomerase. The dehydrogenase component oxidizes the hydroxyl group on pregnenolone to a ketone and requires the oxidizing agent cofactor NAD⁺.

The isomerase then carries out two tautomerism reactions, enolization to a dienol followed by production of the more stable conjugated ketone.

“How does aldol condensation proceed via enolate anions? FAQ explained”

The enzyme provides a base (B:) and an acid (A–H) via appropriate amino acid side chains on the enzyme to facilitate proton removal and supply.

• A fascinating aspect is that the proton removed from the methylene (steroid position 4) by the base is then donated back to position 6. The base is suitably positioned to serve both sites in the steroid.

An exactly analogous enzymic transformation is encountered during the formation of oestrogen and androgen sex hormones, for example., estradiol and testosterone respectively, where dehydroepiandrosterone is oxidized to androstenedione.

The isomerization reaction is also encountered in chemical manipulations of steroids.

• Thus, many natural steroids contain a 5-en-3-ol combination of functionalities, for example., cholesterol.

Treatment of cholesterol with an oxidizing agent (aluminium isopropoxide is particularly suitable) leads to cholest-4-en-3-one, the tautomerism occurring spontaneously under the reaction conditions.

Halogenation:

Aldehydes and ketones undergo acid- and base-catalysed halogenation in the α position. This is also dependent on enolization or the formation of enolate anions.

Thus, bromination of acetone may be achieved by using bromine in sodium hydroxide solution, and this is rationalized mechanistically through the formation of the enolate anion, which then attacks the polarized bromine electrophile.

“Early warning signs of gaps in understanding enolate anion basics: Common questions”

There are two ways of representing this, according to which resonance form of the enolate anion is used.

• Although the preferred resonance form (charge located on the oxygen atom) should be used as the nucleophile because carbon is acting as the nucleophile and a new C–Br bond is formed, the less-favoured resonance form is frequently employed in mechanistic pathways.
• This makes mechanism drawing rather easier but is technically incorrect.
• Kinetic data show us that the rate of reaction is dependent upon two variables, i.e. the carbonyl in acid:
• substrate concentration and the concentration of base. These are the two components necessary for the formation of the enolate anion, which is the slow step in the sequence.
• After the formation of the enolate anion, nucleophilic attack on bromine is rapid; therefore, the bromine concentration does not figure in the rate equation. A related mechanism can be drawn for acid-catalysed halogenation.

Again, the halogen concentration does not figure in the rate equation, and the rate of enolization controls the rate of reaction.

If we wish to synthesize a monohalogenated product, then we have to use an acid-catalysed reaction; base catalysis leads to multiple halogenations. This relates to the acidity of intermediates.

• Thus, each successive halogenation introduces an electron-withdrawing substituent, which increases acidity and facilitates enolate anion formation.
• On the other hand, an electron-withdrawing halogen substituent destabilizes the protonated carbonyl compound and consequently disfavours enolization.

base-catalysed reaction: consider acidity and enolate anion formation
acid-catalysed reaction: consider basicity and conjugate acid formation

Alkylation Of Enolate Anions

Though this topic is treated here under a separate heading, alkylation of enolate anions is nothing other than enolate anions acting as carbanion nucleophiles in S_N2 reactions.

• We deferred this topic from, at that stage we had not encountered the concept of enols and enolate anions.

By treating the 1,3-dicarbonyl compound acuity acetone with methyl iodide in the presence of potassium carbonate, one observes alkylation at the central carbon.

“Asymptomatic vs symptomatic effects of ignoring enolate anion principles: Q&A”

This is easily rationalized via the initial formation of an enolate anion under the basic conditions, followed by an S_N2 reaction on the methyl iodide.

• The enolate anion is the nucleophile and iodide is displaced as the leaving group.
• The enolate anion could be drawn with a charge on carbon or oxygen; the latter is preferred, as discussed above, in that the charge is preferentially located on the electronegative oxygen atom.
• It is feasible, therefore, that either carbon or oxygen could be the nucleophilic atom, and we might expect more chance of oxygen participating.
• Despite this, it is observed that, in almost all cases, alkylation occurs on carbon, not on oxygen, so it does not present a problem.

Two mechanisms could be drawn for the reaction, depending on whether the enolate anion has a charge on the carbon or oxygen.

• Since carbon is eventually the nucleophilic centre, it is permissible to use the carbanion version of the enolate (as, in general, we shall do), though this is strictly not correct, and purists would use the alternative version starting with charge on the oxygen.
• Now for some interesting features of the reaction, though they become fairly obvious with a little thought.
• First, the central methylene contains the more acidic protons (pK_a 9) since it is flanked by two carbonyls, so the enolate anion formed involves this carbon.

In other words, alkylation occurs on the central carbon of acetylacetone, not on the terminal carbons.

• Second, it is possible to use carbonyl compounds such as acetone as a solvent without these reacting under the reaction conditions.
• Acetone will have similar acidity (pK_a 19) to the acetyl groups of acetylacetone, so likewise will not form an enolate anion under conditions that only ionize the central methylene of a 1,3-dicarbonyl compound.
• Furthermore, the product formed still contains an acidic proton on a carbon flanked by two carbonyls, so it can form a new enolate anion and participate in a second S_N2 reaction.
• The nature of the product will thus depend on electrophile availability.

With 1 mol of methyl iodide, a monomethylated compound will be the predominant product, whereas with 2 mol of methyl iodide, the result will be mainly the dimethylated compound.

A further twist is that it is possible to use this reaction to insert two different alkyl groups.

This requires treating first with 1 mol of an alkylating agent, allowing the reaction to proceed, then supplying 1 mol of a second, but different, alkylating agent.

Of course, minor products might be produced, including monoalkylated products and dialkylated products (in which the two alkyl groups are the same), depending on the conditions and how near to completion the reaction proceeds.

• Note that we cannot use aryl halides in these reactions; rear side attack is impossible and we do not get S_N2 reactions at sp²-hybridized carbon.
• 1,3-Dicarbonyl compounds, like acetylacetone, are reasonably acidic (pK_a 9) and the formation of enolate anions is achieved readily. Potassium carbonate is basic enough to ionize acetylacetone in the above example.

However, if we are presented with a substrate having only a single carbonyl group, for example.,. acetone (pK_a 19), then it follows that we must use a stronger base to remove the correspondingly less acidic protons. Strong bases that might be used include sodium hydride and sodium amide.

“Can targeted interventions improve outcomes using enolate anion knowledge? FAQs provided”

These compounds ionize and act as sources of hydride and amide ions respectively, which can remove α-protons from carbonyl compounds.

• These ions are the conjugate bases of hydrogen and ammonia respectively, compounds that are very weak acids indeed.
• What becomes important here is that enolate anion formation becomes essentially irreversible; the enolate anion formed is insufficiently basic to be able to remove a proton from either hydrogen or ammonia.
• This is in marked contrast to the earlier examples of enolate anion formation that were reversible. We now have a means of preparing the enolate anion, rather than relying upon an equilibrium reaction.

Accordingly, reactions are usually done in two stages, preparation of the enolate anion followed by the addition of the alkylating agent electrophile.

In the example shown, alkylation of the ketone is readily accomplished using such a two-stage process with 1 mol of alkyl halide.

• Note that the specificity of this reaction relies on one of the α-carbons having no acidic hydrogens so that only one enolate anion can be formed.
• Another strong base routinely employed in synthetic procedures to prepare enolate anions is lithium diisopropylamide (LDA).
• The diisopropyl amide anion is formed by removing a proton from diisopropylamine using the organometallic derivative n-butyllithium.
• Because of the highly reactive nature of n-butyllithium (it reacts explosively with air) this reaction has to be conducted in an oxygen-free atmosphere and at a very low temperature.

The ionization works because although the acidity of diisopropyl lamine is not great (pK_a 36), the other product formed, i.e. butane, is significantly less acidic (pK_a 50). The reaction is essentially irreversible.

When the carbonyl compound is added to this base, the abstraction of a proton and formation of the enolate anion follow, as seen with sodium hydride or sodium amide above.

• Again, this reaction is essentially irreversible because the other product is the weak base diisopropylamine (pK_a 36).
• So far, there does not seem any particular advantage in using LDA rather than sodium hydride or sodium amide, and the manipulations required are very much more difficult and dangerous.
• The real benefit is that LDA is a very strong base, and because of its quite large size it is also a relatively poor nucleophile.

This reduces the number of competing reactions that might occur where nucleophilicity competes with basicity.

In symmetrical structures such as cyclohexanone, ionization at α-positions occurs readily and allows the preparation of alkylated products.

• In unsymmetrical structures, the sheer size of LDA as a base may allow selectivity by preferential removal of certain α-protons.
• Thus, the ketone pentane-2-one will undergo preferential removal of a proton from the terminal methyl in the generation of an enolate anion.

This allows selective alkylation to be achieved.

“Differential applications of thermodynamic vs kinetic enolates: Notes explained”

Addition–Dehydration: The Aldol Reaction

We now have examples of the generation of enolate anions from carbonyl compounds, and their potential as nucleophiles in simple S_N2 reactions.

• However, we must not lose sight of the potential of a carbonyl compound to act as an electrophile.
• This section, the aldol reaction, is concerned with the enolate anion aldol reaction formation of nucleophiles attacking carbonyl electrophiles to give addition compounds, though it is usual for such addition compounds to then lose water, i.e. addition–dehydration.
• The namesake aldol reaction is the formation of an additional compound, aldol, from two molecules of acetaldehyde, when this aldehyde is treated with aqueous sodium hydroxide.

The terminology aldol comes from the functional groups in the product, aldehyde and alcohol.

This is easily formulated as the production of an enolate anion followed by a nucleophilic attack of this anion onto the carbonyl group of a second molecule of acetaldehyde.

• Aldol is then produced when the addition anion abstracts a proton from solvent. The reaction is reversible, and it is usually necessary to disturb the equilibrium by some means.
• Removal rather messy mixture of products containing at least four different components. This is because both starting materials might feature as nucleophiles or as electrophiles.

In the reverse reaction, the addition anion reforms the carbonyl group by expelling the enolate anion as a leaving group. This reverse aldol reaction is sufficiently important in its own right, and we shall meet examples.

• Note that, as we saw with simple aldehyde and ketone addition reactions, aldehydes are better electrophiles than ketones.
• This arises from the extra alkyl group in ketones, which provides a further inductive effect and extra steric hindrance.

Accordingly, the aldol reaction is more favourable with aldehydes than with ketones. With ketones, it is essential to disturb the equilibrium in some way.

• The aldol reaction as formulated above involves two molecules of the starting substrate. However, by a consideration of the mechanism, one can see that different carbonyl compounds might be used as nucleophiles or electrophiles.
• This would be termed a mixed aldol reaction or crossed aldol reaction. However, if one merely reacted, say, two aldehydes together under basic conditions, one would get a rather messy mixture of products containing at least four different components.
• This is because both starting materials might feature as nucleophiles or as electrophiles.

Mixed Aldol Reaction

⇒ \(\mathrm{RCH}_2 \mathrm{CHO}+\mathrm{R}^{\prime} \mathrm{CH}_2 \mathrm{CHO} \longrightarrow 4 \text { products }\)

Nucleophile And Electrophilic

⇒ \(\mathrm{RCH}_2 \mathrm{CHO}+\mathrm{RCH}_2 \mathrm{CHO}\)

⇒ \(\mathrm{RCH}_2 \mathrm{CHO}+\mathrm{R}^{\prime} \mathrm{CH}_2 \mathrm{CHO}\)

⇒ \(\mathrm{R}^{\prime} \mathrm{CH}_2 \mathrm{CHO}+\mathrm{RCH}_2 \mathrm{CHO}\)

⇒ \(\mathrm{R}^{\prime} \mathrm{CH}_2 \mathrm{CHO}+\mathrm{R}^{\prime} \mathrm{CH}_2 \mathrm{CHO}\)

For the mixed aldol reaction to be of value in synthetic work, it is necessary to restrict the number of combinations. This can be accomplished as follows.

• First, if one of the materials has no α-hydrogens, then it cannot produce an enolate anion, and so cannot function as the nucleophile.
• Second, in aldehyde plus ketone combinations, the aldehyde is going to be a better electrophile, so reacts preferentially in this role.
• A simple example of this approach is the reaction of benzaldehyde with acetone under basic conditions.

Such reactions are synthetically important as a means of increasing chemical complexity by forming new carbon-carbon bonds.

“Steps to apply enolate anion chemistry in organic synthesis: Design vs optimization: Notes guide”

Benzaldehyde has no α-hydrogens, so it cannot be converted into an enolate anion to become a nucleophile. Acetone has α-hydrogens, so it can form an enolate anion and become a nucleophile.

• We now have two possible electrophiles, i.e. one an aldehyde and the other a less reactive ketone.
• The preferred reaction is thus acetone as an enolate anion nucleophile, with benzaldehyde as the preferred electrophile, giving the additional product shown.
• This is not isolated, since it readily dehydrates to give the unsaturated ketone benzalacetone.
• The additional product from aldol reactions frequently dehydrates by heating in acid or in base to give the corresponding α,β-unsaturated carbonyl compound.

Under basic conditions, this occurs readily, even though hydroxide is a poor leaving group, because of the acidity of the α-proton and the conjugation stabilization in the product.

There is evidence that this is not an E2 mechanism under basic conditions, but a so-called E1cb mechanism.

• This stands for elimination–unimolecular–conjugate base, and proceeds via initial removal of the acidic proton to give the conjugate base (enolate anion).
• The reaction is unimolecular because it is the loss of a leaving group from the conjugate base which is the rate-determining step. Removal of the acidic proton is faster than loss of the hydroxide ion.
• Since E1cb reactions are rare (this is the only one we shall consider), we deliberately chose not to include it under general elimination reactions.
• The conditions of the reaction are often sufficient to cause dehydration of the addition product as it is formed, and it is normally extremely difficult to isolate the addition product.

It turns out that the addition reaction (equilibrium) is slow, whereas the elimination reaction (non-reversible) is faster.

• This usually disturbs the equilibrium in an aldol reaction, especially if the product is stabilized by even further conjugation, as in the case of benzalacetone above, where the benzene ring also forms part of the conjugated system.
• An alternative approach to mixed aldol reactions, and the one usually preferred, is to carry out a two-stage process, forming the enolate anion first using a strong base like LDA.
• The first step is essentially irreversible, and the electrophile is then added in the second step.
• An aldol reaction between butane-2-one and acetaldehyde exemplifies this approach.

Note also that the large base LDA selectively removes a proton from the least-hindered position, again restricting possible combinations.

Aldol And Reverse Aldol Reactions In Biochemistry: Aldolase, Citrate Synthase:

Both the aldol and reverse aldol reactions are encountered in carbohydrate metabolic pathways in biochemistry.

• One reversible transformation can be utilized in either carbohydrate biosynthesis or carbohydrate degradation, according to a cell’s particular requirement.
• D-Fructose 1,6-diphosphate is produced during carbohydrate biosynthesis by an aldol reaction between dihydroxyacetone phosphate, which acts as the enolate anion nucleophile, and D-glyceraldehyde 3-phosphate, which acts as the carbonyl electrophile; these two starting materials are also interconvertible through keto-enol tautomerism, as seen earlier.

The biosynthetic reaction may be simplified mechanistically as a standard mixed aldol reaction, where the nature of the substrates and their mode of coupling is dictated by the enzyme. The enzyme is called aldolase.

During carbohydrate metabolism in the glycolytic pathway, fructose 1,6-diphosphate is cleaved to give dihydroxyacetone phosphate and glyceraldehyde 3-phosphate.

• This is a reverse aldol reaction, in which a carbonyl group is formed at the expense of carbon-carbon bond cleavage with expulsion of an enolate anion leaving group.
• The additional functional groups present in the substrates would seriously limit any base-catalysed chemical aldol reaction between these substrates, but this reaction is enzyme-mediated, allowing reaction at room temperature and near-neutral conditions.

The aldol and reverse aldol reactions just described accommodate the chemical changes observed, though we now know that nature uses a slightly different approach via enamines.

• This does not significantly alter our understanding of the reactions, but it does remove the requirement for a strong base and also accounts for the bonding of the substrate to the enzyme.
• A similar aldol reaction is encountered in the Krebs cycle in the reaction of acetyl-CoA and oxaloacetic acid. This yields citric acid and is catalysed by the enzyme citrate synthase.

This intermediate provides the alternative terminology for the Krebs cycle, namely the citric acid cycle.

The aldol reaction is easily rationalized, with acetyl-CoA providing an enolate anion nucleophile that adds to the carbonyl of oxaloacetic acid. We shall see later that esters and thioesters can also be converted into enolate anions.

“Role of enolate anions in forming carbon-carbon bonds: Questions answered”

One interesting feature here is that both acetyl-CoA and oxaloacetic acid have the potential to form enolate anions and that oxaloacetic acid is more acidic than acetyl-CoA, in that the two carbonyl groups are flanking the methylene.

• That citrate synthase achieves the aldol reaction as shown reflects that the enzyme active site must have a basic residue appropriately positioned to abstract a proton from acetyl-CoA rather than oxaloacetic acid, thus allowing acetyl-CoA to act as the nucleophile.
• The obvious product of the aldol reaction would be the thioester citryl-CoA. However, the enzyme citrate synthase also carries out hydrolysis of the thioester linkage, so that the product is citric acid; hence the terminology.

The hydrolysis of the thioester is responsible for disturbing the equilibrium and driving the reaction to completion.

• We should also consider occasions when there are two carbonyl groups in the same molecule.
• We then have the possibility of an intramolecular aldol reaction, and this offers a convenient way of synthesizing ring systems.

Rings with five or six carbons are particularly favoured. Thus, treatment of octan-2,7-dione with base gives good yields of the cyclopentene derivative shown.

The reaction is readily formulated. Note that there are two potential products from the aldol addition, one of which is five-membered and the other seven-membered.

• The five-membered product is more favourable than the seven-membered one simply based on ring strain.
• However, if both products form, they will be in equilibrium as shown. It is the next step, dehydration, that drives the six-membered ring and seven-membered ring not favoured the reaction giving the more stable product, the cyclopentene.
• Any seven-membered addition product can then equilibrate to give more of the five-membered compound.

A similar reaction with heptane 2,6-dione would lead to the methylcyclohexane product, and not the sterically unfavourable four-membered ring alternative.

Note also that if the substrate has both aldehyde and ketone functions the aldehyde will act as the electrophile.

The ketoaldehyde shown forms the one product in good yield, there now being restrictions on preferred ring size and the regiochemistry of the mixed aldol reaction.

If a five- or six-membered ring can form, then intramolecular aldol reactions usually occur more rapidly than the corresponding intermolecular reactions between two molecules of substrate. This provides a very useful route to cyclic compounds.

Other Stabilized Anions As Nucleophiles: Nitriles And Nitromethane

An enolate anion behaves as a carbanion nucleophile, the carbonyl group stabilizing the anion by delocalization of charge.

• Both cyano (nitrile) and nitro groups can fulfil the same role as a carbonyl by stabilizing a carbanion, so we see similar enhanced acidity of α-protons in simple nitrile and nitro compounds.
• pK_a values for nitriles are about 25, whereas aliphatic nitro compounds have pK_a of about 10. Nitro compounds are thus considerably more acidic than aldehydes and ketones (pK_a about 20).

Accordingly, it is possible to generate analogues of enolate anions containing cyano and nitro groups and to use these as nucleophiles towards carbonyl elec trophiles in aldol-like processes. Simple examples are shown.

“How does Michael addition utilize enolate anions? FAQ explained”

As with many aldol reactions, addition is usually followed by the elimination of water, generating a conjugated system with the cyano or nitro group. The presence of extended conjugation through aromatic substituents enhances this process.

These reactants introduce either nitrile or nitro groups into the product. These groups may be converted into carboxylic acids or amines, as shown.

Enamines As Nucleophiles:

We met enamines as products from addition–elimination reactions of secondary amines with aldehydes or ketones.

Enamines are formed instead of imines because no protons are available on nitrogen for the final deprotonation step, and the nearest proton that can be lost from the iminium ion is that at the β-position.

“Early warning signs of complications from ignoring enolate anion protocols: Common questions”

There is a distinct relationship between keto-enol tautomerism and the iminium–enamine interconvert H sion; it can be seen from the above scheme that enamines are nitrogen analogues of enols.

• Their chemical properties reflect this relationship. It also leads us to another reason why enamine formation is a property of secondary amines, whereas primary amines give imines with aldehydes and ketones.
• Enamines from primary amines enamine from primary amine imine favoured enamine from secondary amine would undergo rapid conversion into the more stable imine tautomers (compare enol and keto tautomers); this isomerization cannot occur with enamines from secondary amines, and such enamines are, therefore, stable.

The most prominent property of enamines is that the β-carbon can behave as a carbon nucleophile.

This resonance form can then act as a nucleophile, in much the same way as an enolate anion can. However, there is a marked difference, and this is what makes enamines such useful synthetic intermediates.

• Generation of an enolate anion requires the treatment of a carbonyl compound with a base, sometimes a very strong base.
• This is a consequence of resonance; the overlap of lone pair electrons from the nitrogen provides an iminium system, with the negative counter-charge on the β-carbon.
• The formation of the enamine resonance form is a property of the enamine and requires no base. A simple S_N2 alkylation reaction serves as an example.

As we have already seen, treating cyclohexanone with LDA gives the enolate anion, which can then be allowed to react with methyl iodide to give 2-methyl cyclohexanone.

“Asymptomatic vs symptomatic effects of outdated enolate practices: Answered”

Alternatively, cyclohexanone may initially be transformed into an enamine with a secondary amine, here pyrrolidine. This intermediate enamine can act as a nucleophile and can be alkylated at the β-position using methyl iodide.

• Finally, 2-methylcyclohexanone may be generated by hydrolysis of the iminium system, effectively a reversal of enamine formation.
• This gives us two routes to 2-methylcyclohexanone, a short process using the very strong base LDA and a longer route that involves no strong base and relatively mild conditions.
• The latter synthesis may well be preferred, depending upon the nature of any other functional groups in the starting substrate.
• The essential feature of enamines is that they are nitrogen analogues of enols and behave as enolate anions.
• They effectively mask a carbonyl function while activating the compound towards nucleophilic substitution.

Enamine Reactions In Biochemistry: Aldolase

We saw that an aldol-like reaction could be used to rationalize the biochemical conversion of dihydroxyacetone phosphate (nucleophile) and glyceraldehyde 3-phosphate (electrophile) into fructose 1,6-diphosphate by the enzyme aldolase during carbohydrate biosynthesis.

The reverse reaction, used in the glycolytic pathway for carbohydrate metabolism, was formulated as a reverse aldol reaction.

In a postscript, we noted that nature avoided the use of a strong base to catalyse the reaction by involving an enzyme. Here, we see how this is achieved through an enamine.

• Enzymes are very sophisticated systems that apply sound chemical principles. The side chains of various amino acids are used to supply the necessary bases and acids to help catalyse the reaction.
• Thus, the enzyme aldolase binds the dihydroxyacetone phosphate substrate by reacting the ketone group with an amine, part of a lysine amino acid residue.

This forms an imine that becomes protonated under normal physiological conditions.

“Can preventive measures reduce risks of side reactions in enolate chemistry? FAQs provided”

A basic group removes a proton from the β-carbon of the iminium and forms the enamine.

• This enamine then reacts as a nucleophile towards the aldehyde group of glyceraldehyde 3-phosphate in a simple addition reaction, and the proton necessary for neutralizing the charge is obtained from an appropriately placed amino acid residue.
• Finally, the iminium ion loses a proton and hydrolysis releases the product from the enzyme.
• The reaction is exactly analogous to the chemical aldol reaction (also shown), but it utilizes an enamine as the nucleophile, and it can thus be achieved under typical enzymic conditions, i.e. around neutrality and at room temperature.
• There is one subtle difference though, in that the enzyme produces an enamine from a primary amine.
• We have indicated that enamine formation is a property of secondary amines, whereas primary amines react with aldehydes and ketones to form imine. Thus, a further property of the enzyme is to help stabilize the enamine tautomer relative to the imine.

The Mannich Reaction:

We saw that imines and iminium ions could act as carbonyl analogues and participate in nucleophilic addition reactions.

Iminium ion acting as carbonyl analogue for nucleophilic addition reaction

One simple example was the hydrolysis of imines back to carbonyl compounds via nucleophilic attack of water.

• The Mannich reaction is only a special case of nucleophilic addition to iminium ions, where the nucleophile is an enol system, the equivalent of an enolate anion.
• We have to say ‘the equivalent of an enolate anion’ because conditions that favour iminium cations are not going to allow the participation of negatively charged nucleophiles. The Mannich reaction is best discussed via an example.
• A mixture of dimethylamine, formaldehyde and acetone under mildly acidic conditions gives N, N dimethyl-4-aminobutan-2-one.
• This is a two-stage iminium ion acting as a carbonyl analogue for the nucleophilic addition reaction process, beginning with the formation of an iminium cation from the amine and the more reactive of the two carbonyl compounds, in this case, the aldehyde.
• This iminium cation then acts as the electrophile for the addition of the nucleophile acetone.
• Now it would be nice if we could use the enolate anion as the nucleophile, as in the other reactions we have looked at, but under the mild acidic conditions we cannot have an anion, and the nucleophile must be portrayed as the enol tautomer of acetone.

The addition is then unspectacular, and, after the loss of a proton from the carbonyl, we are left with the product.

“Differential applications of acid-catalyzed vs base-catalyzed enolate formations: Notes explained”

This is a fairly general reaction and requires an amine plus an aldehyde (usually, but not necessarily, formaldehyde) together with an enolizable ketone, which together a β-aminoketone via an iminium system.

The Mannich reaction is surprisingly important in biochemical processes, especially in the biosynthetic formation of alkaloids. We shall also see several examples in heterocyclic chemistry.

Mannich Reaction: The Synthesis Of Tropine

The Mannich reaction was used for the first synthesis of tropine, the parent alcohol of the tropane alkaloids.

• One of the natural tropane alkaloids used medicinally is hyoscyamine, sometimes in its racemic form atropine.
• Hyoscyamine is an anticholinergic, competing with acetylcholine for the muscarinic site of the parasympathetic nervous system, thus preventing the passage of nerve impulses.

The synthesis involved a reaction of methylamine, succindialdehyde and acetone under mild acid conditions, and although yields were poor, tropinone was formed. This could then be reduced with sodium borohydride to give tropine.

It is instructive to formulate a mechanism for this reaction; note that two Mannich reactions are involved. The scheme below shows the sequence of events, though not all the steps are shown.

Biosynthesis Of Tetrahydroisoquinolines

Mannich and Mannich-like reactions are widely used for the chemical synthesis of heterocycles, and in alkaloid biosynthesis in plants.

One such reaction important in nature is a biological equivalent of the Pictet–Spengler tetrahydroisoquinoline synthesis, and offers a slight twist, in that the enol nucleophile is a phenol.

“Steps to incorporate AI into analyzing enolate anion reactions: Questions and answers”

Thus, the reaction of 2-(3-hydroxyphenyl)ethylamine with an aldehyde generates initially an imine that will become protonated to an iminium ion.

• The resonance effect from the phenol group will increase electron density at the ortho and para positions in the aromatic ring.
• With the para resonance form, this is equivalent to having a nucleophile located adjacent to the iminium ion and allows the formation of a favourable six-membered ring via the Mannich-like reaction, the nucleophile attacking the C=N.
• Alternatively, we may consider the phenol to be simply a conjugated enol that is participating in a Mannich reaction.
• The final step is the loss of a proton, and this comes from the position para to the oxygen substituent because this allthe ows regeneration of the aromatic ring and phenol group.
• In a chemical reaction, a racemic product will be formed, but enzyme-controlled biochemical reactions normally produce just one enantiomer.

For a simple specific example, the tetrahydroisoquinoline alkaloid salsolinol is found in some plants, and it can also be detected in the urine of humans as a product of dopamine and acetaldehyde.

Acetaldehyde is typically formed after ingestion of alcohol. Since the urine product is racemic, it would appear that a chemical Pictet–Spengler synthesis is being observed here rather than an enzymic one.

• We saw that tetrahydroisoquinoline alkaloids with appropriate phenol substituents could be involved in radical coupling processes.
• The complex alkaloids tubocurarine and morphine are derived in nature from simpler tetrahydroisoquinoline alkaloids.

Enolate Anions From Carboxylic Acid Derivatives:

The α-hydrogens of carboxylic acid derivatives show enhanced acidity, as do those of aldehydes and ketones, and for the same reasons, that the carbonyl group stabilizes the conjugate base.

Thus, we can generate enolate anions from carboxylic acid derivatives and use these as nucleophiles in much the same way as we have already seen with enolate anions from aldehydes and ketones.

Unfortunately, there are some limitations in the carboxylic acid group of compounds, and the derivatives most often used to form enolate anions are esters. However, esters are less acidic than the corresponding aldehydes or ketones.

“Role of digital tools in improving precision with enolate reaction tracking: FAQs explained”

• Whereas the pK_a for the α-protons of aldehydes and ketones is in the region 17–19, for esters such as ethyl acetate it is about 25.
• This difference must relate to the presence of the second oxygen in the ester since resonance stabilization in the enolate anion should be the same.
• To explain this difference, the overlap of the non-carbonyl oxygen lone pair is invoked. Because this introduces charge separation, it is a form of resonance stabilization that can occur only in the neutral ester, not in the enolate anion.
• It thus stabilizes the neutral ester, reduces carbonyl character, and there is less tendency to lose a proton from the α-carbon to produce the enolate. Note that this is not a new concept; we used the same reasoning to explain why amides were not basic like amines.

The α-hydrogens in thioesters are more acidic than in oxygen esters, comparable in fact to those in the equivalent ketone. This can be rationalized by the larger size of sulfur.

• The sulfur lone pair is located in a 3p orbital, whereas oxygen lone pairs are in 2p orbitals; there is consequently less overlap of orbitals. There can be relatively little contribution from this type of resonance stabilization in thioesters.
• Accordingly, normal enolate anion stabilization is not affected.
  Note that acids and primary and secondary amides cannot be employed to generate enolate anions.
• With acids, the carboxylic acid group has pK_a of about 3–5, so the carboxylic proton will be lost much more easily than the α-hydrogens. In primary and secondary amides, the N–H (pK_a about 18) will be removed more readily than the α-hydrogens.
• Their acidity may be explained by of resonance stabilization of the anion. Tertiary amides might be used, however, since there are no other protons that are more acidic.

Coenzyme A And Acetyl-CoA

The increased acidity associated with thioesters is one of the reasons that biochemical reactions tend to involve thioesters rather than oxygen esters. The most important thiol encountered in such thioesters is coenzyme A.

This is a complex molecule, made up of an adenine nucleotide (ADP-3 -phosphate), pantothenic acid (vitamin B5), and cysteamine (2-mercaptoethylamine), but for mechanism purposes can be thought of as a simple thiol, HSCoA. Pre-eminent amongst the biochemical thioesters is the thioester of acetic acid, acetyl-coenzyme A (acetyl-CoA).

• This compound plays a key role in the biosynthesis and metabolism of fatty acids, as well as being a building block for the biosynthesis of a wide range of natural products, such as phenols and macrolide antibiotics.
• Acetyl-CoA is a good biochemical reagent for two main reasons. First, the α-protons are more acidic than those in ethyl acetate, comparable in fact to a ketone, and this increases the likelihood of generating an enolate anion.
• As explained above, this derives from sulfur being larger than oxygen, so that electron donation from the lone pair that would stabilize the neutral ester is considerably reduced. This means it is easier for acetyl-CoA to lose a proton and become a nucleophile.
• Second, acetyl-CoA is a better electrophile than ethyl acetate, in that it has a better leaving group; thiols (pK_a 10–11) are stronger acids than alcohols (pK_a 16). Acetyl-CoA is thus rather well suited to participate in aldol and Claisen reactions.

“How do advancements in technology enhance enolate anion research? Notes guide”

We shall see later that nature can employ yet another stratagem to increase the acidity of the α-protons in thioesters, by converting acetyl-CoA into malonyl-CoA.

• An enolate anion generated from a carboxylic acid derivative may be used in the same sorts of nucleophilic reactions that we have seen with aldehyde and ketone systems.
• It should be noted, however, that the base used to generate the enolate anion must be chosen carefully. If sodium hydroxide were used, then hydrolysis of the carboxylic derivative to the acid would compete with enolate anion formation.

However, the problem is avoided by using the same base, for example., ethoxide, as is present in the ester hydrogen exchange in the α-position function, so that the ester is not hydrolysed.

• Larger bases, for example., tert-butoxide, may also be valuable, in that they can remove α-protons but tend to be too large to add to the carbonyl group and form a tetrahedral intermediate.

Using ethoxide as a base, we can get hydrogen exchange by equilibration in a labelled solvent; but, because of the lower acidity of the α-protons compared with aldehydes and ketones, this process is less favourable.

Racemization Of Hyoscyamine To Atropine

The base-catalysed racemization of the alkaloid (−)-hyoscyamine to (±)-hyoscyamine (atropine) is an example of enolate anion participation.

• Alkaloids are normally extracted from plants by using bases, thus liberating the free alkaloid bases from salt combinations.
• (−)-Hyoscyamine is found in belladonna (Atropa belladonna) and stramonium (Datura stramonium) and is used medicinally as an anticholinergic.
• It competes with acetylcholine for the muscarinic site of the parasympathetic nervous system, thus preventing the passage of nerve impulses.
• However, with careless extraction using too much base the product isolated is atropine, which has only half the biological activity of (−)-hyoscyamine, since the enantiomer (+)-hyoscyamine is essentially inactive.
• The racemization process involves the removal of the α-hydrogen to form the enolate anion, which is favoured by both the enolate anion resonance plus additional conjugation with the aromatic ring.
• Since the α protons in esters are not especially acidic, the additional conjugation is an important contributor to enolate anion formation.

The proton may then be restored from either side of the planar system, giving a racemic product.

Note that the alcohol portion of hyoscyamine, namely tropine, also contains two chiral centres, but it is a symmetrical molecule and is optically inactive; it can be considered a mesostructure.

• Thus, the optical activity of hyoscyamine stems entirely from the chiral centre in the acid portion, tropic acid.
• Racemization of hyoscyamine may also be brought about by heating, and it is probable that, under these conditions, there is involvement of the enol form, rather than the enolate anion.
• The enol is also stabilized by the additional conjugation that the aromatic ring provides.
• The importance of this additional conjugation is emphasized by the observation that littorine, an alkaloid from Anthocercis littorea, is not readily racemized by either heat or base.
• The esterifying acid in littorine is phenyl-lactic, and the aromatic ring would not be in conjugation with the double bond of the enol or enolate anion.

Racemization depends entirely on the acidity associated with the isolated ester function.

“Early warning signs of outdated methods in enolate anion studies: Common questions”

Additionally, note that base hydrolysis of hyoscyamine gives (±)-tropic acid and tropine, with racemization preceding hydrolysis.

Base hydrolysis of littorine gives optically pure phenyl-lactic acid, so we deduce that hydrolysis is a more favourable process than racemization.

Epimerization Of L-Amino Acids To D-Amino Acids During Peptide Biosynthesis:

Many natural peptide structures, especially peptide antibiotics such as dactinomycin and ciclosporin, contain one or more D-amino acids along with L-amino acids in their structures.

• This contrasts with most proteins, where all the amino acid constituents are of the L-configuration.
• It is now known that the biosynthetic precursors of the D-amino acids are the corresponding L-analogues and that an enzymic epimerization process through an enol-type intermediate is involved.
• However, this does not appear to involve epimerization of the free L-amino acid followed by incorporation of the D-amino acid into the growing peptide chain. There are good reasons for this.
• Enolization in base does not occur, since ionization of the carboxylic acid group predominates. Enolization in acid is also prevented because the basic amino group would be protonated rather than the carbonyl.
• Epimerization appears to take place after the L-amino acid has been incorporated into the peptide and is thus occurring on an amide substrate.
• A simple example is the tripeptide precursor of the penicillin antibiotics, called ACV, an abbreviation for δ-(L-α-aminoacyl)-L-cysteinyl-D-valine.

The amino acid precursors for ACV are L-α-aminoadipic acid (an unusual amino acid derived by modification of L-lysine), L-cysteine, and L-valine (not D-valine).

During ACV formation, the stereochemistry of the valine component is changed.

“Differential applications of traditional vs cutting-edge enolate techniques: Notes explained”

• ACV is the linear tripeptide that leads to isopenicillin N, the first intermediate with the fused ring system found in the penicillins.
• Note, that we are using the D and L convention for amino acid stereochemistry rather than the fully systematic R and S.
• This is one occasion where the use of D and L is advantageous, in that the sulfur atom in L-cysteine means this compound has the R configuration, whereas the other L-amino acids have the S configuration.
• Evidence points to the most likely explanation for the epimerization of L- to D-amino acids being the involvement of an enol-like intermediate.
• The carbonyl form is an amide in this example; but, from the comments made earlier, such a transformation could not be achieved chemically in solution, since the N–H proton would be more acidic and would, therefore, be preferentially removed using a base.
• However, this is an enzymic reaction, thus allowing selectivity determined by the functional groups at the enzyme’s binding site.
• A basic residue is responsible for removing the α-hydrogen to generate the enol-like structure, and then a reverse process allows it to be delivered back, though from the opposite side of the planar structure.
• Since this is an enzymic reaction, the product is also produced in just one configuration, rather than as an equimolar mixture of the two configurations typical of a chemical process.

Metabolic Racemization Of Ibuprofen:

The analgesic ibuprofen is supplied for drug use in its racemic form. However, only the (S)-(+)-enantiomer is the biologically active species; the (R)-(−)-form is inactive.

(S)-(+)- isomer active

(R)-(-)- isomer inactive

some metabolic conversion of R → S via racemization

Nevertheless, the racemate provides considerably more analgesic activity than expected, since in the body there is some metabolic conversion of the inactive (R)-isomer into the active (S)-isomer.

• This can be rationalized readily through an enolization mechanism. As we have indicated under D-amino acid formation above, a simple base-catalysed chemical conversion is ruled out by preferential ionization of the carboxylic acid group, though this may have little bearing on a metabolic process.
• An enzyme-mediated process may involve both basic and acidic amino acid side-chains (see D-amino acid formation above), and we could consider the biological transformation as either base-catalysed or acid-catalysed, as shown below.
• Either would generate a planar enediol intermediate, and the reverse process would account for racemization.

The enediol also benefits from favourable conjugation with the aromatic ring.

Thus, when (±)-ibuprofen is supplied to the body, the active (+)-isomer can be utilized, with the remaining (−)-isomer then being racemized to provide more of the active isomer.

• Theoretically, almost all of the (−)-isomer could be converted as the (+)-isomer is gradually removed by the body.
• For example, since the racemate contains 50% inactive isomer, racemization of this provides another 25% active isomer, then further racemization of the remaining 25% inactive would leave 12.5%, and so on.
• In practice, transport and excretion differences do not allow total usage of all the material.
• Alkylation of the α-position of suitable carboxylic acid derivatives may be achieved using the enolate anion as a nucleophile in a typical S_N2 reaction.

In the example shown, the base used is LDA. This is a strong base that easily removes the weakly acidic α-proton, but because of its size it is a poor nucleophile and so does not affect the ester function.

Nucleophilic addition of an enolate anion from a carboxylic acid derivative onto an aldehyde or ketone is simply an aldol-type reaction.

A simple example is shown; again, LDA is used to generate the enolate anion, and addition to the ketone is carried out as a second step.

Addition-To Carbonyl Of Aldehydes/Ketones

Acylation Of Enolate Anions: The Claisen Reaction

In the aldol reaction, we saw an enolate anion acting as a nucleophile leading to an addition reaction with aldehydes and ketones.

However, if there is a leaving group present, then instead of the intermediate alkoxide anion abstracting a proton from the solvent giving the aldol product, the leaving group may be expelled with the regeneration of the carbonyl group.

Now this is the same situation we encountered when we compared the reactivity of aldehydes and ketones with that of carboxylic acid derivatives.

• The net result here is acylation of the nucleophile, and in the case of acylation of enolate anions, the reaction is termed a Claisen reaction.
• It is important not to consider aldol and Claisen reactions separately, but to appreciate that the initial addition is the same, and differences in products merely result from the absence or presence get acylation of enolate anion − Claisen reaction of a leaving group.
• This is just how we ratio analyzed the different reactions of aldehydes and ketones compared with carboxylic acid derivatives.
• The Claisen reaction (sometimes Claisen con densation) is formally the base-catalysed reaction between two molecules of ester to give a β-ketoester.

Thus, from two molecules of ethyl acetate, the product is ethyl acetoacetate.

To participate in this sort of reaction, the carboxylic acid derivative acting as a nucleophile must have α-hydrogens to generate an enolate anion.

• In practice, esters are most commonly employed in Claisen-type reactions.
• The Claisen reaction may be visualized as the initial formation of an enolate anion from one molecule of ester, followed by a nucleophilic attack of this species onto the carbonyl group of a second molecule.

The additional anion then loses ethoxide as the leaving group, with the reformation of the carbonyl group.

However, the reaction is not quite that simple, and to understand and utilize the Claisen reaction we have to consider pK_a values again.

• Loss of ethoxide from the addition anion is not favourable, since ethoxide is not a particularly good leaving group.
• This is because ethoxide is a strong base, the conjugate base of a weak acid. So far then, the reaction will be reversible.
• What makes it proceed further is the fact that ethoxide is a strong base, and able to ionize acids.
• The ethyl acetoacetate product is a 1,3-dicarbonyl compound and has relatively acidic protons on the methylene between the two carbonyls.
• NaOEt a pK_a of about 11 makes ethyl acetoacetate the most acidic compound in the sequence.
• Ionization of ethyl acetoacetate, generating a resonance-stabilized enolate anion, removes the product from the reaction mixture and shifts the equilibrium to the right.
• This also explains why, in the simple equation above, two reagents are shown on the arrows, first base and then acid.
• The acid is required in the workup to liberate the β-ketoester from the enolate anion.

The importance of ionization of the β-ketoester product can be illustrated by the attempted Claisen reaction between two molecules of ethyl 2-methyl propionate.

“Can computational chemistry revolutionize enolate anion mechanisms? FAQs provided”

Using sodium ethoxide as a base, the reaction does not proceed. This can be ascribed to the nature of the β-ketoester product, which contains no protons sandwiched between two carbonyls and, therefore, no protons that are sufficiently acidic for the final equilibrium-disturbing step.

• The reaction can be made to proceed, however, and the solution is simple: use a stronger base.
• In this way, the base used is sufficiently powerful to remove a less acidic proton from the product, removing it from the reaction mixture and disturbing the equilibrium.
• Any of the strong bases sodium hydride, sodium amide, or LDA might be employed. Although such bases will produce the enolate anion irreversibly, it is still necessary to ionize the product to overcome the effect of the poor leaving group.
• In the β-ketoester product, the pK_a of the only acidic proton is about 20, so this requires a strong base to achieve an equilibrium disturbing ionization.

Claisen And Aldol Reactions In Nature: Hmg-Coa And Mevalonic Acid:

In nature, the biologically active form of acetic acid is acetyl-coenzyme A (acetyl-CoA).

• Two molecules of acetyl-CoA may combine in a Claisen-type reaction to produce acetoacetyl-CoA, the biochemical equivalent of ethyl acetoacetate.
• This reaction features as the start of the sequence to mevalonic acid (MVA), the precursor in animals of the sterol cholesterol.

Later, we shall see another variant of this reaction that employs malonyl-CoA as the nucleophile.

Three molecules of acetyl-CoA are used to form MVA, a third molecule being incorporated via a stereospecific aldol addition to give the branched-chain ester β-hydroxy-β-methylglutaryl-CoA (HMG-CoA).

• This third acetyl CoA molecule appears to be bound to the enzyme via a thiol group, and this linkage is subsequently hydrolysed to form the free acid group of HMG-CoA.
• It should be noted that, on purely chemical grounds, acetoacetyl-CoA is the more acidic substrate in this reaction, and might be expected to act as the nucleophile rather than the third acetyl-CoA molecule.
• The enzyme thus achieves what is a less favourable reaction. There is a rather similar reaction in the Krebs cycle, where acetyl-CoA adds on to oxaloacetate via an aldol reaction, again with the enzymic reaction employing the less acidic substrate as the nucleophile.
• The subsequent conversion of HMG-CoA into MVA involves a two-step reduction of the thioester group a primary alcohol and provides an essentially irreversible and rate-limiting transformation.
• Drug-mediated inhibition of this enzyme, HMG-CoA reductase (HMGR), can be used to regulate the biosynthesis of the steroid cholesterol.

High levels of blood cholesterol are known to contribute to the incidence of coronary heart disease and heart attacks.

The statins, for example., pravastatin, are a group of HMGR inhibitors that possess functionalities that mimic the half-reduced substrate evaluate hemithioacetal.

• The affinity of these agents towards HMG-CoA reductase is some 104-fold more than the natural substrate, making them extremely effective inhibitors of the enzyme, and powerful drugs in coronary care.
• Should there be two ester functions in the same molecule, then it is possible to achieve an intramolecular Claisen reaction, particularly if this results in a favourable five- or six-membered ring.
• This reaction is usually given a separate name, a Dieckmann reaction, but should be thought of as merely an intramolecular extension of the Claisen reaction.
• As we have seen previously, intramolecular reactions are favoured over intermolecular reactions when the reaction is carried out at high dilution, conditions that minimize the interaction of two separate molecules.

A simple example involving the transformation of diethyl adi pate into a cyclic β-ketoester is shown.

We saw the possibilities for a mixed aldol reaction above, in which the reaction could become useful if we restricted the number of couplings possible.

• The same considerations can be mixed with the Claisen reaction applied to the Claisen reaction.

Thus, it is possible to have four products from two esters, depending on which ester became the nucleophile and which was acting as the electrophile.

Mixed Claisen Reaction

⇒ \(\mathrm{RCH}_2 \mathrm{CO}_2 \mathrm{Et}+\mathrm{R}^{\prime} \mathrm{CH}_2 \mathrm{CO}_2 \mathrm{Et} \longrightarrow 4 \text { products }\)

Nucleophile And Electrophile

⇒ \(\begin{aligned}\mathrm{RCH}_2 \mathrm{CO}_2 \mathrm{Et}+\mathrm{RCH}_2 \mathrm{CO}_2 \mathrm{Et}\)

⇒ \(\mathrm{RCH}_2 \mathrm{CO}_2 \mathrm{Et}+\mathrm{R}^{\prime} \mathrm{CH}_2 \mathrm{CO}_2 \mathrm{Et}\)

⇒ \(\mathrm{R}^{\prime} \mathrm{CH}_2 \mathrm{CO}_2 \mathrm{Et}+\mathrm{RCH}_2 \mathrm{CO}_2 \mathrm{Et}\)

⇒ \(\mathrm{R}^{\prime} \mathrm{CH}_2 \mathrm{CO}_2 \mathrm{Et}+\mathrm{R}^{\prime} \mathrm{CH}_2 \mathrm{CO}_2 \mathrm{Et}\)

To be synthetically useful, a mixed Claisen reaction (crossed Claisen reaction) needs one ester with no α-hydrogens so that it cannot become the nucleophile. Such reactants include oxalate, formate and benzoate esters. An example is shown below.

However, one might expect that the product from two molecules of ethyl propionate could also be formed.

• In practice, ethyl oxalate, because of its second electron-withdrawing carboxylate group, is a more reactive electrophile, so the major product is as shown.
• Formates are also more susceptible to nucleophilic attack; they lack the electron-donating inductive effect of an alkyl group and provide no steric hindrance.
• Benzoates are not as reactive as formates and oxalates, but the phenyl ring is electron-withdrawing and they also lack α-hydrogens.
• To minimize self-condensation of the nucleophilic reagent, it helps to add this gradually to the electrophilic species, so that the latter is always present in excess.
• Alternatively, and much more satisfactory from a synthetic point of view, it is possible to carry out a two-stage process, forming the enolate anion first.

We also saw this approach with a mixed aldol reaction. Thus, ethyl acetate could be converted into its enolate anion by reaction with the strong base LDA in a reaction that is essentially irreversible.

This nucleophile can then be treated with the electrophile. This could be a second ester, but there is an even better idea.

• If one is going to use a two-stage process, one can now employ an electrophile with a better-leaving group than ethoxide, and also get over the final ionization problem.

It would not be possible to use an acyl halide in a one-pot reaction because it would be quickly attacked by a base. An acyl halide could be used in a two-stage reaction, as shown here.

Ester–Ketone Condensations: Predicting The Product:

Let us use a systematic approach to consider what product is most likely to result when a mixture of an ester and a ketone, both capable of forming enolate anions, is treated with base.

For example, consider an ethyl acetate–acetone mixture treated with sodium hydride in ether solution.

Four reactions and products can be considered, involving either ketone or ester as the nucleophile, with ketone as the electrophile (aldol reactions) or ester as the electrophile (Claisen reactions).

• Both aldol and Claisen reactions are equilibria, and product formation is a result of disturbing these equilibria. This would be dehydration in Aldol reactions and ionization in Claisen reactions.
• Ionization would be the more immediate determinant. On that basis, it is obvious that the 1,3-dicarbonyl products from Claisen reactions are going to be more acidic than the aldol products, which possess just one carbonyl group.
• Now let us look at the ease of forming the enolate anion nucleophiles. Ketones are more acidic than esters.

Taken together, these factors mean the more favoured product is going to be the β-diketone (acetylacetone), formed from a ketone nucleophile by a Claisen reaction with an ester. This is the reaction observed.

The product is β-diketone acetylacetone

• ketone is more acidic than ester − ketone enolate favoured
• β-diketone is the most acidic of the four possible products

Aldol And Claisen Reactions In The Biosynthesis Of Phenols:

Many natural aromatic compounds are produced from the cyclization of poly-β-keto chains by enzymic aldol and Claisen reactions.

Examples include simple structures like orsellinic acid and phloracetophenone, and more complex highly modified structures of medicinal interest, such as mycophenolic acid, used as an immunosuppressant drug, the antifungal agent griseofulvin, and antibiotics of the tetracycline group, for example., tetracycline itself.

The more complex structures are inappropriate for consideration here, but the two compounds orsellinic acid and phloracetophenone exemplify nicely the enolate anion mechanisms we have been considering, as well as the concept of keto-enol tautomerism.

• A multifunctional enzyme complex is responsible for producing a poly-β-keto chain via a sequence of several Claisen reactions, together with subsequent reactions that achieve cyclization and aromatization.
• The C8 poly-β-keto chain shown is bonded to the enzyme through a thioester linkage.
• Because of the number of functional groups in this molecule, it is very reactive, and the enzyme plays a significant role in stabilizing it and preventing any unwanted chemical reactions.
• In addition, the enzyme binds the substrate in a folded conformation, allowing the atoms to be held in positions approximating to those occupied in the desired product.
• There are various possibilities for undergoing intramolecular aldol or Claisen reactions, dictated by the nature of the enzyme and how the substrate is folded on the enzyme surface.

Methylenes flanked by two carbonyl groups are the more acidic, allowing the formation of enolate anions.

• These may then participate in intramolecular reactions with ketone or ester carbonyl groups, with a natural tendency to form strain-free six-membered rings.
• To produce the compounds orsellinic acid and phloracetophenone, we can envisage the same substrate being folded in two different ways. Which folding occurs will be dependent on the organism and the enzyme it contains.
• With folding A, ionization of the α-methylene allows aldol addition onto the carbonyl six carbons distant along the chain, giving the tertiary alcohol.
• Dehydration occurs as in most chemical aldol reactions, giving the conjugated system, and enolization follows to attain the stability conferred by the aromatic ring.
• The thioester bond is then hydrolysed to produce orsellinic acid, at the same time releasing the product from the enzyme.

Alternatively, folding B allows a Claisen reaction to occur, which, although mechanistically analogous to the aldol reaction, is terminated by expulsion of the leaving group and direct release from the enzyme. Enolization of the cyclohexanone produces phloracetophenone.

“Asymptomatic vs symptomatic effects of ignoring new trends in enolate anion research: Answered”

Essentially the same sort of enolate anion aldol and Claisen reactions occur in the production of the more complex structures mycophenolic acid, griseofulvin, and tetracycline. However, the final structure is only obtained after a series of further modifications.

Reverse Claisen Reactions:

The driving force for the Claisen reaction is the formation of the enolate anion of the β-ketoester product. If this cannot form, the reverse reaction controls the equilibrium.

This means that a reverse Claisen reaction can occur if a β-ketoester is treated with base. This is most likely to occur if we attempt to hydrolyse the β-ketoester to give a β-ketoacid using aqueous base.

• Note that the alcoholic base used for the Claisen reaction does not affect the ester group, since the nucleophile is the same as the leaving group.

Aqueous base treatment of a β-ketoester will, however, result in both ester hydrolysis and a reverse Claisen reaction, and poses a problem if one only wants to hydrolyse the ester.

The reverse Claisen reaction is common, especially with cyclic β-ketoesters, such as one gets from the Dieckmann reaction.

• If one only wants to hydrolyse the ester, it thus becomes necessary to use the rather less effective acid-catalysed hydrolysis method.
• Cleavage of β-diketones, the products of a mixed Claisen reaction between an ester electrophile and a ketone nucleophile, behave similarly towards the base, and a reverse Claisen reaction ensues.

Again, this is prevalent with cyclic systems.

b-diketone

Nevertheless, as we shall, it is also possible to exploit the reverse Claisen reaction to achieve useful transformations.

Decarboxylation Reactions:

• Hydrolysis of the ester function of the β-ketoester Claisen product under acidic conditions yields a β-ketoacid, but these compounds are especially susceptible to loss of carbon dioxide, i.e. decar boxylation.
• Although β-ketoacids may be quite stable, decarboxylation occurs readily on mild heating and is ascribed to the formation of a six-membered hydrogen-bonded transition state.
• Decarboxylation is represented as a cyclic flow of electrons, leading to an enol product that rapidly reverts to the more favourable keto tautomer.

Reverse Claisen reaction in biochemistry: β-oxidation of fatty acids:

Perhaps the most important example of the reverse Claisen reaction in biochemistry is that involved in the β-oxidation of fatty acids, used to optimize energy release from storage fats, or fats ingested as food.

In common with most biochemical sequences, thioesters rather than oxygen esters are utilized.

β-oxidation Of Fatty Acids

The β-oxidation sequence involves three reactions, dehydrogenation, hydration, and then oxidation of a secondary alcohol to a ketone, thus generating a β-keto thioester from a thioester.

We shall study these reactions in more detail later. The β-keto thioester then suffers a reverse Claisen reaction, initiated by nucleophilic attack of the thiol coenzyme A.

The leaving group is the enolate anion of acetyl-CoA, and the reaction thus cleaves off a two-carbon fragment from the original fatty acyl-CoA.

• Since the nucleophile is coenzyme A, the other product is also a coenzyme A ester.
• The reaction generates a new fatty acyl-CoA, shorter by two carbons, which can re-enter the β-oxidation cycle.

Most natural fatty acids have an even number of carbons, so the process continues until the original fatty acid chain is cleaved completely into acetyl-CoA fragments.

We can now see how a number of the reactions recently studied fit together.

Decarboxylation Of Β-Ketoacids In Biochemistry: Isocitrate Dehydrogenase:

The enzyme isocitrate dehydrogenase is one of the enzymes of the Krebs or citric acid cycle, a major feature in carbohydrate metabolism.

• This enzyme has two functions, the major one being the dehydrogenation (oxidation) of the secondary alcohol group in isocitric acid to a ketone, forming oxalosuccinic acid.

This requires the cofactor NAD⁺. For convenience, we are showing non-ionized acids here, for example., isocitric acid, rather than anions, for example., isocitrate.

The second function, and the one pertinent to this section, is the decarboxylation of oxalosuccinic acid to 2-oxoglutaric acid.

• This is simply a biochemical example of the ready decarboxylation of a β-ketoacid, involving an intramolecular hydrogen-bonded system.

This reaction could occur chemically without an enzyme, but it is known that isocitric acid, the product of the dehydrogenation, is still bound to the enzyme isocitrate dehydrogenase when decarboxylation occurs.

It is appropriate here to look at the structure of oxaloacetic acid, a critical intermediate in the Krebs cycle, and to discover that it too is a β-ketoacid.

• In contrast to oxalosuccinic acid, it does not suffer decarboxylation in this enzyme-mediated cycle but is used as the electrophile for an aldol reaction with acetyl-CoA.
• Decarboxylation of 1,1-diacids (gem-diacids) is a similar reaction involving a hydrogen-bonded transition state.

1,1-Diacids may be stable entities, for example., malonic acid, but they are susceptible to decarboxy location upon heating; malonic acid decarboxylates at 150 ◦C.

gem-Diacids are typical products that might be obtained from synthetic sequences using esters of malonic acid, for example., diethyl malonate, a 1,3-dicarbonyl compound.

• Since the methylene group in diethyl malonate is sandwiched between two carbonyls, the enol keto protons are considerably more acidic than those in ethyl acetate.

The pK_a is of the order of 13, compared with about 24 for ethyl acetate, so it becomes much easier to form the enolate anion.

These decarboxylation reactions must not be viewed as unwanted processes that complicate reactions, but reactions that can be put to very good use. There were hints in the last paragraph.

• Two carbonyl groups in a 1,3 relationship increase the acidity of the α-protons between the two groups compared with protons adjacent to just one carbonyl group. It is easier to form enolate anions and then carry out nucleophilic reactions.

Therefore, since we may subsequently remove an ester function by hydrolysis and decarboxylation, we can view an ester group as a useful and temporary activating group. This is exemplified by the two sequences below.

Diethyl malonate can be converted into its enolate anion, which may then be used to participate in an S_N2 reaction with an alkyl halide.

• Ester hydrolysis and mild heating leads to production of an alkylated acetic acid.
• The same product might be obtained by starting with ethyl acetate, but this would be less efficient and possibly require a stronger base because the lower acidity of the α-protons makes the generation of the enolate anion less effective.

One of the ester groups in diethyl malonate can thus be regarded as a temporary activating group to increase the acidity of the α-protons.

The same viewpoint can taken for the ester function in a β-ketoester such as ethyl acetoacetate.

• Again, the acidity of the α-protons is increased because there are two carbonyl groups, and the generation of an enolate anion is facilitated.

Although mono- or di-alkylation of a ketone might be achieved through enolate anions, it would be easier to use the more acidic β-ketoester and follow this with hydrolysis and decarboxylation.

In general terms, a β-ketoester like ethyl acetoacetate can be considered as a pathway to substituted ketones, and diethyl malonate is a source of substituted acids.

“Success rate of interventions using modern enolate anion techniques: FAQ”

Note also that we can even make good use of the reverse Claisen reaction. Thus, alkylation of ethyl acetoacetate followed by suitable base treatment to effect a reverse Claisen reaction would also generate a substituted acid.

• An alcoholic base would be used for the enolate anion chemistry, whereas an aqueous base would initiate the reverse Claisen reaction and ester hydrolysis.
• In this sequence, we are using the acyl group as a temporary activating group.
• Back we saw two methods of synthesizing 2-methylcyclohexanone, i.e. by direct alkylation of the enolate anion derived from cyclohexanone and by using an enamine derivative as the nucleophilic species.
• The latter route had the advantage of not using a strong base to generate the nucleophile.
• We can now add a further approach for synthesis of the same compound, via a β-ketoester.

This also has the advantage of proceeding smoothly and, although it does use base to generate the enolate anion, the base required would be considerably less strong than for the ketone route.

On several occasions, we have noted that reactions involving enolate anions could be improved significantly by utilizing strongly basic reagents, such as sodium hydride, sodium amide, or LDA, and carrying out the reaction in two stages.

This stratagem removed the constrictions imposed by unfavourable equilibria, by preparing the enolate anion in an essentially irreversible reaction, then adding the electrophile that could have a more reactive leav ing group.

This is further exemplified by the synthesis of a β-diketone from a β-ketoester, as shown below, again exploiting a decarboxylation reaction.

Claisen Reactions In Nature Involving Malonyl-CoA:

We saw that nature employs a Claisen reaction between two molecules of acetyl-CoA to form acetoacetyl-CoA as the first step in the biosynthesis of mevalonic acid and subsequently cholesterol.

• This was a direct analogy for the Claisen reaction between two molecules of ethyl acetate.
• In fact, in nature, the formation of acetoacetyl-CoA by this particular reaction using the enolate anion from acetyl-CoA is pretty rare.
• We have just seen that diethyl malonate can be used instead of ethyl acetate as a nucleophile.
• The second ester group is effectively used to activate the system for producing a nucleophile and then is removed when the required reaction has been achieved.

Would it surprise you to know that, concerning this strategy, nature got there first?

“Is enolate-related risk reversible if addressed promptly? Answer provided”

The nucleophile in biological Claisen reactions that effectively adds on acetyl-CoA is almost always malonyl CoA.

• This is synthesized from acetyl-CoA by a reaction that utilizes a biotin-cyt–enzyme complex to incorporate carbon dioxide into the molecule.
• This has now flanked the α-protons with two carbonyl groups and increases their acidity. The enzymic Claisen reaction now proceeds, but, during the reaction, the added carboxyl is lost as carbon dioxide.
• Having done its job, it is immediately removed. In contrast to the chemical analogy, a carboxylated intermediate is not formed.
• Mechanistically, one could perhaps write a concerted decarboxylase tion–nucleophilic attack, as shown.
• An alternative rationalization is that decarboxylation of the malonyl ester is used by the enzyme to effectively generate the acetyl enolate anion without the requirement for a strong base.
• Malonyl-CoA is used as the nucleophilic species in the biosynthesis of fatty acids and a whole host of other natural products, including aromatic compounds.

Nucleophilic Addition To Conjugated Systems: Conjugate Addition And Michael Reactions:

We are familiar with the concept that the reactivity of a carbonyl group can be ascribed to the difference in electronegativity between carbon and oxygen, and the resultant unequal sharing of electrons.

The polarization δ+/δ− can be considered as a contribution from the resonance form having full charge separation.

Now let us go a step further, and conjugate the carbonyl group with a double bond.

If we polarize the carbonyl as before, then conjugation allows another resonance form to be written, in which the β-carbon now carries a positive charge.

Thus, as well as the carbonyl carbon being electrophilic, the β-carbon is also an electrophilic centre.

Conjugation of the carbonyl with a double bond transfers the electronic characteristics δ+/δ− of the carbonyl group along the carbon chain.

• The alkene would normally be nucleophilic and react with electrophiles. When conjugated with a carbonyl, it now becomes electrophilic and reacts with nucleophiles.
• A typical nucleophilic attack on the β-position is now shown, resulting in a transfer of negative charge onto the carbonyl. The product is a resonance form of an enolate anion with charge on the oxygen.

Abstraction of a proton from solvent will thus ultimately result in the production of the more favourable keto tautomer, and restoration of the carbonyl group.

It is possible to get either the typical addition reaction onto the carbonyl group, termed a 1,2- addition, or this form of conjugate addition, termed

1,4-addition, terminology that is understandable if the enol tautomer is considered as the product formed first.

“Treatment scope in academia vs industry for enolate anion applications: FAQs”

The less-reactive sodium borohydride may reduce unsaturated aldehydes by 1,2-addition, whereas unsaturated ketones tend to undergo conjugate addition. This allows selective reduction processes to be exploited.

For example, in the unsaturated ketone shown, we may achieve a reduction of the carbonyl using LAH, a reduction of the double bond via catalytic hydrogenation, or conjugate reduction using sodium borohydride.

The conjugate addition of a thiol, methanethiol, to the α, β-unsaturated aldehyde acrolein may be used in the synthesis of the amino acid methionine.

• Under basic conditions, the nucleophile will be the thiolate anion, and 1,4-addition leads to this aldehyde.
• Methionine may then be obtained via the Strecker synthesis, a sequence that involves imine formation, then nucleophilic attack of cyanide on this imine/carbonyl analogue.

The reaction is completed by acidic hydrolysis of the nitrile function to a carboxylic acid.

It should also be noted, as we have seen earlier, that other electron-withdrawing groups, for example., esters and nitriles, can achieve the same end as aldehydes or ketones.

“Cost of ignoring enolate anion principles vs benefits of systematic approaches: Q&A”

Conjugate addition can be observed when such groups are conjugated with a double bond.

Flavonoids: Conjugate addition And Heterocyclic Ring Formation:

Flavonoids are natural plant phenols containing a six-membered oxygen heterocyclic ring.

• Considerable quantities of flavonoids are consumed daily in our vegetable diet, and there is growing belief that they have beneficial properties, acting as antioxidants and giving protection against cardiovascular disease, and perhaps cancer.
• Their polyphenolic nature enables them to scavenge injurious free radicals, such as superoxide and hydroxyl radicals, which can cause serious cell damage.
• In particular, flavonoids in red wine and tea have been demonstrated to be effective antioxidants.

One of the simplest natural flavonoids is flavanone liquiritigenin, a material that contributes to the bright yellow colour of liquorice root.

Liquiritigenin may be synthesized readily, as shown, by a two-stage process starting from the phenolic ketone and aldehyde.

The conjugate addition of enolate anions onto α, β-unsaturated systems is an important synthetic reaction and is termed the Michael reaction, though this terminology may often be used in the broader context for the other conjugate additions considered above.

A typical example of the Michael reaction is the base-catalysed reaction of ethyl acetoacetate with the α, β-unsaturated ester ethyl acrylate.

“Why are enolate anion mechanisms often misunderstood in practice? Questions answered”

The nucleophile will be the enolate anion from ethyl acetoacetate, which attacks the β-carbon of the electrophile, generating an addition complex that then acquires a proton at the α-position with restoration of the carbonyl group.

• The product is an δ-ketoester with an ester side-chain that has a β-relationship to the keto group.
• This group may thus be removed by a sequence of acid-catalysed hydrolysis, followed by thermal decarboxylation. The final product in this sequence is therefore a δ-ketoacid, i.e. a 1,5-dicarbonyl compound.

Other examples of the Michael reaction are shown below. Note the relatively mild bases that are employed in these reactions where the nucleophiles are 1,3-dicarbonyl compounds.

Michael’s Reaction: The Robinson Annulation

A rather nice example of enolate anion chemistry involving the Michael reaction and the aldol reaction is provided by the Robinson annulation, a ring-forming sequence used in the synthesis of steroidal systems (Latin: annulus, ring).

In the partial synthesis shown, there are two reagents, the α, β-unsaturated ketone methyl vinyl ketone and the 1,3-diketone 2-methylcyclohexa-1,3-dione.

“Most common complications of poorly understood enolate anion concepts: FAQs”

These are reacted together in a basic solution. It can be deduced that the 1,3-diketone is more acidic than the monotone substrate, so will be ionized by the removal of a proton from the carbon between the two carbonyls to give the enolate anion as a nucleophile.

• This attacks the α, β-unsaturated ketone in a Michael reaction. Understandably, this large nucleophile prefers to attack the unhindered β-position rather than the more congested ketone carbonyl.
• The product from the Michael reaction will be a triketone. Now this substrate has four potential sites for proton removal, all flanked by a single ketone group, and thus all hydrogens are of similar acidity.
• The reaction that occurs is the intramolecular reaction that generates a strain-free six-membered ring system.
• This involves generating an enolate anion through the loss of a proton from the terminal methyl of the sidechain, followed by an aldol reaction involving the appropriate ring carbonyl as electrophile.
• Dehydration follows to generate the conjugated system, and it is this dehydration that disturbs the equilibrium. This annulation process was of considerable value in early approaches to steroid synthesis.
• The structural relationship of the bicyclic product obtained here to the male sex hormone testosterone is immediately apparent.
• Further, the non-conjugated carbonyl is now activating the adjacent carbon that subsequently features in building up the third ring system.

Michael Acceptors Can Be Carcinogens:

The Michael reaction involves conjugate addition of a nucleophile onto an α, β-unsaturated carbonyl compound, or similar system.

• Such reactions take place in nature as well, and some can be potentially dangerous to us.
• For example, the α, β-unsaturated ester ethyl acrylate is a cancer suspect agent.
• This electrophile can react with biological nucleophiles and, in so doing, bind irreversibly to the nucleophile, rendering it unable to carry out its normal functions.
• A particularly important enzyme that can act as a nucleophile is DNA polymerase, which is responsible for the synthesis of strands of DNA, especially as part of a DNA repair mechanism.

The nucleophilic centre is a thiol grouping, and this may react with ethyl acrylate as shown.

All is not doom and gloom, however, in that nature has provided in our bodies an alternative nucleophile to react with stray electrophiles like Michael acceptors.

This rather important compound is the tripeptide glutathione, a combination of glutamic acid, cysteine, and glycine.

It is the thiol group in glutathione that reacts with a carcinogenic α, β-unsaturated carbonyl compound in the same way as did the thiol group of DNA polymerase.

• As a result, the carcinogen becomes irreversibly bound to glutathione, and can no longer interact with other biochemicals.
• Furthermore, as a result of the amino acid functionalities, the inactivated carcinogen now has increased polarity compared with the original compound.
• This compound is likely to be water-soluble, and can thus be excreted from the body. We have also seen glutathione inactivating other electrophiles, such as toxic epoxides.
• Glutathione is also implicated in the removal of toxic metabolites from the analgesic paracetamol (USA: acetaminophen).

The oxidative metabolism of paracetamol produces an N-hydroxy derivative, and this readily loses water to generate a reactive and toxic quinone imine, which interacts with proteins to cause cell damage.

“Difference between aldol condensation and Michael addition: Notes explained”

Glutathione normally deactivates this reactive electrophile through a conjugate addition reaction.

• This time, we see conjugate addition onto an unsaturated imine rather than an unsaturated ketone. Rearomatization produces a non-toxic paracetamol–glutathione adduct.
• Unfortunately, if someone takes a large overdose of paracetamol, there may be insufficient glutathione available to detoxify all the metabolites.
• This can precipitate cell damage, particularly to the liver. Paracetamol is a safe analgesic unless taken in overdose.
• Multiple conjugate additions: anionic polymerization and superglue We have seen several reactions in which alkene derivatives can be polymerized.

Radical polymerization is the usual process of producing industrial polymers, but we also saw the implications of cationic polymerization.

• Here we see how an anionic process can lead to polymerization, and that this is an example of multiple conjugate additions.
• Alkene polymers such as poly(methyl methacrylate) and polyacrylonitrile are easily formed via anionic polymerization because the intermediate anions are resonance stabilized by the additional functional group, the ester or the nitrile.
• The process is initiated by a suitable anionic species, a nucleophile that can add to the monomer through conjugate addition in Michael fashion.
• The intermediate resonance-stabilized addition anion can then act as a nucleophile in further conjugate addition processes, eventually giving a polymer.

The process will terminate by proton abstraction, probably from solvent.

Methyl cyanoacrylate combines the anion-stabilizing features of both an ester group and a nitrile.

• Addition anions form very easily because of their enhanced resonance stability, and this polymerization process forms the basis of superglue.
• Traces of moisture on surfaces (including fingers) initiate anionic polymerization and the bonding together of almost any material.
• Superglue now has some value in surgery, bonding tissue without the need for stitches.

The post Nucleophilic Reactions Involving Enolate Anions appeared first on BDS Notes.

As described, water constitutes approximately 60% of the healthy adult human body. Body water is divided into two compartments (i.e., intracellular fluid and extracellular fluid [ECF]), which are in osmotic equilibrium.

“What is the regulation of body fluid osmolality?”

• Water intake into the body generally occurs orally, and the water ingested is absorbed into the ECF by the gastrointestinal tract via a mechanism similar to that which mediates water absorption by the proximal tubule.
• However, in clinical situations, intravenous infusion is an important route of water entry. Regardless of the route of entry (oral versus intravenous), water first enters the ECF and then equilibrates with the intracellular fluid. The
• kidneys are responsible for regulating water balance and under most conditions is the major route for the elimination of water from the body (Table 5-1). Other routes of water loss from the body include evaporation from the cells of the skin and respiratory passages.
• Collectively, water loss by these routes is termed insensible water loss because people are unaware of its occurrence. The production of sweat accounts for the loss of additional water.
• Water loss by this mechanism can increase dramatically in a hot environment, with exercise, or in the presence of fever. Finally, water can be lost from the gastrointestinal tract.
• Fecal water loss is normally small (~100 mL/day) but can increase dramatically with diarrhea (for example., 20 L/day in persons with cholera). Vomiting also can cause gastrointestinal water loss.

Normal Routes of Water Gain and Loss in Adults at Room Temperature (23°C):

“Understanding the role of water balance in human physiology”

Effect of Environmental Temperature and Exercise on Water Loss and Intake in Adults:

“How does the body regulate osmolality and water balance?”

Although water loss from sweating, defecation, and evaporation from the lungs and skin can vary depending on the environmental conditions or during pathologic conditions, the loss of water by these routes cannot be regulated.

• In contrast, the renal excretion of water is tightly regulated to maintain whole-body water balance. The maintenance of water balance requires that water intake and loss from the body are precisely matched. If intake exceeds losses, a positive water balance exists.
• Conversely, when intake is less than losses, a negative water balance exists.
  When water intake is low or water losses increase, the kidneys conserve water by producing a small volume of hyperosmotic urine concerning plasma.
• When water intake is high, a large volume of hypoosmotic urine is produced. In a healthy person, the urine osmolality (U_osm) can vary from approximately 50 to 1200 mOsm/kg H₂O, and the corresponding urine volume can vary from approximately 18 to 0.5 L/day.
• It is important to recognize that disorders of water balance are manifested by alterations in the body fluid osmolality, which usually are measured by changes in

“Importance of maintaining body fluid osmolality”

When plasma osmolality (P_osm) is reduced (i.e., hypos- molality), water moves from the extracellular fluid into cells, causing them to swell.

• Symptoms associated with hypo osmolality are related primarily to swelling of brain cells. For example, a rapid decrease in P_osm can alter neurologic function and thereby cause nausea, malaise, headache, confusion, lethargy, seizures, and coma.
• When P_osm is increased (i.e., hyperosmolality), water is lost from cells, causing them to shrink. The symptoms of an increase in P_osm also are primarily neurologic and include lethargy, weakness, seizures, coma, and even death.
• The symptoms associated with changes in body fluid osmolality vary depending on how quickly osmolality is changed. Rapid changes in osmolality (i.e., over hours) are less well tolerated than changes that occur more gradually (i.e., over days to weeks).
• Indeed, when alterations in body fluid osmolality have developed over an extended period, such persons may be entirely asymptomatic.
• This situation reflects the ability of cells over time either to eliminate intracellular osmoles, as occurs with hyperosmolality, or to generate new intracellular osmoles in response to hyperosmolality and thus minimize changes in cell volume of the neurons.
• This ability has important clinical implications when treating a patient with an abnormal plasma osmolality. For example, rapid correction of the osmolality of a person who has had long-standing hyperosmolality of the body fluids can lead to the development of osmotic demyelination syndrome.

“Common mechanisms of water balance regulation explained”

The syndrome can result in paralysis of multiple muscle groups and can be fatal.

“Role of antidiuretic hormone (ADH) in osmolality regulation”

Plasma osmolality (P_osm). Because the major determinant of plasma osmolality is Na⁺ (with its anions Cl- and HCO^–₃), these disorders also result in alterations in the plasma [Na⁺]

• When an abnormal plasma [Na⁺] is observed in an individual, it is tempting to suspect a problem in Na⁺ balance. However, the problem usually is related to water balance, not Na⁺ balance.
• As described, changes in Na⁺ balance result in alterations in the volume of the ECF, not its osmolality.
• Under steady-state conditions, the kidneys control water excretion independently of their ability to control the excretion of various other physiologically important substances such as Na⁺, K⁺, and urea
• Indeed, this ability is necessary for survival because it allows water balance to be achieved without upsetting the other homeostatic functions of the kidneys.
• This chapter discusses the mechanisms by which the kidneys maintain water balance by excreting either hypoosmotic (dilute) or hyperosmotic (concentrated) urine.

The control of arginine vasopressin (AVP) secretion and its important role in regulating the excretion of water by the kidneys are also explained.

Arginine Vasopressin

AVP, also known as antidiuretic hormone, acts on the kidneys to regulate the volume and osmolality of the urine.

• When plasma AVP levels are low, a large volume of urine is excreted (diuresis), diluting the urine.
• When plasma levels are high, a small volume of urine is excreted (antidiuresis), and the urine is concentrated.
• Illustrates the effect of AVP on the urine flow rate and osmolality. The excretion of

“How does ADH affect water reabsorption in the kidneys?”

As already noted, AVP does not appreciably alter the excretion of solute, which underscores the fact that AVP controls water excretion and maintains water balance without altering the excretion and homeostatic control of other substances.

• AVP is a small peptide that is nine amino acids in length. It is synthesized in neuroendocrine cells located within the supraoptic and paraventricular nuclei of the hypothalamus.
• The synthesized hormone is packaged in granules that are transported down the axon of the cell and stored in the nerve terminals located in the neurohypophysis (posterior pituitary).
• The anatomy of the hypothalamus and pituitary gland. The secretion of AVP by the posterior pituitary can be influenced by several factors.
• The two primary physiologic regulators of AVP secretion are the osmolality of the body fluids (osmotic) and the volume and pressure of the vascular system (hemodynamic). Other factors that can alter AVP secretion include nausea

Arginine Vasopressin At The Cellular Level: The gene for arginine vasopressin (AVP) is found on chromosome 20. It contains approximately 2000 base pairs with three exons and two introns.

• The gene codes for a 145 amino acid prohormone that consists of a signal peptide, the AVP molecule, neurophysin, and a glycopeptide (copeptin).
• As the cell processes the prohormone, the signal peptide is cleaved off in the rough endoplasmic reticulum. Once packaged in neurosecretory granules, the preprohormone is further cleaved into AVP, neurophysin, and copeptin molecules.
• The neurosecretory granules are then transported down the axon to the posterior pituitary and stored in the nerve endings until released.
• When the neurons are stimulated to secrete AVP, the action potential opens Ca++ channels in the nerve terminal, which raises the intracellular [Ca++] and causes exocytosis of the neurosecretory granules.
• All three peptides are secreted in this process. Neurophysin and copeptin do not have an identified physiologic function.
• (stimulates), atrial natriuretic peptide (inhibits), and angiotensin II (stimulates). Several drugs, prescription and nonprescription, also affect AVP secretion. For example, nicotine stimulates secretion, whereas ethanol inhibits secretion.

“Impact of aldosterone on osmolality and water balance”

Osmotic Control Of Arginine Vasopressin Secretion:

Changes in the osmolality of body fluids play the most important role in regulating AVP secretion; changes as minor as 1% are sufficient to alter it significantly.

• Although the neurons in the supraoptic and paraventricular nuclei respond to changes in body fluid osmolality by altering their secretion of AVP, it is clear that separate cells exist in the anterior hypothalamus that sense changes in body fluid osmolality and regulate the activity of the AVP-secreting neurons.
• These cells, termed osmoreceptors, appear to sense changes in body fluid osmolality by either shrinking or swelling.

“Biomechanics of osmosis in body fluids explained”

The osmoreceptors respond only to solutes in plasma that are effective osmoles. For example, urea is an ineffective osmole when the function of osmoreceptors is considered.

• Thus elevation of the plasma urea concentration alone has little or no effect on AVP secretion.
• When the effective osmolality of the plasma increases, the osmoreceptors send signals to the AVP synthesizing/secreting cells located in the supraoptic and paraventricular nuclei of the hypothalamus, and AVP synthesis and secretion are stimulated.
• Conversely, when the effective osmolality of the plasma is reduced, secretion is inhibited.
• Because AVP is rapidly degraded in the plasma, circulating levels can be reduced to zero within minutes after secretion is inhibited. As a result, the AVP system can respond rapidly to fluctuations in body fluid osmolality.

Illustrates the effect of changes in plasma osmolality on circulating AVP levels. The set point of the system is the plasma osmolality value at which AVP secretion begins to increase.

• Below this set point, virtually no AVP is released. Above this set point, the slope of the relationship is quite steep and accounts for the sensitivity of this system. The set point varies among individuals and is genetically determined.
• In healthy adults, it varies from 275 to 290 mOsm/kg H₂O (average ~280 to 285 mOsm/kg H₂O). As described later in this chapter, the set point shifts in response to changes in blood volume and pressure.
• It also shifts during pregnancy, with the osmolality of the mother’s body fluids decreasing during the third trimester.
• The reasons for the shift of the set point during pregnancy are not completely known but likely involve hormones (for example., relaxin) whose circulating levels are elevated at this stage of pregnancy.

Hemodynamic Control Of Arginine Vasopressin Secretion:

A decrease in blood volume or pressure also stimulates AVP secretion.

The receptors responsible for this response are located in both the low-pressure (left atrium and large pulmonary vessels) and the high-pressure (aortic arch and carotid sinus) sides of the circulatory system.

“Comparison of osmolality regulation in health vs disease”

Because the low-pressure receptors are located in the high-compliance side of the circulatory system (i.e., venous) and the majority of blood is in the venous side of the circulatory system, these low-pressure receptors can be viewed as responding to overall vascular volume.

• The high-pressure receptors respond to arterial pressure. Both groups of receptors are sensitive to a stretch of the wall of the structure in which they are located (for example., the cardiac atrial wall and the wall of the aortic arch) and are termed baroreceptors.
• Signals from these receptors are carried in afferent fibers of the vagus and glossopharyngeal nerves to the brainstem (solitary tract nucleus of the medulla oblongata), which is part of the center that regulates heart rate and blood pressure.
• Signals then are relayed from the brainstem to the AVP secretory cells of the supraoptic and paraventricular hypothalamic nuclei.
• The sensitivity of the baroreceptor system is less than that of the osmoreceptors, and a 5% to 10% decrease in blood volume or pressure is required before AVP secretion is stimulated.

This phenomenon is illustrated, in B. Several substances have been shown to alter the secretion of AVP through their effects on blood pressure.

• These substances include bradykinin and histamine, which lower pressure and thus stimulate AVP secretion, and norepinephrine, which increases blood pressure and inhibits AVP secretion.
• Alterations in blood volume and pressure also affect the response to changes in body fluid osmolality.
• With a decrease in blood volume or pressure, the set point is shifted to lower osmolality values and the slope of the relationship is steeper.
• In terms of survival of the individual, this means that when faced with circulatory collapse, the kidneys continue to conserve water, even though by doing so they reduce the osmolality of the body fluids.
• With an increase in blood volume or pressure, the opposite occurs. The set point is shifted to higher osmolality values, and the slope is decreased.

Arginine Vasopressin Actions On The Kidneys:

The primary action of AVP on the kidneys is to increase the permeability of the collecting duct to water.

• In addition, and notably, AVP increases the permeability of the medullary portion of the collecting duct to urea.
• Lastly, AVP stimulates sodium chloride (NaCl) reabsorption by the thick ascending limb of Henle’s loop, the distal tubule, and the cortical portion of the collecting duct.

Inadequate release of arginine vasopressin (AVP) from the posterior pituitary results in the excretion of large volumes of dilute urine (polyuria).

• To compensate for this loss of water, the individual must ingest large volumes of water (polydipsia) to maintain constant body fluid osmolality.
• If the individual is deprived of water, the body fluids become hyperosmotic. This condition is called central diabetes insipidus or pituitary diabetes insipidus.
• Central diabetes insipidus can be inherited, although this situation is rare. It occurs more commonly after head trauma and with brain neoplasms or infections.

“Mechanisms of water reabsorption in the nephron”

Persons with central diabetes insipidus have a urine-concentrating defect that can be corrected by the administration of exogenous AVP.

• The inherited (autosomal dominant) form of central diabetes insipidus is caused by a variety of mutations in the AVP gene.
• In patients with this form of central diabetes insipidus, mutations have been identified in all regions of the AVP gene (i.e., AVP, copeptin, and neurophysin).
• The most common mutation is found in the neurophysin portion of the gene. In each of these situations, defective trafficking of the peptide occurs, with abnormal accumulation in the endoplasmic reticulum.

It is believed that this abnormal accumulation in the endoplasmic reticulum results in the death of the AVP secretory cells of the supraoptic and paraventricular nuclei.

• The syndrome of inappropriate antidiuretic hormone (ADH) secretion (SIADH) is a common clinical problem characterized by plasma AVP levels that are elevated above what would be expected based on body fluid osmolality and blood volume and pressure—hence the term inappropriate ADH secretion.
• In addition, the collecting duct overexpresses water channels, thus augmenting the effect of AVP on the kidney. Persons with SIADH retain water, and their body fluids become progressively hypoosmotic.

In addition, their urine is more hyperosmotic than expected based on the low body fluid osmolality. SIADH can be caused by infections and neoplasms of the brain, drugs (for example., antitumor drugs), pulmonary diseases, and carcinoma of the lung.

• Many of these conditions stimulate AVP secretion by altering neural input to the AVP secretory cells. However, small cell carcinoma of the lung produces and secretes several peptides, including AVP.
• Recently, nonpeptide vasopressin receptor antagonists (for example., conivaptan and tolvaptan) have been developed that can be used to treat SIADH and other conditions in which AVP-dependent water retention by the kidneys occurs (for example., congestive heart failure and hepatic cirrhosis).

“How do the kidneys maintain osmolality balance?”

The actions of AVP on the water permeability of the collecting duct have been studied extensively. AVP binds to a receptor on the basolateral membrane of the cell. This receptor is termed the V2 receptor (i.e., vasopressin 2 receptor).

• Binding to this receptor, which is coupled to adenylyl cyclase through a stimulatory G protein (Gs), increases the intracellular levels of cyclic adenosine monophosphate (cAMP).
• The rise in intracellular cAMP activates protein kinase A, which ultimately increases the number of aquaporin (AQP)-2 water channels in the apical membrane of the cell and the synthesis of more AQP-2.
• With the removal of AVP, the number of AQP-2 water channels in the apical membrane is reduced, thereby rendering the membrane impermeable to water.
• Because the basolateral membrane is freely permeable to water because of the presence of AQP-3 and AQP-4 water channels, any water that enters the cell through apical membrane water channels exits across the basolateral membrane, resulting in net absorption of water from the tubule lumen.

“Role of the loop of Henle in concentrating urine”

AVP also increases the permeability of the terminal portion of the inner medullary collecting duct to urea. This increase in permeability results in an increase in urea reabsorption and an increase in the osmolality of the medullary interstitial fluid.

• The inner medullary collecting duct expresses two different urea transporters (UTs: UT-A1 and UT-A3. UT-A1 is found in the apical membrane and UT-A3 is found in the basolateral membrane.
• AVP, acting through adenylyl cyclase and the cAMP/protein kinase A cascade, increases the permeability of the apical membrane to urea.
• This increase in permeability is associated with phosphorylation of UT-A1 and UT-A3. Increasing the osmolality of the interstitial fluid of the renal medulla also increases the permeability of the collecting duct to urea.
• This effect is mediated by the phospholipase C pathway and involves protein kinase C phosphorylation. Thus this effect is separate from and additive to that of AVP.
• AVP also stimulates the reabsorption of NaCl by the thick ascending limb of Henle’s loop and by the distal tubule and cortical segment of the collecting duct.
• It is thought that stimulation of thick ascending limb NaCl transport, in particular, may help maintain the hyperosmotic medullary interstitium that is necessary for the absorption of water from the medullary portion of the collecting duct (discussed later in this chapter).

Arginine Vasopressin At The Cellular Level

The gene for the V₂ receptor is located on the X chromosome. It codes for a 371-amino-acid protein that is in the family of receptors that have seven membrane-spanning domains and are coupled to heterotrimeric G proteins.

• As binding of AVP to its receptor on the basolateral membrane activates adenylyl cyclase.
• The increase in intracellular cyclic adenosine monophosphate (cAMP) then activates protein kinase A, which results in phosphorylation of aquaporin (AQP)-2 water channels, which reduces the endocytic removal of AQP-2 from the apical membrane and also results in increased transcription of the AQP-2 gene through activation of a cAMP response element.
• AVP also increases the rate of insertion of vesicles containing AQP-2 into the apical membrane by facilitating their movement along microtubules driven by the molecular motor dynein.

Once near the apical membrane, proteins called SNAREs interact with vesicles containing AQP-2 and facilitate the fusion of these vesicles with the membrane.

• The net addition of AQP-2 to the apical membrane, resulting from reduced endocytosis and increased insertion, allows more water to enter the cell driven by the osmotic gradient (lumen osmolality < cell osmolality).
• The water then exits the cell across the basolateral membrane through AQP-3 and AQP-4 water channels, which are constitutively present in the basolateral membrane.
• When the V⁺ receptor is not occupied by AVP, clathrin-mediated endocytosis of AQP-2 is enhanced and the exocytic insertion of AQP-2 is reduced, which decreases the total number of AQP-2 channels in the apical membrane, rendering the apical membrane once again impermeable to water.

Recently, persons have been found who have activating (gain-of-function) mutations in the V₂ receptor gene. Thus the receptor is constitutively activated even in the absence of AVP.

• These persons have laboratory findings similar to those seen in the syndrome of inappropriate antidiuretic hormone secretion (SIADH), including reduced plasma osmolality, hyponatremia (reduced plasma [Na⁺]), and urine more concentrated than would be expected from the reduced body fluid osmolality.
• However, unlike persons with SIADH, in whom circulating levels of AVP are elevated and thus responsible for water retention by the kidneys, these persons have undetectable levels of AVP in their plasma. This new clinical entity has been termed “nephrogenic syndrome of inappropriate antidiuresis.”
• The collecting ducts of some persons do not respond normally to arginine vasopressin (AVP). These persons cannot maximally concentrate their urine and consequently have polyuria and polydipsia.

“Impact of glomerular filtration rate (GFR) on water balance”

This clinical entity is termed nephrogenic diabetes insipidus to distinguish it from central diabetes insipidus. Nephrogenic diabetes insipidus can result from several systemic disorders and, more rarely, occurs as a result of inherited disorders.

• Many of the acquired forms of nephrogenic diabetes insipidus are the result of decreased expression of aquaporin-2 (AQP-2) in the collecting duct.
• Decreased expression of AQP-2 has been documented in the urine-concentrating defects associated with hypokalemia, lithium ingestion (some degree of nephrogenic diabetes insipidus develops in 35% of persons who take lithium for bipolar disorder), ureteral obstruction, a low-protein diet, and hypercalcemia. The inherited forms of nephrogenic diabetes insipidus reflect mutations in the AVP receptor (V₂ receptor) gene or the AQP-2 gene.
• Approximately 90% of hereditary forms of nephrogenic diabetes insipidus are the result of mutations in the V₂ receptor gene, with the other 10% being the result of mutations in the AQP-2 gene.

Because the gene for the V₂ receptor is located on the X chromosome, these inherited forms are X-linked. Most of these mutations result in trapping of the receptor in the endoplasmic reticulum of the cell; only a few cases result in the surface expression of a V₂ receptor that does not bind AVP.

• The gene coding for AQP-2 is located on chromosome 12 and is inherited as both an autosomal recessive and an autosomal dominant defect. As noted in Chapters 1 and 4, aquaporins exist as homotetramers.
• This homotetramer formation explains the difference between the two forms of nephrogenic diabetes insipidus. In the recessive form, heterozygotes produce both normal AQP-2 and defective AQP-2 molecules.
• The defective AQP-2 monomer is not delivered to the plasma membrane, and thus the homotetramers that do form contain only normal AQP-2 molecules.
• Accordingly, mutations in both alleles would be required to produce nephrogenic diabetes insipidus.
• In the autosomal dominant form, the defective monomers can form tetramers with normal monomers, as well as defective monomers. However, these tetramers cannot be delivered to the plasma membrane.

“How does the renal system respond to dehydration?”

Thirst

In addition to affecting the secretion of AVP, changes in plasma osmolality and blood volume or pressure lead to alterations in the perception of thirst.

• When body fluid osmolality is increased or the blood volume or pressure is reduced, a person perceives thirst. Of these stimuli, hypertonicity is the more potent.
• An increase in plasma osmolality of only 2% to 3% produces a strong desire to drink, whereas decreases in blood volume and pressure in the range of 10% to 15% are required to produce the same response.
• As already discussed, people have a genetically determined threshold for AVP secretion (i.e., a body fluid osmolality above which AVP secretion increases).
• Similarly, people have a genetically determined threshold for triggering the sensation of thirst. However, the thirst threshold is higher than the threshold for AVP secretion.
• On average, the threshold for AVP secretion is approximately 285 mOsm/kg H₂O, whereas the thirst threshold is approximately 295 mOsm/kg H₂O. Because of this difference, thirst is stimulated at a body fluid osmolality at which AVP secretion is already stimulated.
• The neural centers involved in regulating water intake (the thirst center) are located in the same region of the hypothalamus involved with regulating AVP secretion.
• However, it is not certain if the same cells serve both functions. Indeed, the thirst response, like the regulation of AVP secretion, occurs only in response to effective osmoles (for example., NaCl).
• Even less is known about the pathways involved in the thirst response to decreased blood volume or pressure, but it is believed that the pathways are the same as those involved in the volume- and pressure-related regulation of AVP secretion.
• Angiotensin II, acting on cells of the thirst center (subfornical organ), also evokes the sensation of thirst. Because angiotensin II levels are increased when blood volume and pressure are reduced, this effect of angiotensin II contributes to the homeostatic response that restores and maintains the body fluids at their normal volumes.
• The sensation of thirst is satisfied by the act of drinking even before sufficient water is absorbed from the gastrointestinal tract to correct the plasma osmolality.
• Oropharyngeal and upper gastrointestinal receptors appear to be involved in this response. However, relief of the thirst sensation by these receptors is short-lived, and thirst is completely satisfied only when the plasma osmolality or blood volume or pressure is corrected.
• It should be apparent that the AVP and thirst systems work in concert to maintain water balance. An increase in plasma osmolality evokes drinking and, through AVP action on the kidneys, the conservation of water.
• Conversely, when the plasma osmolality is decreased, thirst is suppressed and, in the absence of AVP, renal water excretion is enhanced.
• However, most of the time fluid intake is dictated by cultural factors and social situations, which is especially the case when thirst is not stimulated. In this situation, maintaining a normal body fluid osmolality relies solely on the ability of the kidneys to excrete water.
• How the kidney accomplishes this task is discussed in detail in the following sections of this chapter.

“Disorders of osmolality and water balance explained”

Renal Mechanisms For Dilution And Concentration Of The Urine

Under normal circumstances, the excretion of water is regulated separately from the excretion of solutes.

• For this separate regulation to occur, the kidneys must be able to excrete urine that is either hypoosmotic or hyperosmotic concerning the body fluids.
• This ability to excrete urine of varying osmolality in turn requires that solute be separated from water at some point along the nephron. As discussed, the reabsorption of solute in the proximal tubule results in the reabsorption of a proportional amount of water.
• Hence solute and water are not separated in this portion of the nephron. Moreover, this proportionality between proximal tubule water and solute reabsorption occurs regardless of whether the kidneys excrete dilute or concentrated urine.
• Thus the proximal tubule reabsorbs a large portion of the filtered solute and water but does not produce dilute or concentrated tubular fluid. The loop of Henle, in particular the thick ascending limb, is the major site where solute and water are separated.
• Thus the excretion of both dilute and concentrated urine requires normal function of the loop of Henle.

With adequate access to water, the thirst mechanism can prevent the development of hyperosmolality. Indeed, it is this mechanism that is responsible for the polydipsia seen in response to the polyuria of both central and nephrogenic diabetes insipidus.

“Role of osmolality regulation in diagnosing diseases”

• Water intake also is influenced by social and cultural factors. Thus persons ingest water even in the absence of the thirst sensation. Normally the kidneys can excrete this excess water because they can excrete up to 18 L/day of urine.
• However, in some instances, the volume of water ingested exceeds the kidneys’ capacity to excrete water, especially over short periods. When this situation occurs, the body fluids become hypoosmotic.
• An example of how water intake can exceed the capacity of the kidneys to excrete water is found in long-distance runners.
• A study of participants in the Boston Marathon found that hyponatremia developed in 13% of the runners during the race.

This finding reflected the practice of some runners of ingesting water, or other hypotonic drinks, during the race to remain “well hydrated.”

• In addition, water is produced from the metabolism of glycogen and triglycerides used as fuels by the exercising muscle.
• Because throughout the race they ingested and generated more water through metabolism than their kidneys were able to excrete, hyponatremia developed. In some racers, the hyponatremia was severe enough to elicit the neurologic symptoms described previously.
• Throughout the popular media, one can find articles urging us to drink eight 8-oz glasses of water a day (the 8 × 8 recommendation). Drinking this volume of water is said to provide innumerable health benefits.
• As a result, it seems that everyone now has a water bottle as his or her constant companion.
• Although ingesting this volume of water over a day (approximately 2 L) does not harm most persons, no scientific evidence exists to support the beneficial health claims ascribed to the 8 x 8 recommendation. t Indeed, most persons get adequate amounts of water through the foods they ingest and the fluids taken with those meals.
• The maximum amount of water that can be excreted by the kidneys depends on the amount of solute excreted, which in turn depends on food intake.

“How does dehydration affect osmolality?”

For example, with maximally dilute urine (urine osmolality [U_osm] = 50 mOsm/kg H₂O), the maximum urine output of 18 L/day is achieved only if the solute excretion rate is 900 mmol/day.

U_osm = Solute excretion/Volume excreted 50 mOsm/kg H₂O = 900 mmol/18 L

• If solute excretion is reduced, as commonly occurs in elderly people with reduced food intake, the maximum urine output decreases.
• For example, if solute excretion is only 400 mmol/day, a maximum urine output (at U_osm = 50 mOsm/kg H₂O) of only 8 L/day can be achieved. Thus persons with reduced food intake have a reduced capacity to excrete water.

The excretion of hypoosmotic urine is relatively easy to understand. The nephron simply must reabsorb solute from the tubular fluid and not allow water reabsorption to occur as well.

• The reabsorption of solute without concomitant water reabsorption occurs in some portions of the descending limb and along the entire ascending limb of Henle’s loop.
• Under appropriate conditions (i.e., in the absence of AVP), the distal tubule and collecting duct also dilute the tubular fluid.
• The excretion of hyperosmotic urine is more complex and thus more difficult to understand. This process in essence involves removing water from the tubular fluid without solute.

“Complications of overhydration and hyponatremia”

Because water movement is passive, driven by an osmotic gradient, the kidney must generate a hyperosmotic compartment that then reabsorbs water osmotically from the tubular fluid.

• The compartment in the kidney where this reabsorption occurs is the interstitial space of the renal medulla.
• It has long been recognized that Henle’s loop is associated with the kidneys’ ability to excrete hyperosmotic urine.
• Indeed, only birds and mammals can excrete hyperosmotic urine, and among vertebrates, only the avian and mammalian kidneys have loops of Henle.

Moreover, some animals, such as desert rodents, have extremely long loops of Henle and excrete urine with an osmolality that can exceed 5000 mOsm/kg H₂O.

• This extraordinary ability to concentrate the urine allows the animals to survive without the need to drink water because they obtain sufficient water in the food (for example., seeds) that they ingest.
• For more than 50 years our understanding of how the loop of Henle can generate a hyperosmotic environment within the renal medulla was focused on the process of countercurrent multiplication.
• By this process, solute (principally NaCl) is reabsorbed without water from the ascending limb of Henle’s loop into the surrounding medullary interstitium.

This reabsorption decreases the osmolality in the tubular fluid and raises the osmolality of the interstitium at this point.

• The increased osmolality of the interstitium then causes water to be reabsorbed from the descending limb of Henle’s loop, thus increasing the tubular fluid osmolality in this segment.
• Thus at any point along the loop of Henle, the fluid in the ascending limb has an osmolality less than fluid in the adjacent descending limb. This osmotic difference was termed the single effect.
• Because of the countercurrent flow of tubular fluid in the descending (fluid flowing into the medulla) and ascending (fluid flowing out of the medulla) limbs, this single effect could be multiplied.

“Techniques for monitoring osmolality levels”

The multiplication of this single effect results in an osmotic gradient within the medullary interstitium, where the tip of the papilla has an osmolality of 1200 mOsm/kg H₂O, compared with 300 mOsm/kg H₂O at the corticomedullary junction.

• Although it is simple in concept, it is now clear that countercurrent multiplication cannot fully explain the process by which the loop of Henle generates a hyperosmotic medullary interstitium.

Given our evolving understanding, specifically of the urine-concentrating mechanism, what follows is a simplified explanation that highlights several key concepts:

1. Urine is concentrated by the AVP-dependent reabsorption of water from the collecting duct.
2. Reabsorption of NaCl from the ascending limb of Henle’s loop generates a high [NaCl] in the medullary interstitium (up to 600 mmol/L at the tip of the papilla), which then drives water reabsorption from the collecting duct.
3. Urea accumulates in the medullary interstitium (up to 600 mmol/L), which allows the kidneys to excrete urine with the same high urea concentration. This phenomenon allows large amounts of urea to be excreted with relatively little water.

Summarizes the essential features of the mechanisms whereby the kidneys excrete either dilute or concentrated urine.

First, how the kidneys excrete dilute urine (water diuresis) when AVP levels are low or zero is considered. The following numbers refer to those encircled.

1. Fluid entering the descending thin limb of the loop of Henle from the proximal tubule is isosmotic concerning plasma.

• This state reflects the essentially isosmotic nature of the solute and water reabsorption in the proximal tubule. (Note: Water is reabsorbed from the segments of the proximal tubule via AQP-1).

2. Depending on the nephron type (i.e., short-looped nephrons versus long-looped nephrons, some water will be reabsorbed by the thin descending limb.

• Importantly, this water reabsorption is limited to the outer medulla and the outermost portion of the inner medulla.
• By con-fining water reabsorption to these outer portions of the medulla, less water is added to the deepest part of the inner medullary interstitial space, thus preserving the hyperosmolality of this region of the medulla.

3. In the inner medulla, the terminal portion of the descending thin limb and all of the thin ascending limb are impermeable to water.

• These same nephron segments express the Cl^– transporter ClC-K1, which mediates Cl^– reabsorption with Na⁺ following passively via the paracellular pathway.
• This passive reabsorption of NaCl without concomitant water reabsorption begins the process of diluting the tubular fluid.

“Pathophysiology of osmolality imbalances explained”

“Emerging research on osmolality regulation mechanisms”

4. The thick ascending limb of the loop of Henle also is impermeable to water and actively reabsorbs NaCl from the tubular fluid thereby diluting it further.

• Dilution occurs to such a degree that this segment often is referred to as the diluting segment of the kidney.
• Fluid leaving the thick ascending limb is hypoosmotic concerning plasma (approximately 150 mOsm/kg H₂O).

5. The distal tubule and cortical portion of the collecting duct actively reabsorb NaCl. In the absence of AVP, these segments are not permeable to water (i.e., AQP-2 is not present in the apical membrane of the cells).

• Thus when AVP is absent or present at low levels (i.e., decreased plasma osmolality), the osmolality of tubule fluid in these segments is reduced further because NaCl is reabsorbed without water.
• Under this condition, fluid leaving the cortical portion of the collecting duct is hypo-motif concerning plasma (approximately 50-100 mOsm/kg H₂O).

6. The medullary collecting duct actively reabsorbs NaCl. Even in the absence of AVP, this segment is slightly permeable to water, and some water is reabsorbed.

7. The urine has an osmolality as low as approximately 50 mOsm/kg H₂O and contains low concentrations of NaCl. The volume of urine excreted can be as much as 18 L/day or approximately 10% of the glomerular filtration rate (GFR).

• Next, how the kidneys excrete concentrated urine (antidiuresis) when plasma osmolality and plasma AVP levels are high is considered. The following numbers refer to those encircled.
• These steps are similar to those for the production of dilute urine. An important point in understanding how concentrated urine is produced is to recognize that while reabsorption of NaCl by the ascending thin and thick limbs of the loop of Henle dilutes the tubular fluid, the reabsorbed NaCl accumulates in the medullary interstitium and raises the osmolality of this compartment.
• The accumulation of NaCl in the medullary interstitium is crucial for the production of urine hyperosmotic to plasma because it provides the osmotic driving force for water reabsorption by the medullary collecting duct.
• As already noted, AVP stimulates NaCl reabsorption by the thick ascending limb of Henle’s loop.
• This action is thought to maintain the medullary interstitial gradient at a time when water is being added to this compartment from the medullary collecting duct, which would tend to dissipate the gradient.

“Case studies on osmolality and water balance outcomes”

Because of NaCl reabsorption by the ascending limb of the loop of Henle, the fluid reaching the collecting duct is hypoosmotic concerning the surrounding interstitial fluid.

Thus an osmotic gradient is established across the collecting duct.

• In the presence of AVP, which increases the water permeability of the last half of the distal tubule and the collecting duct by increasing the number of AQP-2 water channels in the luminal membrane of the cells, water diffuses out of the tubule lumen, and the tubule fluid osmolality increases.
• This diffusion of water out of the lumen of the collecting duct begins the process of urine concentration.

The maximum osmolality that the fluid in the distal tubule and cortical portion of the collecting duct can attain is approximately 290 mOsm/kg H₂O (i.e., the same as plasma), which is the osmolality of the interstitial fluid and plasma within the cortex of the kidney.

• As the tubular fluid descends deeper into the medulla, water continues to be reabsorbed from the collecting duct, increasing the tubular fluid osmolality to 1200 mOsm/kg H₂O at the tip of the papilla.
• The urine produced when AVP levels are elevated has an osmolality of 1200 mOsm/kg H₂O and contains high concentrations of urea and other non-absorbed solutes. The urine volume under this condition can be as low as 0.5 L/day.
• Under most conditions, a relatively constant volume of tubular fluid is delivered to the AVP-sensitive portions of the nephron (late distal tubule and collecting duct).

Plasma AVP levels then determine the amount of water that is reabsorbed by these segments. When AVP levels are low, a relatively small volume of water is reabsorbed by these segments, and a large volume of hypoosmotic urine is excreted (up to 10% of the filtered water).

• When AVP levels are high, a large volume of water is reabsorbed by these same segments, and a small volume of hyperosmotic urine is excreted (<1% of filtered water).
• During antidiuresis, most of the water is reabsorbed in the distal tubule and cortical and outer medullary portions of the collecting duct.
• Thus a relatively small volume of fluid reaches the inner medullary collecting duct, where it is then reabsorbed.

This distribution of water reabsorption along the length of the collecting duct (i.e., cortex > outer medulla > inner medulla) allows for the maintenance of a hyperosmotic interstitial environment in the inner medulla by minimizing the amount of water entering this compartment.

Renal Mechanisms At The Cellular Level:

Water movement across the various segments of the nephron occurs through water channels.

• The proximal tubule and portions of some thin descending limbs of Henle’s loop are highly permeable to water, and these segments express high levels of AQP-1 in both the apical and basolateral membranes.
• The vasa recta also are highly permeable to water and express AQP-1. AQP-7 and AQP-8 also are expressed in the proximal tubule.
• As already discussed, AQP-2 is responsible for arginine vasopressin (AVP)-regulated water movement across the apical membrane of principal cells of the late distal tubule and collecting duct, and AQP-3 and AQP-4 are responsible for water movement across the basolateral membrane.
• Mice lacking the AQP-1 gene have been created. These mice have a urine-concentrating defect with increased urine output. Several persons have been found who also lack the normal AQP-1 gene.
• Interestingly, these persons do not have polyuria. However, when challenged by water deprivation, they can concentrate their urine to only approximately half of what is seen in a healthy person.

“Global prevalence of osmolality disorders”

Role Of Urea

As noted, a hyperosmotic renal medullary interstitium is critically important in concentrating the urine and provides the driving force for the reabsorption of water from the collecting duct.

• The principal solutes within the renal medullary interstitium are NaCl and urea, but the concentration of these solutes is not uniform throughout the medulla (i.e., a gradient exists from cortex to papilla).
• Other solutes also accumulate in the medulla (for example., ammonium [NH⁺₄] and K⁺), but the most abundant solutes are NaCl and urea. For simplicity, this discussion assumes that NaCl and urea are the only solutes.
• At the junction of the medulla with the cortex, the interstitial fluid has an osmolality of approximately 300 mOsm/kg H₂O, with virtually all osmoles attributable to NaCl. The concentrations of both NaCl and urea increase progressively with increasing depth into the medulla.

When maximally concentrated urine is excreted, the medullary interstitial fluid osmolality is approximately 1200 mOsm/kg H₂O at the papilla.

• Of this value, approximately 600 mOsm/ kg H₂O is attributed to NaCl, and 600 mOsm/kg H₂O is attributed to urea. As described later, NaCl is an effective osmole in the inner medulla and thus is responsible for driving water reabsorption from the medullary collecting duct. T
• The high urea concentration of the medullary interstitial fluid allows this solute to be excreted at a high concentration (600 mmol/L) in a small volume of urine, thus limiting the amount of water that otherwise would be needed to excrete the daily load of bread
• The medullary gradient for NaCl results from the accumulation of NaCl reabsorbed by the segments of Henle’s loop (see the previous discussion).
• Urea accumulation within the medullary interstitium is more complex and occurs most effectively when hyperosmotic urine is excreted (i.e., antidiuresis). When dilute urine is produced, especially over extended periods, the osmolality of the medullary interstitium declines.
• This reduced osmolality is almost entirely caused by a decrease in the concentration of urea. This decrease reflects washout by the vasa recta (discussed in a later section of this chapter) and diffusion of urea from the interstitium into the tubular fluid within the medullary portion of the collecting duct.

(Note: The cortical and outer medullary portions of the collecting have a low permeability to urea, whereas the inner medullary portion has a relatively high permeability because of the presence.)

• Urea is not synthesized in the kidney but is generated by the liver as a product of protein metabolism. It enters the tubular fluid via glomerular filtration.
• Approximately half of this filtered urea is reabsorbed by the proximal tubule. During antidiuresis, water reabsorption by the cortical and outer medullary portions of the collecting duct leads to an increase in the urea concentration of the tubular fluid.
• When this fluid reaches the portion of the inner medullary collecting duct that expresses UT-A1 and UT-A3, urea is reabsorbed.
• The reabsorption of urea is further enhanced by the high levels of AVP, which increase the expression of the UTs. Some of this reabsorbed urea is secreted into thin descending limbs of Henle’s loops via UT-A2, and some enter vasa recta via UT-B.

The urea that is secreted into the descending thin limbs of Henle’s loops is then trapped in the nephron until it again reaches the medullary collecting duct, where it can reenter the medullary interstitium.

• Thus urea recycles from the interstitium to the nephron and back into the interstitium. This process of recycling facilitates the accumulation of urea in the medullary interstitium, where it can attain a concentration at the tip of the papilla of 600 mmol/L.
• It is the high concentration of urea in the interstitial fluid that prevents the diffusion of urea out of the lumen of the inner medullary collecting duct into the interstitium, thereby facilitating urea excretion in the urine.
• As described, the hyperosmotic medulla is essential for concentrating the tubular fluid within the collecting duct.
• Because water reabsorption from the collecting duct is driven by the osmotic gradient established in the medullary interstitium, urine can never be more concentrated than that of the interstitial fluid in the papilla.
• Thus any condition that reduces the medullary interstitial osmolality impairs the ability of the kidneys to maximally concentrate the urine.

However, because the inner medullary collecting duct is highly permeable to urea, especially in the presence of AVP, urea cannot drive water reabsorption across this nephron segment (i.e., urea is an ineffective osmole).

• Instead, the urea in the tubular fluid and medullary interstitium equilibrate and a small volume of urine with a high concentration of urea is excreted.
• It is the medullary interstitial NaCl concentration that is responsible for reabsorbing water from the inner medullary collecting duct and thereby concentrating the nonurea solutes (for example., NH⁺ salts, K⁺ salts, and creatinine) in the urine.

At The Cellular Level

The expression of the urea transporter (UT­A1) in the inner medullary collecting duct is increased by arginine vasopressin (AVP) via a cyclic adenosine monophosphate–mediated mechanism. UT­A1 expression also is increased by hyperosmolality.

• This effect is mediated by changes in intracellular Ca++ and protein kinase C activity. Thus the effects of AVP and hyperosmolality are separate and additive.
• The expression of UT­A3 and UT­A2 also is increased by AVP. Knockout mice have been created that lack the UT­A1/UT­A3 collecting duct transporters, the UT­A2 thin descending limb transporter, or the UT­B vasa recta transporter.
• All of these animals have some degree of impairment of urinary concentration. Humans with genetic loss of UT­B exhibit a similar urinary concentrating defect as the knockout mouse model.

Vasa Recta Function

The vasa recta, the capillary networks that supply blood to the medulla, are highly permeable to solute and water. As with the loop of Henle, the vasa recta form a parallel set of hairpin loops within the medulla.

• Not only do the vasa recta bring nutrients and oxygen to the medullary nephron segments but, more importantly, they also remove the water and solute that is continuously added to the medullary interstitium by these nephron segments.
• The ability of the vasa recta to maintain the medullary interstitial gradient is flow-dependent. A substantial increase in vasa recta blood flow dissipates the medullary gradient. Alternatively, decreased blood flow reduces oxygen delivery to the nephron segments within the medulla.
• Because the transport of salt and other solutes requires oxygen and adenosine triphosphate, reduced medullary blood flow decreases salt and solute transport by nephron segments in the medulla. As a result, the medullary interstitial osmotic gradient cannot be maintained, which also reduces the ability to concentrate the urine.

Assessment Of Renal Diluting And Concentrating Ability

Assessment of renal water handling includes measurements of urine osmolality and the volume of urine excreted. The range of urine osmolality is from 50 to 1200 mOsm/kg H₂O.

• The corresponding range in urine volume is 18 to as little as 0.5 L/day. These ranges are not fixed, but they vary from person to person and, as noted previously, depend on the amount of water ingested and lost from nonrenal routes, as well as the amount of solute excreted.
• As emphasized in this chapter, the ability of the kidneys to dilute or concentrate the urine requires the separation of solute and water.
• This separation of solute and water in essence generates a volume of water that is “free of solute.” When the urine is dilute, solute-free water is excreted from the body.
• When the urine is concentrated, solute-free water is returned to the body (i.e., conserved).
• The concept of free water clearance (CH₂O) provides a way to calculate the amount of solute-free water generated by the kidneys, either when dilute urine is excreted or when concentrated urine is formed.
• As its name denotes, CH₂O is directly derived from the concept of renal clearance discussed.

“Complications of ignoring osmolality issues”

To calculate CH₂O, the clearance of total solute by the kidneys must be calculated. This clearance of total solute (i.e., osmoles, whether effective or ineffective) from plasma by the kidneys is termed the osmolar clearance (C_osm) and can be calculated as follows:

⇒ \(\mathrm{C}_{\mathrm{OSM}}=\frac{\mathrm{U}_{\mathrm{OSM}} \times \dot{\mathrm{V}}}{\mathrm{P}_{\mathrm{OSM}}}[latex]

where U_osm is the urine osmolality, V is the urine flow rate, and P_osm is the osmolality of plasma. Cosm has units of volume/unit time. CH₂O is then calculated as follows:

⇒ [latex]\mathrm{C}_{\mathrm{H}_2 \mathrm{O}}=\dot{\mathrm{V}}-\mathrm{C}_{\mathrm{osm}}\)

By rearranging equations 5-3; it should be apparent that

⇒ \(\dot{\mathrm{V}}=\mathrm{C}_{\mathrm{H}_2 \mathrm{O}}+\mathrm{C}_{\mathrm{osm}}\)

In other words, it is possible to partition the total urine output (V) into two hypothetical components. One component contains all the urine solutes and has an osmolality equal to that of plasma (i.e., U_osm = P_osm).

• This volume is defined by Cosm and represents a volume from which there has been no separation of solute and water. The second component is a volume of solute-free water (i.e., CH₂O).
• When dilute urine is produced, the value of CH₂O is positive, indicating that solute-free water is excreted from the body. When concentrated urine is produced, the value of CH₂O is negative, indicating that solute-free water is retained in the body.
• By convention, negative CH₂O values are expressed as T^CH₂O (tubular conservation of water).
• Calculating ^CH₂O and T^CH₂O can provide important information about the function of the portions of the nephron involved in producing dilute and concentrated urine.

Whether the kidneys excrete or reabsorb free water depends on the presence of AVP. When AVP is absent or AVP levels are low, solute-free water is excreted. When AVP levels are high, solute-free water is reabsorbed.

The following factors are necessary for the kidneys to excrete a maximal amount of solute-free water (CH₂O):

1. AVP must be absent. Without AVP, the collecting duct does not reabsorb a significant amount of water.
2. The tubular structures that separate solute from water (i.e., dilute the luminal fluid) must function normally. In the absence of AVP, the following nephron segments can dilute the luminal fluid:
   • Thin ascending limb of Henle’s loop
   • Thick ascending limb of Henle’s loop
   • Distal tubule
   • Collecting duct
3. Because of its high transport rate, the thick ascending limb is quantitatively the most important of these segments involved in the separation of solute and water.
   • An adequate amount of tubular fluid must be delivered to the aforementioned nephron sites for maximal separation of solute and water.
   • Factors that reduce delivery (for example., decreased GFR or enhanced proximal tubule reabsorption) impair the kidneys’ ability to excrete solute-free water.

Similar requirements also apply to the conservation of water by the kidneys (TCH₂O). For the kidneys to conserve water maximally, the following conditions must exist:

1. An adequate amount of tubular fluid must be delivered to the nephron segments in which separation of solute from water occurs. The important segment in the separation of solute and water is the thick ascending limb of Henle’s loop. Delivery of tubular fluid to Henle’s loop depends on GFR and proximal tubule reabsorption.
2. Reabsorption of NaCl by the nephron segments must be normal; again, the most important segment is the thick ascending limb of Henle’s loop.
3. A hyperosmotic medullary interstitium must be present. The interstitial fluid osmolality is maintained by NaCl reabsorption by Henle’s loop (con-ditions 1 and 2) and by effective accumulation of urea. Urea accumulation in turn depends on ade-quate dietary protein intake.
4. Maximum levels of AVP must be present and the collecting duct must respond normally to AVP.

The concept of free-water clearance as just described does not distinguish between effective and ineffective osmoles, either in the plasma or in the urine.

However, urea, which can account for half of the total urine osmoles, is not an effective osmole when the movement of water between intracellular fluid and extracellular fluid is considered.

Accordingly, when one wants to understand how the handling of water by the kidneys contributes to the maintenance of whole-body water balance, it is more appropriate to consider only the solutes that are effective osmoles. For plasma (i.e., extracellular fluid), the effective osmoles are Na⁺ and its attendant anions. For urine, they are the nonurea solutes.

The importance of using effective osmoles in determining the impact of renal water handling on whole-body water balance (i.e., body fluid osmolality) is illustrated by the following example.

A patient has an elevated plasma [urea], and his plasma [Na⁺] also is increased to 150 mEq/L. His total plasma osmolality (including urea) is 320 mOsm/kg H₂O, but his effective plasma osmolality (calculated as 2 x plasma [Na⁺]) is only 300 mOsm/kg H₂O.

His urine osmolality is 600 mOsm/kg H₂O, with 300 mOsm/ kg H₂O related to urea and 300 mOsm/kg H₂O related to nonurea solutes. His urinary flow rate is 3 L/day.

According to equations 5-2 and 5-3, his total osmolar clearance (C_osm) and free-water clearance (C_H2O) are as follows:

⇒ \(\mathrm{C}_{\mathrm{oSM}}=\frac{600 \mathrm{mOsm} / \mathrm{kg} \mathrm{H}_2 \mathrm{O} \times 3 \mathrm{~L} / \text { day }}{320 \mathrm{mOsm} / \mathrm{kg} \mathrm{H}_2 \mathrm{O}}=5.6 \mathrm{~L} / \text { day }\)

⇒ \(\mathrm{C}_{\mathrm{H}_2 \mathrm{O}}=3 \mathrm{~L} / \text { day }-5.6 \mathrm{~L} / \text { day }=-2.6 \mathrm{~L} / \text { day }\left(\mathrm{T}_{\mathrm{H}_2 \mathrm{O}}^{\mathrm{C}}\right)\)

Thus it appears that the kidneys are conserving 2.6 L/day of solute-free water, which would be an appropriate response to correct the elevated plasma osmolality.

However, when C_osm and C_H2O are analyzed from the perspective of effective osmoles, the following results are obtained:

⇒ \(\text { Cosm }=\frac{300 \mathrm{mOsm} / \mathrm{kg} \mathrm{H}_2 \mathrm{O} \times 3 \mathrm{~L} / \text { day }}{300 \mathrm{mOsm} / \mathrm{kg} \mathrm{H}_2 \mathrm{O}}=3 \mathrm{~L} / \text { day }\)

⇒ \(\mathrm{C}_{\mathrm{H}_2 \mathrm{O}}=3 \mathrm{~L} / \text { day }-3 \mathrm{~L} / \text { day }=0 \mathrm{~L} / \text { day }\)

When viewed from the more appropriate perspective of effective osmoles, it thus is apparent that the kidneys are not reabsorbing solute-free water and the patient’s kidneys are not correcting the hyperosmolality.

The post Regulation Of Body Fluid Osmolality Regulation Of Water Balance appeared first on BDS Notes.

“What is the regulation of extracellular fluid volume?”

The major solutes of the extracellular fluid (ECF) are the salts of Na⁺. Of these, sodium chloride (NaCl) is the most abundant.

• Because NaCl is also the major determinant of ECF osmolality, alterations in Na⁺ balance commonly are assumed to disturb ECF osmolality.
• However, under normal circumstances, this is not the case because the arginine vasopressin (AVP) and thirst systems maintain body fluid osmolality within a very narrow range.
• For example, the addition of NaCl to the ECF (without water) increases the Na+ concentration and osmolality of this compartment (intracellular fluid osmolality also increases because of osmotic equilibration with the ECF).

“Understanding the role of NaCl in fluid balance”

This increase in osmolality in turn stimulates thirst and the release of AVP from the posterior pituitary.

• The increased ingestion of water in response to thirst, together with the AVP-induced decrease in water excretion by the kidneys (so-called antidiuresis), quickly restores ECF osmolality to normal.
• However, the volume of the ECF increases in proportion to the amount of water ingested, which in turn depends on the amount of NaCl added to the ECF.
• Thus in the new steady state, the addition of NaCl to the ECF is equivalent to adding an isosmotic solution, and the volume of this compartment increases. Conversely, a decrease in the NaCl content of the ECF lowers the volume of this compartment.

The kidneys are the major route for the excretion of NaCl from the body. Only about 10% of the Na⁺ lost from the body each day is lost by nonrenal routes (for example., in perspiration and feces).

“How does the body regulate ECF volume and NaCl balance?”

Thus the kidneys are critically important in regulating the volume of the ECF.

• Under normal conditions, the kidneys keep the volume of the ECF constant by adjusting the excretion of NaCl to match the amount ingested in the diet.
• If ingestion exceeds excretion, ECF volume increases above normal, whereas the opposite occurs if excretion exceeds ingestion.
• It has been observed that the kidneys excrete sodium chloride (NaCl) more quickly when the NaCl is administered orally versus by an intravenous infusion.

This observation has led to the search for factors within the gastrointestinal tract that might modulate the renal excretion of NaCl. Indeed, neuroendocrine cells that produce the peptide hormones uroguanylin and guanylin in response to NaCl ingestion have been identified in the intestine.

• These hormones increase NaCl and water excretion by the kidneys (uroguanylin > guanylin) by inhibiting Na⁺ reabsorption in both the proximal tubule and collecting duct.
• Interestingly, the kidneys also produce uroguanylin and guanylin, suggesting that they also might play a paracrine role in the intrarenal regulation of NaCl excretion.
• The potential importance of these peptides in regulating renal NaCl excretion is seen in mice lacking the uroguanylin gene. These mice have a blunted natriuretic response to an oral NaCl load, and they also have increased blood pressure.

The typical diet contains approximately 140 mEq/ day of Na⁺ (8 g of NaCl), and thus daily Na⁺ excretion is also about 140 mEq/day. However, the kidneys can vary the excretion of Na⁺ over a wide range.

• Excretion rates as low as 10 mEq/day can be attained when persons are placed on a low-salt diet. Conversely, the kidneys can increase their excretion rate to more than 1000 mEq/day when challenged by the ingestion of a high-salt diet.
• These changes in Na+ excretion can occur with only modest changes in the ECF volume and steady-state Na⁺ content of the body.
• The response of the kidneys to abrupt changes in NaCl intake typically takes several hours to several days, depending on the magnitude of the change. During this transition period, the intake and excretion of Na⁺ are not matched as they are in the steady state.

“Importance of maintaining extracellular fluid volume”

Thus the individual experiences either positive Na⁺ balance (intake > excretion) or negative Na⁺ balance (intake < excretion). However, by the end of the transition period, a new steady state is established, and intake once again equals excretion.

• Provided that the AVP and thirst systems are intact and normal, alterations in Na+ balance change the volume, but not the Na⁺ concentration, of the ECF. Changes in ECF volume can be monitored by measuring body weight because 1 L of ECF equals 1 kg of body weight.
• In this chapter, the physiology of the receptors that monitor ECF volume is reviewed and the various signals that act on the kidneys to regulate NaCl excretion and thereby ECF volume are explained.
• In addition, the responses of the various portions of the nephron to these signals are considered. Finally, the pathophysiologic mechanisms involved in the formation of edema are presented, with emphasis on the role of NaCl handling by the kidneys.

Concept Of Effective Circulating Volume

As described, the ECF is subdivided into two compartments: blood plasma and interstitial fluid. Plasma volume is a determinant of vascular volume and thus blood pressure and cardiac output.

• The maintenance of Na⁺ balance, and thus ECF volume, involves a complex system of sensors and effector signals that act primarily on the kidneys to regulate the excretion of NaCl.
• As can be appreciated from the dependence of vascular volume, blood pressure, and cardiac output on ECF volume, this complex system is designed to ensure adequate tissue perfusion.
• Because the primary sensors of this system are located in the large vessels of the vascular system, changes in vascular volume, blood pressure, and cardiac output are the principal factors regulating renal NaCl excretion (described later in this chapter).

In a healthy person, changes in ECF volume result in parallel changes in vascular volume, blood pressure, and cardiac output. Thus a decrease in ECF volume, a situation termed volume contraction, results in reduced vascular volume, blood pressure, and cardiac output.

• Conversely, an increase in ECF volume, a situation termed volume expansion, results in increased vascular volume, blood pressure, and cardiac output.
• The degree to which these cardiovascular parameters change depends on the degree of volume contraction or expansion and the effectiveness of cardiovascular reflex mechanisms.
• When a person is in a negative Na⁺ balance, ECF volume is decreased and renal NaCl excretion is reduced. Conversely, with a positive Na⁺ balance, an increase in ECF volume occurs, which results in enhanced renal NaCl excretion (i.e., natriuresis).

However, in some pathologic conditions (for example., congestive heart failure and hepatic cirrhosis), the renal excretion of NaCl is not reflective of the ECF volume.

• In both of these situations, the volume of the ECF is increased. However, instead of increased renal NaCl excretion, as would be expected, a reduction in the renal excretion of NaCl occurs.
• To explain renal Na+ handling in these situations, it is necessary to understand the concept of effective circulating volume (ECV). Unlike the ECF, the ECV is not a measurable and distinct body fluid compartment.
• The ECV refers to the portion of the ECF that is contained within the vascular system and is “effectively” perfusing the tissues (effective blood volume is another commonly used term).

“Common mechanisms of NaCl balance regulation explained”

More specifically, the ECV reflects the perfusion of those portions of the vascular system that contain the volume sensors (described later in this chapter).

• In healthy persons, ECV varies directly with the volume of the ECF and, in particular, the volume of the vascular system (arterial and venous), the arterial blood pressure, and cardiac output.
• However, as noted, this is not the case in certain pathologic conditions. In the remaining sections of this chapter, the relationship between ECF volume and renal NaCl excretion in healthy adults, where changes in ECV and ECF volume occur in parallel, is examined.
• Patients with congestive heart failure frequently have an increase in the volume of the extracellular fluid (ECF), which is manifested as an accumulation of fluid in the lungs (pulmonary edema) and peripheral tissues (peripheral edema).

This excess fluid is the result of sodium chloride (NaCl) and water retention by the kidneys. The kidneys’ response (i.e., retention of NaCl and water) appears paradoxical because the ECF volume is increased.

• However, because of poor cardiac performance, perfusion of the portions of the vascular system that contain the volume sensors is reduced (i.e., decreased effective circulating volume).
• Therefore the volume sensors misinterpret these signals as indicative of ECF volume contraction and respond by increasing NaCl and water retention by the kidneys, thereby exacerbating a vicious cycle of impaired cardiac function and increased NaCl and water reabsorption.
• Large volumes of fluid accumulate in the peritoneal cavity of patients with advanced hepatic cirrhosis. This fluid, called ascites, is a component of the ECF and results from NaCl and water retention by the kidneys.

Again, the response of the kidneys in this situation seems paradoxical if only ECF volume is considered.

• With advanced hepatic cirrhosis, blood pools in the splanchnic circulation (i.e., the damaged liver impedes the drainage of blood from the splanchnic circulation by the portal vein).
• Thus volume and pressure are reduced in the portions of the vascular system where the volume sensors are found and, as in the case of congestive heart failure, the volume sensors interpret reduced effective circulating volume as decreased ECF volume and respond accordingly.
• Hence the kidneys respond as they normally would to ECF volume contraction, resulting in NaCl and water retention and an increase in ECF volume, which results in the accumulation of ascites fluid.

“Role of aldosterone in regulating NaCl balance”

Volume Sensing Systems

The ECF volume (or ECV) is monitored by multiple sensors. A number of the sensors are located in the vascular system, and they monitor its fullness and pressure.

• These receptors typically are called volume receptors; because they respond to pressure-induced stretch of the walls of the receptor (for example., blood vessels or cardiac atria), they also are referred to as baroreceptors.
• The sensors within the liver and central nervous system (CNS) are less well understood and do not seem to be as important as the vascular sensors in monitoring the ECF volume.

Volume Sensors In The Low-Pressure Cardiopulmonary Circuit:

Volume sensors (i.e., baroreceptors), which are located within the walls of the cardiac atria, right ventricle, and large pulmonary vessels, respond to the distention of these structures.

• Because the low-pressure venous side of the circulatory system has high compliance, these sensors respond mainly to the “fullness” of the vascular system.
• These baroreceptors send signals to the brainstem through afferent fibers in the glossopharyngeal and vagus nerves. The activity of these sensors modulates both sympathetic nerve outflow and AVP secretion.
• For example, a decrease in the filling of the pulmonary vessels and cardiac atria increases sympathetic nerve activity and stimulates AVP secretion. Conversely, distention of these structures decreases sympathetic nerve activity.

In general, 5% to 10% changes in blood volume and pressure are necessary to evoke a response.

• The cardiac atria possess an additional mechanism related to the control of renal NaCl excretion. The myocytes of the atria synthesize and store a peptide hormone.
• This hormone, termed atrial natriuretic peptide (ANP), is released when the atria are distended, which, by mechanisms outlined later in this chapter, reduces blood pressure and increases the excretion of NaCl and water by the kidneys.
• The ventricles of the heart also produce a natriuretic peptide termed brain natriuretic peptide (BNP), so named because it was first isolated from the brain. Like ANP, BNP is released from the ventricular myocytes by distension of the ventricles. Its actions are similar to those of ANP.

“How does ADH affect extracellular fluid volume?”

Volume Sensors In The High-Pressure Arterial Circuit:

Baroreceptors also are present in the arterial side of the circulatory system; they are located in the wall of the aortic arch, carotid sinus, and afferent arterioles of the kidneys.

• The aortic arch and carotid barorecep- tors send input to the brainstem through afferent fibers in the glossopharyngeal and vagus nerves. The response to this input alters sympathetic outflow and AVP secretion.
• Thus a decrease in blood pressure increases sympathetic nerve activity and AVP secretion. An increase in pressure tends to reduce sympathetic nerve activity (and activate parasympathetic nerve activity).
• The sensitivity of the high-pressure baroreceptors is similar to that in the low-pressure side of the vascular system; 5% to 10% changes in pressure are needed to evoke a response.

The juxtaglomerular apparatus of the kidneys, particularly the afferent arteriole, responds directly to changes in pressure. If perfusion pressure in the afferent arteriole is reduced, renin is released from the myocytes.

• Renin secretion is suppressed when perfusion pressure is increased. As described later in this chapter, renin determines blood levels of angiotensin II and aldosterone, both of which play an important role in regulating renal NaCl excretion.
• Of the two classes of baroreceptors, those on the high-pressure side of the vascular system appear to be more important in influencing sympathetic tone and AVP secretion.
• For example, patients with congestive heart failure often have an increased vascular volume with dilation of the atria and ventricles, which would be expected to decrease sympathetic tone and inhibit AVP secretion via the low-pressure baroreceptors.

Constriction of a renal artery by an atherosclerotic plaque, for example, reduces perfusion pressure to that kidney.

• This reduced perfusion pressure is sensed by the afferent arteriole of the juxtaglomerular apparatus and results in the secretion of renin.
• The elevated renin levels increase the production of angiotensin II, which in turn increases systemic blood pressure by its vasoconstrictor effect on arterioles throughout the vascular system.
• The increased systemic blood pressure is sensed by the juxtaglomerular apparatus of the contralateral kidney (i.e., the kidney without stenosis of its renal artery), and renin secretion from that kidney is suppressed.

“Impact of renin-angiotensin-aldosterone system (RAAS) on ECF volume”

In addition, the high levels of angiotensin II act to inhibit renin secretion by the contralateral kidney (negative feedback).

• The treatment of patients with constricted renal arteries includes surgical repair of the stenotic artery, administration of angiotensin 2 receptor blockers, or administration of an inhibitor of angiotensin-converting enzyme.
• The angiotensin-converting enzyme inhibitor blocks the conversion of angiotensin 1 to angiotensin 2.
• However, the sympathetic tone often is increased and AVP secretion often is stimulated in these patients (the renin-angiotensin-aldosterone system also is activated).
• This phenomenon reflects the activation of baroreceptors in the high-pressure arterial circuit in response to reduced blood pressure and cardiac out-put secondary to the failing heart (i.e., the high-pres-sure baroreceptors detect a reduced ECV and misinterpret this signal as indicative of reduced ECF volume).

Hepatic Sensors:

The liver also contains volume sensors that can modulate renal NaCl excretion, although they are not as important as the vascular sensors.

• One type of hepatic sensor responds to pressure within the hepatic vasculature and therefore functions in a manner similar to the baroreceptors in the low- and high-pressure vascular circuits.
• A second type of sensor also appears to exist in the liver. This sensor responds to [Na⁺] of the portal blood entering the liver.
• Afferent signals from both types of sensors are sent to the same area of the brainstem where afferent fibers from both the low- and high-pressure circuit baroreceptors converge.
• Increased pressure within the hepatic vasculature or an increase in portal blood [Na⁺] results in a decrease in efferent sympathetic nerve activity.

As described later in this chapter, this decreased sympathetic nerve activity leads to an increase in renal NaCl excretion.

Central Nervous System Na⁺ Sensors:

As with the hepatic sensors, the CNS sensors do not appear to be as important as the vascular sensors in monitoring the ECF volume and controlling renal NaCl excretion.

• Nevertheless, alterations in the [Na⁺] of blood carried to the brain in the carotid arteries or the [Na⁺] of the cerebrospinal fluid modulate renal NaCl excretion.
• For example, if the [Na⁺] in either the carotid artery blood or the cerebrospinal fluid is increased, a decrease in renal sympathetic nerve activity occurs, which in turn leads to an increase in renal NaCl excretion.
• The hypothalamus appears to be the site where these sensors are located. Angiotensin II and natriuretic peptides are generated in the hypothalamus.

These locally generated signals, together with systemically generated angiotensin II and natriuretic peptides, appear to play a role in modulating the CNS Na⁺-sensing system.

• Of the volume and Na+ sensors just described, those located in the vascular system are better understood. Moreover, their function in health and disease explains quite effectively the regulation of renal NaCl excretion.
• Therefore the remainder of this chapter focuses on the vascular volume sensors (i.e., baroreceptors) and their role in regulating renal NaCl excretion.

“Biomechanics of sodium reabsorption in the nephron”

Volume Sensor Signals:

When the vascular volume sensors have detected a change in ECV, which under normal conditions reflects ECF volume, they send signals to the kidneys, which result in appropriate adjustments in NaCl and water excretion.

• Accordingly, when the ECF volume is expanded, renal NaCl and water excretion are increased.
• Conversely, when the ECF volume is concentrated, renal NaCl and water excretion are reduced.
• The signals involved in coupling the volume sensors to the kidneys are both neural and hormonal. These signals are summarized, as are their effects on renal NaCl and water excretion.

Renal Sympathetic Nerves:

As described, sympathetic nerve fibers innervate the afferent and efferent arterioles of the glomerulus, as well as the nephron cells.

With negative Na⁺ balance (i.e., ECF volume contraction), baroreceptors in both the low- and high-pressure vascular circuits stimulate the sympathetic input to the kidneys.

This stimulation has the following effects:

The afferent and efferent arterioles constrict in response to a-adrenergic stimulation.

• This vaso-constriction predominantly affects the afferent arteriole, effectively reducing hydrostatic pressure within the glomerular capillary lumen and decreasing glomerular filtration.
• The resulting reduction in the glomerular filtration rate (GFR) reduces the filtered load of Na⁺ to the nephrons.
• Renin secretion is stimulated by the cells of the afferent arterioles in response to p-adrenergic receptor stimulation. As described later, renin ultimately increases the circulating levels of angiotensin II and aldosterone.

NaCl reabsorption along the nephron is directly stimulated by a-adrenergic stimulation, effectively reducing the fraction of filtered Na⁺ that is ultimately excreted.

• Quantitatively, the most important segment influenced by sympathetic nerve activity is the proximal tubule.
• As a result of these combined actions, increased renal sympathetic nerve activity decreases net NaCl excretion, an adaptive response that works to restore ECF volume to normal, which is a state termed euvolemia.
• With positive Na⁺ balance (i.e., ECF vol-ume expansion), renal sympathetic nerve activity is reduced, which generally reverses the effects just described.

Volume Sensing Systems At The Cellular Level:

A new “renal hormone” has been discovered recently, a flavin adenine dinucleotide-dependent amine oxidase named renalase.

• Renalase is similar in structure to monoamine oxidase and breaks down catechol amines (for example., epinephrine and norepinephrine).
• Several tissues (for example., skeletal muscle, heart, and small intestine) express renalase, but the kidneys secrete the enzyme into the circulation.

Because persons with chronic renal failure have very low levels of renalase in their plasma, the kidney is probably the primary source of the circulating enzyme.

• In experimental animals, infusion of renalase decreases blood pressure and heart contractility.
• Although the precise role of renalase in cardiovascular function and blood pressure regulation is not known, it may be important in modulating the effects of the sympathetic nervous system and especially the effects of the sympathetic nerves on the kidney.

“Steps in restoring NaCl balance during dehydration”

Renin-Angiotensin-Aldosterone System

Cells in the afferent arterioles (juxtaglomerular cells) are the site of synthesis, storage, and release of the proteolytic enzyme renin. Three factors are important in stimulating renin secretion:

1. Perfusion pressure. When perfusion pressure to the kidneys is reduced, renin secretion by the afferent arteriole is stimulated. Conversely, an increase in perfusion pressure inhibits renin release by the afferent arteriole.
2. Sympathetic nerve activity. Activation of the sympathetic nerve fibers that innervate the afferent arterioles increases renin secretion via P-adrenergic receptor stimulation. Renin secretion is decreased as renal sympathetic nerve activity is decreased.
3. Delivery of NaCl to the macula densa. Delivery of NaCl to the macula densa regulates the GFR by a process termed tubuloglomerular feedback.
   • In addition, the macula densa plays a role in renin secretion. When NaCl delivery to the macula densa is decreased, renin secretion is enhanced. Conversely, an increase in NaCl delivery inhibits renin secretion.
   • It is likely that macula densa-mediated renin secretion helps to maintain systemic arterial pressure under conditions of a reduced intravascular volume.
   • For example, when intravascular volume is reduced, perfusion of body tissues (including the kidneys) decreases, which in turn decreases the GFR and the filtered amount of NaCl.
   • The reduced delivery of NaCl to the macula densa then stimulates renin secretion, which acts through angiotensin II (a potent vasoconstrictor) to increase blood pressure and thereby maintain tissue perfusion.

Although many tissues express renin (for example., brain, heart, and adrenal gland tissues), the primary source of circulating renin is the kidneys. Renin is secreted by juxtaglomerular cells located in the afferent arteriole.

• At the cellular level, renin secretion is mediated by the fusion of renin-containing granules with the luminal membrane of the cell.
• This process is stimulated by a decrease in intracellular [Ca⁺⁺], a response opposite to that of most secretory cells where secretion is normally stimulated by an increase in intracellular [Ca⁺⁺].
• Renin release is also stimulated by an increase in intracellular cyclic adenosine monophosphate levels.

Thus anything that increases intracellular [Ca⁺⁺] inhibits renin secretion, which includes stretch of the afferent arteriole (myogenic control of renin secretion), angiotensin 2 (feedback inhibition), and endo- thelin.

• Conversely, anything that increases intracellular cyclic adenosine monophosphate stimulates renin secretion, which includes norepinephrine acting through p-adrenergic receptors and prostaglandin E₂.
• Increases in intracellular cyclic guanosine monophosphate have been shown to stimulate renin secretion in some situations and inhibit secretion in others.
• Notably, two substances that increase intracellular cyclic guanosine monophosphate are natriuretic peptides and nitric oxide.
• Nitric oxide stimulates renin secretion, whereas atrial natriuretic peptide and brain natriuretic peptide are inhibitory.

The control of renin secretion by the macula densa may involve paracrine factors such as prostaglandin E₂ (which stimulates renin secretion when NaCl delivery to the macula densa is decreased) and adenosine (which inhibits renin secretion when NaCl delivery to the macular densa is increased).

“Mechanisms of sodium reabsorption in the nephron”

Summarizes the essential components of the renin-angiotensin-aldosterone system. Renin alone does not have a physiological function; it functions as a proteolytic enzyme.

• Its principal substrate is a circulating protein, angiotensinogen, which is produced by the liver. Angiotensinogen is cleaved by renin to yield a 10-amino-acid peptide, angiotensin 1.
• Angiotensin 1 also has no known physiological function, and it is further cleaved to an 8-amino-acid peptide, angiotensin 2, by a converting enzyme (angiotensin-converting enzyme [ACE]) found on the surface of vascular endothelial cells.

1. Pulmonary and renal endothelial cells are important sites for the bioconversion of angiotensin 1 to angiotensin 2. ACE also degrades bradykinin, a potent vasodilator. Angiotensin 2 has several important physiologic functions, including:
2. Stimulation of aldosterone secretion by the adrenal cortex
3. Arteriolar vasoconstriction, which increases blood pressure
4. Stimulation of AVP secretion and thirst
5. Enhancement of NaCl reabsorption by the proximal tubule, thick ascending limb of Henle’s loop, the distal tubule, and even the collecting duct; of these segments, the effect on the proximal tubule is quantitatively the largest

Angiotensin 2 is an important secretagogue for aldosterone. An increase in the plasma K⁺ concentration is the other important stimulus for aldosterone secretion.

• Aldosterone is a steroid hormone produced by the glomerulosa cells of the adrenal cortex. Aldosterone acts in a number of ways on the kidneys.
• With regard to the regulation of the ECF volume, aldosterone reduces NaCl excretion by stimulating its reabsorption by the thick ascending limb of the loop of Henle, portions of the distal tubule, and the collecting duct.
• (The portions of the distal tubule that functionally respond to aldosterone together with the collecting duct are referred to as the aldosterone-sensitive distal nephron [ASDN].)

The effect of aldosterone on renal NaCl excretion depends mainly on its ability to stimulate Na+ reabsorption in the ASDN.

• Aldosterone has many cellular actions in cells of the ASDN.
• Notably, it increases the abundance of the apical membrane Na⁺– Cl^– symporter in the cells of the distal tubule (DCT2 segment; see previous
• At the Cellular Level box) and the abundance of the epithelial Na+ channel in the apical membrane of principal cells in the late portion of the distal tubule and collecting duct.

Volume Sensing System At The Cellular Level:

The distal tubule can be divided into three distinct segments based on the presence of specific membrane transporters.

• The first segment after the macula densa (DCT1) expresses a Na⁺-Cl^– symporter, which is specifically inhibited by the thiazide class of diuretics.
• The next segment (DCT2) expresses the Na⁺-Cl^– symporter and the epithelial Na⁺ channel. The last segment of the distal tubule (connecting tubule), like the collecting duct, expresses only the epithelial Na+ channel.
• Aldosterone selectivity and sensitivity are conferred by the presence of mineralocorticoid receptors, as well as the presence of the enzyme 11 β-hydroxysteroid dehydrogenase 2 (11β-HSD2).

Because the mineralocorticoid receptor also binds glucocorticoids, 11β- HSD2 is required for aldosterone specificity because it metabolizes glucocorticoids and thus prevents them from binding to the mineralocorticoid receptor.

• The mineralocorticoid receptor is found throughout the distal tubule and collecting duct. However, 11β- HSD2 is only found in the DCT2, the connecting tubule, and the collecting duct.
• Thus the aldosterone-sensitive distal nephron consists of the DCT2 and connecting tubule (collectively termed the late distal tubule) and the collecting duct. Accordingly, the DCT1 segment is referred to as the early distal tubule.
• By this action, Na⁺ entry into the cells across the apical membrane is increased. Extrusion of Na+ from the cell across the basolateral membrane occurs via the Na⁺-K⁺-adenos- ine triphosphatase (ATPase) pump, the abundance of which is also increased by aldosterone.

Thus aldosterone increases net reabsorption of Na+ from the tubular fluid by ASDN segments, and reduced levels of aldosterone decrease the amount of Na+ reabsorbed by these segments.

• As noted, aldosterone also enhances Na⁺ reabsorption by cells of the thick ascending limb of the loop of Henle.
• This action probably reflects the increased entry of Na⁺ into the cell across the apical membrane (probably by the apical membrane Na⁺-K⁺-2Cl^– symporter) and increased extrusion from the cell by the basalt- eral membrane Na⁺-K⁺-ATPase pump.
• Diseases of the adrenal cortex can alter aldosterone levels and thereby impair the ability of the kidneys to maintain Na⁺ balance and euvolemia.

“How do the kidneys maintain ECF volume balance?”

With decreased secretion of aldosterone (hypoaldosteronism), the reabsorption of Na⁺ by the aldosterone-sensitive distal nephron (late distal tubule and collecting duct) is reduced, and sodium chloride (NaCl) is lost in the urine.

• Because urinary NaCl loss can exceed the amount of NaCl ingested in the diet, negative Na⁺ balance ensues, and the extracellular fluid (ECF) volume decreases.
• In response to the ensuing ECF volume contraction, sympathetic tone is increased, and levels of renin, angiotensin 2, and arginine vasopressin are elevated.
• With increased aldosterone secretion (hyperaldosteronism), the opposite effects are observed: Na⁺ reabsorption by the aldosterone-sensitive distal nephron is enhanced and excretion of NaCl is reduced.

Consequently, ECF volume is increased, sympathetic tone is decreased, and the levels of renin, angiotensin 2, and arginine vasopressin are decreased.

• As described later in this chapter, atrial natriuretic peptide and brain natriuretic peptide levels also are elevated in this setting.
• As summarized, activation of the renin-angiotensin-aldosterone system, as occurs with ECF volume depletion, decreases the excretion of NaCl by the kidneys.
• Conversely, this system is suppressed by ECF volume expansion, thereby enhancing renal NaCl excretion.

Natriuretic Peptides

The body produces a number of substances, including ANP and BNP, that act on the kidneys to increase Na⁺ excretion.

• Of these substances, natriuretic peptides produced by the heart and kidneys are best understood and are the focus of the following discussion.
• The heart produces two natriuretic peptides. Atrial myocytes primarily produce and store the peptide hormone ANP, and ventricular myocytes primarily produce and store BNP.
• Both peptides are secreted in response to myocardial wall stretch (i.e., during cardiac dilatation that accompanies volume expansion and/or heart failure), and they act to relax vascular smooth muscle and promote NaCl and water excretion by the kidneys.
• The kidneys also produce a related natriuretic peptide termed urodilatin. Its actions are limited to promoting NaCl excretion by the kidneys.
• In general, the actions of these natriuretic peptides, as they relate to renal NaCl and water excretion, antagonize those of the renin-angiotensin-aldosterone system.

Natriuretic Peptide Actions Include:

1. Afferent arteriolar vasodilation and efferent arteriolar vasoconstriction within the glomerulus, increase the GFR and the filtered amount of Na⁺.
2. Inhibition of renin secretion by the juxtaglomerular cells of the afferent arterioles.
3. Inhibition of aldosterone secretion by the glomerulus cells of the adrenal cortex. This inhibition occurs by two mechanisms:
   • Inhibition of renin secretion by the juxtaglomerular cells, thereby reducing angiotensin II-induced aldosterone secretion, and
   • Direct inhibition of aldosterone secretion by the glomerulosa cells of the adrenal cortex.
4. Inhibition of NaCl reabsorption by the collecting duct, which also is caused in part by reduced levels of aldosterone. However, the natriuretic peptides also act directly on the collecting duct cells.
   • Through the second messenger, cyclic guanosine monophosphate, natriuretic peptides inhibit Na⁺ channels in the apical membrane and thereby decrease Na⁺ reabsorption.
   • This effect occurs predominantly in the medullary portion of the collecting duct.
5. Inhibition of AVP secretion by the posterior pituitary and AVP action on the collecting duct. These effects decrease water reabsorption by the collecting duct and thus increase the excretion of water in the urine.

“Role of the loop of Henle in NaCl concentration”

• These effects of the natriuretic peptides increase the net excretion of NaCl and water by the kidneys.
• Hypothetically, a reduction in the circulating levels of these peptides would be expected to decrease NaCl and water excretion, but convincing evidence for this effect has not been reported.

Arginine Vasopressin:

• As discussed, a decreased ECF volume stimulates AVP secretion by the posterior pituitary.
• The elevated levels of AVP decrease water and NaCl excretion by the kidneys, which serve to reestablish euvolemia.

Control Of Renal Nacl Excretion During Euvolemia

The maintenance of Na⁺ balance and therefore euvolemia requires the precise matching of the amount of NaCl ingested and the amount excreted from the body.

• As already noted, the kidneys are the major route for NaCl excretion. Accordingly, in a euvolemic person, we can equate daily urine NaCl excretion with daily NaCl intake.
• The amount of NaCl excreted by the kidneys can vary widely. Under conditions of salt restriction (i.e., a low NaCl diet), virtually no Na⁺ appears in the urine.
• Conversely, in persons who ingest large quantities of NaCl, renal Na⁺ excretion can exceed 1000 mEq/day. The kidneys require several days to respond maximally to variations in dietary NaCl intake.

During the transition period, excretion does not match intake, and the person is in either positive (intake > excretion) or negative (intake < excretion) Na⁺ balance.

• This phenomenon is illustrated. When Na⁺ balance is altered during these transition periods, the ECF volume changes in parallel.
• Water excretion, regulated by AVP, also is adjusted to keep plasma osmolality constant, effectively resulting in isosmotic changes in ECF volume.
• Thus with positive Na⁺ balance, the ECF volume expands, whereas with negative Na⁺ balance, the ECF volume contracts. In both cases, no change in plasma [Na⁺] occurs.

These changes in ECF volume can be detected by monitoring changes in body weight. Ultimately, renal excretion reaches a new steady state and NaCl excretion once again is matched to intake.

• The time course for the adjustment of renal NaCl excretion varies (from hours to days) and depends on the magnitude of the change in NaCl intake.
• Adaptation to large changes in NaCl intake requires a longer time than adaptation to small changes in intake.
• The general features of Na⁺ handling along the nephron must be understood to comprehend how renal Na⁺ excretion is regulated. (for the cellular mechanisms of Na⁺ transport along the nephron.)

“Impact of glomerular filtration rate (GFR) on ECF volume”

Most (67%) of the filtered amount of Na⁺ is reabsorbed by the proximal tubule. An additional 25% is reabsorbed by the thick ascending limb of the loop of Henle, and the remainder is largely reabsorbed by the distal tubule and collecting duct.

In a normal adult, the filtered amount of Na⁺ is approximately 25,000 mEq/day.

Filtered amount of Na⁺ = (GFR)(plasma [Na⁺])

= (180 L/day)(140 mEq/L)

= 25,200 mEq/day

With a typical diet, less than 1% of this filtered amount is excreted in the urine (approximately 140 mEq/day). Because of the large amount of filtered Na⁺, small changes in Na⁺ reabsorption by the nephron can profoundly affect Na⁺ balance and thus the volume of the ECF.

• For example, an increase in Na⁺ excretion from 1% to 3% of the filtered amount represents an additional loss of approximately 500 mEq/day of Na⁺.
• Because the ECF Na⁺ concentration is 140 mEq/L, such a Na⁺ loss would decrease the ECF volume by more than 3 L (i.e., water excretion would parallel the loss of Na⁺ to maintain body fluid osmolality constant: [500 mEq/day]/[140 mEq/L] = 3.6 L/ day of fluid loss).
• Such fluid loss in a person weighing 70 kg would represent a 26% decrease in the ECF volume.

In euvolemic subjects, the nephron segments distal to the loop of Henle, namely the distal tubule and collecting duct, are the main nephron segments where Na⁺ reabsorption is adjusted to maintain excretion at a level appropriate for dietary intake.

• However, this does not mean that the other portions of the nephron are not involved in this process.
• Because the reabsorptive capacity of the distal tubule and collecting duct is limited, the upstream segments of the nephron (i.e., the proximal tubule and loop of Henle) must reabsorb the bulk of the filtered amount of Na⁺.

Thus during euvolemia, Na⁺ handling by the nephron can be explained by two general processes:

1. Na⁺ reabsorption by the proximal tubule and loop of Henle is regulated so that a relatively constant portion of the filtered amount of Na⁺ is delivered to the distal tubule.
   • The combined action of the proximal tubule and loop of Henle reabsorbs approximately 92% of the filtered amount of Na⁺, and thus 8% of the filtered amount is delivered to the distal tubule.
2. Reabsorption of this remaining portion of the filtered amount of Na⁺ by the distal tubule and collecting duct is regulated so that the amount of Na⁺ excreted in the urine closely matches the amount ingested in the diet at a steady state.
   • Thus these later nephron segments make final adjustments in Na⁺ excretion to maintain the euvolemic state.

Mechanisms For Maintaining Constant Na⁺ Delivery To The Distal Tubule:

A number of mechanisms maintain delivery of a constant fraction of the filtered amount of Na⁺ to the beginning of the distal tubule.

• These processes are autoregulation of the GFR (a mechanism that keeps the filtered amount of Na⁺ constant), glomerulotubu-lar balance, and load dependence of Na⁺ reabsorption by the loop of Henle.
• Autoregulation of the GFR allows maintenance of a relatively constant filtration rate over a wide range of perfusion pressures. Because the filtration rate is constant, the delivery of filtered Na⁺ to the nephrons also is kept constant.

Despite the autoregulatory control of the GFR, small variations in GFR occur. If these changes were not compensated for by an appropriate adjustment in Na⁺ reabsorption by the nephron, Na⁺ excretion would change markedly.

“How does the renal system respond to sodium imbalance?”

• Fortunately, Na⁺ reabsorption in the euvolemic state, especially by the proximal tubule, changes in parallel with changes in the GFR. This phenomenon is termed glomerulotubular (G-T) balance.
• Thus if the GFR increases, the amount of Na⁺ reabsorbed by the proximal tubule increases proportionately. The opposite occurs if the GFR decreases.

The final mechanism that helps maintain the constant delivery of Na⁺ to the beginning of the collecting duct involves the ability of the loop of Henle to increase its reabsorptive rate in response to increased delivery of Na⁺.

Regulation Of Distal Tubule And Collecting Duct Na⁺ Reabsorption:

When delivery of Na⁺ is constant, small adjustments in the distal tubule and, to a lesser degree, collecting duct Na⁺ reabsorption is sufficient to balance excretion with intake.

• (As already noted, as little as a 2% change in fractional Na⁺ excretion produces more than a 3 L change in the volume of the ECF.) Aldosterone is the primary regulator of Na⁺ reabsorption by the distal tubule and collecting duct and thus of Na⁺ excretion under this condition.
• When aldosterone levels are elevated, Na⁺ reabsorption by these segments is increased (excretion is decreased). When aldosterone levels are decreased, Na⁺ reabsorption is decreased (excretion is increased).

In addition to aldosterone, a number of other factors, including natriuretic peptides, prostaglandins, uroguanylin, adrenomedullin, and sympathetic nerves, alter Na⁺ reabsorption by the distal tubule and collecting duct.

• However, the relative effects of these other factors on the regulation of Na⁺ reabsorption by these segments during euvolemia are unclear.
• As long as variations in the dietary intake of NaCl are minor, the mechanisms previously described can regulate renal Na⁺ excretion appropriately and thereby maintain euvolemia.
• However, these mechanisms cannot effectively handle significant changes in NaCl intake.
• When NaCl intake changes significantly, ECF volume expansion or ECF volume contraction occurs.
• In such cases, additional factors are invoked to act on the kidneys to adjust Na⁺ excretion and thereby reestablish the euvolemic state.

The excretion rate of Na⁺ by the kidneys can be quantitated in the following way:

⇒ \(\mathrm{U}_{\mathrm{Na}^{+}} \times \dot{\mathrm{V}}=\mathrm{GFR} \times \mathrm{P}_{\mathrm{Na}^{+}}-\mathrm{R}\)

where U_Na+ × V is the excretion rate in mEq/time (U_Na+ is the urine [Na⁺] and V is the urine flow rate), GFR X P_Na+ is the filtered amount of Na⁺ (GFR is the glomerular filtration rate and P_Na+ is the plasma [Na⁺]), and R is the amount of Na⁺ reabsorbed by the nephron.

“Pathophysiology of ECF volume imbalances explained”

Control Of Na⁺ Excretion With Volume Expansion

During ECF volume expansion, baroreceptors in both the high- and low-pressure vascular circuits send signals to the kidneys. These signals result in increased excretion of NaCl and water.

The signals acting on the kidneys include:

1. Decreased activity of the renal sympathetic nerves
2. Increased release of ANP and BNP from the heart and urodilatin by the kidneys
3. Inhibition of AVP secretion from the posterior pituitary and decreased AVP action on the collecting duct
4. Decreased renin secretion and thus decreased production of angiotensin 2
5. Decreased aldosterone secretion, which is a consequence of reduced angiotensin II levels, and elevated natriuretic peptide levels The integrated response of the nephron to these signals is illustrated.

Three general responses to ECF volume expansion occur (the numbers correlate with those circled:

“Emerging research on ECF volume regulation mechanisms”

The GFR increases. The GFR increases mainly as a result of the decrease in sympathetic nerve activity.

• Sympathetic fibers innervate the afferent and efferent arterioles of the glomerulus and control their diameter. Decreased sympathetic nerve activity leads to arteriolar dilation.
• Because afferent arteriolar dilation is greater than efferent dilation, the hydrostatic pressure within the glomerular capillary is increased, thereby increasing the filtration pressure and the GFR.
• Note that the corresponding filtration fraction decreases because the renal plasma flow increases to a greater degree than the GFR.

Natriuretic peptides, which are increased during ECF volume expansion, also promote an increase in GFR via differential direct effects on the afferent (vasodilation) and efferent (vasoconstriction) arterioles.

• With the increase in the GFR, the filtered amount of Na⁺ increases. The reabsorption of Na⁺ decreases in the proximal tubule and loop of Henle.
• Several mechanisms act to reduce Na⁺ reabsorption by the proximal tubule, but the precise role of each of these mechanisms remains unresolved.
• Because activation of the sympathetic nerve fibers that innervate this nephron segment stimulates Na⁺ reabsorption, the decreased sympathetic nerve activity that results from ECF volume expansion decreases Na⁺ reabsorption.

In addition, angiotensin 2 directly stimulates Na⁺ reabsorption by the proximal tubule. Because angiotensin 2 levels also are reduced by ECF volume expansion, proximal tubule Na⁺ reabsorption decreases accordingly.

• Increased hydrostatic pressure within the glomerular capillaries also increases the hydrostatic pressure within the peritubular capillaries.
• In addition, the decrease in filtration fraction reduces the peritubular oncotic pressure.
• These alterations in the capillary Starling forces reduce the absorption of solute (for example., NaCl) and water from the lateral intercellular space and thus reduce proximal tubular reabsorption.

Both the increase in the filtered amount of NaCl and the decrease in NaCl reabsorption by the proximal tubule result in the delivery of more NaCl to the loop of Henle.

• Because activation of the sympathetic nerves and aldosterone stimulates NaCl reabsorption by the loop of Henle, the reduced nerve activity and low aldosterone levels that occur with ECF volume expansion serve to reduce NaCl reabsorption by this nephron segment.
• Thus the fraction of the filtered amount delivered to the distal tubule is increased.
• Na⁺ reabsorption decreases in the distal tubule and collecting duct. As noted, the amount of Na⁺ delivered to the distal tubule exceeds that observed in the euvolemic state (the amount of Na⁺ delivered to the distal tubule varies in proportion to the degree of ECF volume expansion).

This increased amount of delivered Na⁺ can overwhelm the reabsorptive capacity of the distal tubule and the collecting duct, an effect heightened by the reduced reabsorptive capacity of these segments associated with increased circulating natriuretic peptides and decreased circulating aldosterone levels.

• The final component in the response to ECF vol-ume expansion is the excretion of water. As Na⁺ excretion increases, plasma osmolality begins to fall, which decreases the secretion of AVP.
• AVP secretion also is decreased in response to the elevated levels of natriuretic peptides. In addition, these natriuretic peptides inhibit the action of AVP on the collecting duct.
• Together, these effects decrease water reabsorption by the collecting duct and thereby increase water excretion by the kidneys.

Thus the excretion of Na⁺ and water occurs in concert; euvolemia is restored, and body fluid osmolality remains constant.

• The time course of this response (hours to days) depends on the magnitude of the ECF volume expansion.
• Thus if the degree of ECF volume expansion is small, the mechanisms just described generally restore euvolemia within 24 hours. However, with larger degrees of ECF volume expansion, the response can take several days.

In brief, the renal response to ECF volume expansion involves the integrated action of all parts of the nephron:

1. The filtered amount of Na⁺ is increased,
2. The proximal tubule and loop of Henle reabsorption is reduced (the glomerular filtration rate is increased and proximal reabsorption is decreased, and thus G-T balance does not occur under this condition), and
3. The delivery of Na⁺ to the distal tubule is increased. This increased delivery, along with the inhibition of distal tubule and collecting duct reabsorption, results in the excretion of a larger fraction of the filtered amount of Na⁺ and thus restores euvolemia.

“Case studies on ECF volume and NaCl balance outcomes”

Control Of Na⁺ Excretion With Volume Contraction

During ECF volume contraction, volume sensors in both the high- and low-pressure vascular circuits send signals to the kidneys that reduce NaCl and water excretion. The signals that act on the kidneys include:

1. Increased renal sympathetic nerve activity
2. Increased secretion of renin, which results in elevated angiotensin II levels and thus increased secretion of aldosterone by the adrenal cortex
3. Stimulation of AVP secretion by the posterior pituitary

The integrated response of the nephron to these signals is illustrated. The general response is as follows

“Global prevalence of ECF volume disorders”

• The GFR decreases. Afferent and efferent arteriolar constriction occurs as a result of increased renal sympathetic nerve activity.
• The effect is greater on the afferent than on the efferent arteriole. This vasoconstriction causes the hydrostatic pressure in the glomerular capillary to fall and thereby decreases the GFR.
• The filtration fraction increases because the renal plasma flow decreases more than the GFR, but the absolute decrease in the GFR reduces the filtered load of Na⁺.

Na⁺ reabsorption by the proximal tubule and loop of Henle is increased. Several mechanisms augment Na⁺ reabsorption in the proximal tubule.

• For example, increased sympathetic nerve activity and angiotensin II levels directly stimulate Na⁺ reabsorption.
• The decreased hydrostatic pressure within the glomerular capillaries also leads to a decrease in the hydrostatic pressure within the peritubular capillaries.
• In addition, the increased filtration fraction results in an increase in the peritubular oncotic pressure.

These alterations in the capillary Starling forces facilitate the movement of fluid from the lateral intercellular space into the capillary and thereby stimulate the reabsorption of solute (for example., NaCl) and water by the proximal tubule.

• The reduced amount of filtered Na⁺ and enhanced proximal tubule reabsorption decrease the delivery of Na⁺ to the loop of Henle.
• Increased sympathetic nerve activity, as well as elevated levels of angiotensin 2 and aldosterone, stimulate Na⁺ reabsorption by the thick ascending limb.
• Because sympathetic nerve activity is increased and angiotensin 2 and aldosterone levels are elevated during ECF volume contraction, increased Na⁺ reabsorption by this segment is expected. Thus less Na⁺ is delivered to the distal tubule.

Na⁺ reabsorption by the distal tubule and collecting duct is enhanced. The small amount of Na⁺ that is delivered to the distal tubule is almost completely reabsorbed because transport in this segment and the collecting duct is enhanced.

• This stimulation of Na⁺ reabsorption by the distal tubule and collecting duct is induced by increased angiotensin 2 and aldosterone levels (increased sympathetic nerve activity also will stimulate Na⁺ reabsorption).
• Finally, water reabsorption by the late portion of the distal tubule and the collecting duct is enhanced by AVP (AVP also stimulates limited Na⁺ reabsorption in the late distal tubule and collecting duct), the levels of which are elevated through activation of the low high-pressure vascular volume sensors and by the elevated levels of angiotensin 2.
• As a result, water excretion is reduced.

Because both water and Na+ are retained by the kidneys in equal proportions, euvolemia is reestablished and body fluid osmolality remains constant.

• The time course of this expansion of the ECF (hours to days) and the degree to which euvolemia is attained depend on the magnitude of the ECF volume contraction and the dietary intake of Na+.
• Thus the kidneys reduce Na+ excretion and euvolemia can be restored more quickly if additional NaCl is ingested in the diet.

In brief, the nephron’s response to ECF volume contraction involves the integrated action of all its segments:

1. The filtered amount of Na+ is decreased,
2. Proximal tubule and loop of Henle reabsorption is enhanced (the GFR is decreased and proximal reabsorption is increased and thus G-T balance does not occur under this condition), and
3. The delivery of Na+ to the distal tubule is reduced. This decreased delivery, together with enhanced Na+ reabsorption by the distal tubule and collecting duct, virtually eliminates Na+ from the urine.

Edema

Edema is the accumulation of excess fluid within the interstitial space. As described, Starling forces across the capillary wall determine the movement of fluid into and out of the vascular compartment in exchange with the extravascular interstitial compartment.

• Alterations of these forces under pathologic conditions can lead to increased movement of fluid from the vascular space into the interstitium, resulting in edema formation.
• The role of the kidneys in the formation of edema can be appreciated by recognizing that the interstitial compartment typically must contain 2 to 3 L of excess fluid before edema is clinically evident (for example., swelling of the ankles).

The source of this fluid is the vascular compartment (i.e., plasma), which has a volume of 3 to 4 L in healthy persons.

• Alterations in the Starling forces that would accompany a 2 to 3 L fluid shift out of the vascular compartment into the interstitial compartment would be predicted to limit such marked fluid movement and the decline in blood pressure that would attend such a marked fluid shift.
• However, retention of NaCl and water by the kidneys maintains intravascular compartment volume, thereby maintaining the blood pressure and facilitating interstitial fluid redistribution and edema development.

Alterations In Starling Forces:

In the Starling forces and their effect on fluid movement across the capillary wall were explained.

• Edema results from changes in the Starling forces that alter these fluid dynamics.
• Recall that fluid movement across a capillary wall is driven by hydrostatic and oncotic pressure gradients:

Filtration rate = K_f[(P_c — P_i) — a( π_c —π_i)]

where K_f is the filtration coefficient of the capillary wall (a measure of the intrinsic wall permeability and the surface area available for fluid flow), and P_c and P_i are the hydrostatic pressures within the lumen of the capillary.

The interstitium, respectively, σ is the reflection coefficient for protein across the capillary wall (approximately 0.9 for skeletal muscle), and π_c and π_i are the oncotic pressures generated by protein within the capillary lumen and the interstitium, respectively.

Capillary Hydrostatic Pressure (P_c)

Increasing the Pc favors the movement of fluid out of the capillary or retards its movement into the capillary, thereby promoting edema formation.

• Normally the resistance of the precapillary arteriole is well regulated such that changes in systemic blood pressure do not result in marked alterations in P_c.
• However, postcapillary resistance is not regulated to the same degree, and thus alterations in the pressure within the venous side of the circulation have significant effects on P_c.
• Consequently, an increase in the venous pressure elevates P_c, which increases the movement of fluid into the interstitium, resulting in the accumulation of edema fluid.
• Common causes for increased venous pressure include venous thrombosis and congestive heart failure.

Plasma Oncotic Pressure (π_c):

A decrease in π_c would be expected to favor the movement of fluid out of the capillary lumen and inhibit its reabsorption from the interstitium.

• Because albumin is the most abundant plasma protein, alterations in nc result primarily from changes in the plasma [albumin].
• However, it is important to remember that changes in plasma protein concentration result in parallel changes in the protein concentration of the interstitial fluid.
• This phenomenon reflects the fact that the reflection coefficient for protein is 0.9 and thus proteins can cross the capillary wall.
• Because of the parallel changes in capillary and interstitial fluid protein concentration, the oncotic pressure gradient across the capillary wall (π_c – π_i) may not change appreciably.

Lymphatic Obstruction:

As noted , the lymphatic system serves to return interstitial fluid formed by capillary filtration to the vascular system.

• Obstruction of a lymphatic duct interferes with this process, and as a result, interstitial fluid accumulates in the portion of the body drained by the obstructed duct (i.e., edema forms).
• As this interstitial fluid accumulates, the interstitial hydro-static pressure increases, and eventually a new steady state is reached where the Starling forces are once again balanced and no additional fluid accumulates.
• However, unless the obstruction is corrected, the area. Edema can be classified as localized or generalized.

Localized edema, as the name denotes, represents the abnormal accumulation of interstitial fluid in a specific area or region of the body.

• Common causes of localized edema include insect stings and lymphatic obstruction.
• The venom of many stinging or biting insects contains substances that either directly increase capillary permeability or cause the release of mediators of inflammation that have a similar effect.
• In addition, the venom or inflammatory mediators may cause vasodilation. Increasing the permeability of the capillary, or in some cases the postcapillary venule, increases the filtration coefficient (Kf) and also can decrease the protein reflection coefficient.

Both effects can increase fluid movement out of the capillary, with the latter effect also altering the Starling forces by changing the protein oncotic pressure gradient.

• Starling forces are further altered in response to the vasodilation (i.e., capillary hydrostatic pressure [Pc] is increased).
• The net effect of these changes is that more fluid moves out of the capillary into the interstitium and localized swelling occurs.

Lymphatic obstruction often accompanies surgical treatment of tumors. For example, in some women with breast cancer, regional lymph nodes that drain the affected breast are surgically removed.

• When those located in the axilla are removed, the draining of lymph from that arm may be impaired. As a result, edema may develop in the arm.
• Generalized edema results when Starling forces across all capillary beds are altered. Edema may be present in the lungs (i.e., pulmonary edema) or throughout the systemic circulation (i.e., peripheral edema).

Peripheral edema is most commonly observed in the feet, ankles, and legs, where the force of gravity magnifies the changes in Starling forces (i.e., further increases P_c) and thereby causes more fluid to leave the capillary and enter the interstitium.

• One of the most common causes of generalized edema is congestive heart failure.
• In this condition, blood accumulates in the venous side of the circulation, raising P_c, which in turn causes fluid to move out of the capillary into the interstitium.

Generalized edema is also seen with renal diseases associated with the nephrotic syndrome.

• In the nephrotic syndrome, glomerular capillary permeability is altered, allowing large quantities of albumin to be lost in the urine (albuminuria).
• If the rate of loss exceeds the rate at which albumin is synthesized by the liver, the plasma [albumin] falls.

The reduction in plasma protein concentration, and thus nc, was thought to be the primary cause of edema formation in patients with nephrotic syndrome.

• Because the oncotic pressure gradient across the capillary wall may not change appreciably (i.e., interstitial protein oncotic pressure also falls), it is likely that other factors are responsible for, or at least contribute to, the abnormal accumulation of fluid in the interstitial compartment.
• Supporting this notion is the observation that edema does not spontaneously develop in rats deficient in albumin. It is now known that one of these other factors is primary NaCl retention by the distal tubule and collecting duct.

With damage to the glomerular filtration barrier, the serum protein plasminogen enters the renal tubules where it is cleaved to form plasmin by the serine protease urokinase (produced by proximal tubule cells).

• Plasmin, also a serine protease, then cleaves the y-subunit of the epithelial Na⁺ channels present in the apical membrane of cells in the late distal tubule and collecting duct, thereby increasing the open time of these channels.
• This phenomenon results in increased Na⁺ (and Cl^–) reabsorption. The ensuing retention of NaCl (along with water) increases vascular volume and thereby leads to an increase in P_c, increased movement of fluid into the interstitial compartment, and thus edema formation.

Drained by the obstructed lymphatic duct remains edematous even in this new steady state.

Capillary Permeability:

• An increase in capillary permeability favors increased movement of fluid across the capillary wall and thus accumulation of excess fluid in the interstitial compartment.
• The increased permeability also can alter the capillary reflection coefficient for protein(s), allowing more protein across the capillary and thus altering the protein oncotic pressure gradient
  (π_c-π_i).

Role Of The Kidneys:

The role of the kidneys in edema-forming states is best illustrated by considering the situation that exists with heart failure. Because of decreased cardiac performance, venous pressure is elevated, and perfusion of the kidneys is impaired.

“Complications of ignoring ECF volume issues”

• The increase in venous pressure alters the Starling forces (i.e., increased Pc) and causes fluid to accumulate in the interstitium.
• At the same time, decreased cardiac performance (decreased cardiac output and blood pressure) reduces the ECV, which is misinterpreted by the body’s vascular volume sensors as a decrease in ECF volume.
• The fall in ECF volume activates the renal sympathetic nerves and the renin-angiotensin-aldo-sterone system and causes AVP secretion. In response to these signals, the kidneys retain NaCl and water, as already described.

This retention of isotonic fluid expands the ECF volume and thus blood volume, thereby helping perpetuate a vicious cycle of fluid accumulation that can further exacerbate congestive heart failure.

• Intravascular volume expansion also contributes to the increased Pc, increased interstitial fluid accumulation, and edema formation.
• As fluid begins to accumulate in the interstitium, it is taken up by the lymphatics and returned to the systemic circulation. As noted, thoracic duct and right lymphatic duct flow is approximately 1 to 4 L/day.
• The lymphatic system can increase this flow up to 20 L/day. Because a significant amount of lymph returns to the circulation at the level of regional lymph nodes, the actual amount of interstitial fluid returned to the systemic circulation by the lymphatic system can exceed 20 L/day.

Nevertheless, the capacity of the lymphatic system has a limit. When this limit is reached, edema fluid begins to accumulate.

• The importance of NaCl retention by the kidneys in edema formation provides two approaches for treatment. The first involves dietary manipulation. The ultimate source of NaCl is the diet.
• Thus if dietary intake of NaCl is restricted, the amount that can be retained by the kidneys is reduced and edema formation is limited. The second approach is to inhibit the kidneys’ ability to retain NaCl.
• This inhibition is accomplished clinically by the use of diuretics, which, as described, inhibit Na⁺ transport mechanisms in the nephron. Thus NaCl excretion is increased and NaCl retention is blunted.

The post Regulation Of Extracellular Fluid Volume And Nacl Balance appeared first on BDS Notes.

The process of gene expression is not over when the genetic code has been used to create the sequence of amino acids that constitutes a protein.

• To be useful to the cell, this new polypeptide chain must fold up into its unique three-dimensional conformation, bind any small molecule cofactors required for its activity, be appropriately modified by protein kinases or other protein-modifying enzymes, and assemble correctly with the other protein subunits with which it functions.
• Thus, the polypeptide that emerges from the ribosome is inactive, and before taking on its functional role in the cell.

It Must Undergo At Least The First Of The Following Four Types Of Post Translational Processing:

1. Protein folding
2. Proteolytic cleavage
3. Chemical modification
4. Inteine splicing

Protein Folding

The proteome is the final product of genome expression and comprises all the proteins present in a cell at a particular time.

• A ‘typical’ mammalian cell, for example, a hepatocyte, is thought to contain 10,000-20,000 different proteins, about 8 × 10⁹ individual molecules in all, representing approximately 0.5ng of protein or 18 – 20% of the total cell weight (airmen, 2002).
• A protein, like a DNA molecule, is a linear unbranched polymer. The monomeric subunits of a protein are called amino acids and the resulting polymers, or polypeptides, are rarely more than 2000 units in length.

Four Levels Of Protein Structure: Proteins are found to have four distinct levels of structure. These levels are hierarchical, the protein being built up stage by stage, with each level of structure depending on the one below it.

“Understanding post-translational modifications through FAQs: Q&A explained”

Primary structure: The primary structure of a protein is formed by joining amino acids into a polypeptide.

• The amino acids are linked by peptide bonds which are formed by a condensation reaction between the carboxyl group of one amino acid and the amino group of a second amino acid.
• The two ends of the polypeptide are chemically distinct: one has a free amino group and is called the amino, NH₂, – or N – terminus; the other has a free carboxyl group and is called the carboxyl, COOH – or C – terminus.
• The direction of the polypeptide can therefore be expressed as either N → C (left to right) or C → N (right to left).

“Importance of studying post-translational modifications for biology students: Questions explained”

Secondary Structure: The secondary structure of protein refers to the different conformations that can be taken up by the polypeptide.

• The two types of secondary structures are the β-helix and α-sheet, both of which are stabilized by hydrogen bonds that form between different amino acids in the polypeptide.
• Most polypeptides are long enough to be folded into a series of secondary structures one after another along the molecule.

Tertiary Structure: The tertiary structure of protein results from folding the secondary structural components of the polypeptide into a three-dimensional configuration.

• The tertiary structure is stabilized by various chemical forces, notably hydrogen bonding between individual amino acids, and hydrophobic forces, which dictate that amino acids with non-polar (i.e., water-hating or hydrophobic) side groups must be shielded from water by embedding within the internal regions of the protein.
• There may also be covalent linkages called disulphide bridges between cysteine amino acids at various places in the polypeptide.

“Common challenges in understanding post-translational modifications effectively: FAQs provided”

Quaternary structure: The quaternary structure of proteins involves the association of two or more polypeptides, each folded into its tertiary structure, into a multi-subunit protein.

• Not all proteins form quaternary structures, but it is a feature of many proteins with complex functions, including several involved in genome expression.
• Some quaternary structures are held together by disulphide bridges between different polypeptides, but many proteins comprise looser associations of subunits stabilized by hydrogen bonding and hydrophobic effects.
• However, according to the functional requirements, quaternary proteins can revert to their component polypeptides, or change their subunit composition.
• All the information that a polypeptide needs to adopt its correct three-dimensional structure is contained within its amino acid sequence. This is one of the central principles of molecular biology.
• The notion that the amino acid sequence contains all the information needed to fold the polypeptide into its correct tertiary structure derives from experiments carried out on ribonuclease in the 1960s (Anfinsen, 1973).
• The enzyme ribonuclease is a small protein, just 124 amino acids in length, containing four disulphide bridges and with a tertiary structure that is made up predominantly of α-sheet, with very little β-helix.

• Experiment showing spontaneous folding pathways for proteins: Studies of folding of proteins were carried out with ribonuclease that had been purified from cow pancreas and resuspended in butler.
• The addition of urea, a compound that disrupts hydrogen bonding, resulted in a decrease in the activity of the enzyme (measured by testing its ability to cut RNA) and an increase in the viscosity of the solution, indicating that the protein was being denatured by unfolding into an unstructured polypeptide chain.
• The critical observation was that when the urea was removed from the experimental solution, the viscosity decreased and the enzyme activity reappeared. The conclusion is that the protein refolds spontaneously when the denaturant (i.e., urea) is removed.
• In these initial experiments, the four disulphide bonds remained intact because they were not disrupted by urea but the same result occurred when the urea treatment was combined with the addition of a reducing agent to break the disulphide bonds: the activity was still regained on renaturation.
• This shows that the disulphide bonds are not critical to the protein’s ability to refold, they merely stabilise the tertiary structure once it has been adopted.

“Why is early learning of post-translational modifications critical for molecular biology? Answered”

A more detailed study of the spontaneous folding pathways for ribonuclease and other small proteins has led to the following two sets of the process:

1. The secondary structural motifs along the polypeptide chain form within a few milliseconds
   of the denaturant being removed.
2. During the next few seconds or minutes, the secondary structural motifs interact with one another and the tertiary structure gradually takes shape, often via a series of intermediate conformations.

• In other words, the protein follows a folding pathway. However, there may be more than one possible pathway that a protein can follow to reach its correctly folded structure. Such in vitro folding pathways were found true for only the small proteins; large proteins fail to refold.
• Experiments have demonstrated that once a protein domain in a multidomain protein emerges from the ribosome, it forms a compact structure within a few seconds that contains most of the final secondary structure β-helices and α-sheets) aligned in roughly the right way.
• For many protein domains, this unusually open and flexible structure, which is called a molten globule, is the starting point for a relatively slow process in which many side chain adjustments occur that eventually form the correct tertiary structure.
• Nevertheless, because it takes several minutes to synthesize a protein of average size, a great deal of the folding process is completed by the time the ribosome releases the C-terminal end of a protein (Federov and Baldwin, 1997).

Role Of Molecular Chaperones In Folding Of Proteins: Most of our current understanding of protein folding in the cell is based on the discovery of proteins that help other proteins to fold. These are called molecular chaperones and have been studied most vividly in E. coli.

• Both eukaryotes and archaea possess equivalent proteins (chaperones), although some of the details of the way they work are different (Hartl, 1996; Slavotinck and Biesecker, 2001).
• Molecular chaperones in E.coli. The molecular chaperones in E.coli can be divided into two groups: Hsp 70 chaperones (Hsp = heat shock proteins) and chaperonins.

• The Hsp70 chaperones, include the proteins called HSP70 (coded by the dnak gene and sometimes called Dnak protein), Hsp40 (coded by dnaJ) and Grp E.
• The chaperonins, the main version of which in E.coli is the GroEL- GroES complex. Molecular chaperones do not specify the tertiary structure of a protein, they merely help the protein find that correct structure.
• The two types of chaperones do this in different ways. For example, the Hsp70 family (of chaperones) bind to hydrophobic regions of proteins, including proteins that are still being translated.

“Steps to explain types of post-translational modifications: Phosphorylation vs glycosylation: Q&A guide”
“Is post-translational modification-related risk reversible if addressed promptly? Answer provided”

• They prevent protein aggregation by holding the protein in an open conformation until it is completely synthesized and ready to fold.
• The Hsp70 chaperones are also involved in other processes that require shielding of hydrophobic regions in proteins, such as transport through membranes and disaggregation of proteins that have been damaged by heat stress.
• The chaperonins work in quite a different way. GroEL and GroES form a multi-subunit structure that looks like a hollowed-out bullet with a central cavity Molecular chaperones facilitate correct folding of protein subunits.
• A single unfolded protein enters the cavity and emerges folded. The mechanism for this is not known but it is postulated that GroEL/GroES acts as a cage that prevents the unfolded protein from aggregating with other proteins and that the inside surface of the cavity changes from hydrophobic to hydrophilic in such a way as to promote the burial of hydrophobic amino acids within the protein.

• It is later found that the cavity unfolds proteins that have folded incorrectly, passing these unfolded proteins back to the cytoplasm so that they can have a second attempt at adopting their correct tertiary structure Molecular chaperones and chaperons in eukaryotes.
• Eukaryotic cells have at least two families of molecular chaperones, known as the hsp60 and hsp70 proteins.
• Different family members function in different organelles. For example, mitochondria contain their own hsp60 and hsp70 molecules that are distinct from those that function in the cytosol (where hsp60 is called TCP-1 in vertebrate cells), and a special hsp70 (called BIP) helps to fold proteins in the endoplasmic reticulum.
• The hsp70 machinery acts early in the life of many proteins, binding to a string of about seven hydrophobic amino acids before protein leaves the ribosome.
• The hsp60-like proteins form a large barrel-shaped structure that acts later in the proteins’ life after it has been fully synthesized.
• This type of chaperone forms an “isolation chamber” into which misfolded proteins are fed, preventing their aggregation and providing them with a favourable environment in which they attempt to refold.

“Role of ubiquitination in protein degradation: Questions answered”

Proteolytic Cleavage

Proteolytic cleavage has the following two functions in the post-translational processing of proteins.

1. It is used to remove short pieces from the N – and/or C – terminal regions of polypeptides, leaving a single shortened molecule that folds into the active protein.
2. It is used to cut polyproteins into segments, all or some of which are active proteins. These events are relatively common in eukaryotes but less frequent in bacteria. Not all proteins undergo proteolytic cleavage.

Cleavage Of The Ends Of Polypeptides: Processing by proteolytic cleavage is common with secreted polypeptides whose biochemical activities might be injurious to the cell producing the protein.

• An example is provided by melittin, the most abundant protein in bee venom and the one responsible for causing cell lysis after injection of the bee sting into the person or animal being stung.
• Melttin (protein) lyses cells in bees as well as animals so have to be initially synthesized as inactive. precursor. This precursor, called pronielittin, has 22 amino acids at its N-terminus.
• The pre-sequence is removed by an extracellular protease that cuts it at all eleven positions, releasing the active venom protein.
• The protease does not cleave within the active sequence because its mode of action is to release dipeptides with the sequence X – Y, where X is alanine, aspartic acid or glutamic acid, and Y is alanine or proline, these motifs are not found in the active sequence.

“How does acetylation regulate gene expression? FAQ explained”

Amino Acid Abbreviation:

“Early warning signs of gaps in understanding post-translational modification basics: Common questions”

• Insulin also undergoes processing similar to melittin. It is a chromonal protein made in the islets of Langerhans in the vertebrate pancreas and responsible for controlling blood sugar levels. Insulin is synthesised as proproinsulin, which is 105 amino acids in length.
• The processing pathway involves the removal of the first 24 amino acids to give proinsulin, followed by two additional cuts which excise the central segment leaving two active parts of the protein, the A and B chains, which link together by the formation of two disulphide bonds to the form mature insulin.
• The first segment to be removed, the 24 amino acids from the N-terminus, is a signal peptide, a highly hydrophobic stretch of amino acids that attaches the precursor protein to a membrane before transport across that membrane and out of the cell.

Proteolytic Cleavage Of Polyprotein: In the examples, proteolytic processing results in a single mature protein. This does not happen every time.

• Some proteins are initially synthesized as polyproteins, long polypeptides that contain a series of mature proteins linked together in a head-to-tail fashion. Cleavage of the polyprotein releases the individual proteins, which may have very different functions from one another.
• Polyproteins are very common in eukaryotes.
• Several types of viruses that infect eukaryotic cells use them as a way of reducing the sizes of their genomes, a single polyprotein gene with one promoter and one terminator taking up less space than a series of individual genes.
• Polyproteins are also involved in the synthesis of peptide hormones in vertebrates.
• For example, the polyprotein called pro-opiomelanocortin, made in the pituitary gland, contains at least ten different peptide hormones.
• These are released by proteolytic cleavage of the polyprotein, but not all can be produced at once because of overlaps between individual peptide sequences. Instead, the exact cleavage pattern is different in different cells.

“Asymptomatic vs symptomatic effects of ignoring post-translational modification principles: Q&A”

Chemical Modification

The genome can code for 21 different amino acids: the 20 specified by the standard genetic code, and selenocysteine, which is inserted into polypeptides by the context-dependent reading of a 5′ – UGA – 3′ codon.

• This range is increased dramatically by posttranslational chemical modification of proteins, which results in an immense supply of different types of amino acids.
• The most simple sort of chemical modifications involve the addition of a small chemical group (for example., an acetyl, methyl or phosphate group;) to an amino acid side chain, or the amino or carboxyl groups of the terminal amino acids in a polypeptide (Bradshaw et ah, 1998).
• More than 150 different modified amino acids have been reported in different proteins; each modification is found to be carried out in a highly specific manner, i.e., each amino acid being modified in a highly specific manner and the same amino acids being modified in the same way in every copy of the proteins, for example., histone H3.

Examples Of Post-Translational Chemical Modifications:

“Can targeted interventions improve outcomes using post-translational modification knowledge? FAQs provided”

• We have observed how acetylation and methylation of histone H3 and other histones have an important influence on chromatin structure and hence on genome expression.
• Other types of chemical modification have important regulatory roles, for example., phosphorylation is used in transduction.
• Glycosylation is a complex type of modification of amino acids. It involves the attachment of large carbohydrate side chains to polypeptides (Drickamer and Taylor, 1998).

There Are Following Two General Types Of Glycosylation:

1. O-linked glycosylation. It is the attachment of a sugar side chain via the hydroxyl group of a serine or threonine amino acid.
2. N-linked glycosylation. It involves attachment through the amino group on the side chain of asparagine.

Glycosylation results in attachment to the proteins of grand structures comprising branched networks of 10 to 20 sugar units of various types.

• These side chains help to target proteins to particular sites in the cells and determine the stability of proteins circulating in the bloodstream.
• Another category of large-scale modification is acylation which involves the attachment of long-chain lipids, often to serine or cysteine amino acids. Acylation occurs with many proteins that become associated with membranes.
• A less common modification is biotinylation, in which a molecule of biotin is attached to a small number of enzymes that catalyze the carboxylation of organic acids such as acetate and propionate (Chapman, Smith and Cronan, 1999).

Inteine Splicing

The final type of post-translational processing is inteine splicing. It is a protein version of the more extensive intron splicing that occurs in the context of pre-RNAs.

• Inteins are internal segments of proteins that are removed soon after translation, the two external segments or exteins becoming linked together.
• The first intein was discovered in 1990 in S. cerevisiae and more than 100 inteins have been reported till today.
• Most interns are known in bacteria, archaea and lower eukaryotes. In a few cases, there is more than one intein in a single protein.

“Differential applications of reversible vs irreversible modifications: Questions answered”

• Most inteins are approximately 150 amino acids in length and like pre-mRNA introns, the sequences at the splice junctions of inteins have some similarity in most of the known examples.
• The first amino acid of the downstream extein is cysteine, serine or threonine.
• A few other amino acids within the intein sequence are also conserved. These conserved amino acids are involved in the splicing process, which is catalyzed by the intein itself.

Characteristics Of Inteins: Self-catalysis. The first interesting feature of inteins was discovered when the structure of two inteins was determined by X-ray crystallography (Duan et al., 1997; Klabunde et al.,
1998).

• These structures are similar in some respects to that of a Drosophila protein called Hedgehog, which is involved in the development of the segmentation pattern of the embryo of fruit fly. Hedgehog is an autoprocessing protein that cuts itself into two.
• The structural similarity with inteins lies in the part of the Hedgehog protein that catalyzes its self-cleavage.
• Molecular biologists believe that possibly the same protein structure has evolved twice, or possibly inteins and Hedgehog protein shared a common link at some stage in the evolutionary past.

Intein Homing: The second interesting feature of intein is that with some inteins the excised segment is a sequence-specific endonuclease.

• The intein cuts DNA at the sequence corresponding to its insertion site in a gene coding for an intein-free version of the protein from which it is derived.
• If the cell also contains a gene coding for the intein-containing protein, then the DNA sequence for the intein can jump into the cut site converting the intein-minus gene into an intein-plus version, a process called intein homing (Pietrokovski, 2001).

Monitoring And Control Of Protein Quality

A protein that has a sizable exposed patch of hydrophobic amino acids on its surface is usually abnormal: it has either failed to fold correctly after leaving the ribosome, suffered an accident that partly unfolded it at a later time, or failed to find its normal partner subunit in a larger protein complex.

• Such a protein is useless to the cell. Further, such protein complexes can be dangerous. Many proteins with an abnormally exposed hydrophobic region can form large aggregates, precipitating out of solution.
• We shall see that in rare cases, such aggregates form and cause severe human diseases. But in the vast majority of cells, powerful protein quality control mechanisms prevent such disasters.
• Cells have evolved elaborate mechanisms that recognize and remove the hydrophobic patch proteins. Two of the mechanisms depend on the molecular chaperones just discussed, which bind to the patch and attempt to repair the defective protein by giving it another chance to fold.

“Difference between phosphorylation and glycosylation in proteins: Q&A explained”

• At the time, by covering the hydrophobic patches, these chaperones transiently prevent protein aggregation. Proteins that very rapidly fold correctly on their own do not display such patches and are therefore bypassed by chaperones.
• All of the options of quality control that a cell makes for a difficult-to-fold newly synthesized protein have been outlined.
• As indicated, when attempts to refold a protein fail, a third option is called into play that destroys the protein by proteolysis.
• The proteolytic pathway begins with the recognition of an abnormal hydrophobic patch on the surface of a protein and it ends with the delivery of the entire protein to a protein destruction machine, a complex protease called proteosome.
• As described next, this process depends on an elaborate protein marking system that also carries out other central functions in the cell by destroying selected normal proteins.

Protein Degradation: The protein synthesis and processing events that we have studied so far result in new proteins that take up their place in the cell’s proteome. These proteins either replace existing ones that have reached the end of their working lives or provide new protein functions in response to the changing requirements of the cell.

• The concept that the proteome of a cell can change over time requires not only de novo protein synthesis but also the removal of proteins whose functions are no longer required.
• This removal must be highly selective so that only the correct proteins are degraded, and must also be rapid to account for the abrupt changes that occur under certain conditions, for example during key transitions in the cell cycle (Hunt, 1997).
• In eukaryotes, most breakdown involves a single system, involving ubiquitin and the proteasome. A link between ubiquitin and protein degradation was first established in 1975 when it was shown that this abundant 76-amino acid protein is involved in energy-dependent proteolysis reactions in rabbit cells (Varshavsky, 1997).
• Subsequent research identified a series of three enzymes that attach ubiquitin molecules, singly or in chain, to lysine amino acids in proteins that are targeted for breakdown.
• Whether or not a protein becomes ubiquitinated depends on the presence or absence within it of amino acid motifs that act as degradation-susceptible signals.

These signals have not been completely characterized but yeast is known to contain about 10 different such signals. These signals include the following two categories.

1. The N-degron, a sequence element present at the N-terminus of a protein;
2. PEST sequence, internal sequences that are rich in proline (P), glutamic acid (E), serine (S) and threonine (T).

These sequences are permanent features of the proteins that contain them and so cannot be straightforward ‘degradation signal’-ifthey were so then these proteins would be broken down as soon as they are synthesized.

• Instead, they must determine susceptibility to degradation and hence the general stability of a protein in the cell.
• How this might be linked to the controlled breakdown of selected proteins at specific times, for instance during the cell cycle, is little understood.

“Most common complications of poorly understood post-translational modifications: FAQs”

• The second component of the ubiquitin-dependent degradation pathway is the proteosome the structure within which ubiquitinated proteins are broken down.
• In eukaryotes, the proteasome is a large, multi-subunit structure with a sedimentation coefficient of26S, comprising a hollow cylinder of 20S and two “caps” of 19S (Groll et al., 1997; Ferrell et al., 2000).
• Archaea also have proteasomes of about the same size but these are less complex, being composed of multiple copies of just two proteins; eukaryotic proteasomes contain 14 different types of protein subunits.
• The entrance into the cavity within the proteasome is narrow, and a protein must be unfolded before it can enter. This unfolding probably occurs through an energy-dependent process and may involve a structure similar to chaperonins.
• After unfolding, the protein can enter the proteasome within which it is cleaved into short peptides 4-10 amino acids in length. These are released back into the cytoplasm where they are broken down into individual amino acids which can be reutilized in protein synthesis.

Abnormal Protein Folding Causes Destructive Human Diseases: When all of a cell’s protein quality controls fail, large protein aggregates tend to accumulate in the affected cell.

• Some of these aggregates, by adsorbing critical macromolecules to them, can severely damage cells and even cause cell death.
• The protein aggregates released from dead cells tend to accumulate in the extracellular matrix that surrounds the cells in a tissue, and in extreme cases, they can also damage tissues.
• Since the brain is composed of a highly organised collection of nerve cells, it is especially vulnerable. These protein aggregates primarily tend to cause diseases of neurodegeneration.
• Prominent among these are Huntington’s disease and Alzheimer’s disease – the latter causing age-related dementia in more than 20 million people in today’s world (Alberts et al., 2002).
• A protein aggregate, which has to survive, grow and damage an organism, must be highly resistant to proteolysis both inside and outside the cell.
• Many of the protein aggregates that cause problems from fibrils built from a series of polypeptide chains that are layered one over the other as a continuous stack of p-sheets.
• These are so-called cross beta filaments, which tend to be highly resistant to proteolysis. This resistance presumably explains why this structure is observed in so many of the neurological disorders caused by protein staining deposits, called amyloid.

“Why are post-translational modification mechanisms often misunderstood in practice? Questions answered”

• One particular variety of these neurological diseases is quite notorious. These are prion diseases. Unlike Huntington’s or Alzheimer’s disease, a prion disease can spread from one organism to another, providing that the second organism eats a tissue containing the protein aggregate.
• A set of diseases- called scrapie in sheep, Creutzfeldt- Jacob disease (CJD) in humans, and bovine spongiform encephalopathy (BSE) in cattle-are caused by a misfolded, aggregated form of a protein called PrP (for prion protein).
• The PrP is normally located on the outer surface of the plasma membrane, most prominently in neurones.
• Its normal function is not known. However, PrP has the unfortunate property of being convertible to a very special abnormal conformation.
• This conformation not only forms protease-resistant, cross-beta filaments; it also is “infectious” because it converts normally folded molecules of PrP to the same form.
• This property creates a positive feedback loop that produces the abnormal form of PrP, called PrP and thereby allows PrP to spread rapidly from cell to cell in the brain, causing death of both animals and humans.
• It is dangerous to eat the tissues of animals that contain PrP*, as witnessed most recently by the spread of BSE (commonly referred to as the “mad cow disease”) from cattle to humans in Great Britain.

Signal Hypothesis (Protein Targeting)

Ribosomes are either free in the cytoplasm or associated with membranes, depending on the type of protein being synthesised. Membrane-bound ribosomes, which are indistinguishable from free ribosomes, synthesise proteins that enter membranes.

• These proteins either become a part of the membrane or in eukaryotes, either pass into membrane-bound organelles (for example., the Golgi apparatus, mitochondria, chloroplasts, vacuoles) or transported outside the plasma membrane.
• The signal hypothesis of Gunter Blobcl, a 1999 Nobel laureate, and his colleagues, explains the mechanism for membrane attachment. The mechanism applies to both prokaryotes and eukaryotes. Here, the signal hypothesis has been described for mammals).

“Success rate of interventions using modern post-translational modification techniques: FAQ”

• The signal for membrane insertion is in the form of a polypeptide. It is coded into the first one to three dozen amino acids of membrane-bound proteins. This signal peptide takes part in a chain of events that leads the ribosome to attach to the membrane and insertion of the protein. The first step occurs when the signal peptide becomes exposed on the outside of the ribosome.
• A ribonucleoprotein particle, called signal recognition particle (SRP), which consists of six different proteins and a 7S RNA (about 300 nucleotide long), recognizes the signal peptide.
• In the second step, the complex of signal recognition particle, ribosome, and signal peptide then passes to a membrane, where the SRP binds to a receptor called a docking protein (DP) or signal recognition particle receptor.
• During this time, protein synthesis or translation halts. The ribosome is brought into direct contact with the membrane, and other proteins which help anchor the ribosome.
• In the third step, the protein synthesis resumes, with the nascent protein usually passing directly into a translocation channel (called translocon). Once through the membrane, the signal peptide is cleaved from the protein by an enzyme called signal peptidase.
• Evidence for the signal hypothesis came about through recombinant DNA techniques.
• A signal sequence was placed in front of the a-globin gene, whose protein product is normally not transported through a membrane. When this gene was translated, the ribosome became membrane-bound, and the protein passed through the membrane.

Since different proteins enter different membrane-bound compartments (for example the Golgi apparatus), some mechanism must direct a nascent protein to its proper membrane. This specificity seems to depend on the exact signal sequence and membrane-bound glycoproteins called signal sequence receptors.

• After the ribosome binds to the docking protein, the signal peptide interacts with a signal sequence receptor, which presumably determines whether that protein is specific for that membrane. If it is, the remaining processes continue.
• If not, the ribosome may be released from the membrane. The signal peptide does not seem to have a consensus sequence similar to transcription or translation recognition boxes.
• Rather similarities (at least for the endoplasmic reticulum and bacterial membrane-bound proteins) include a positively charged (basic) amino acid (commonly lysine or arginine) near the beginning (N- terminal end), followed by about a dozen hydrophobic (nonpolar) amino acids, commonly alanine, isoleucine, leucine, phenylalanine and valine.

“Cost of ignoring post-translational modification principles vs benefits of systematic approaches: Q&A”

The Signal Peptide Of The Bovine Prolactin Protein:

NH₂– Met Asp Ser Lys Gly Ser Ser Gin Lys Gly Ser Arg Leu Leu Leu Leu Leu Val Ser Asn Leu Leu Leu Cys Gin Gly Val Val Ser/Thr Pro Val .Asn Asn Cys – COOH

Post Translational Processing Multiple Choice Questions And Answers

Question 1. Peptide linkage is

1. —CO—NH—
2. —CO—NH₂
3. —COOH—NH₂
4. —CH—N

Answer: 1. —CO—NH—

Question 2. The tertiary structure of proteins having amino acids cysteine is achieved through

1. Ionic bonds
2. Covalent bonds
3. Disulphide bonds
4. Hydrogen bonds

Answer: 3. Disulphide bonds

The post Post-translational Modifications of the Protein appeared first on BDS Notes.

The majority of genes are spaced out more or less randomly along the length of the DNA molecule. In some cases, however, they are grouped into distinct clusters.

• Sometimes the individual genes in a cluster are unrelated and there is no apparent reason or advantage to having them organized in this way.
• More commonly, clusters are made of genes that contain related units of biological information, for example., operons and multigene families.

Operons

Operons are quite common aspects of the organization of genes in bacteria. An operon is a cluster of genes coding for a series of enzymes that work in concert in an integrated biochemical pathway.

• The first operon to be discovered was the lactose or lac operon of E.coli which is a cluster of three genes each coding for one of the three enzymes involved in the conversion (catabolism) of the disaccharide lactose into its monosaccharide units glucose and galactose.
• These enzymes are not required all the time, only when lactose is available.
• But when needed they are required together, for example., the three units of biological information carried by Lac must be read at the same time.
• A sophisticated system enabling the lac genes to be expressed together and only when needed has evolved around the fact that the genes are clustered.

“Understanding multigene families through FAQs: Q&A explained”

Multigene Families

Operons have no direct counterparts in organisms other than bacteria. In contrast, multigene families are found in many organisms, mainly eukaryotic organisms.

• A multigene family or gene family is also a cluster of related genes.
• Still, in multigene families, the individual genes have identical or similar nucleotide sequences and therefore contain identical or similar pieces of biological information.

Why do IViultigene Families are Required?

Some proteins in the cell are required in small quantities, but other proteins may be required in large quantities so that the demands can not be easily met. These demands can be met in either of the following two ways:

• In some cases, a solitary gene is present in the cell but is transcribed repeatedly to produce a large number of messenger RNA molecules, and these mRNA molecules act as templates for many rounds of protein synthesis.
• For example, although the gene coding for silkworm protein fibroin is present in only one copy per haploid complement, it keeps on making silk actively.
• Similarly, in a cell (for example., erythroblasts) engaged in exclusive synthesis of hemoglobin, a solitary copy of the gene produces large quantities of a and (3 polypeptide chains of hemoglobin.
• However, although a whole family of related globin genes are found in each cell, different but solitary globin genes function at different stages of development.
• In other cases, multiple copies of a gene are found in the same cell, all taking part in transcription within a cell, giving rise to large quantities of the gene product as in the case of ribosomal RNA (rRNA) genes, histone genes, tRNA genes, heat shock protein genes, etc.

Types Of Multigenes: Members of multigene families share DNA sequence homology, descend from a single ancestral gene, and their gene products frequently have similar functions. Members of multigene families are often, but not always, found together in a single location along a chromosome.

Dispersed Family Of Gene: In some multigene families, the individual members are clustered, as with the globin genes, but in others, the genes are dispersed around the genome.

• An example of a dispersed family is the five human genes for aldolase, an enzyme involved in energy generation, which is located on chromosomes 3, 9,10, 16, and 17.
• The important point is that, even though dispersed, the members of the multigene family have sequence similarities that point to a common evolutionary origin.

Multigene Families Are Of The Following Main Types:

1. Simple Multigene Families:
   • In these gene families, all of the genes are exactly or virtually the same. For example, most higher organisms have multiple copies of the gene for the 5S rRNA (a component of the ribosome), probably because large quantities of 5S rRNA molecules must be synthesized at certain times, creating a demand that just one of a few genes would be unable to satisfy.
   • Rather than being spread randomly throughout the chromosomes, the 5S rRNA genes are clustered into a multigene family. In humans, for example, there is a family of about two thousand 5S rRNA genes in chromosome 1.
   • The 5S rRNA genes are present independently of the rDNA which is localized at the NOR in eukaryotes. However, in prokaryotes and yeast, 5S rRNA genes are present in close vicinity of rDNA. The 5S rRNA genes are also organized in tandem repeats, each repeat consisting of a gene 120 bp long and a spacer region. The length of complete repeat is 375 bp in Drosophila.
   • In wheat, there are two loci for 5S rRNA having repeats of different lengths, 480 bp and 500 bp. These repeat units may sometimes carry pseudogenes (instead of functional genes), which are not used for the synthesis of any RNA.
2. Complex Multigene Families:
   • A multigene family is a group of similar, but not necessarily identical sequences, each sequence representing a gene, so that the gene is present in multiple copies. Complex multigene families are of the following types:

Multigene Families With Divergent Members

Globin Gene Family In The Human Genome: The human α-globin and β-globin genes are subfamilies of the globin gene superfamily and are two of the most intensively studied regions of the human genome.

• There is a cluster of three α-globin β-genes on the short arm of chromosome 16 and another with five β- globin genes on the short arm of chromosome 11.
• Members of both subfamilies share nucleotide sequence similarity, but members of the same subfamily have the greatest sequence similarity.

“Importance of studying multigene families in biology: Questions explained”

Haemoglobin is a tetramer, containing twoα- and two β- polypeptides. Each polypeptide incorporates a haem group that reversibly binds oxygen.

• Within each subfamily, genes are coordinately turned on and turned off during embryonic, fetal, and adult stages of development.
• For both the alpha and beta subfamilies, this expression occurs in the same order in which the genes are arranged on the chromosome.
• The alpha subfamily spans more than 30 kb and contains three genes: the (zeta) gene, expressed only in the early embryonic stage, and two copies of the gene, expressed during the fetal (α₁) and adult stages (α₂).
• In addition, two nonfunctional pseudogenes (ψ z and ψ) are present in the cluster. Pseudogenes are designated by the prefix ψ (psi), followed by the symbol of the gene that they most resemble. Thus, the designation ψ α₁ indicates a pseudogene of the adult α, gene.

• Pseudogenes are non-functional versions of genes that resemble other gene sequences but contain significant nucleotide substitutions, deletions, and duplications that prevent their expression.
• The organization of the alpha subfamily members and the location of their introns and exons reveal several interesting features.
• First, as is common in eukaryotes, the DNA encoding the three functional genes occupies only a small portion of the region containing the subfamily. Most of the DNA in this region is intergenic spacer.
• Second, each functional gene in this subfamily contains two introns at precisely the same positions.
• Third, the nucleotide sequences within corresponding exons are nearly identical in the z and genes.
• Both genes encode polypeptide chains of 141 amino acids. However, their intron sequences are highly divergent, even though they are about the same size. Significantly, much of the nucleotide sequence of each gene is contained in these noncoding introns.

“Common challenges in understanding multigene families effectively: FAQs provided”

• The human β- globin gene cluster is longer than the α- globin cluster and contains five genes spaced over 60 kb of DNA. As with the alpha subfamily, the order of genes on the chromosome parallels their order of expression during development.
• Of the five genes, three are expressed before birth. The ε(epsilon) gene is expressed only during embryogenesis, while the two nearly identical γ(gamma) genes (Gγ and Aγ) are expressed only during fetal development.
• The polypeptide products of the two y-genes differ only by a single amino acid. The two remaining genes, δ(delta) and β(beta), are expressed following birth. Finally, a single pseudogene ψβ₁ is present within the subfamily.

All five functional genes encode proteins with 146 amino acids and have two similarly sized introns at the same positions. The second intron in the P gene is significantly larger than its counterpart in the functional a genes.

• These similarities reflect the evolutionary history of each subfamily and the events such as gene duplication, nucleotide substitution, and chromosome translocations that produced the present-day globin superfamily.
• It has been shown that in these sequences (cluster genes and p cluster genes), the coding regions show little divergence, but spacers in between the genes show considerable diversity as in the case in many other gene families.
• The only explanation for this is that the protein function puts a constraint on the evolution of the coding sequence, while no such constraint is exercised in the spacer region which is fast evolving. Based on the degree of similarity in amino acid sequences of globin of different mammals, evolutionary trees have been prepared in the past.

Actin Gene Family In Eukaryotes: Actin is a cytoskeleton protein. Actin genes represent a multigene family, where the members are homologous, but non-identical, giving rise to slightly different variants so that different members function either at different times in the same tissue or in different tissues at the same time.

“Steps to explain types of multigene families: Simple vs complex: Q&A guide”

For example, a minimum of six closely related polypeptides are synthesized in different cells at different times in mammals; the number may be larger in some flowering plants. The actions may be broadly classified as a,(3 and y chains.

• Of these, actins make the contractile apparatus of the muscle, while [3 and y actins are found together in almost all nonmuscle cells.
• There may be variants in each of these three classes, making up to 20-30 actin-related sequences in the human genome; humans have six genes for actin.
• In several plant systems (for example., tobacco, potato, etc.) also, more than 20 related but variant actin sequences have been reported.
• Actin is extraordinarily well-conserved among eukaryotes. The amino acid sequence of actions from different species is usually about 90% identical. Yeast actin and Drosophila muscle actin are 89% identical.

Other multigene families with divergent members: Other multigene families have divergent members; for example.,

1. Albumin, a fetoprotein (major component of blood plasma), serine proteases, the interferons (for defense against a viral infection), and the immunoglobulins;
2. Chorion proteins make the major components of egg shells in insects.

Multigene families with identical genes: In some cases, for producing large quantities of a gene product, many copies of identical genes are required. These genes may occur as repeat units, each of which comprises

1. A coding region, which is highly conserved to produce the same gene product and
2. A spacer region may show divergence. Examples of these multigene families are the following

Multiple copies of histone genes. The organization of the histone genes is a variation on the theme established in the globin gene family.

“Role of gene duplication in multigene family evolution: Questions answered”

• Here a cluster of five related, but nonidentical genes, separated from each other by highly divergent intergenic spacer regions, is tandemly repeated many times.
• As we already know histones are positively charged (basic) proteins which interact with the negatively charged phosphate groups of DNA to form nucleosomes, the chromatin units.
• In rapidly diving cells (for example., a cleaving fertilized egg of a frog), histone synthesis must keep pace with DNA replication, and the necessary quantity of histones must appear each time, as one cell cycle runs into the next.
• Multiple copies of the histone Histones, cluster allow the coordinate synthesis of DNA and histone during the S phase of the cell cycle. Many interesting correlations support this idea.
• Yeast cells, with much less DNA than other eukaryotes, have only two clusters of four histone genes(they lack histone HI).
• In contrast, clusters of five histone genes are tandemly repeated from 10 to 800 times in many complex organisms. Mitotic cell division is very rapid during development in sea urchins and newts and these organisms have one of the largest set of histone gene clusters.

Numbers Of Histone Gene Clusters In Selected Eukaryotes:

“Early warning signs of gaps in understanding multigene family basics: Common questions”

Polarity differences in transcription: Beyond its tandem arrangement, the histone gene family shows several other differences from other families.

• First, almost all histone genes lack introns. Second, individual genes within clusters of a given species often orient in opposite directions concerning transcription.
• As shown, the arrangement of genes and their polarity of transcription, shown by the direction of the arrow, vary in the sea urchin and Drosophila, two organisms where this cluster has been extensively studied. Studies also show polarity differences in other organisms.

• Despite polarity differences, the amino acid sequences of the various histones (reflecting the DNA encoding each one) are remarkably similar in highly divergent organisms. The homology of histone genes is one of the best examples of sequence conservation through evolution.
• Another interesting feature of the multigene family of histone genes observed in Drosophila is the presence of an ‘ attachment site’1 per repeat unit; 100 such repeat units being present in each haploid genome.
• With the help of the attachment site, the genes (DNA) remain attached to the nuclear scaffold. These attachment sites help in transcription.
• In mice and humans, members of the histone gene family are clustered but do not form tandem arrays. A region at the distal tip of the long arm of human chromosome 7 contains all copies of the histone gene family, interspersed with other nonhistone genes.
• Therefore, no single pattern of histone gene organization applies to all organisms.
• Ribosomal RNA (rRNA) genes in tandem arrays. For the production of about 10 million ribosomes per eukaryotic cell, rRNA genes are present in multiple copies at nucleolar organizing regions(NOR) of specific satellited chromosomes.

“Asymptomatic vs symptomatic effects of ignoring multigene family principles: Q&A”

The number of these genes may vary from 50 to 30,00 in a cell and this number may be unequally distributed on NORs if more than one such loci are present.

The DNA comprising these genes is called rI)NA (ribosomal DNA), which is repetitive. Each repeat unit has

1. A coding region with genes that specify 18S, 5.8S and 28S rRNA molecules;
2. A spacer region called intergenic spacer (IGS) and
3. Internal transcribed spacers (ITS) one each between 18S and 5.8S genes and between 5.8S and 28S genes. Because parts of the intergenic spacer (IGS) adjoining region, known as an external transcribed spacer (ETS), is also transcribed, the use of the term non-transcribed spacer (NTS) for the whole spacer region has been considered to be inappropriate. The NTS makes only a part of IGS, the remaining part of IGS being ETS.

The IGS region in its turn has a region consisting of a tandem array of variable number of subrepeats ranging from 100 – 300 bp (bp = base pairs) in length. The variation in the number and size of the sub-repeats in IGS is responsible for the variation in the length of the repeat units (IGS + coding region).

The rRNA repeat units are usually looped off from the main chromosome fibers, in the form of extended threads at the nucleolar organizing region. These loops, in association with specific proteins, form the nucleoli, where rRNA synthesis and processing take place.

“Can targeted interventions improve outcomes using multigene family knowledge? FAQs provided”

• The number of nucleoli and the corresponding number of nucleolar organizing chromosomes in an organism may vary from one to several.
• At each locus (NOR), rDNA repeat units may evolve independently both in length and also in the nucleotide sequence of the spacer region.
• As a result of this, the length of the repeat unit including coding and spacer regions (non-transcribed spacer or NTS; external transcribed spacer or ETS and internal transcribed spacers or ITS) varies from about 7 kb (kilobase) pairs to 14 kb. In wheat and barley, it is usually 9 kb to 10 kb.

Transfer RNA (tRNA) Mulligan Families: The genes for each of the different tRNAs are also found in multiple copies to meet the heavy demands of the cell for the production of tRNA molecules. Ten to several hundred genes for each IRNA are present in each haploid genome.

• While in some cases with fewer copies of a gene, these copies are dispersed, in other cases such as in Xenopus (a load, an amphibian), tandem repeats of long sequences (each repeat having genes for several different tRNAs) are found.
• In Xenopus, for example, a 3.2 kb repeat length has 8 genes, 2 for tRNA and six others, for six different 3′ tRNA molecules.

• In still other cases (for example., Drosophila) genes for completely different tRNA species arc clustered over a length of several thousand base pairs. Sometimes, tRNA pseudogenes are also found.
• Small nuclear RNA (Sn RNA) genes. Small nuclear RNAs (snRNAs) are found in abundance in the nuclei of all eukaryotes and represent neither IRNA nor rRNA.
• Six snRNAs, which are usually found, range from 100 (U6) to 215 (U3) nucleotides in length and are involved in RNA processing through the formation of snRNP (small nuclear ribonucleoproteins).
• SnRNAs are encoded by multiple copies of identical genes organized in tandem arrays of repeat units, each SnRNA gene being flanked on either side of spacer DNA ranging from 800 bp to 45,000 bp in length in different organisms.

“Steps to incorporate AI into analyzing multigene family evolution: Questions and answers”

• DNA of genes of Sn RNAs is reported to have a higher ratio( 10:1) of pseudogenes than functional genes. In humans, for example, there are 30 genes, for U1 (an SnRNA) on chromosome 1 and 500 to 1000 pseudogenes distributed throughout the genome.
• Multigene families for storage proteins in crop plants. Storage proteins of several crop plants have been reported to be encoded by multigene families and are represented mainly as prolamins ( major storage proteins in cereals; soluble in aqueous alcohol) and oxglobulins (major storage proteins in legumes; soluble in salt).
• Three to ten genes for each of the ten prolamin families in wheat; and 18 genes belonging to three families of globulin genes are known in pea.
• Homologies in genes coding for prolamins and globulins in different crop plants have been reported. Their multiplicity appropriately meets the demand for rapid synthesis of these proteins in the developing seeds.

“Role of digital tools in improving precision with multigene family tracking: FAQs explained”

Concerted Evolution Of Multigene Families

Multigene families are found to have mechanisms that prevent the individual copies from accumulating mutations and hence diverging away from the functional sequence. This is called concerted evolution.

Thus, if one copy of the family acquires an advantageous mutation then that mutation can spread throughout the family until all members possess it. Following genetic mechanisms may be involved in such homogenization of concerted evolution:

1. Gene conversion: Gene conversion can result in the sequence of one copy of a gene being replaced with all or part of the sequences of a second copy. Multiple gene conversion events could therefore maintain identity among the sequences of the individual members of a multigene family.
2. Unequal crossing over Unequal crossing over may lead to duplications or deletions leading to homogenization, i.e., either mutant member will be deleted or multiply and spread throughout the gene family. In yeast, it has been reported that the rates of gene conversion and unequal crossing-over are higher than the rate of mutations so that homogeneity can be maintained.

Further, certain specific sequences are also located in the multiple gene families which stimulate higher rates of recombination in a multigene family. In rDNA, the NTS (non-transcribed spacer) region is a hot spot of recombination thus facilitating homogenization.

The post Multigene Families – Types, Evolution and Examples appeared first on BDS Notes.

Bacteria are exposed to a wide range of environmental conditions. For example, E. coli may encounter rapidly changing growth conditions as they pass from the mammalian intestinal tract to sewer systems to polluted rivers, lakes, ponds, and so on.

• Each of these ecological niches will provide different organic molecules for use as energy sources. From this fact, it can be concluded that natural selection will have preserved those organisms that have evolved ways of adapting; to the wide range of environmental conditions encountered f during their evolution.
• Indeed, the available information f indicates that most prokaryotes such as E. coli exhibit remarkable capacities to adapt to diverse environmental; conditions.
• To a great extent, the adaptability of bacteria and other? prokaryotes depend on their ability to “turn on” and “turn off’ the expression of specific sets of genes in response to the specific demands of the environmental milieu (i.e., surroundings).
• In other words, prokaryotes exhibit an outstanding ability to regulate the expression of specific genes in response to environmental signals.

The expression of particular genes is “turned on” when the products of these genes are needed for growth in a given environment. Their expression is “turned old when their products are no longer needed for growth in the existing environmental surroundings.

• The ability of an organism to regulate gene expression in this way will increase its overall “fitness’ (i.e., its ability to grow and leave progeny under a variety of environmental conditions).
• The synthesis of gene transcripts (i.e., various types of RNA molecules) and translation products (i.e., proteins) require the expenditure of a great amount of energy.
• The chromosome of the bacterium Escherichia coli, a single-celled organism, consists of a single circular DNA molecule of about 4.6 × 10⁶ nucleotide pairs. This DNA encodes approximately 4300 proteins, although only a fraction of these are made at any one time.
• The expression of many of them is regulated according to the available food in the environment (see Alberts et al, 2002).

By “turning off the expression of genes when their products are not needed, an organism can avoid wasting energy to synthesize products that maximize the growth rate in the existing environmental surroundings.
Here, one can ask three sorts of questions:

1. What are the mechanisms by which these organisms regulate gene expression in response to changes in the environment?
2. Is there a single mechanism by which the expression of different genes or sets of genes are regulated?
3. Or are different genes controlled by different mechanisms?

Some genes, for example, the genes specifying ribosomal RNAs, ribosomal proteins and transfer RNAs, are certainly expressed at some time in virtually all cells regardless of the environmental conditions. The products of these genes are required for the growth of all cells in all environments.

• However, the products of many other genes are required for growth only in certain environments, and the expression of these genes is regulated such that the products are synthesized only when they are needed.
• As a result, the expression of these genes is continually being “turned on” and “turned off’ in response to changes in the environment.
• From this discussion, one can conclude that gene expression can be (and is) regulated at several different levels: for example, transcription, mRNA processing, mRNA turnover, translation, and enzyme function.
• However, extensive data indicate that the regulation of transcription is the most important mode of control of gene expression, at least in prokaryotes.
• The regulatory mechanisms of transcription in both prokaryotes and eukaryotes are of two basic types. The first, and best-understood, category includes mechanisms involved in the rapid turn-on and turn-off of gene expression in response to environmental changes.
• Such sort of mechanisms are very significant in microorganisms (bacteria) because these organisms are exposed to sudden changes in the environment.

“Understanding bacterial gene regulation through FAQs: Q&A explained”

They provide microorganisms with a great deal of plasticity, and an ability to rapidly adjust their metabolic processes to achieve maximal growth and reproduction under highly variable environmental conditions.

• These quick-responding on-off switches seem to be less important in higher eukaryotes. This might be expected since the circulatory systems of higher eukaryotes buffer their cells against many sudden environmental changes.
• The second major category of regulatory mechanisms includes the so-called preprogrammed circuits of gene expression. In these cases, some event (for example., infection by a virus) triggers the expression of one set of genes.
• The product (or products) of one (or more) of these genes function by turning off the transcription of the first set of genes and/or turning on the transcription of the second set of genes.
• In turn, one or more of the products of the second set act by turning on a third set, and so on. In these cases, the sequential expression of genes is genetically preprogrammed, and the genes usually cannot be turned on out of sequence. Such preprogrammed sequences of gene expression in viral infections are well worked out.
• In most of these preprogrammed sequences, it seems the circuit is cyclical. For example, in viral infections, some event associated with the packaging of the viral DNA or RNA inside the protein coat somehow seems to reset the program so that the first set of genes will again be expressed when a progeny virus subsequently infects another host cell.

Discovery Of Prokaryotic Gene Regulatory Proteins: Genetic analyses in bacteria carried out in the 1950s provided the first evidence for the existence of gene regulatory proteins that turn specific sets of genes on or off. One of these regulators, the lambda repressor, is encoded by a bacterial virus, the bacteriophage lambda.

• The repressor shuts off the viral genes that code for the protein components of new virus particles and thereby enables the viral genome to remain a silent passenger in the bacterial chromosome, multiplying with the bacterium when conditions are favorable for bacterial growth. The lambda repressor was among Gene regulatory proteins.
• the first gene regulatory proteins to be characterized. Other bacterial regulators respond to nutritional conditions by shutting off genes encoding specific sets of metabolic enzymes when they are not needed.
• For example, lac repressor, the first of these bacterial proteins to be recognized, turns off the production of the proteins responsible for lactose metabolism when this sugar is absent from the medium.
• The first step toward understanding gene regulation was the isolation of mutant strains of bacteria and bacteriophage lambda that were unable to shut off specific sets of genes.

“Importance of studying gene expression regulation in bacteria for biology students: Questions explained”

• It was proposed at the time, and later proven, that most of these mutants were deficient in proteins acting as specific repressors for these sets of genes.
• Because, these proteins, like most gene regulator proteins, are present in small quantities, it was difficult and time-consuming to isolate them. They were eventually purified by fractionating cell extracts.
• Once isolated, the proteins were shown to bind to specific DNA sequences close to the genes that they regulate.
• The precise DNA sequences that they recognized were then determined by a combination of classical genetics, DNA sequencing, and DNA footprinting experiments.

Constitutive Genes And Inducible Genes

Certain gene products, such as tRNA molecules, rRNA molecules, ribosomal proteins, RNA polymerase components (polypeptides), and other enzymes catalyzing metabolic processes that are commonly known for cellular “housekeeping” functions, are essential components of almost all living cells.

• Genes that specify products of this type are continually being expressed in most cells. Such genes are said to be expressed constitutively and are called constitutive genes.
• The rest of the gene products are needed for cell growth only under certain environmental conditions. Constitutive synthesis of such gene products would be wasteful, using energy that could otherwise be utilized for more rapid growth and reproduction under the existing environmental conditions.
• The evolution of regulatory mechanisms that would provide for the synthesis of such gene products only when and where they were needed would provide organisms with a selective advantage over organisms lacking these mechanisms.
• This undoubtedly explains why presently existing organisms, including the “primitive” bacteria and viruses, exhibit highly developed and very efficient mechanisms for the control of gene expression.
• E.coli and most other bacteria are capable of growth using any one of several carbohydrates (for example., glucose, sucrose, galactose, arabinose, lactose) as an energy source. If glucose is present in the environment, it will be preferentially metabolized by E. coli cells. In the absence of glucose, however, E. coli cells can grow very well on other carbohydrates.

• Cells of E. coli growing in a medium containing the sugar lactose, for example, as the sole carbon source synthesize two enzymes, β-galactosidase and β-gaIactoside permease, that they are uniquely required for the catabolism of lactose (Note: A third enzyme, α-galactoside transacetylase is also synthesized).
• The enzyme β- galactosidase cleaves lactose into glucose and galactose, and the enzyme β-galactoside permease pumps α-galactosides into the cell.
• Neither of these enzymes is of any use to E. coli cells when present in an environment not containing lactose. The synthesis of these two enzymes, of course, requires the utilization of a great amount of energy (in the form of ATP and GTP).
• Thus, E. coli cells have evolved a regulatory mechanism by which the synthesis of these lactose catabolic enzymes is turned on in the presence of lactose and turned off in its absence. In natural environments (such as intestinal tracts and sewers), E. coli cells probably encounter an absence of glucose and the presence of lactose relatively rarely.
• Most of the time, therefore, E. coli cells growing on a carbohydrate other than lactose are transferred to a medium containing lactose as the only carbon source, they rapidly begin synthesizing the enzymes required for lactose utilization.
• This process, by which the expression of genes is turned on in response to a substance in the environment, is called induction. Genes whose expression is so regulated are called inducible genes.

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• The substances or molecules responsible for induction are known as inducers (for example., in this case, lactose is inducer).
• Enzymes that are involved in catabolic (i.e., degradative) pathways, such as in lactose, galactose, or arabinose utilization, are characteristically inducible. Research made it evident that induction occurs at the level of transcription.
• Induction alters the rate of synthesis of enzymes, not the activity of existing enzyme molecules. Induction should not be confused with enzyme activation, in which the binding of a small molecule to an enzyme increases the activity of the enzyme (but does not affect its rate of synthesis).
• Further, E. coli and other bacteria possess the metabolic capacity to synthesize most of the organic molecules (such as amino acids, purines, and vitamins) required for their growth.
• For example, E. coli has five genes coding for enzymes that are required in the synthesis of tryptophan (an amino acid).

These five genes must be expressed in E. coli cells growing in an environment devoid of tryptophan to provide adequate amounts of this amino acid for ongoing protein synthesis.

• When E. coli cells are present in an environment containing concentrations of tryptophan sufficient to support optimal growth, the continued synthesis of the tryptophan biosynthetic enzymes would be a waste of energy, because these bacteria can take in external tryptophan.
• Thus, a regulatory mechanism has evolved in E. coli by which the synthesis of the tryptophan is present in the external milieu (surrounding environment).
• This process of “turning off’ the expression of sets of genes is called repression. A gene whose expression has been turned off in this way is said to be repressed; when its expression is turned on, a gene of this type is said to be depressed.
• Enzymes that are components of anabolic (biosynthetic) pathways are frequently subject to repression (are repressible).
• Repression, like induction, occurs at the level of transcription. However, repression should not be confused with feedback inhibition, in which the binding of an end product to the first enzyme in a biosynthetic pathway inhibits the activity of the enzyme (but does not affect its synthesis).

Transcriptional Control Mechanisms

In bacteria, there occur several mechanisms of gene regulation at the level of transcription. A notable method depends on whether the enzymes being regulated act in catabolic (degradative) or anabolic (synthetic) metabolic pathways.

• For example, in a multistep catabolic system, the availability of the molecule to be degraded commonly determines whether the enzymes in the pathway will be synthesized. In contrast, in a biosynthetic pathway, the final product is often the regulatory molecule.
• Even when a single protein molecule is translated from a monocistronic mRNA molecule, the protein may be autoregulated, i.e., the protein itself may inhibit initiation of transcription and high concentrations of the protein may cause less transcription of the mRNA that encodes the protein.
• The molecular mechanisms for each of the regulatory patterns differ greatly and are of the following two types – negative regulation and positive regulation. In a negatively regulated system, an inhibitor is present in the cell and prevents transcription.

• An antagonist of the inhibitor, called an inducer, is needed to allow initiation of transcription. In a positively regulated system, an effector molecule (which may be a protein, a small molecule, or a molecular complex) activates a promoter; no inhibitor must be abolished.
• Negative and positive regulation are not mutually exclusive, and some systems are both positively and negatively regulated, utilizing two regulators to respond to different conditions in the cell. Thus, a catabolic system may be regulated positively or negatively.
• In a biosynthetic (anabolic) pathway, the final product usually regulates negatively its synthesis; in the simplest type of negative regulation, the absence of the product increases its synthesis, and the presence of the product decreases its synthesis.

The Operon Model: Induction and repression of gene expression can be accomplished by essentially the same mechanism.

• This mechanism was first accurately described in 1961 when Francois Jacob and Jacques Monod, both 1965 Nobel Prize recipients, proposed the operon model to explain the regulation of genes encoding the enzymes required for lactose utilization in E. coli.
• Jacob and Monod proposed that the transcription of one or a set of contiguous structural genes (i.e. genes coding for polypeptides) is regulated by two controlling elements.
• One of these controlling elements called the regulator gene (or repressor gene), codes for a protein called the repressor; under the appropriate conditions, the repressor binds to the second element, the operator.
• The operator is always located contiguous to the structural gene or genes whose expression it regulates.

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• When the repressor is bound to the operator, transcription of the structural genes cannot occur, We now know that this results because the binding of the repressor to the operator stoanettlly prevents RNA polymerase (enzyme) from binding at the promoter site (the RNA polymerase binding site), which is always located contiguous with the operator sequence.
• The operator is usually located between the promoter and structural genes. (Note: The promoter was not recognized at the time of Jacob and Monod’s proposal but has since been shown to be an essential component of an operon).
• Thus, the complete contiguous unit, including the structural genes, the operator, and the promoter is called the operon (Gardner et al, 2002).
• In other words, an operon is a unit of prokaryotic gene expression that includes co-ordinately regulated (structural) genes and control elements (promoter operator) which are recognized by regulatory gene products (furnace et al., 2000).

• Whether the repressor will bind to the operator and turn off the transcription of the structural genes in an operon is determined by the presence or absence of effector molecules (small molecules such as amino acids and sugars) in the environment.
• In the case of inducible operons (for example., lac operon), these effector molecules are called inducers. Those active on repressible operons (for example., trp operon) are called co-repressors and act by binding to (or forming a complex with) the repressors.

The only essential difference between inducible operons and repressible operons is whether the naked repressor or the repressor effector molecule complex is active in binding to the operator.

1. In the case of an inducible operon, the free repressor binds to the operator, turning it off. When the effector molecule or the inducer (for example., allolactose in lac operon) is present, it binds to the repressor, that is, the repressor-inducer complex cannot bind to the operator.
   • Thus, the addition of an inducer turns on (or induces) the transcription of the structural genes in the operon.
2. In the case of a repressible operon (for example., trp operon), the situation is just reversed. The free repressor- cannot bind to the operator. Only the repressor-effector molecule (co-repressor) complex is active in binding to the operator.
   • Thus, transcription of the structural genes in the repressible operon is turned on in the absence of and turned off in the presence of the effector molecule (co-repressor). Except for this difference in the operator, repressible operons are comparable.

• A single mRNA transcript carries the coding information of an entire operon. Thus, the mRNAs of operons consisting of more than one structural gene are polygenic or polycistronic.
• For example, the tryptophan operon mRNA of E.coli is a huge macromolecule carrying the coding sequences that specify five different polypeptides because of their cotranscription, all the structural genes in an operon are coordinately expressed.

“Steps to explain operon systems: Lac operon vs Trp operon: Q&A guide”

Examples of Operons

Lac Operons (Inducible System): Lactose (milk sugar, a disaccharide) is a β-galactoside that E. coli can use for energy and as a carbon source after it is broken down into glucose and galactose. The enzyme that performs the breakdown is β-galactosidase:

There are very few molecules of β-galactosidase enzyme in a wild-type E. coli cell grown in the absence of lactose. Within minutes after adding lactose to the medium, however, this enzyme appears in large quantities within the bacterial cell.

When the synthesis of β-galactosidase (encoded by the lacZ or z gene) is induced, the production of two additional enzymes is also induced, β-galactoside permease (encoded by the lacY or y gene) and β-galactoside acetyltransferase (encoded by the lac A or a gene).

The permease (enzyme) is involved in transporting lactose into the cell. The transferase is believed to protect the cell from the build-up of toxic products created by β-galactosidase (enzyme) acting on sugars other than lactose, the transferase prevents β-galactosidase from cleaving.

• The regulator gene. Not only are three lac genes (z, y, and a) induced together, but they are adjacent to one another in the E. coli chromosome; they are transcribed on a single polycistronic messenger RNA.
• Induction involves the protein product of another gene, the regulator gene, or gene (lac I). Although the regulator gene is located adjacent to the three other lac genes (i.e., three structural genes), it is an independent transcriptional entity.
• The regulator gene specifies a protein, the repressor, that interferes with the transcription of the genes involved in lactose metabolism.
• The operator. For the repressor protein to exert its influence over transcription, there must be a control element (receptor site) located near the beginning of the P-galactosidase (lacZ) gene.

“Role of repressors and activators in bacterial gene regulation: Questions answered”

This control element is a region referred to as the operator, or operator site. the operator site is a sequence of DNA that the product of the regulator gene, the repressor, recognizes. When the repressor is bound to the operator, it interferes with RNA polymerase binding or prevents the RNA polymerase from achieving the open complex.

• In either case, transcription of the operon is prevented. The repressor is released when it combines with an inducer, a derivative of lactose called allolactose.
• Note that the promoter is not only recognized by RNA polymerase but also has elements near the initiation site of transcription. At this stage, an operon can be redefined as a sequence of adjacent genes all under the transcriptional control of the same promoter and operator.
• The nucleotide sequence of the lac operator region. The operator is referred to as the primary operator, O1, centered at +11. Two other operator sequences have been found.
• One, O2, is centered at +412. The third operator, O3, overlaps the C-terminal end of the i gene and is centered at 82.
• The structure of the repressor and its interaction with the operator site was worked out recently with X-ray crystallography. The functional repressor is a homotetramer of the protein product of a gene; i.e., it is formed from four identical copies of the repressor protein.

• Since each operator site has two-fold symmetry, two repressor monomer proteins bind to each operator site. The monomer is shaped so that it fits into the major groove of the DNA to locate the exact base sequence of the operator; it then binds at the point through electrostatic forces.
• A tetramer can bind to two of the operator sites at the same time, presumably O1 and O3 or O1 and O2. In the process, the DNA is formed into a loop.

• Induction of the lac operon. Before the operon can be “turned on” to produce lactose utilizing enzymes, the repressor will have to be removed from the operator.
• The repressor is an allosteric protein; when it binds with one particular molecule, it changes the shape of the protein, which changes its ability to react with a second particular molecule.
• Here the first molecule is the inducer allolactose and the second molecule is the operator DNA. When allolactose is bound to the repressor, it causes the repressor to change shape and lose its affinity for operator sequences.

• With allolactose (inducer) bound to the repressor, the ability of the repressor to bind to the operator is greatly reduced, by a factor of 10³. Since no covalent bonds are involved, the repressor simply dissociates from the operator. After the repressor is released from the operator, RNA polymerase can now begin transcription.
• The three structural genes of the lac operon are then transcribed and subsequently translated into their respective proteins. This system of control is very efficient.
• The presence of the lactose molecule permits transcription of the structural genes of the lac operon, which acts to break down the lactose.
• After all the lactose is metabolized, the repressor molecule returns to its original shape and can again bind to the operator. The system is “turned off”.

Lac Operon Mutants (Genetic Manipulations Of Lac Operon): Verification of the lac operon system came about through the use of mutants and partial diploids (merozygotes) of the lac operon well before DNA sequencing techniques had been developed.

• The structural (i.e., enzyme specifying) genes of the lac operon, z, y, and a, all have known mutant forms in which the particular enzyme does not perform its function. These mutant forms of the enzymes are z⁺, y⁺, and a⁺.
• Merozygotes or partial diploids in E. coli can be created through sexduction because some strains of E. coli have the lac operon incorporated into a factor.
• Since the F⁺ strain can pass the F^‘ particle into the F^– strain, lac operon diploids can be formed. By careful manipulation, various combinations of mutations can be looked at in a diploid state.

“How does feedback inhibition regulate bacterial genes? FAQ explained”

Constitutive Mutants are mutants in which the three lac operon structural genes are transcribed at all times, i.e., they are not turned off even in the absence of lactose. Constitutive production of enzymes can come about in several ways.

• A defective repressor, produced by a mutant regulator gene, will not turn the system off, nor will a mutant operator that will no longer bind the normal repressor.
• The regulator constitutive mutants are designated f; the operator constitutive mutants are designated Cf. Both types of mutants produce the same phenotype: constitutive expression of the three lac operon genes.
• When a new mutant is isolated, it is possible to determine whether it is caused by a regulator or operator mutation.
• For example, we can determine the exact location of a mutation on the bacterial chromosome by standard mapping techniques or, more recently, by DNA sequencing.
• Alternatively, the Jacob and Monod model predicts different modes of action for the two types of mutations.
• In merozygotes, a constitutive operator mutation affects only the operon it is physically a part of. Operator mutations are therefore called cis-dominant.
• However, a constitutive i-gene mutation, since it works through an altered protein, is recessive to a wild-type regulator gene in the same cell, regardless of which operon (chromosomal or F’ factor) the mutation is on.
• Constitutive regulator mutations are, therefore, trans-acting. (Note: If two mutations are on the same piece of DNA, they are in the cis configuration.
• If they are on different pieces of DNA, they are in the trans configuration). Transacting mutations usually work through a protein product that diffuses through the cytoplasm. C/sacting mutants are changes in recognition sequences on the DNA.

• Thus, when the bacterium has a regulator constitutive mutation (f); the cell has constitutive production of the operon.
• If the wild-type regulator is introduced in an F’ plasmid, the normal (i.e., inducible) phenotype is restored because the F’ i⁺ allele is dominant to the chromosomal mutation- the i⁺ gene regulates both the chromosomal and F’ operons.
• Hence, both operons are inducible. We do not need to be bothered about the other components of the F’ plasmid, because it carries a z^–allele; only the activity of the chromosomal operon will be observed. When the chromosomal operon carries an operator constitutive mutation; the cell also has constitutive production of the operon.
• When a wild-type operator is introduced into the cell in an F’ plasmid, the cell still has the constitutive phenotype because the operator allele on the F’ plasmid does not control the bacterial operon; the lac operon bacterial chromosome will be continually transcribed.
• The chromosomal operon has a cls-domain operator mutation that has a constitutive phenotype. One should also note that only the bacterial chromosome determines the phenotype because the introduced F’ plasmid has a z^–allele.

Other types of lac operon control mutations: Jacob and Monod’s operon model has been supported by some other types of mutations. For example, a super-repressed mutation was discovered. This mutation represses the operon even in the presence of large quantities of the inducer.

• Thus, the repressor seems to have lost the ability to recognize the inducer. The gene product is acting as a constant repressor, rather than as an allosteric protein.
• In an i^S/i⁺ merozygote, both operons are repressed because the i^S repressor binds to both operators. Another mutation reproduces much more of the repressor than normal and presumably represents a mutation of the promoter region of the I gene (see Tamarin, 2002).
• In 1966, W. Gilbert and B.MnHcr- Hill isolated the late repressor and thereby provided final proof of the validity of the method. M. Ptasline and his colleagues in 1992 isolated the repressor for phage λ operons.

Catabolite Repression Of Lac Operon (Glucose Effect)

An unusual feature of the lac operon and other operons that code for enzymes that catabolize certain sugars (e.g., arabinose, galactose) is that they are all repressed in the presence of glucose.

• That is, glucose is catabolized in preference to other sugars. This is called the glucose effect or catabolic repression.
• The mechanism of catabolic repressor involves cyclic AMP(cAMP). In eukaryotes, cAMP acts as a second messenger, an intracellular messenger regulated by certain extracellular hormones.

• Geneticists were surprised to discover cAMP in E. coli, where it works in association with another regulatory protein, the catabolite activator protein (CAP), to control the transcription of certain operons.
• When glucose is not present, cAMP combines with CAP, and the CAP-cAMP complex binds to a distal part of the promoter of operons with CAP sites (for example., the lac operon;).
• This binding increases the affinity of RNA polymerase for the promoter because, without the binding of the CAP-cAMP complex to the promoter, the transcription rate is very low.
• The uptake of glucose by E.coli cells causes the loss of cAMP from the cell, probably by inhibiting adenyl cyclase, and thus lowers the CAP-cAMP level.
• The transcription rate of operons with CAP sites will be reduced. The same reduction of transcription rates has been observed in mutant strains of E. coli when this part of the distal end of the promoter is deleted.

“Early warning signs of gaps in understanding bacterial gene regulation basics: Common questions”

• The binding of CAP-cAMP to the CAP site causes the DNA to bend more than 90 degrees. This bending by itself, may enhance transcription, making the DNA more available to the RNA polymerase enzyme.
• During the process of initiation of transcription, the CAP is in direct contact with RNA polymerase.
• This was shown by photocrosslinking studies in which CAP was treated with a cross-linking agent that bound the subunit of RNA polymerase when irradiated with UV light. (Photocrosslinking is a technique used to determine which moieties (proteins, DNA) are nearby during a particular process).
• For the two proteins to cross-link, they must be in direct contact during the initiation of transcription. Catabolite repression is an example of positive regulation: The binding of the CAP-cAMP complex at the CAP site enhances the transcription rate of the transcriptional unit.
• Thus, the lac operon is both positively and negatively regulated; the repressor exerts negative control, and the CAP-cAMP complex exerts positive control of transcription.

TRP Operon: The catabolic operons or inducible operons are activated when the substrate that is to be catabolized enters the cell. Anabolic operons function in the reverse manner. They are turned off (repressed) when their end product accumulates beyond the needs of the cell.

• Two entirely different mechanisms seem to control the transcription of repressible operons. The first mechanism follows the basic scheme of inducible operons and involves the end product of the pathway.
• The second mechanism involves a secondary structure in messenger RNA transcribed from an attenuator region of the operon.
• Tryptophan synthesis. Tryptophan operon is an example of a repressible system. It contains the five genes that code for the synthesis of the enzymes that build tryptophan amino acid, starting from chorismic acid. It has a promoter operator sequence (p, o) as well as its regulator gene (trpR).

Operator Control: In trp popcorn, the product of the trpR gene is called a repressor. It is inactive by itself it does not recognize the operator sequence of the operon. The repressor only becomes active when it combines with tryptophan.

• Thus, when tryptophan builds up, enough is available to bind with and activate the repressor. Tryptophan is, thus, called a corepressor. The compressor-repressor complex then recognizes the operator, binds to it, and prevents transcription by RNA polymerase.

“Asymptomatic vs symptomatic effects of ignoring bacterial gene regulation principles: Q&A”

• After the available tryptophan in the cell is used up, the diffusion process causes tryptophan to leave the repressor, which then detaches from the trip operator. Since the transcription process no longer is blocked, so proceeds normally (i.e., the operon is now derepressed).
• Transcription continues until enough of the various enzymes are synthesized to again produce an excess of tryptophan. Some tryptophan molecules become available to bind to the repressor and make a functional complex, and the operon is again shut off.
• This process is repeated to ensure that tryptophan is being synthesized as needed. This regulation is modified, however, by the existence of the second mechanism for regulating repressible operons- attenuation.

Attenuator-controlled trp operon: Details of the second control mechanism of repressible operons have been explained primarily by C. Yanofsky and his colleagues (1984), who worked with the tryptophan operon in E. coli.

This type of person is controlled by its attenuator region and a similar system is also found in operons involved in the synthesis of other amino acids such as leucine and histidine. (Attenuation means “premature termination”).

Leader Transcript: In the top operon, an attenuator region lies between the operator and the first structural gene. The messenger RNA (mRNA) transcribed from the attenuator region, termed the leader transcript, has been sequenced, revealing two surprising and interesting facts.

• Four subregions of the mRNA have base sequences that are complementary to each other so that three different stem-loop structures can form in the mRNA.
• Depending on conditions, regions 1-2 and 3-4 can form two stem-loop structures, or regions 2-3 can form a single stem-loop.
• When one stem-loop structure is formed, the others are acquired. As we will observe, the particular combination of stem-loop structures determines whether transcription continues.

Leader peptide gene: Another fact has been obtained by sequencing the leader transcript and it has been found that there is a small gene coding information for a peptide from base 27 to 68. The gene for this peptide is called the leader peptide gene.

It codes for fourteen amino acids including two adjacent tryptophan. These adjacent tryptophan codons are critically important in attenuator regulation. The proposed mechanism for this regulation follows.

Assuming that the operator site is available to RNA polymerase, transcription of the attenuator region will begin. As soon as the 5′ end of the mRNA for the leader peptide gene has been transcribed, a ribosome attaches and begins translating this mRNA. Depending on the levels of amino acids in the cell, three different outcomes can take place.

Excess Tryptophan: If the concentration of tryptophan in the cell is such that abundant tryptophan-tRNAs exist, translation proceeds down that leader peptide gene. The moving ribosome overlaps regions 1 and 2 of the transcript and allows stem-loop 3-4 to form.

• This stem-loop structure is called a terminator or attenuator stem, causing transcription to be terminated. Note that stem-loop 3-4, the terminator stem, followed by a series of uracil (U)- containing bases, is a rho-independent transcription terminator.
• Hence, when existing quantities of tryptophan, in the form of tryptophan-tRNA, are adequate for translation of the leader peptide ‘gene’, the transcription is terminated.

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Tryptophan Starvation: If the quantity of tryptophan tRNA is lowered, the ribosome must wait at the first tryptophan codon until it acquires a Trp-tRNATrp. The stalled ribosome will permit stem-loop 2-3 to form, which precludes the formation of the terminator stem-loop (3-4).

• In this configuration, transcription is not terminated, so that eventually, the whole operon is transcribed and translated raising the level of tryptophan in the cell. The stem-loop 2-3 structure is referred to as the preemptor stem.
• This is important that the preemptor stem is not an independent transcription terminator and thus, without the rho protein present, will not terminate transcription.

General Starvation: A final configuration is possible. Here, no ribosome interferes with stem formation, and presumable stem-loops 1-2 and 3-4 (terminator) form. This configuration also terminates transcription because of the terminator stem.

• It is believed that this configuration occurs if the ribosome is stalled on the 5′ side of the trp codons, which happens when the cell is starved for other amino acids. Presumably, it makes no sense to manufacture tryptophan when other amino acids are in short supply.
• Hence, the cell can carefully bring up the levels of the various amino acids in the most efficient manner. The tryptophan operon in bacilli such as Bacillus subtilis, is also controlled by attenuation, but a secondary structure in the mRNA transport is induced by binding not the ribosome, but a trp RNA-binding attenuation protein (TRAP) (Antson et al, 1999).

The Ara Operon: The almost complete mechanism by which the lac and trp operons of E. coli are regulated, is known and supported by an extensive body of experimental data. However, the arabinose (ara) operon of E. coli exhibits much more complex patterns of regulation that are still not completely understood.

• In the lac and tip operons, the product of the regulator gene, the repressor, functions in a negative manner, turning off transcription of the operon.
• On the other hand, the catabolic activator protein (CAP) exerts a positive control over the lac operon by stimulating transcription of the operon.
• The major regulatory protein of the ara operon exhibits both negative and positive regulatory effects on the transcription of the structural genes of the operon, depending on the environmental conditions.
• Moreover, the regulatory components that control transcription of the ara operon include one element that acts from a distance of over 200 nucleotide pairs away from the promoter that it helps to control.

“Difference between inducible and repressible operons in bacteria: Q&A explained”

• The arabinose (ara) operon of E. coli contains three structural genes (araB, araA, and araD) that encode the three enzymes involved in the catabolism of arabinose. These three genes are cotranscribed on a single mRNA that is initiated at a promoter called PBad.
• Active transport of arabinose into cells is carried out by the products of genes araE, araF, and araG. These genes are located at sites quite distant from the araBAD operon of interest here and will not be further discussed.
• The major regulatory protein of the ara operon (i.e., the araCprotein) is produced from a transcript that is initiated at a promoter called Pc.
• The Pc promoter is only slightly over 100 nucleotide pairs away from P_BAD, but the two promoters initiate transcription in opposite directions.
• The araC protein acts as a negative regulator (a repressor) of transcription of the araB, araA, and araD structural genes from the P_BAD promoter in the absence of arabinose and cAMP. It acts as a positive regulator (an activator) of transcription of these genes from the P_BAD promoter when arabinose and cAMP are present.
• Thus, depending on the presence or absence of the effector molecules arabinose and cAMP, the araC regulatory gene products may exert either a positive or a negative effect on the transcription of the araB, araA, and araD structural genes.

Since the operon is subject to catabolite repression like the lac operon and thus to positive control by CAP and cAMP, induction of the ara operon depends on the positive regulatory effects of two proteins, the araC protein and CAP.

• The binding sites for these two proteins and RNA polymerase all appear to be located in a region of the ara operon historically called oral (I for induction), located between the three structural genes of the operon and the regulatory gene (araC). ara Once geneticists working on the problem of regulation of the ara operon thought that all the binding sites for the araC regulatory protein and cAMP-CAP complex were in the oral region.
• The surprising discovery was that repression of the ara operon depended on the binding of ofaraC protein at a site called araO₂ (O for operator, 2 because it was the second ara operator identified) located 211 nucleotide-pairs upstream (relative to the direction of the transcription from P_BAD) from the araC protein binding site in oral. (Note: operator araO₂– the first ara operator to be identified – controls the transcription of the araC regulator gene initiated at Pc).
• The currently accepted model for the repression of the ara operon is that the araC protein must bind (as a dimer) at both the aral site and the araO₂ site and that these proteins then bind to each other to form a DNA loop.
• When the loop structure is formed, it must prevent or interfere with the binding of RNA polymerase at the adjacent promoter (P_BAD) of the operon. In the presence of arabinose and cAMP, the ara operon is induced. Moreover, under these conditions, the ara C protein has been shown to become an activator of transcription of the operon.
• The arabinose- are protein complex and the cAMP-CAP complex must open the loop by binding at their aral sites. This, in turn, must permit RNA polymerase to bind at the PBAD site and initiate transcription of the ara structural genes.

Mode Of Working Of Gene Regulatory Proteins: Gene regulatory proteins must recognize specific nucleotide sequences embedded within their structure.

• It was originally thought that these proteins might require direct access to the hydrogen bonds between base pairs in the interior of the double helix to distinguish between one DNA sequence and another.
• It is now clear, however, that the outside of the double helix is studded with DNA sequence information that gene regulatory proteins can recognize without having to open the double helix.

Molecular Recognition: Gene regulatory proteins are found to contain structural motifs that read DNA sequences. Molecular recognition in biology generally relies on an exact fit between the surfaces of two molecules, and the study of gene regulatory proteins has provided some of the clearest examples of this principle.

• A gene regulatory protein recognizes a specific DNA sequence because the surface of the protein is extensively complementary to the specific surface features of the double helix in that region.
• In most cases, the protein makes a large number of contacts with the DNA, involving hydrogen bonds, ionic bonds, and hydrophobic interactions.
• Although individual contact is weak, the 20 or so contacts that are typically formed at the protein-helix interface add together to ensure that the interaction is both highly specific and very strong.
• DNA-protein interactions include some of the highest and most specific molecular interactions known in biology. DNA-protein interactions are of the following two types.

Helix-turn-Helix Motif: The first DNA-binding protein motif to be recognized was the helix-tum-helix. This was originally identified in bacterial proteins; but later on, this motif has been reported in hundreds of DNA-binding proteins from both eukaryotes and prokaryotes.

• This protein is constructed from two α-helices connected by a short extended chain of amino acids, which constitutes the “turn”. The two helices are held at a fixed angle, primarily through interactions between the two helices.
• The C-terminal helix is called the recognition helix because it fits into the major grooves of DNA; its amino acid side chains, which differ from protein to protein, play an important role in recognizing the specific DNA sequence to which the protein binds.
• Examples of helix-tum-helix proteins include tryptophan repressor, lambda cro, lambda repressor fragment, CAP fragment, etc.

“Most common complications of poorly understood bacterial gene regulation: FAQs”

Homeodomain Protein: Soon after the discovery of gene regulatory proteins in bacteria, genetic analyses in the fruitfly Drosophila led to the characterization of an important class of genes, the homeotic selector genes, that play a critical part in coordinating fly development.

• Mutations in these genes cause one body part in the fly to be converted into another, showing that the proteins they encode control critical developmental decisions.
• When the nucleotide sequence of several homeotic selector genes of Drosophila and higher animals was determined in the early 1980s, each proved to contain an almost identical stretch of 60 amino acids that defines this class of proteins and is termed the homeodomain.
• When the three-dimensional structure of the homeodomain was determined, it was shown to contain a helix-tum-helix motif related to that of the bacterial gene regulatory proteins, providing one of the first indications that the principle of gene regulation established in bacteria is relevant to higher organisms as well.
• More than 60 homeodomain proteins now have been discovered in Drosophila alone, and homeodomain proteins have been identified in virtually all eukaryotic organisms that have been studied, from yeasts to plants to humans.

The structure of a homeodomain that is bound to its specific DNA sequence. Whereas the helix-tum-helix motif of bacterial gene regulatory proteins is often embedded in different structural contexts, the helix-turn-helix motif of homeodomains is always surrounded by the same structure, suggesting that the motif is always presented in the same way.

Other Types Of Motifs Of Gene Regulatory Proteins Include The Following:

1. DNA-binding zinc finger motifs;
2. β-sheet proteins;
3. Leucine zipper motif; and
4. Heterodimerization.

Translational Control

In prokaryotic gene regulation at the translation level, the lifetime of a mRNA molecule may be genetically determined. Enzymatic degradation of mRNA is from the 5′ to the 3′ end, i.e., the end of the RNA that is first synthesized is also the end that is first degraded.

• The average lifetime of many mRNA molecules of E. coli is only two minutes at 37°C. The specific nucleotide sequences at the 5′ end may influence its susceptibility to enzymatic digestion.
• Further, catabolic enzymes are denied access to the mRNA when the ribosomes are coated at their 5′ ends (i.e., in the case of polyribosomes).
• Hence, the lifetime of mRNAs may also be correlated with the number of free ribosomes available at any given moment to translate mRNA molecules.

“Why are bacterial gene regulation mechanisms often misunderstood in practice? Questions answered”

Bacteria vary their rates of protein synthesis by varying their ribosomal content rather than by varying the translational rate. Example: In the lactose system of E. coli, there are three structural genes under the control of a common operator locus determining the production of

1. β- galactosidase,
2. Galactoside permease and
3. Galactoside acetylase.

These three proteins are produced in the respective ratios l.T/2:l/5, reflecting their respective locations relative to the 5′ (operator) end of the polycistronic mRNA in which they are coded (these differences are examples of translation regulations).

• Thus, there is a polarity gradient within the polycistronic mRNA that reduces the probability of cistron translation as a function of its distance from the 5′ end.
• It is hypothesized that ribosomes attach to different starting points (ribosome-binding sites) along the polycistronic mRNA at different rates as reflected by the amounts of the three proteins synthesized.

Post-translational control (Feedback Inhibition or End Product Inhibition): The expression of genes also can be regulated after proteins have been synthesized.

• This is called post-translational control of gene action. Feedback inhibition is a regulatory mechanism that does not affect enzyme synthesis, but rather inhibits enzyme activity.
• The end product of a biosynthetic pathway may combine loosely (if in high concentration) with the first enzyme in the pathway.
• This union does not occur at the catalytic site, but it does modify the tertiary structure of an enzyme and, hence, inactivates the catalytic site.
• This allosteric transition of protein molecule blocks its enzymatic activity and prevents the overproduction of end products and their intermediate metabolites.

Example: The studies on isoleucine synthesis in E. coli (Umbarger, 1961) demonstrated that the addition of isoleucine (the end product of a five-step conversion of threonine) to a culture of the bacteria resulted in immediate blocking of the threonine-ÿisoleucine pathway.

• In the presence of added isoleucine, the cells preferentially use this exogenous end product (i.e., isoleucine) and their isoleucine synthesis ceases.
• Moreover, the production of each of the five enzymes is not interfered with, but the action of an enzyme responsible for the deamination of threonine to aketobutyrate is inhibited by the end product, isoleucine.

“Cost of ignoring bacterial gene regulation principles vs benefits of systematic approaches: Q&A”

Operons Of Phage Lambda (Regulation Of Phage λ Life Cycles)

When a bacteriophage infects a bacterial cell, it has to express its genes in an orderly fashion; some gene products are needed early in infection, and other products are required during the later part of infection.

• Early genes usually control phage DNA replication; late genes usually determine phage coat proteins and the lysis of bacterial cells.
• A bacteriophage is considered most efficient if it expresses early genes first and late genes last in the infection process.
• Also, temperate phages have the option of entering into lysogeny with the cell, here, control processes determine which path is taken.

One generalization has been made regarding the phages that their genes are clustered into early and late operons, with separate transcriptional control mechanisms for each.

1. Lysis: Bursting of a cell by the destruction of plasma membrane following an infection by a virus.
2. Lysogenic: The state of a bacterial cell that has an integrated phage (prophage) in its chromosome.
3. Lysogenic Bacteria: Bacteria harboring temperate bacteriophages.
4. Temperate Phage: A phage (virus) that invades but may not destroy (lyse) the host
5. (bacterial Cell): however, it may continue into the lytic cycle.
6. Virulent Phage: A phage (vims) that destroys the host (bacterial) cell.

Phage λ (= lambda) is one of the best-studied bacteriophages. It has a double-stranded DNA (chromosome) of about 48,500 base pairs. Since it is a temperate phage, it can exist either vegetatively or as a prophage, integrated into the host chromosome.

• The life cycle of this phage is suitable for our analysis since its choice of pattern of life cycle is determined in an interesting and complex way.
• It provides a model system of operon controls. Here, complexity arises from having two conflicting life-cycle choices: one choice favors the lytic cycle and another choice prefers the nonlytic (i.e., lysogenic) phage of the life cycle.
• The expression of one of the two life-cycle alternatives, lysogenic or lytic cycles, depends on whether two repressors, Cl and cro, have access to the operator site. The Cl repressor acts to favor lysogeny: it expresses the lytic cycle and represses lysogeny.
• The operator sites, when bound by either Cl or cro, can either enhance or repress transcription. Certain other control mechanisms are involved in determining aspects of λ life cycle such as antitermination and multiple promoters for the same genes (see Ptashne and coworkers 1982; Ptashne, 1987, 1989).

Structure Of Phage λ Operons: Phage lambda (A.) shows a complex system of controls of both early and late operons, as well as controls for the decision of lytic infection versus lysogenic integration.

• The X genes are grouped into four operons: left and right, late, and repressor.
• The left and right operons contain the genes for DNA replication recombination and phage integration. The late operon contains the genes that determine phage head and tail proteins and lysis of the host cell. The sequence of events following phage infection is relatively well known.
• The genetic map of phage X is a circle, but the X chromosome has two linear stages in its life cycle. (Note: circular form of DNA is infective cellular form).
• In one linear form, its DNA is packed within the phage head, and in another linear form, the DNA of the phage becomes integrated into the host chromosome to form a prophage. These two linear forms of DNA do not have the same ends.

• The mature DNA which is packed within the phage heads before lysis of the cell, is flanked by cos sites. It results from a break in the circular map between the A and R loci. The prophage is integrated at the att site and the circular map is thus broken there at integration.
• The homologous integration sites on both X and the E.coli chromosome consist of a 15bp core sequence (called “0” in both), flanked by different sequences on both sides in both the bacterium and the phage.
• In the phage, the region is referred to as POP’ where P and P’ (P for phage) arc two different regions flanking the O core on the phage DNA.

In the bacterium, the region is called BOB’ where B and B’ (B for bacterium) arc two different regions flanking the O E.coli chromosome.

• Integration, which is a part of the lysogenic life cycle, requires the product of the λ int gene, a protein known as integrase, and is referred to as site-specific recombination.
• Later excision of the prophage, during the introduction, when the phage leaves the host chromosome to enter the lytic cycle, requires both the integrase and the protein product of the neighboring xis gene, the excisional (enzyme).

Following infection of the E.coli cell by a λ phage DNA circularizes, using the plementarity of the cos sites. Transcription begins, and within a very short time, the phage is producing virus progeny or entering the lysogenic cycle and integrating into the host chromosome.

Early And Late Transcription: When the phage first infects an E. coli cell, transcription of the left and right side operons is started at the left (P_L) and right (P_R) promoters, respectively:

• The N(left) and CRO (right) genes are transcribed and then translated into their respective proteins. Then, transcription stops on both operons at rho-dependent terminators (t_RI, tLI).
• Transcription cannot continue until the protein product of the N gene is produced. This protein is called an antitermination protein.
• When it binds at sites upstream from the terminators, called nutL and nutR (nut stands for N utilization; L and R stands for left and right), the polymerase reads through the terminators and continues to transcribe the left and right operons. (Note: It is still not clear why antitermination has evolved here, it is believed that it gives the phage better control over the timing of events; see Tamarin, 2002)

• Then transcription continues along the left and right operons through the ell and cIII genes. At a later stage, if the lytic response is followed by phage, the Q gene, which codes for a second antitermination protein in the right operon, has the same effect on the late operon as the N gene did on the two early operons.
• Thus, without the Q gene product, transcription of the late operon proceeds about two hundred nucleotides and then terminates.
• With the Q gene product, the late operon is transcribed. Hence, in phage λ, proteins that allow RNA polymerase to proceed past termination signals mediate general control of transcription.
• If only the previously described events were to take place, the lytic cycle pathway would always be followed. However, a complex series of events can occur in the repressor region that may lead to a “decision” to follow the lysogenic cycle instead.

Repressor Transcription: The cIII-gene product inhibits a host cell protease, called FtsH, that would normally break down the ell-gene product.

• The ell-gene product binds at two promoters, enhancing their availability to RNA polymerase just as (the CAP-cAMP product enhances the transcription of the lac operon.
• The cell protein binds to (lie promoters for cl transcription and in gene) and cl (repressor proteins) produced, favoring lysogeny, as well as the cm gene product, the antirepressor, which is a repressor of and therefore favors the lytic pathway, (cro stands for control of repression and other things.
• These mutants can only undergo lysis without the possibility of lysogeny. Normal λ infections produce turbid plaques, accounted for by lysogenic bacterial growth within the plaques).

“Is bacterial gene regulation-related risk reversible if addressed promptly? Answer provided”

Maintenance of Repression: The cl gene, with the aid of the ell-gene product, is transcribed from a promoter known as P_RE, (the RE stands for repression establishment;)

• Once cl is transcribed, it is translated into a protein called the X repressor, which interacts at the left and right operators, O, and OR of the left and right operons.
• When these operators are bound by cl protein, transcription of the left and right options (and therefore also the late operons) ceases. There are several subdivisions of the repression.
• First, lysogeny can be initiated because the int gene has been transcribed at the early stage of infection. Second, since ell and clll are no longer synthesized, cl transcription from the pRE promoter stops.

• However, P_RM (RM stands for repression maintenance), allows low levels of transcription of the cl gene. The cl gene is found to control its concentration in the cell.
• When the right and left operators were sequenced, each was discovered to have three sites of repressor recognition.
• On the right operator, for example, the rightmost site (Ogl) was found to be most efficient at binding repressor. When the repressor was bound at this site, the right operon was repressed, and transcription of cl was enhanced (it is similar to the enhancement of transcription by the binding of CAP-cAMP at the CAP site in the lac operon).
• Excess repressor, when present, however, was also bound by the other two sites within OR. The above process results in the repression of the cl gene itself.
• Hence, maintenance levels of cl can be kept within a very narrow limit. A third subdivision of repression is the prevention of superinfection.
• That is, bacteria lysogenic for λ phage are protected from further infection by other λ phages because the repressor is already present in the cell.
• Thus, the excess of repressor controls new invading λ phages. (Note: for this situation, it is normally said that bacterial cells lysogenic for phage λ are immune from infection by additional λ phage).
• These bacteria are also protected from infection by T₄ phage with all mutants. The rex-gene product, the product of the other gene in the repressor operon, controls the protection.

• Further, the promoters for maintenance and establishment of repression differ markedly in their control of repressor gene expression. When P_RE is active, a veiy high level of repressor is present whereas P_RM produces only a low level of repressor.
• The level of repressor is due to the length of the leader RNA transcribed on the 5′ side of the cl gene.
• The P_RE promoter transcribes a very long leader RNA and is very efficient at translation of the cl region. In contrast, the P_RM promoter starts transcription at the initiation codon of the protein. This leaderless mRNA is translated very’ inefficiently in Cl.
• The λ repressor is a dimer of two identical subunits. Each subunit is composed of two domains, or “ends”.
• The carboxyl- and amino-terminal ends are separated by a relatively open region which is susceptible to attack of protease enzyme.
• The alpha-helical parts of the amino-terminal ends interdigitate into the major groove of the DNA to locate specific sequences making up the left and right operator sequences. As described earlier in context with lac operon, the operator, O_R1 O_R2 and O_R3 each have two-fold symmetry.
• The binding of the λ repressor in O_R1 enhances the binding of another molecule or repressor into O_R2. Together, they enhance P_RM transcription, presumably through contact with polymerase. The repressors also block P_R transcription.

Lysogenic versus Lytic Response: Now let us see how does λ phage turns toward the lytic cycle. In this context, the control is exerted by the cro-gene product, another repressor molecule that works at the left and right operators in a manner antagonistic to the way the Cl repressor works.

• In other words, using the right operator as an example, cro-gene product binds preferentially to the left of most of the three sites within oR and represses cl but enhances the transcription of cro.
• The cro-gene product can direct the cell toward a lytic response if it occupies the oR and oL sites before the λ repressor, or if the λ repressor is removed.
• From the point of view of phage λ, when would be a good time for the Cl repressor to be removed? From an evolutionary point of view, it is expected that a prophage might be at an advantage if it left a host’s chromosome and began the lytic cycle when it “sensed” damage to the host.
• One ofthe best ways to induce a prophage to enter the lytic cycle is to direct ultraviolet (UV) light at the host bacterium. UV light causes DNA damage and induces several repair systems. One, called SOS repair makes use of the protein products of the recA gene.
• Among the activities of this enzyme is to cleave the λ repressor in the susceptible region between domains. The cleaved repressor falls free of the DNA, making the operator sites available for the cro-gene product. The lytic cycle then follows. Various elements involved in phage λ infection have been tabulated.

Elements In Phage λ Infection:

• Under initial infection, the choice between lysogeny and lytic cycle depends primarily on the ell protein, which gauges the health activity of the host.
• After lysogeny is established, it can be reversed by the processes that inactivate the cl protein, indicating genetic damage to the bacterium (the SOS response), or an abundance of other hosts in the environment (zygotic induction).
• In zygotic induction, the lytic cycle is induced during conjugation, presumably when an Hfr cell sends a copy of the F^– prophage into an F^– cell. At that point, without a repressor present, the prophage can reassess whether to continue lysogeny or enter the lytic cycle.

“Success rate of interventions using modern bacterial gene regulation techniques: FAQ”

Questions And Answers

Question 1. How can inducible and repressive enzymes of microorganisms be distinguished?
Answer: Both enzyme types are distinguished by studying the synthesis or lack of synthesis of the enzyme in cells grown on chemically defined media. If the enzyme is synthesized only in the presence of a certain metabolite or a particular set of metabolites, it is probably inducible. If it is synthesized in the absence but not in the presence of a particular metabolite or group of metabolites, it is probably repressible.

Question 2. Distinguish between

1. Repression and
2. Feedback inhibition is caused by the end product of the biosynthesis pathway. How do these two regulatory mechanisms complement each other to provide for the efficient regulation of metabolism?

Answer: Repression occurs at the level of transcription during enzyme synthesis. The end product, or a derivative of the end product, of a repressible system acts as an effector molecule that usually, if not always, combines with the product of one or more regulator genes to turn off the synthesis of the enzymes in the biosynthesis.

Question 3. In the lac operon of E. coli, what is the function of each of the following genes or sites:

1. Regulator,
2. Operator,
3. Promoter,
4. Structural genes z, and
5. Structural gene y?

Answer:

Question 4. What would be the result of inactivation by mutation ofthe following genes or sites in the E. coli lac operon:

1. Regulator;
2. Operator;
3. Promoter;
4. Structural genes and
5. Structural gene y?

Answer:

1. Constitutive synthesis of the lac enzymes;
2. Constitutive synthesis of the lac enzymes;
3. Uninducibility of the lac enzymes;
4. No β-galactosidase activity;
5. No β -galactoside permease activity

Question 5. Of what biological significance is the phenomenon of catabolite repression?
Answer: Catabolite repression has evolved to assure the use of glucose as a carbon source when this carbohydrate is available, rather than less efficient energy sources.

Question 6. Describe the role of cyclic AMP in transcriptional control in E. coli.
Answer: Cyclic AMP, combined with CAP protein, attaches to CAP sites enhancing transcription of nonglucose, sugar metabolizing operon in E. coli. Glucose inhibits its formation by inhibiting adenyl cyclase.

Question 7. J. Beckwith isolated point mutations that were simultaneously uninducible for the lac, ara, mal, and gal operons, even in the absence of glucose. Provide two different functions that could be missing in these mutations.
Answer: One should have an idea about how these operons are controlled. Each operon requires an inducer and also the catabolic repression activation system. These mutants could be unable to make cAMP because the adenyl cyclase gene is defective. Alternatively, they could be making a defective catabolite-activating protein (CAP).

Question 8. Describe the interaction of the attenuator and the operator control mechanisms in the trp operon of E. coli under varying concentrations of tryptophan in the cell. How does attenuator control react to a shortage of other amino acids?
Answer:

• The E. coli trp operon functions as a normal repressible operon. In addition, attenuation, based on secondary structure and stalling of the ribosome on the leader transcript, can further prevent transcription.
• Attenuator control can be exerted based on other amino acids if their codons appear in the leader transcript, causing ribosome stalling.

Question 9. What are the three different physical forms that the phage λ chromosome can take?
Answer:

• The λ chromosome has one circular and two linear forms. The circular form is the infective cellular form.
• A break at one point (cos site) takes place during packaging into the phage head, and a break at another point forms the linear integrative prophage.

Multiple Choice Questions Answers

Question 1. In the operon concept, the regulator gene functions as

1. Inhibitor
2. Regulator
3. Repressor
4. All of these

Answer: 3. Repressor

Question 2. The operator gene of the lac operon is “turned on” when the lactose molecule binds to

1. Operator gene
2. Repressor gene
3. Promotor site
4. mRNA

Answer: 2. Repressor gene

Question 3. An example of an inducible operon is

1. Lac-operon
2. Try-operon
3. Arginine operon
4. Both 1 and 2

Answer: 1. Lac-operon

Question 4. How many promoters control the transcription of E.coli lac operon?

1. One
2. Two
3. Three
4. Four

Answer: 2. Two

Question 5. Which one of the following is not a component of the lac-operon model?

1. Promoter
2. Structural gene
3. Primer gene
4. Regulator gene

Answer: 3. Primer gene

Question 6. In E.coli, the presence of tryptophan amino acid causes

1. Activation of operon
2. Closure of operon
3. Both of above
4. None

Answer: 2. Closure of operon

The post Regulation of Gene Expression in Bacteria Notes appeared first on BDS Notes.

Ribosomal RNA (rRNA)

Ribosomes are cell organelles, that function as a cell’s protein factories.

• They provide for the sites of protein synthesis involving the translation of the genetic information encoded in the messenger RNA (mRNA).
• When not engaged in protein synthesis, the ribosomes in a bacterial cell are always free, but in an eukaryotic cell, these may be found either free in the cytosol or attached to the membrane of ER (called rough endoplasmic reticulum).
• The initial progress in understanding the detailed structure of the ribosome came not from observing them with the electron microscope but by analyzing their components by ultracentrifugation.
• Ribosomes, as well as other small particles and molecules, are measured in units that describe their rate of sedimentation during density gradient centrifugation in sucrose.

“Understanding tRNA and rRNA through FAQs: Q&A explained”

• This technique gives information on size and shape (due to the speed of sedimentation) while simultaneously isolating the molecules.
• Isolation by centrifugation in sucrose is a relatively gentle isolation technique; the molecule still retains its biological properties and can be used for further experimentation.
• In the 1920s, physical chemist T. Svedberg developed ultracentrifugation, giving his name to the unit of sedimentation: the Svedberg unit, S.
• In sucrose density-gradient centrifugation, the gradient is formed by layering on decreasingly concentrated sucrose solutions.
• In a related technique, cesium chloride density-gradient centrifugation, the gradient develops during centrifugation.

The sucrose centrifugation is stopped after a fixed time, whereas in the cesium chloride technique, the system spins until it reaches equilibrium.

• The sucrose method tends to be more rapid. Samples can be isolated from a sucrose gradient by punching a hole in the bottom of the tube and collecting the drops in sequentially numbered containers.
• The first (lowest-numbered) containers will contain the heaviest molecules (with the highest S values).

Components Of Ribosome: Intact ribosomes have sedimentation coefficients of 80S for eukaryotes and 70S for bacteria, and each can broken down into smaller components:

1. Each ribosome comprises two subunits of unequal size. In eukaryotes these subunits are 60S and 40S, in bacteria they are 50S and 30S.
2. Note that sedimentation coefficients are not additive because they depend on shape as well as mass; it is perfectly acceptable for the intact ribosome to have an S value less than the sum of its two subunits.
3. The large subunit contains three rRNAs in eukaryotes (the 28S, 5.8S, and 5S rRNAs). In bacteria, the equivalent of the eukaryotic 5.8 rRNA is contained within the 23S rRNA.
4. The small subunit contains a single rRNA in both types of organisms : an 18S rRNA in eukaryotes and a 16S rRNA in bacteria.
5. Both subunits contain a variety of ribosomal proteins, the numbers.

“How do tRNA and rRNA work together in protein synthesis? FAQ answered”

• The ribosomal proteins of the small subunit are called SI, S2, S3, etc.; those of the large subunit are called L1, L2, etc. There is just one of each protein per ribosome, except for L7 and LI2, which are present as dimers.

Investigation Of The Fine Structure of Ribosome: Once the basic composition of eukaryotic and bacterial ribosomes had been worked in water, attention was focused on how the various molecules of rRNAs and proteins fit together.

• Important information was provided by the first rRNA sequences, comparisons between these identifying conserved regions that can base-pair to form complex two-dimensional structures.
• This suggested that the rRNAs provide a scaffolding within the ribosome, to which the proteins are attached, an interpretation that under-emphasizes the active role that rRNAs play in protein synthesis.

“Importance of studying tRNA and rRNA for biology students: Questions explained”

Much of the subsequent research has concentrated on the bacterial ribosome, which is smaller than the eukaryotic version and available in large amounts from extracts of cells grown to high density in liquid cultures.

The Following Technical Approaches Have Been Used To Study The Bacterial Ribosome:

1. Nuclease protection studies enable contacts between rRNAs and proteins to be identified.
2. Protein-protein crosslinking identifies pairs or groups of proteins that are located close to one another in the ribosome.
3. Electron microscopy has gradually become more sophisticated, enabling the overall structure of the ribosome to be resolved in greater detail.
   • Innovations such as immunoelectron microscopy, in which ribosomes are labeled with antibodies specific for individual ribosomal proteins before examination, have been used to locate the positions of these proteins on the surface of the ribosome.
4. Site-directed hydroxyl radical probing makes use of the ability of Fe ions to generate hydroxyl radicals that cleave RNA phosphodiester bonds located within any of the sites of radical production.
   • This technique was used to determine the exact positioning of ribosomal protein S5 in the ribosome of E. coli.
   • Different amino acids within S5 were labeled with Fe(II) and hydroxyl radicals induced in the reconstituted ribosomes. The positions at which 16S rRNA was cleaved were then used to infer the topology of the rRNA in the vicinity of the S5 protein (Heilek and Noller, 1996).
5. X-ray crystallography. In recent years discussed techniques have been increasingly supplemented by X-ray crystallography which has been responsible for the most exciting insights into ribosome structure.

“Common challenges in understanding tRNA and rRNA effectively: FAQs provided”

Analyzing the bulk amounts of X-ray diffraction data that are produced by crystals of an object as large as a ribosome is an enormous task, particularly at the level needed to obtain a structure that is detailed enough to be informative about how the ribosome works (Pennisi, 1999).

• In E. coli, all three ribosomal RNA segments are transcribed as a single long piece of RNA that is then cleaved and modified to form the final three pieces of RNA (i.e., 16S, 23S, and 5S).
• The region of DNA that contains the three ribosomal RNA molecules also contains genes for four tRNAs. There occur about ten copies of this region in each chromosome of E. coli.
• The occurrence of three r RNA segments on the same piece of RNA ensures a final ratio of 1:1:1, the ratio needed for ribosomal construction.

Nucleolus And Transcription In Eukaryotes: In eukaryotes, all rRNAs (i.e., 5.8S, 18S, and 28S) but the 5S ribosomal RNA section are transcribed as part of the same piece of RNA. However, eukaryotic cells have many copies of these rRNA genes, depending on the species.

• For example, the fruit fly, Drosophila melanogaster, has about 130 copies of the DNA region in the larger segments of ribosomal RNA that are transcribed.
• These regions occur in tandem on the X and Y sex chromosomes and are known collectively as the nucleolar organizer.
• The smallest ribosomal RNA (i.e., 5S rRNA) is also produced from a duplicated gene, but at a different point in the genome.
• For example, in D. melanogaster, the 5S rRNA is produced on chromosome It is still not known why 5S rRNA is transcribed separately.

• As we know eukaryotes have three RNA polymerases (enzymes).
• Eukaryotic RNA polymerase 1 (or polymerase A) transcribes only the nucleolar organizer DNA (i.e., most rRNAs except 5S).
• RNA polymerase 2 (or polymerase B) transcribes most genes (i.e., genes for mRNAs).
• RNA polymerase 3 (or polymerase C) transcribes small genes, primarily the 5S rRNA gene and tRNA genes.

“Why is early learning of tRNA and rRNA critical for molecular biology? Answered”

• Primase does primer synthesis during DNA replication. In addition, mitochondria, chloroplasts, and some phages have other RNA polymerases.
• At the nucleolar organizer, the nucleolus forms a dark globule found in eukaryotic nuclei.

• The nucleolus is the place where ribosomes are assembled.
• The various ribosomal proteins that have been synthesized in the cytoplasm migrate to the nucleus and eventually to the nucleolus, where, with the final forms of the rRNA, they are assembled into ribosomes.
• In the nucleolar organizer, an untranscribed region of spacer DNA separates each repeat of the large ribosomal DNA gene.

• In the electron micrograph, the polarity of transcription is evident from the short RNA at one end of the transcribing segment and the long RNA at the other ends, with a uniform gradation between.
• Notice that many RNA polymerases are transcribing each region at the same time.
• The regions between the transcribed DNA segments are the spacer DNA regions.
• Because each RNA gene has a fixed initiation site (promoter) and a fixed termination site, the transcripts adopt the characteristic Christmas tree configuration.

“Steps to explain tRNA and rRNA functions: Decoding vs structural roles: Q&A guide”

Processing of Ribosomal RNAs: Three of four rRNAs (185, 5.8, and 28S ) are made by chemically modifying and cleaving a single large (45S) precursor rRNA.

• Extensive chemical modifications occur in the 13,00-nucleotide- long precursor rRNA before the rRNAs are cleaved out of it and assembled into ribosomes. (Note. The 5SrRNA does not require chemical modification).
• Such modifications include about 10 methylations of the 2’-OH positions on nucleotide sugars and 100 isomerizations of uridine nucleotides to pseudouridine.

• The functions of these modifications are not understood in detail, but they probably aid in the folding and assembly of the final rRNAs and may also delicately alter the function of ribosomes.
• Each modification is made at a specific position in the precursor RNA.
• These positions are specified by several hundred “guide RNAs”, which locate themselves through base pairing to the precursor rRNA and thereby bring an RNA modifying enzyme to the appropriate position.

“Role of rRNA in ribosome structure and function: Questions answered”

• Other guide RNAs promote, probably by causing conformational changes in the precursor RNA, the cleavage of the precursor rRNA into the rRNAs, All of these guide RNAs are members of a large class of RNA, called small nucleolar UN As (or snoRNAs).
• So named because these RNAs perform their functions in a sub-compartment of the nucleus, the nucleolus.
• Many snoRNAs are encoded in the introns of other genes especially those encoding ribosomal proteins.
• They are therefore synthesized by RNA polymerase 2 and processed from excised intron sequence.

Role Of Nucleolus In Transcription: In addition to the important role in ribosome biogenesis, the nucleolus is also the site where other RNAs are produced and other RNA protein complexes are assembled.

• For example, the U6 snRNP which functions in pre-mRNA splicing, comprises one RNA molecule and at least seven proteins. The U6 snRNA is chemically modified by snoRNAs in the nucleolus before its final assembly there into the U6 snRNP.
• Other important RNA protein complexes including telomerase and the signal recognition particle, are also believed to be assembled at the nucleolus.
• Finally, the tRNAs are processed as well in the nucleolus.
• Thus the nucleolus can be thought of as a large factory at which many different noncoding RNAs are processed and assembled with proteins to form a large variety of ribonucleoprotein complexes.

Ribosomal RNA Multiple Choice Questions And Answer

Question 1. The function of nucleolus is the synthesis of

1. DNA
2. mRNA
3. rRNA
4. tRNA

Answer: 3. rRNA

Question 2. The functional unit in the synthesis of protein is

1. Peroxisome
2. Dictyosome
3. Lysosome
4. Polysome

Answer: 4. Polysome

Question 3. An anticodon of tRNA represents

1. Wobble hypothesis
2. Template hypothesis
3. Gene flow hypothesis
4. Richmond and Long effect

Answer: 1. Wobble hypothesis

“How does tRNA decode mRNA during translation? FAQ explained”

Question 4. Which of the following RNAs picks up specific amino acids from the amino acid pool in the cytoplasm to the ribosome during protein synthesis?

1. tRNA
2. mRNA
3. rRNA
4. All of them

Answer: 1. tRNA

Question 5. Which site of the tRNA molecule hydrogen bonds to a mRNA molecule?

1. Codon
2. Anticodon
3. 5’end of tRNA
4. 3’end of tRNA

Answer: 2. Anticodon

Question 6. The cloverleaf model belongs to

1. tRNA
2. DNA
3. Centriole
4. Flagella

Answer: 1. tRNA

Question 7. An anticodon is complementary to the nucleotide triplet in

1. tRNA
2. rRNA
3. mRNA
4. cDNA

Answer: 3. mRNA

Question 8. Which of the characters does not apply to tRNA?

1. It is the smallest of the RNAs
2. It acts as an adapter for amino acids
3. It has a clover leaf structure
4. It is the largest of the RNAs
5. It bears anticodon

Answer: 4. It is the largest of the RNAs

Question 9. According to Wobble’s hypothesis

1. The first base is unstable
2. The second base is unstable
3. The third base is unstable
4. The process of polypeptide chain elongation has been established

Answer: 3. Third base is unstable

“Early warning signs of gaps in understanding tRNA and rRNA basics: Common questions”

Question 10. The codon for anticodon 3’UUA 5′ is

1. 5’AAU 3′
2. 3’AAU5′
3. 5’AAT 3′
4. 3′ AAC5′

Answer: 1. 5’AAU 3′

Question 11. tRNA recognizes aminoacyl synthetase enzymes by

1. Anticodon
2. DHU loop
3. T C loop
4. AA-site

Answer: 2. DHU loop

Question 12. Shape of 3 – D view of tRNA is

1. Z- shaped
2. X-shaped
3. Y-shaped
4. L-shaped

Answer: 4. L-shaped

“Asymptomatic vs symptomatic effects of ignoring tRNA and rRNA principles: Q&A”

Question 13. The amino acid binding site in tRNA is

1. 5′ end
2. Anticodon loop
3. CCA 3’end
4. DHU loop

Answer: 3. CCA 3’end

Question 14. Genetic information for the synthesis of ribosomal RNA is coded in

1. DNA present in the nucleus
2. Nucleolar associated chromatin
3. Granular zone of nucleolus
4. Amorphous zone of nucleolus

Answer: 2. Nucleolar associated chromatin

The post Transfer RNA And Ribosomal RNA Notes appeared first on BDS Notes.

Messenger RNA (mRNA) acts as the intermediate molecule between the gene and the polypeptide translation product. Its existence was postulated by Crick and his associates during the 1950s.

• Among the indirect evidence for mRNA at that time was the knowledge that in eukaryotes the genes reside on the chromosomes in the nucleus, whereas protein synthesis occurs in ribosomes in the cytoplasm.
• The physical separation of genes and ribosomes means that some sort of messenger molecule must carry the biological information from the nucleus to the cytoplasm.
• In bacteria, the physical separation of DNA and ribosomes is less distinct but a messenger molecule is still necessary.
• Evidence that this messenger is RNA came first from Elliot Volkin and Lazarus Astrachan in 1956, but more convincingly from Sol Spiegelman and Benjamin D. Hall in 1961.
• Both groups demonstrated that after infection of a culture of bacteria with a bacteriophage, the new RNA that is synthesized is related in sequence to the phage DNA, suggesting that the phage genes are copied into RNA before synthesis of the phage proteins occurs.
• Shortly afterward two independent groups Brenner, Francois Jacob, and Mathew Meselson, who did the crucial experiment at the California Institute of Technology) and James Watson’s group at Harvard- directly identified mRNA molecules in E. coli cells.

“Understanding coding vs non-coding RNAs through FAQs: Q&A explained”

Life Span Of mRNA

Messenger RNA molecules are not generally long-lived in the cell, i.e., most of them are unstable.

• Most bacterial mRNAs have a half-life of only a few minutes and so are turned over very rapidly.
• In eukaryotic cells, mRNA molecules have longer half-lives (for example., 6 hours for many mammalian mRNAs) but are still subject to turnover.

“Importance of studying coding and non-coding RNAs in biology: Questions explained”

• Turnover of mRNA is important because it means that the absolute amount of a particular mRNA in the cell can be controlled by adjusting the rate at which the relevant gene is transcribed.
• If the transcription rate for the gene decreases then the level of the mRNA in the cell also decreases until a new steady state is reached.
• In fact, in both bacteria and eukaryotes mRNAs are known to be resistant to cytoplasmic ribonuclease enzymes and survive for long periods.
• For example, mRNA with a lifetime of six hours has been detected in the bacterium Bacillus cents at a time when the cells are induced to become long-lived spores.

“Common challenges in understanding RNA types effectively: FAQs provided”

• Likewise, in differentiating eukaryotic cells mRNAs with a lifetime of days have been detected.
• For example, in the immature red blood cells (reticulocytes) of mammals the mRNA is synthesized originally by the nucleus in early stages and expelled to the cytoplasm.
• In later stages, the nuclei of maturing reticulocytes degenerate but the mRNA exists for up to 2 days for prolonged utilization in the synthesis of globin protein of hemoglobin.
• Further, in extreme cases, such as in the state of dormancy adopted by many animal eggs and plant sips, M, mRNA is maintained in a stable form for months or even years.

Self-Splicing And Ribozyimes

Sidney Altman in 1981 showed that RNA can have catalytic properties. In 1982, Thomas Cech and Sidney Altman discovered self-splicing by RNA; both were awarded the 1989 Nobel Prize in Chemistry.

• Working with an intron in the 35S ribosomal RNA precursor in the single-cell eukaryote (ciliated protozoan) Tetrahymena thermophile.
• Cech and his colleagues found that they could induce intron removal in vitro with no proteins present. A guanine-containing nucleotide (GMP; GDP or GTP) had to be present.
• The diagram depicts how self-splicing occurs. The intron acts as an enzyme; we call an RNA with catalytic or enzymatic properties a ribozyme.
• During self-splicing, the U-A bond at the left (5′) side of the intron is transferred to the GTP.
• The U that is now unbounded displaces the G at the right (3′) side of the intron, reconnecting the RNA with a U-U connection and releasing the intron.

“Steps to explain the structure of coding RNAs: mRNA vs tRNA: Q&A guide”

• Since all bonds are reversible transfers (transesterifications) rather than new bonds, no external source of energy is required. Self-splicing introns of this type are called group I introns.
• Although the first enzymatic activity of the ribozyme is its removal, its secondary structure after removal gives it the ability to further catalyze reactions.

“Role of non-coding RNAs in gene regulation: Questions answered”

• The reactions that ribozymes catalyze are transesterifications and the hydrolysis reaction of splitting an RNA molecule into two parts.
• Ribozymes can also perform other functions, including peptide bond formation. Currently, at least seven different classes of ribozymes are known, based on their enzymatic properties.
• A ribozyme that can split other RNAs and that occurs in small plant pathogens (viruses) is called a hammerhead ribozyme because of its shape.
• Self-splicing has also been found in genes in the mitochondria of yeast.

“How do microRNAs (miRNAs) influence protein synthesis? FAQ explained”

• These introns are referred to as group II introns because they use a different mechanism of splicing that does not require an external nucleotide.
• Instead, the first bond is transferred within the intron to an adenosine, forming a lariat structure.
• For the lariat to form, the ribose of the adenosine must make three phosphodiester bonds.

Examples Of Ribozymes:

“Early warning signs of gaps in understanding RNA basics: Common questions”

Structure of Ribozymes: There appear to be at least two kinds of ribozymes.

• Some ribozymes have folded structures and catalyze reactions on themselves, a process called intramolecular catalysis.
• Other ribozymes act on other molecules, without themselves being changed, a process called intermolecular catalysis.
• The ribozyme consists of a catalytic core made up of two domains,-each one comprising two of the base-paired regions, with the splice sites brought into proximity by an interaction between two other parts of the secondary structure.
• Although this RNA structure is sufficient for splicing, it is possible that with some introns the stability of the ribozyme is enhanced by non-catalytic protein factors that bind to it.
• For example, Group 1 introns in organelle genes, many of these containing an ORF coding for a protein called a maturase that appears to play a role in splicing.

Functions Of Ribozymes

“Role of non-coding RNAs in cancer research: Questions answered”

Those ribozymes that are known today carry out three types of biochemical reactions:

1. Self-cleavage: as displayed by the self-splicing group 1, 2, and 3 introns and by some virus genomes.
2. Cleavage of other RNAs: as carried out by RNase P (discovered by Altman et al, 1983)
3. Synthesis of peptide bonds: by the rRNA component of the ribosome (discovered by Harry Noller and his colleagues in 1992).

In the test tube, synthetic RNA molecules have been shown to carry out other biologically relevant reactions, such as;

1. Synthesis of ribonucleotides (Unran and Bartel, 1998).
2. Synthesis and copying of RNA molecules (Exland and Bartel, 1996: Johnston et al, 2001)
3. Transfer of an RNA-bound amino acid forming a dipeptide, in a manner analogous to the role of tRNA in protein synthesis (Lohse and Szostak, 1996).

The discovery of these catalytic properties solved the polynucleotide-polypeptide dilemma by showing that the first biochemical systems could have been centered entirely on RNA (Bartel and Unran, 1990).

Role of Ribozymes in the Origin of Life: Ideas about the RNA world during chemical evolution and the origin of life have taken shape in recent years (Robertson and Ellington, 1998).

• Biologists now envisage that RNA molecules initially replicated slowly and haphazardly simply by acting as templates for binding of complementary nucleotides which polymerized spontaneously.
• This process would have been very accurate so a variety of RNA sequences would have been generated, eventually leading to one or more with incipient ribozyme properties that were able to direct their own, more accurate self-replication.
• It is possible that a form of natural selection operated so that the most efficient replicating systems began to predominate.
• Greater accuracy in replication would have enabled RNAs to increase in length without losing their sequence specificity, providing the potential for more sophisticated catalytic properties,
• Possibly culminating in structures as complex as present-day Group-J introns (for example. Tetrahymena tRNA intron) and ribosomal RNAs.

“Asymptomatic vs symptomatic effects of ignoring RNA functions: Q&A”

Chances Of Errors In Gene Splicing

Certain topological problems might pose chances of errors in gene splicing.

• The following two types of errors can commonly occur during the splicing of a gene:
• The first of these is the substantial distance that might lie between splice sites, possibly a few tens of kb, representing 100nm or more if the mRNA is in the form of a linear chain.
• A means is therefore needed to bring the splice sites into proximity.
• The second topological problem concerns the selection of the correct splice site.

“Steps to apply RNA knowledge in biotechnology: Gene therapy vs diagnostics: Q&A guide”

• All splice sites are similar, so if a pre-mRNA contains two or more introns then there is the possibility that the wrong splice sites could be joined, resulting in exon skipping, i.e., the loss of an exon from the mature mRNA.
• Equally unfortunate would be a selection of a cryptic splice site, a site within an intron or exon that has sequence similarity with consensus motifs of real splice sites.
• Cryptic sites are present in most pre-mRNAs and must be ignored by the splicing apparatus.

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