[Organic Reaction Mechanisms Series] Organic Reaction Mechanisms || Reactions of Acids and their Derivatives

CHAPTER 2

Reactions of Acids and their Derivatives

A. J. KIRBY

University Chemical Laboratory, Cambridge

CARBOXYLIC ACIDS . Tetrahedral Intermediates . Intermolecular Catalysis and Reactions .

Reactions in Hydroxylic Solvents . Reactions in Aprotic Solvents .

Intramolecular Catalysis and Neighbouring-group Participation Association-prefaced Catalysis . Metal-ion Catalysis . Enzymic Catalysis .

Serine Proteinases . Thiol Proteinases . . . Acid Proteinases . Metallo-proteinases . Other Enzymes . .

Decarboxylation . . NON-CARBOXYLIC ACIDS . Phosphorus-containing Acids .

Non-enzymic Reactions .

Sulphur-containing Acids . References .

Enzymic Reactions . . .

. 29

. 29

. 32

. 32

. 40

. 42

. 50

. 54

. 55

. 56

. 60

. 61

. 62

. 63

. 66

. 68

. 68

. 68

. a1

. a5

. aa

CARBOXYLIC ACIDS

Tetrahedral Intermediates

Guthriel has measured the equilibrium constant for the addition of sodium methoxide in methanol to methyl trifluoroacetate, using IgF-NMR spectroscopy. From the value

0- I I

CFsCOOMe + MeO- CF3-C-OMe

OMe

obtained (7 ~ - 1 a t 25"), a value of 2 x 10-4 M - ~ can be calculated for the similar addition of methanol to the ester. This work clears up an apparent contradiction in the literature : Bender, in a much quoted paper,Z observed negligible addition of methoxide to methyl trifluoroacetate, but i t is now clear that his infrared technique (1953) was barely sensitive enough to detect the rather low conversion expected at the concentrations of ester and

29

Organic Reaction Mechanisms 1976 Edited by A. R. Butler, M. J. Perkins

Copyright © 1977 by John Wiley & Sons, Ltd.

30 Organic Reaction Mechanisms 1976

methoxide that he used. The new results, together with a determination of the equilibrium constant (0.06 M-1) for the formation of methyl trifluoroacetate in aqueous methanol, allow calculations of the free energies of formation of trifluoro-orthoacetic acid and its various methyl esters under the same conditions; and hence of the standard free energy changes for the addition of water and methanol to trifluoroacetic acid and its methyl ester. Comparison with similar data for formic and acetic acids by means of Taft plots shows that the p* values for these additions to carboxyl groups are significantly higher than for the same reactions of carbonyl groups. For example, the p* obtained for the addition of water to RCOOH, RCOOMe and RCOMe are 2.9, 2.1 and 1.7, respectively.

Deslongchamps has reviewed3 the evidence for his theory of stereoelectronic control of the cleavage of tetrahedral intermediates.4 New results5 concern a more detailed study of the hydrolysis of imidate salts (1) and (2). anti-Imidate salts (1) are hydrolysed to ester

)b-Et

NN+- Me R-C 3 RCOOEt + MeaNH

/

and amine over the whole pH range, but syn-imidates (2) give mixtures of products under basic conditions :

Et \ ,o= RCOOEt+MeaNH

R-C \N+-Me

RCOOEt + MeaNH

(2) + RCONMea+EtOH

/ Me

Only 20-25% of amide plus alcohol are produced from (2; R = Me or cyclohexyl) at pH 11 and above, where the product ratio is apparently pH-dependent, and this is interpreted in terms of stereoelectronic control of the breakdown of the anionic tetrahedral interm'ediate (3).

The crystal structure of the conformationally rigid orthoester ( P ) , shown earlier6 to be unusually resistant to hydrolysis, shows each of the three six-membered rings to be in the expected chair conformation.7

OEt

R-C-NMea I I 0-

For the majority of reactions the kinetic consequences of tetrahedral addition intermediates are only apparent in special cases or under particular conditions. The excep- tions generally involve the displacement of a poor leaving group (carbon, nitrogen) from a carbonyl centre, and the most studied of such reactions is the alkaline hydrolysis of

2 Rmtions of Acids and their Derivatives 31

amides. The change of kinetic order with changing hydroxide concentration is compli- cated by ionization of the NH of primary and secondary amides: a particularly rich example is the hydrolysis of barbituric acid,8 where the order in hydroxide ion varies from fractional, through zero, to second, as the hydroxide concentration is raised from 0.02 to 3 M. The secondary deuterium isotope effect for the hydrolysis of p-nitroacetanilide (5) similarly changes, from 1.08 a t low [OH-] to 0.87 a t high hydroxide concentration,e though with this very good leaving group a change in rate-determining step probably accounts for the variation in k H / k D . Independent evidence for this is available most recently in work on the alkaline hydrolysis'of N-methylacetanilidel0 and formanilide.11 Hammett plots for the alkaline hydrolysis and methanolysis of N-methylformanilideI1 are curved up t o 1 ar-NaOH, and an order in hydroxide greater than two is observed in one case. This is put down, gratefully no doubt, to medium effects.

The lessons learned from the very large amount of mechanistic work on the alkaline hydrolysis of amides are put to good use in a neat procedure for preparative-scale hydrolysis of tertiary amides.12 The reaction of water with three equivalents of potassium tert-butoxide in ether produces a highly nucleophilic, poorly solvated hydroxide, which can hydrolyse most tertiary amides a t room temperature. Reaction is presumed to involve the dianion (6) of the tetrahedral intermediate : i t would be surprising if protonation of the leaving group by tert-butyl alcohol were not also involved.

The aminolysis of benzyl penicillin (at the 8-lactam carbonyl group) is general base- catalysed, with Brensted 8-values close to unity for both [RNHz]Z and [RNH2][OH-] terms. The rate-determining step is thought to be the diffusion of the general base to the tetrahedral intermediate (part structure 7). The reaction with ethylenediamine, with its general base built in, is 30 times faster than expected for a monoamine of the same basicity.l3

Intramolecular cyclizations produce higher concentrations of tetrahedral intermediates than to bimolecular additions in many cases. The hydrolysis of neutral phthalanil- ides has been shown14 to involve breakdown of the tetrahedral intermediate, by generating this independently from the isoimide. The same step is probably also rate- determining in the acid-catalysed hydrolysis of maleanilides15 where the Brernsted coefficient for the leaving group falls from 0.15 a t p H 0 to -0.12 in strong acid.

The cyclization of ethyl 2-(hydroxymethy1)benzoate to phthalide16 is general base- catalysed, with a Brensted coefficient 8 = 0.87. For the same reaction of the 4-nitro- compound (8), /3 = 0.97, within experimental error of unity, the value expected for a rate-determining proton-transfer in the thermodynamically unfavourable direction.


HB+ 0- OEt - so - eo 0 + EtOH

OzN OH OzN ‘ \

(8) (9)

The diffusion process involved is probably formation of the encounter complex (9). Exchange of the carbonyl-oxygen atom with the solvent has been detected17 in the

acid-catalysed hydrolysis of phenyl acetate-carbonyl-180. The ratio kby&exch = 120 for this ester (in 40:60 dioxan-water 1.5 M in HCI) is consistent with the formation of a tetrahedral addition intermediate on the reaction pathway for the A-2 hydrolysis of aryl esters.

Infermolecular Catalysis and Reactions

Reactions in Hydroxylic Solvents

Reviewers have dealt with the chemistry of fl-lactams (several times’s* 9 , the usefulness of the entropy of activation parameter in chemical reactions,zo and reactions of carbonyl and acyl groups.21 A theoretical analysis of the effects of medium acidity on simple acyl transfer reactions has appeared.22

Grunwald has published PMR studies of proton-transfer processes involving the amino-acids lysine and cysteine,23 alanine, phenylalanine, and glycine and its amide and esters24 in water. This work, happily termed by the authors “complimentary” to the early and definitive study of glycine by Sheinblatt and Gutowsky,25 is based on measurements of (1/T2 - 1/T1) for the H2O or collapsed H20-NHi NMR spectra, and provides evidence for the involvement of water molecules in several proton-transfer processes. Such evidence is not, however, available for the intramolecular proton-transfer between carboxyl and a-amino-groups because of the rapid exchange between the COOH and water protons.

Activity coefficients have been measured for the simple alkyl acetates a t 25” in concentrated aqueous solutions (up to 7 molal) of eight 1 : 1 electrolytes.26 Salt effects on the alkaline hydrolysis of ethyl oxalate27 and dielectric constant effects on the alkaline hydrolysis of n-butyl and pentyl acetates28 have been measured in ethylene glycol-water mixtures.

The effects of small amounts of added acetone on the alkaline hydrolysis of ethyl acetate depend on the pressure.20 At low pressures, increasing the amount of the organic co-solvent retards the reaction, but a t 3 kbar the reverse is true.

Alkaline hydrolysis of aspirin is slower than that of phenyl acetate because of the electrostatic effect of the neighbouring carboxylate group. In a study of several solvent mixtures30 the rate ratio was largest in 80% dimethyl sulphoxide, which also has the largest dielectric constant and anion desolvating effect.

DeTar and Tenpas31 present the most successfulattempt yet torelate the empirical Taft steric substituent constants to measurable physical properties of molecules. They use enthalpies of formation of hydrocarbons, which are available, or are readily estimated by the methods of molecular mechanics. The model for the formation of the tetrahedral intermediate from an ester, RCOOEt + RC(OH),OEt, is the formatiori of a neoalkane from an isoalkane: RCHMe2 + RCMe3. The difference in the estimated enthalpies of formation, A AH, reflects the “strain” involved in introducing the fourth heavy-atom

2 Reactions of Acids and their Derivatives 33

substituent a t the tetrahedral carbon and correlates rather well with E , values, The relation obtained:

E, = 0 . 5 5 2 4 4 8 + 4.419

has a standard deviation of 0.4 over a range of 4 in E, and a correlation coefficient of 0.95 for 24 esters.

The steric effects of the 1-adamantyl and l-bicyclo[2.2.2]octyl groups on ester hydrolysis are similar, and larger than that of the tert-butyl group.32 The reaction measured is the alkaline hydrolysis of p-nitrophenyl esters RCOOAr, and the rates for the tertiary R groups of interest decrease in the order R = But > 1-adamantyl -[2.2.2] > Et&, with the pivalate ester about twice as reactive as the adamantyl derivative. Perhaps the simplest possible method of estimating the relative importance of steric effects involves counting the number of carbon atoms a t the various positions starting from the a-carbon at0m,3~ but such a system cannot cope with the complications involved in ring formation.

Other such complications arise in conformational analysis. The rate of base-catalysed ethoxy-group exchange (measured by loss of 1%-label) of 4-substituted cyclohexane- 1,l-dicarboxylate esters (10) is not affected by the size of the alkyl substituent (R = H,

COOEt I‘”

Me or But), but the rate is increased by about 50% for R = Ph.34 This is in contrast to the debenzylation by thiophenoxide of the corresponding dibenzylpiperidinium compounds (ll), where the rate depends on the size of the substituent; though once more the 4-phenyl compound is the fastest (relative rates for R = H, Me, But and Ph are 1.0, 1.3, 2.15, 2.8). The authors emphasize the risks involved in applying the standard methods of conformational analysis using 4-But groups, when the atoms concerned are directly involved in reactions.

The rates of alkaline hydrolysis of a series of substituted 4-hydroxy-1-methylpiperidine acetates (12) in 95% ethanol have been interpreted in terms of steric hindrance to attack on different conformations.35

The same skeleton has been used by Russian workers to investigate the effect of a quaternary ammonium group on the rate of alkaline hydrolysis of a series of 4-nitro- benzoate esters (13). A large number of sidechains R, some rigid and some flexible, carrying the RaN+-group were used,36 and it was found that the group causes a sharp


increase in rate unless it is prevented from approaching the reaction site for steric reasons. A linear dependence of rate on ionic strength is found, up to about 0.5 M.

A short note reports the main conclusions of an extensive investigation of a large number of B A C ~ reactions of carboxylate esters with nucleophiles ;37 rates are correlated with Taft substituent and steric substituent constants, PKa’S of nucleophile and leaving group, and group refractions, separate three-parameter correlations being used for each variable.

The rates of hydrolysis of substituted methyl 1- and 2-naphthoates in acid and alkali are correlated by the Hammett equation,38 but the rates of formation of the esters in methanol apparently are n ~ t . ~ Q More distant substitution has been used in calculating transmission factors for polar substituent effects in two furan systems. PKa’S and rates of alkaline hydrolysis (in 60% aqueous acetone) were measured for a series of acids and

(14) (16)

their methyl esters (14) and (15) and were correlated by the Hammett equation. For the pKa’s of the furoic acids, for example, p = 0.50 in 50% ethanol. The transmission factors obtained were about 0.3 for the furoates40 (0.291 for hydrolysis, 0.329 for pK’s) and for (15) 0.215 for hydrolysis and 0.246 for pK’s.41

The hydrolysis of ethyl acetate in strong acid has been studied,42 as has that of butyl acetate in aqueous HC1 and H2SO4 ;43 similar mechanisms involving an activated complex of ester, H2O and H30+ are proposed.

The esterification of methacrylic acid by butan-1-01 catalysed by H2SO4 is of the first order in each,44 while that of acetic acid by isobutyl alcohol is reported to be of second order in acetic acid under certain conditions.45

A technique is reported for measuring the oxygen-exchange reaction between trifluoroacetic acid and water, by means of 180 and IR matrix-isolation spec t ros~opy.~~

A proton inventory study of neutral hydrolysis of bis-(4-nitrophenyl carbonate shows a linear relation between kobs and the atom fraction of deuterium in H20-DaO mixtures.47 On the usual interpretation, this means that a single proton contributes to the observed solvent deuterium isotope effect, k H / k D = 2.24, for this reaction. That is not consistent with a mechanism involving a single H2O molecule, where both protons must be involved t o the same extent, or with a cyclic two-water transition state, where again two protons must be similarly involved. The result is consistent with the usual48 mechanism for spontaneous ester hydrolysis, where one water molecule acts as a general base to remove a single proton from a second nucleophilic water molecule, as long as the transition state is fairly early and extensive proton transfer has not occurred. This picture is compatible with the normal mechanism. The effects of selected water-structure makers and -breakers on this reaction are small, and the authors suggest that this is evidence against important effects of disrupted water structure in the microenvironment of enzyme active sites.

The reactions of nucleophiles (other than water) withmethy14-nitrophenylcarbonate49 are a little slower than, but otherwise very similar to, the corresponding reactions of 4-nitrophenyl acetate. The difference is much larger for 1-(methoxycarbony1)pyridinium compounds, these being some 200 times less reactive than the acetyl derivatives. The larger difference for the more reactive compounds is not in accord with the Hammond postulate, and there is a suggestion that a change of mechanism may be involved.

Catalysis by pyridine of the hydrolysis of phenyl acetate is potentiated by


irradiation;50 photohydrolysis is up to 120 times faster than the dark reaction, and a quantum yield of 0.17 is estimated; the products are phenol and aceticacid, thoughinthe absence of pyridine the photo-Fries rearrangement supervenes. The catalysis is attributed to an excited state of pyridine, presumably one of increased basicity, and it will be interesting to know if the mechanism has changed from the general base-catalysis of the dark reaction to nucleophilic catalysis under irradiation,

The alkaline hydrolysis of a series of p-substituted benzylidenehydantoins (16) in 0.1 M-KOH at several temperatures in the range 20-95" has been studied. The isokinetic relationship is followed, with /3 = 320 f 10°K. Hammett's p changes from -0.30 to 0.55 over the temperature range, and is close to zero a t 45": AG+ is dominated by the entropy term.51 The rate of the same reaction of 5-substituted hydantoins (17) depends on the cation present.52

(16) (17)

A positive salt effect is observed for the hydrolysis of p-sulphonatophenyl benzoate a t low NaOH(NaC1) concentrations, but, as frequently observed, the second-order rate constant reaches a limiting value a t high salt concentrations.53 The substituent constant is effectively zero for the SO, group a t zero salt concentration, but when the limiting rate constants are used a positive value of u0 (0.37 at 25") is found. This is considered to represent the value for ion-pairs, ArSO-Nag, which are dissociated a t low salt concentrations. (The corresponding p-toluenesulphate esters,54 for which there is no salt effect, show only a positive para-substituent constant for SO, (0.38 a t 50°), consistent with ion-pair formation even at very low ionic strengths).

A conductometric study of the alkaline hydrolysis of isopentyl and n- and isobutyl acetate in 0.02~-NaOH a t various temperatures between 20" and 50" is reported55.

The alkaline hydrolysis of phenyl and p-methoxyphenyl hydrogen malonates goes by the B.4~2 mechanism,56 and not by ElcB as found for nitrophenyl esters.57 True ElcB reactions are found for several urethane derivatives. The isothiocyanate intermediate can be detected by UV spectroscopy in the alkaline hydrolysis of (18),5* and the Brensted (leaving group) plot5gfor the alkaline hydrolysis of esters (19) is biphasic, with a much greater slope for aryl esters than for alkyl esters (19); R = alkyl), as expected if the mechanism changes t o ElcB for the better leaving groups. The hydrolysis of arylureas60 is also considered to go via the isocyanate.

Two variations on the alkyl-oxygen cleavage of esters are: the hydrolysis of 3- anilinophthalides (20) in 20% aqueous ethanol, which involves initial opening of the lactone ring followed by normal hydrolysis of the Schiff base (21) produced;61 and, secondly, the displacement of carboxylate from the sulpholanes (22), which is found62 to involve an elimination-addition pathway. The initial step in the solvolysis (60% acetic


acid) of a-chlorobenzyl benzoates (23) is the loss of chloride ion ( S N ~ reaction) ; electron- withdrawing substituents in Ar’ slow the reaction (p‘ = -0.8).63

The formation of esters by the alkylation of carboxylate-oxygen is an easy reaction in dipolar aprotic solvents. Alkylation of substituted benzoates by hexyl chloride follows the Hammett equation with p = -0.73 (DMSO, 60”).64 Steric effects are small, but interesting: rates increase with increased steric crowding or increasing “rule of six” number. The reaction is fastest for alkyl iodides and Cs+ salts, as might be expected, and in HMPA >> DMSO, 1-methylpyrrolidone > DMF.

The alkylation of substituted cinnamic acids by diphenyldiazomethane has been used to study solvent effects in a dozen alcohols, ethyl acetate and acetone65; good linear free-energy relations are found with the reactions of the corresponding benzoic acids under the same conditions, and a transmission coe5cient of 0.39 is calculated; multiple linear correlations with three solvent parameters (the Kirkwood function of the dielectric constant, the Taft substituent constant for the R group of the alcohol solvent ROH, and the number of y-hydrogen atoms) are presented.

Czech workers report further studies of the alkylation of benzoic acids by ethylene oxide. The reaction (at 600-1070 Torr) is catalyaed by the acid itself and by tertiary amines.66 In hydroxylic solvents reaction is second-order, first-order in ethylene oxide and in acid or base, and the rate depends on the pK of the acid, with Br~lnsted‘s a = 0.62 for the acid- and 0.34 for the base-catalysed process. For the strongest acids (pK c 3.18), reaction is third-order (acid, base and ethylene oxide). The reaction (with terephthalic acid67) is also catalysed by soft nucleophiles (tertiary phosphines, arsines, stibines), and kinetic parameters are similar to those found for the pyridine-catalysed reaction. It is concluded that the same mechanism is involved for all the base and nucleophile-catalysed reactions, namely an initial reaction of the catalyst with ethylene oxide, which generates the zwitterion RsMfCHzCHzO-, and hence the carboxylate anion. The common rate- determining step is the alkylation of the carboxylate anion by ethylene oxide.

The reactions of acyl esters of carbohydrates with ammonia have been reviewed.68 A detailed study of the aminolysis of tri-0-methyl-2-deoxyglucono-S-lactone (24)

shows that the reaction is simpler than expected for the aminolysis of an open-chain alkyl ester; there is no apparent change in rate-determining step with decreasing pH,

- RNHn M e O M S

CONHR M e 0

(24)

and the reaction is fully described69 by terms in OH-, amine, [amineI2, [RNHz] [OH-] and [RNHz] [RNHsf]. The Br~lnsted coefficient for the first-order reaction with a series of amines is 0.85, close to that observed for the corresponding reactions of phenyl acetate. These properties, and the high reactivity of the 8-lactone towards aminolysis (and hydrolysis also), are characteristic of esters with leaving groups as good as, or better than, trifluoroethoxide ; it is therefore suggested that the ring-opening of the tetrahedral intermediate formed from a lactone (relieving the strain associated with the cisoid conformation) is significantly accelerated relative to that from acyclic compounds. The reaction of (24) catalysed by morpholine is not aminolysis, but is general base- catalysis of hydrolysis &$= = 1.9).


The effect of varying temperature and pressure on the benzylaminolysis of three alkyl-substituted y-lactones (25) a t 80-1 10" has been studied.70 Under the conditions used the products are the lactams (26). The steric effects of the group R are unexceptional.

The aminolysis reactions of a-acetoxystyrenes (27) are also similar, both quantitatively and qualitatively, to those of phenyl acetate, suggesting that the pK, of the enol of acetophenone in water is about 11.71 Brsnsted coefficients ,9 for both nucleophile and leaving group are close to unity, as for the aminolysis of aryl acetates,72 and there is no reason to postulate any involvement of the enol double bond in the reaction.

The X-ray structure of the non-planar amide (28)73 and the synthesis of another (59)74 are reported. In (28) the bond on the nitrogen syn to the carbonyl-oxygen is over

30" out of the plane defined by O=C-N. Extended Hiickel calculations of the most stable conformations of methyl penicillin

show the expected similarity to N-acetyl-~-alanyl-D-alanine.~~ Wolfenden has measured the distribution coefficient of acetamide from water to the

gas phase a t 25" by a dynamic technique.76 The amide is more hydrophilic, by a large margin, than any other simple uncharged organic compound measured. It is so much more hydrophilic than ethyl acetate, for example, that solvation may be said to provide the entire driving force for the strongly exergonic ammonolysis of this ester in water.

Charton77 has analysed published data for the acid- and base-catalysed hydrolysis of amides and 1-acylimidazoles and finds that steric effects on the two reactions are closely similar to each other and quantitatively close to those for reactions of the corres sponding esters.

The full account has appeared76 of Williams' work on the hydrolysis of N-acetyl- trialkylammonium ions as models for N-protonated amides.79 The results show rather clearly that the reactivity of an N-protonated amide would not be high enough to support the observed rates of hydrolysis of acetamides in acid; this strengthens the evidence for the generally accepted mechanism of hydrolysis by way of the 0-protonated amide. Giffney and O'ConnorsO advocate the N-protonation mechanism, largely on the grounds that 4-chloroacetanilide is hydrolysed some 100 times faster than 4-chloro-N- methylbenzamide, although their basicities are similar. Since the methyl group is electron-donating compared with 4-chlorophenyl, these authors would expect slower

38 Organic Reaction Mechanisms I976

attack by water on the protonated acetamide; this argument falls, however, because benzoyl derivatives are generally less reactive than acetyl even under conditions where electronic effects are dominant; for example, the rate constant for alkaline hydrolysis of phenyl acetate (60% acetone, 25”) is about 60 times greater than for the hydrolysis of methyl benzoate.dE

Activation parameters and salt effects ( I - ~ M - N ~ C ~ O ~ ) have been measured for a few substituted benzamides in 0.5-5~-HC104 at 95O.81 The acid hydrolysis of o-nitro- formanilide82 involves amide, H3O+ and the acid anion (Cl-, HSO,), but not HzO, in the transition state. An interesting possibility is the involvement of the o-nitro-group in the hydrolysis.

The mechanism of acid- (A-2) and base-catalysed hydrolysis of benzohydroxamic acid is unchanged by ortho- or N-methyl substituents.83

The alkaline hydrolysis of several amides has already been discussed.8-11 The pK’s of a series of formanilides ArNHCHO follow the Hammett equation, with p = -1.70,11 and fall as low as 15.9 for the p-nitro-compound.

Substituent effects and activation parameters for the alkaline hydrolysis of two series of substituted hydantoins (30 and 31) are consistent with rate-determining attack by OH- on the carbonyl group at position 4 of the monoanions [&anions for (31)].84

HN<cooMe o T - C O O H

O A N H H

(80) (all The ratio of C-N to C-S bond cleavage of thioacetamide is independent of tempera-

ture and of pH between pH 9.5 and 10.5.8s The hydrolysis of substituted-phenylureas (at 101 ”) shows a pHindependent region

near pH 7 and relatively weak catalysis by acid and base.86 In 0.5-98% (w/w) Has04 the rate first passes through a maximum, which is not more than about one order of magnitude above the water rate, then falls to a rate (near -Ho = 6) which is lower than the water rate for substituents other than NO2, then increases again in very strong acid. The usual trend of HC1 z H2SO4 > HC104 is observed but is much less marked than for amides and carbamates. Application of the usual criteria of mechanism for acid-catalysed reactions generdly give curved plots : the Zucker-Hammett A-1 equation is followed, but with a slope of only 0.3, so the authors feel that the usual explanation of the rate maximum, an A-2 mechanism of the 0-protonated urea, is not “validated”. They therefore conclude that the 0-protonated urea is stable at low to intermediate acidities. Since anion effects are small, and decreasing water activity appears to be the dominant factor, it nevertheless seems certain that a protonated species is involved, and the authors suggest that the reactive species is the N-protonated urea and propose a mechanism (32) based on BunnettOlsen and Yates r plots and an unknown acidity scale HN. This

0 I I

ArNHCOOH


mechanism raises the problem, common to all mechanisms involving ammonium leaving groups, of how significant electrophilic catalysis can work a t a cationic centre. The most reasonable mechanism, which in this case would involve protonation of the anterior lobe of the C-N bonding orbital as the bond breaks, is geometrically impossible in a six- membered ring (cf. the corresponding sN2 reaction, 6-edo-tet in Baldwin’s notations7).

The pH-independent reaction near pH 7, mentioned above, has been investigated further for 4-methyl- and 4-nitrophenyl-urea.88 As expected for a reaction showing a significant spontaneous hydrolysis, general species catalysis is readily detected, and rate constants for general acid and general base catalysis by various phosphate species are reported.

The acid-catalysed hydrolysis of thiourea89 gives urea as the initial product. A polarographic investigation of the hydrolysis of maleimide defines the plateau in the

pH-rate profile (pH 11-13), and leads to an estimated pK of about 10.0 for the imide proton.90

Kinetics and mechanism in the reactions of amidines have been reviewed;91 the aminolysis of ethyl acetimidate near pH 8 leads, not to the amidine, but to the N - alkylacetimidategz[reaction (I)], which may be hydrolysed or converted by the ammonia

,OEt ,OEt CH3-C \NH +RNHz CH3-C NNR +NH3

into the amidine ; near pH 10 the amidine appears to be produced directly, so that control of pH is important in the reactions of imidate esters with protein amino-groups.

The hydrolysis of “chlordiazepoxide” (33) yields “demoxepam” (34), which is further hydrolysed (pH 1-11) by competing attack a t the C=O and C=N ~en t re s .9~

(W (84)

The alkaline hydrolysis of tetramethylguanidinium perchlorate shows an important term second-order in OH-, whereas only a first-order term is apparent in the hydrolysis of the unsubstituted guanidinium cation.94 When the first-order (in OH-) reactions are compared, guanidine is 750 times more reactive, suggesting that reaction does not involve addition of OH- to the guanidinium cation. Since cyanamide is not an intermediate-it would be hydrolysed more slowly than guanidine under the conditions-an elimination mechanism is ruled out. The authors therefore propose a mechanism involving the attack of water on the free base :

HzO + (NH2)2C=NH (NHz)zC=O + NH3

It seems scarcely possible, under conditions where the guanidinium cation is present, that one if not all of three alternative reactions should not be faster than this; namely, HzO attack on the cation and attack by hydroxide on both guanidine and its conjugate acid. There is an interesting problem here: perhaps steric effects become important with six extra methyl groups in ground and transition states, and results with pentamethyl- guanidine might lead to different conclusions.

40 Organic Reaction Mechanism 1976

Equilibrium constants for the formation of dicarboxylic acids from the anhydrides in acetic acid range over several powers of ten,Q6 but in nearly all cases the aoylation of phenol with the cyclic anhydride in anhydrous acetic acid leads to acetylation of the phenol.96

The hydrolysis of a series of substituted benzoic anhydrides in water-dioxan mixtures, catalysed by 0. IM-HC~ (50-75") shows a change from A-2 to increasing A-1 character as the polarity of the solvent decreases @substituted, and p-But and C1-compounds). The p-Me0-compound shows A-1 behaviour under all conditions.97

The hydrolysis of aryl fluoroformates is faster than that of the corresponding chloro- formates ;9a it probably follows that carbon-halogen bond breaking is not far advanced in the transition state: i.e. that formation of the tetrahedral intermediate is rate- determining, as would be expected. The reaction is slowed by the addition of acetate ions, which displace fluoride to produce the less reactive mixed acetic benzoic anhydride.

The hydrolysis and alcoholysis of mesityl and 2,4,6-trichlorophenyl chloroformate (studied at 10.845O) appear to involve an associative mechanism,QD as do the reactions of the corresponding thio-compounds ArOCSCl.100 The change to a dissociative mechanism is noted in the methanolysis of a series of chlorides RCSC1, for compounds with R = MezN or Ph. For the chloroformate derivatives, when R = Me0 or MeS, the associative mechanism is preferred.101

Pyridines catalyse the hydrolysis of methyl chloroformate by the nucleophilic mechanism. The Brernsted plot for a series of substituted pyridines is biphasic, with a slope j = 0.93 for bases of pK less than3.6, falling to /3 = 0.15 for more basic pyridines ;lo2

this can only be simply explained by a change of rate-determining step, from breakdown to formation of the tetrahedral intermediate (35), and fixes the leaving group capability

0- + I +

A Py + MeO-CO-CI - Py-C-CI __+ PyCOOMe + CI- I

(as) OMe

of chloride as equivalent to that of a pyridine with pK = 3.6. For reactions of weakly basic pyridines with p-nitrophenyl acetate the rates and Brensted j are similar, but p-nitrophenoxide is a much poorer leaving group than chloride, and thus also poorer than any pyridine, so that breakdown is rate-determining in all cases, and the Brensted plot is linear. Similarly the reactions of p-nitrophenoxide, and also of phenoxide and acetate, with methyl chloroformate, involve rate-determining formation of the tetrahedral intermediate.

Reactions in Aprotic Solvents

The superoxide anion is comparable to hydroxide in reactivity towards the carbonyl group. KO2 reacts with esters (but not amides or nitriles) in benzene in the presence of 18-crown-6 under mild conditions to give the hydrolysis products after aqueous acid work-up.103 In some cases the products are oxidized.

The position of the acid-amide equilibrium, set up when 1 : 1 mixtures of a carboxylic acid and butylhexanamide are heated a t 200-260", depends on both electronic and steric effects;104 steric effects control the equilibrium, and rates are highest for the weakest acids.

One of the most important reactions in this group is the aminolysis of various carboxyl


derivatives, particularly active esters. This is the basic reaction of peptide synthesis, and the improvements possible when mechanism and reactivity are properly understood can easily be significant for synthetic procedures. Studies are reported on the effect of solvent polarity on the aminolysis of active esters of N-substituted amino-acids,lo5 on the effect of temperature on the reaction of valine methyl ester with benzyl and aryl esters of (benzyloxycarbonyl)phenylalanine,l06 and on the rates of dipeptide formation from the reaction of polymer-based N-(0-nitrobenzenesulpheny1)amino-acid N-hydroxysuccinimide esters with various amino-acid derivatives in EMF and dichloromethane.107

More specifically mechanistic investigations are generally carried out with simpler substrates. p-Values of 0.67 and 1.31 are found for the reaction of 8-4-chlorophenyl thiobenzoates with butylamine and pyrrolidine, respectively ;lo* p for the leaving group (pyrrolidinolysis) is about 3.0, which is consistent with rate-determining breakdown of the tetrahedral intermediate. The reactions are generally first-order in thioester and amine, but the o-mercaptobenzoate gives a third-order reaction, second-order in amine ; owing to this difference, and the increase of an order of magnitude in rate compared with the p-hydroxy-isomer, it was suggested that the ortho-hydroxy-group is involved in the reaction.

Varying the amine, on reaction with p-nitrophenyl mercaptoacetate in MeCN a t 25", gives a Brsnsted j3 = 0.48.109

Similar investigations in benzene of the aminolysis by butylamine, benzylamine and aniline of aryl mercaptoacetates and benzoates show apparent bifunctional catalysis by carboxylic acids.llo* 111 The polarity of the transition state, as manifested by the effects of the medium on reactivity, depends strongly on the structures of the reactants; the butylaminolysis of p-nitrophenyl acetate, for example, appears to involve a much more polar transition state than that of 2,4-dinitrophenyl acetate.112

2-Pyridones are also bifwctional catalysts, with predominant electrophilic character.113

Halogen exchange of substituted benzoyl chlorides is entropy-controlled and may be interpreted in terms of hardness and softness of nucleophile and leaving group and extended Huckel theory.114 For the reactions of dimethyl- and diethyl-aminocarbonyl chloride, R2NCOC1, the rates are in the usual order C1- > Br- > I-.115

The reactions of anilines with substituted benzoyl chlorides have been studied by the stopped-flow technique in hexamethylphosphoramide.116 For the reaction of aniline with a series of benzoyl chlorides p = 3.4, and for the reaction of substituted anilines with benzoyl chloride p+ = -1.4. When the solvent is changed from chlorobenzene to nitrobenzene the sensitivity to substitution in the aniline falls, while p for the benzoyl chlorides is increased.117 The reaction is catalysed by tertiary amines118 and is faster (with aryl haloformates) for Br than for C1 leaving group and in the more polar solvent119 (nitrobenzene > benzene). The deuterium isotope effect for the acylation of PhzND by 3,5-dinitrobenzoyl chloride in benzene, k H l k D = 2.98 (at 25", falling to 1.52 a t 55"); no isotope effect is observed for alkylation of the amine with methyl iodide ; evidently proton removal is concerted with another step in the acylation, probably addition of amine- nitrogen to the carbonyl group, but it occurs in a rapid separate step in the alkyl' 'L t' ion reaction.120

Acylation of a series of alcohols by substituted benzoyl chlorides in nitrobenzene shows the expected retarding effect of bulky or more electrophilic groups on the alcohol.121 Reactions are third-order, second-order in the alcoho1,122 and are catalysed by triethylamine ; it is suggested that classical general species-catalysis mechanisms do not account for the observed properties of these reactions.123


Acylation of alcohols by phthalic anhydride is also a third-order reaction and faster in aromatic than in aliphatic solvents.124

The rates of the reactions of a series of mono- and di-carboxylic acids with dicyclohexylcarbodi-imide in THF to produce the anhydride depend on the pK of the acid, with maleic the most reactive, acetic acid the least, of a series of eleve2 aoids.lz5

Intramolecular Catalysis and Neighbouring-group Participation

Baldwin's rules87 for cyclization have relatively little effect on the way we think about attack on the carbonyl group, except where the precise geometry of attack on an ester, or particularly an amide, is of interest; and although the geometry of attack at digonal carbon is of intense interest, the disfavoured processes, 3- and 4-exo-dig in Baldwin's notation (36 and 37) will generally be disfavoured in any case for reactions of nitriles, for thermodynamic reasons.

Breslow and McClurel26 have discovered a remarkable change of mechanism in the hydrolysis of several dimethylmaleamic acids, with phenolic hydroxyl groups in the leaving group. For example, the hydrolysis of the amide (38) in acetonitrile 1~ in water,

- Go +ArCHlNHz

COOH 0

buffered below pH 4, gives dimethylmaleic anhydrideF9 as i t does in aqueous solution, some 50% more slowly than the N-benzylmaleamic acid. When the pH of the buffer is raised, so that the carboxyl group of (38) is ionized, the hydrolysis of the N-benzyl compound becomes very slow, presumably proceeding by way of the small amount of the acid form remaining. The hydrolysis of (38) is slowed also, but much less so, so that (the anion of) (38) is now hydrolysed faster than the N-benzyl compound. Moreover, the product is now no longer the anhydride (39), nor is this an intermediate in the reaction. It is suggested, as the most likely alternative rather than on the basis of specific evidence, that the mechanism has changed to intramolecular general base-catalysis (40) with the phenolic hydroxyl group acting as a general acid a t aome point so that the amine can

)&,--$ ->x" -0 COOH \

(40) (41)


leave from (41) as the neutral form.126 Further work will no doubt clarify the details of this intriguing reaction: but it has been suggested by Breslow as a possible model for the catalytic reaction of the enzyme carboxypeptidase (see below, p. 62).

The acid-catalysed hydrolysis of aryl-substituted maleanilic acids in strong acid is dependent on C-N bond strength and the activity of water.15 The reaction, like that of phthalanilic acids,l4shows substantial catalysis by the carboxylgroup. KlugerandChanl5 give a careful analysis of the observed kinetics in terms of a mechanism in which the general acid catalysis found in strongly acidic solutions is ascribed to the kinetically equivalent general base catalysed breakdown of the protonated tetrahedral intermediate (42). The change in the dependence on the pK of the conjugate acid of the leaving group,

from PLG = 0.15 in dilute acid to -0.12 in strong acid, is a consequence of the change in ground state from neutral amide to its conjugate acid.

The dependence on leaving group for the pH-independent hydrolysis of substituted phthalanilic acids is given by p = -1.05, falling to -0.67 for the acid-catalysed reactionl4 (equivalent to values of /?LG of 0.38 and 0.24, respectively, similar to the values found for maleanilic acids). Here too, a rate maximum is found as the acidity of the solutions is increased; on the other hand, the rate passes through a minimum, a t about 50 mole yo water, when the solvent contains increasing amounts of dioxan. With nitroaniline leaving groups, the observed reaction changes to formation of the imide, e.g. (43), and this reaction is favoured also by increasing the fraction of organic co-solvent.

0

(48)

A similar reaction is involved in the hydrolysis of asparagine derivatives with a free a-carboxyl group. For several such N-acyl compounds the observed rate of hydrolysis (0.03-0.4~-HC1 at 100') is accounted for in terms of competing inter- and intra-molecular


catalysis. Rate constants assigned to the intramolecular COOH-catalysed reaction are similar to those for the hydrolysis of succinamic acids.127

Amide hydrolysis is not catalysed by a neighbouring carboxylate group: but maleuric acid (44) finds an alternative intramolecular pathway12s in alkali, rearranging to the aspartic acid derivative (45).

coo-

coo- H

I CONHz

co- CONHCONHz

(44) (45)

,coo- ,coo-

Intramolecular catalysis by the carboxyl group of the hydrolysis of an acylimidazole is very similar to the reaction with normal amides, except that the compounds are much more reactive. The rate-determining step in the hydrolysis of N(-o-carboxybenzoy1)- imidazole (46) up to about pH 9 is the hydrolysis of the phthalic anhydride initially produced.1ze An effective carboxyl group concentration of 8 x 104 M (compared with intermolecular catalysis by acetic acid) is normal for this type of reaction, and so is the mechanism, with one exception. This is a case where the N-protonated amide (47) is probably involved. The only detectable reaction of the anion of (46) is the attack of hydroxide, so here too the carboxylate group does not catalyse the hydrolysis reaction.

A similar mechanism accounts for the cyclization of the carbamoylimidazole (48; R = H) to the N-carboxy-anhydride (49), and hence the formation of oligoglycines a t

(48) (49)

pH 7.O.l3O (48) is formed by the action of carbonylbisimidazole on glycine (R = H) or sarcosine (R = Me), and an ElcB mechanism is ruled out by the similarratesof cyclization of the glycine and sarcosine derivatives.

The hydrolysis of benzhydryl hydrogen phthalate (50) is some 103 times faster than that of the corresponding terephthalate ester, or of methyl hydrogen phthalate.131 The facts appear to be uniquely consistent with intramolecular general acid-catalysis of alkyl- oxygen fission by the neighbouring COOH group (the anion shows no participation by


COO-). The author writes the mechanism, which has not been observed previously, as proton transfer to the alkyl-oxygen of the ester-carboxyl group (51), although proton- transfer to carbonyl-oxygen will explain the facts equally well. General acid catalysis of ester hydrolysis is not normally observed for simple compounds: presumably the

0

timing of the bond-breaking process of theSN1 reaction fits better with the requirements of the general acid catalysis process than for addition to a carbonyl group, although it is possible that the decisive factor is simply the more favourable entropy of activation for the unimolecular process (51).

The equilibrium between aspirin and the mixed salicylic acetic anhydride (52) in several non-polar solvents (EtOAc, benzene, MezCO, MeCN) has been investigated.132 Surprisingly, (52) is formed only on prolonged standing, and the initial equilibrium (reached more rapidly in the presence of EtsN or magnesium hydroxide or carbonate as catalysts) involves the formation of the mixed acetylsalicylic acetic anhydride (53) and

a r H - O - C O C H 3

OCOCH3

IT (58)

salicylic acid. The corresponding anhydride, thought to be the mixed anhydride corresponding to (52), has also been observed (by IR and NMR spectra) in equilibrium with 3,5-dinit.roaspirii in aprotic solvents.133

Intramolecular nucleophilic participation by amide-nitrogen is observed in peptide syntheses, where the peptide with the free amino-group has an unprotected aspartic acid sidechain carboxyl group. Thus, the free COOH group of the terminal Asp-Phe sequence can compete with the amino-group for an active ester reagent to produce a mixed anhydride (54), which acylates the phenylalanine-nitrogen to give the cyclic imide (55).'34

OCOR'

*\N 0 Ph

R" H Gy CON& - " \Ph (54)

I (55)


Amide participation is directed through oxygen in the double intramolecular reaction of the hippuric ester of trans-2-iodocyclopentanol (56),135 but this happens after the rate-determining step, since the corresponding trans-p-toluenesulphonate reacts more slowly than the acetate ester.136

I

Intramolecular reactions are generally more convincing as models for similar enzymic processes the more closely they approach them in rate. The ester (57) was studied many years ago137 as a model for the deacylation step in chymotrypsin catalysis and was found to be hydrolysed near pH 8 with intramolecular general base catalysis by the imidazole group as shown (57). Catalysis is inefficient, however, partly no doubt because (57) can exist in many more conformations than the one which favours the reaction. The effects on the efficiency of catalysis of building in various kinds of conformational restrictionlss are shown for the series of esters (58-60). All are hydrolysed by intramolecular general base catalysis, according to the solvent deuterium isotope effect criterion (kHlkD = 2.5-2.6), but the range of reactivity is not large, aa is usually found for reactions involving intramolecular general base catalysis, and the rate of hydrolysis of the most reactive ester is still over 500 times smaller than the rate of deacylation of acetyl-a- chymotrypsin.

r

(67) krel (60”) = 1.0 (58) 2.8

(69) 0.83 (60) 11.6

A more versatile chymotrypsin model is the N-(imidazolylmethy1)benzohydroxamic acid (61). The high nucleophilicity of the hydroxamate ion makes (61) a powerful nucleophile towards p-nitrophenyl acetate, and in the presence of a large amount of ester a “burst” of p-nitrophenoxide is observed as (61) is rapidly converted into the 0-acetyl derivative (62). Now the imidazole group takes over, acting as a general base to catalyse the hydrolysis of (62) (by a factor of 130), back to the reactive form of the catalyst ; this boosts turnover much as in the chymotrypsin reaction.1~9

2 Reactions of Acids atd their Derivatives 47

COPh ,COPh

P-NPA &"'OH - ___, HN wN

(61) (62)

p-NPA = p-Nitrophenyl acetate

Pyrimidine-nitrogen acts as an intramolecular catalyst in the hydrolysis of the amide (63) derived from 2-thiocytosine.140 The amide can be made by alkylating the thio- pyrimidine with iodoacetamide, but only under basic conditions : in acid the carboxylic acid (64) is the product, and (63) is readily hydrolysed to (64) a t low pH (half-life about 1 h a t pH 3). The authors suggest that the protor ated N-1 of the pyrimidine acts as a general acid to catalyse hydrolysis of the amide, on the (mistaken) grounds tha t the nucleophilic mechanism should be more favourable for a six- than for a five-membered cyclic transition state. In fact the reaction is almost certainly the first example of intramolecular nucleophilic catalysis by pyrimidine-nitrogen, resembling the reaction catalysed by the carboxyl group. In both cases the conjugate acid of the nucleophile has a pK near 4, low enough to co-exist with enough protonated amide for the cyclization 65) to proceed a t a useful rate.

NHz NH2

- 7 - (-J etc. dJ"a H fj f i CONHz

ii" . . HO

H O y NHz NHz

(66)

Quinoline-nitrogen is thought to act as a general base to assist thepiperidinolysisof the esters (66) in chlorobenzene.141

X


The imide (67) undergoes mostly intramolecular aminolysis when kept in dilute solution in pyridine,142 and a similar cyclization is complete in a few minutes when the amine-protecting group is removed from the peptide (68) by hydrogenolysis.143

(69)

Corey, Brunelle and Stork report a study of the mechanism of their very effective procedure for the synthesis of medium-ring lactones. Cyclization of the active ester (69) in benzene a t 80” is first-order in thiolesterl44 and not subject to general acid or base catalysis. The 4-pyridyl S-ester does not give lactone under these conditions. A ketene route is conclusively ruled out, leaving as the most likely possibility the “double- activation” mechanism (70). Reaction is much faster when the more basic imidazole

S N H ~ ( c H ~ ) l & H 0- 9

(69) ‘?f(,CHz) 16

-o/CHa \ (70) Os N + o&z)lb

I H

derivative (71) is used but most of the starting material is diverted to form N-acylimidazole. This side-reaction can be prevented sterically by incorporating a tert-butyl group next to N-3, a t the expense of a decrease in overall reactivity.145 The catalyst in its most highly evolved form, (72), achieves conversions to lactone of up to 80-90% a t 50”.

Me Me I I cI& sco ( CHz) i sOH ~ ~ ~ c o ( c H z ) l r O H

But (71) (72)

The spectacular increases in the rates of acid-catalysed lactonization of hydro- coumarinic acids caused by substitution with a methyl group were explained by Cohen in terms of “stereopopulation control”.l46 The pentamethyl compound (74), for example, is cyclized 1011 times faster than (73). New results147 from Loudon’s laboratory indicate 8 & & &

\ \ \

(78) kre1= 1.0 (74) 1ou (75) 160 (76) 2.4 x lo*


that a large part of this factor has to be accounted for in terms of steric compression in the ground state of (74), which is relieved in the transition state for cyclization. The hydroxy- acids (75) and (76) represent partially and fully “locked” analogues of (7Q), but the effect of conformational restriction on the rate of cyclization is only a fraction of that observed for (74). Evidence for ground-state strain in structures such as (74) is provided by a “steric isotope effect”, of about 10% ( k ~ / b = 1.09 +_ 0.02) for cyclization of the hexadeuterio-cornpound (77), and force-field calculations148 of the strain energy of (73) and (78) and the corresponding lactones.

Fife and Benjamin’s studyla of the general base catalysed lactonization of 2-hydroxy- methy1)benzoate has been mentioned above (p. 31). Lactonization of the corresponding amides is involved in a suggested procedure for making “latent” drugs. The ester amide (79) is hydrolysed in base a t the rate of the ester hydrolysis, because the hydroxymethyl group produced rapidly releases the amine (the active form of the latent compound) by intramolecular nucleophilic catalysis.149

Butylaminolysis of pyrocatechol monobenzoate (80) in acetonitrile is much faster than that of the corresponding o-methoxy-compound (81). The reaction of (81) is second-order in amine, while that of (80), with the free OH group, is of the first order. Evidently, the neighbouring group replaces one of the molecules of amine in the transition state, and the authors150 suggest that the ionized group acts as a general base (82).

OCOPh

0- BuNHs+

BuNHt

0- l


At low amine concentrations (80) reacts up to 16,000 times more rapidly than (81) in acetonitrile a t 25", a factor much larger than any previously attributed to intramolecular general base catalysis. It is suggested that the normal low efficiency of intramolecular general base catalysis151 may be peculiar to aqueous solution, where solvent-catalysed reactions occur a t a significant rate in the absence of general species-catalysts. This is of course true, but the argument applies equally to intermolecular catalysis: in a truly inert solvent any catalysed reaction is by definition infinitely faster. The important comparison is between intramolecular and intermolecular catalysis involving the same mechanism: i.e., in the case of the very interesting reactions of (80) and (81), between the rate of aminolysis of (80) and the rate of the same reaction of (81) catalysed by phenol. Data have been presented150 that show a substantial catalytic effect of butylammonium chloride on the reaction of (al), but this is ascribed to general base catalysis by the chloride anion.

The equilibrium constants for the 1,5-acyl migration (83) + (84) show a maximum for the six-membered rings shown.152 The same reaction in the open-chain system (85) is acid-catalysed.15s

(88) (84) (85)

The o-(hydroxyamin0)-group, shown last year to be an effective intramolecular nucleophile for the ester goup,l54 also attacks the corresponding amides (80).155 The cyclization to the 2,1-benzisoxazolin-3( lH)-one (87) is general acid catalysed and occurs readily in both aqueous and aprotic solvents. The N,N-dimethylamide (86); R = Me)

CONRa A d J + R 2 N H

NHOH

(86) (87)

is hydrolysed about 100 times faster than phthalamic acid (and probably by the same mechanism). Since tertiary amides are generally more reactive than primary in intramolecular reactions catalysed by COOH (in contrast to the simple acid-catalysed reactions)'5&.it was suggested that neighbouring NHOH is a more efficient catalyst than COOH by an order of magnitude or so; this reaction thus becomes an interesting pro- position in protecting-group chemistry, since it is readily generated from the almost inert nitro-group.

Association-prefaced Catalysis

Reactions in reversed micellar systems have been reviewed by Fendler.157 It has been pointed out that a dependence of activity on sub-unit aggregation is a common feature of micellar and enzymic reactions.158

Guthrie and Ueda'sQ have published a full account of the reactions of the cationic functional steroid (88; R = H) with aryl esters.160 At zero ionic strength all anionic

2 Reactions of Acids and th&r Derivatives 51

' V coo- (88) (89)

substrates show positive deviations, and all positively charged substrates show negative deviations, from the linear free-energy relationship betwen log kz for catalysis by (88) and by imidazole. The compound (88 ; R = H) is a good catalyst (twice as good as hydroxide, 20 times better than imidazole) for the hydrolysis of 3-arylpropionate esters (89); but the hydrophobic effect of increasing the group Ar (up to phenanthryl) is not large; this can be increased by using the much less reactive N-isopropyl derivative (88; R = CHMez), but even here the largest factor (with Ar = 3-phenanthryl) is only about 25.

Cram and his colleagues have reviewed the design of host molecules for the complexa- tion of specific types of substrates,lsl with special reference to his work on the chiral recognition of amino-esters. A system (90) which shows such chiral selectivity, has now162 been armed with functional groups (R = CHzSH or CHzOH) and set to work on p-

(91)

nitrophenyl esters of D- and L-amino-acids. Ethanolysis (in 1 :4 (v/v) EtOH-CH2C12, buffered with 0.2 M-HOAC + 0.1 M-MedNf OAc-) of L-amino-esters containing NH; groups [which are known to be complexed by the polyether ring of (SO)] by the dithiol (90; R = CH2SH) is 100-1ooO times faster than the same reaction with the corresponding acyclic polyether (91), although the L-proline ester (NH; group) is solvolysed a t similar rates in the presence of the two dithiols. These differences are orders of magnitude smaller in a more hydrophilic medium (40% aqueous MeCN) where non-specific H-bonding solvation by solvent is more competitive. There is also a strong chiral specificity. I n the non-aqueous medium the cyclic (8)-dithiol reacts with L-amino-ester salts more rapidly


than does the (R)-dithiol, by factors that depend on the size of the side chain of the amino-acid and reach as high as 9.2 when this is isopropyl. This selectivity was (qualitatively) predicted on the basis of an examination of models of the tetrahedral intermediates in the (nucleophilic catalysis) mechanism, which should be close in geometry to the transition states. In the favoured transition statelintermediate (92), the

(92)

side-chain is directed away from the c h i d binaphthalene system. If the sidechain and H are interchanged, giving the D-amino-ester, more severe non-bonded interactions are expected.

The effectiveness of polyethleneimine with various N-alkyl substituents as a catalyst for the hydrolysis of p-nitrophenyl esters of a range of fatty acids depends on the chain- length of the alkyl group of both catalyst and ester, and the binding constants (l/Km) show a correlation with the size of the alkyl group.163

Mellitin (a cationic peptide surfactant from the venom of the honey bee, with 26 amino-acid residues) is an effective catalyst for the hydrolysis of p-nitrophenyl dodecanoate,l64 facilitating both dispersion of aggregated ester in aqueous solvent and its alkaline hydrolysis. An autocatalytic effect is confirmed by the addition of dodecanoate : apparently a complex involving both mellitin and the carboxylate product is a still more effective catalyst.

The reactivity of N-methylmyristohydroxamate (93) towards p-nitrophenyl acetate is enhanced by the addition of hydrophobic miceIles, and much more effectively by

(98) (94)

CTABr. In contrast, the anionic sodium lauryl sulphate depresses its reactivity.la5 The reactivity of (93) in non-ionic micelles is further enhanced by the addition of the hydrophobic methyltrioctylammonium chloride (TMAC) but not by BQN+Br- (which cannot itself form a micelle). Similar enhancements of the activity of the zwitterionic oxime (94) by non-ionic micelles also show the effects of added cationic and anionic surfactants, but not of added TMAC. It is suggested that enhancements of reactivity of nucleophilic anions in cationic micelles are largely due to the formation of desolvated ion-pairs in hydrophobic environments.

Similar high reactivity of desolvated ion-pairs is thought to account for the first efficient (intermolecular) nucleophilic catalysis of amide cleavage under mild conditions. N-Methylaceto-2,4-dinitroanilide is readily cleaved by N-methylmyristohydroxamate (93) in dry dipolar aprotic solvents (DMF, MeCN or CaH6) a t room temperature.166


Small amounts of water depress the rate of the reaction sharply, and the amide is stable to (93) in aqueous CTABr micelles.

The reaction of the long-chain analogue (95; R = n-CleH33) of p-nitrobenzoylcholine (95; R = Me) with hydroxide ion is catalysed by cationic micelles of CTABr and the

RNMezCHzCH20COCeH4NOz-p RNMeSBr- + +

(95) (96)

RNMezCH2CHZOH

(97)

hydroxyethyl compound (96 ; R = HOCHzCHz) to similar extents. Nucleophilic attack of (97) on (95) is a virtual reaction, so the higher reactivity of (97) than of (96) towards other esters, phosphates, alkyl halides and amides can be ascribed with some confidence to nucleophilic attack by the ionized hydroxyethyl group.167

The effect of cationic micelles of varying hydrophobic character on the alkaline hydrolysis of p-nitrophenyl acetate, and of the esters of 3-nitrobenzoic and 3-nitrobenzene- arsonic acid has been investigated.168

Micelles of 3-[ (cetyldimethylammonio)methyl]imidazole (98) are good catalysts for the hydrolysis of p-nitrophenyl acetate and hexanoate (pH 7.2-8.2), partly because of an increased contribution from the imidazole anion reaction in the cationic micelle. But (98) is much less effective than the histidine derivative (99), mostly because of its lower pK.ls9

+

coo- + I

n-ClaHssNMeaCHz CisHz7CONHCHCHz

1) H 1) H

( 9 4 (99)

Alkaline hydrolysis of the p-nitrophenyl ester (100; Ar = p-OzNC&) is catalysed by micelles of cetyltrimethylammonium bromide much more effectively than is that of the phenyl or p-methoxphenyl esters, and it is suggested that the ElcB mechanism is specifically favoured in the micellar-catalysed process.170

PhSCH&OOAr BuOCSSCOBu II II s s

(100) (101)

The catalytic action of alkylammonium carboxylates on the hydrolysis of p-nitrophenyl alkanoates is enhanced by micelle formation in hydrocarbon solvents, increases with the number of +NH groups and is greatest for esters of short-chain acids in aliphatic solvents.171

The decomposition of butyl dixanthogen (101) is strongly catalysed by CTABr a t pH 10.5.172

Cationic, neutral and anionic micelles have little effect on the rates of hydrolysis of 4-methyl- and 4-nitrophenylurea, though the addition of small amounts of the urea causes large increases in the critical micelle concentration (CMC) of CTABr and nonyl- phenoxypoly(oxyethylene)-14-ethanol. On the other hand, reversed micelles of


dodecylammonium propionate in benzene accelerate the decomposition of 4-nitrophenylurea by a factor of 3000,173 compared with the pH-independent reaction a t pH 6.7 in water. (The addition of small amounts of water to the reversed micellar system effectively stops the reaction.) The reaction in benzene is also catalysed well above the CMC by the constituents of the surfactant RNH; -OOCR’, presumably by general acid-base catalysis.

The alkaline hydrolysis of lauroyl chloride is rapid a t 30-60”, and first-order over a t least one half-life, so that the product laurate does not affect the rate.174

A formalism for the treatment of kinetics in micellar systems has been reported.175 The parameters used are the radius of the hydrocarbon core of the (spherical) micelle, the thickness of the reaction surface layer, the surface and zeta potentials, and a distribution coefficient for a hydrophobic substrate. The treatment is applied to the inhibition of orthoester hydrolysis by an anionic surfactant and predicts similar inhibition by a cationic surfactant.

Deacylation of acylcyclodextrins is the slow step in the reactions of cyclodextrins with ester “substrates”. This step is selectively accelerated by amines, and for the deacylation of p-cyclodextrin 0-cinnamate Komiyama and Bender found the most efficient catalyst to be diazabicyclo-octane (DABCO) (> butylamine > quinuclidine > piperidine > triethylamine $ diisobutylamine).176 The DABCO is probably included in the cavity of the cyclodextrin and is considered to act as a general base catalyst. The acylcyclodextrin- DABCO complex is hydrolysed up to 57 times faster than the ester alone at pH 13.6.

X-ray and neutron diffraction studies of several a-cyclodextrin-“substrate” complexes reveal two distinct forms of a-cyclodextrin, termed the “tense” and “relaxed” forms.177 In the “tense” form, found for the empty (apart from just two water molecules) a-cyclodextrin the torus is puckered, with one glucose unit rotated out of the regular arrangement. With substrates included in the cavity the torus is round and regular. It is likely that the “tense” form cannot bind substrate directly, and the possible mechanisms of substrate bindings are simple models for the induced-fit type of enzyme- substrate complex formation.

The binding of p-nitrophenoxide by cyclohepta-amylose is scarcely affected by inethylation of the seven 3-OH groups, or the 14 2- and 6-OH groups. It is thought that if strain were a major factor in binding this “substrate” a substantially larger effect would have been observed.178

Metal-ion Cahlysis

Hydrolysis of the ester (102) is subject to highly efficient catalysis by CO(II) and Ni(n) ions. The mechanism involves intramolecular attack by substrate-bound metal hydroxide

( 102 1 (108)

2 Reactioiis of Acids and their Derivatives 55

(103), and near pH 7 the intramolecular reaction is up to 2 x lo5 times more effective than external catalysis by hydroxide.179 Lewis acid activation of the carbonyl group can be ruled out. The reaction is only about 103 times slower than the reaction of carboxypeptidase with good ester substrates.

The hydrolysis of 5-chloroquinolin-8-yl glucuronide is catalysed by metal ions a t pH 7, to an extent that parallels the stability of the metal-ion-oxime complexes.180

A NMR study of the binding of Zn(II) by L-aspartyl-L-phenylalanyl methyl ester shows that the metal ion is bound too far from the ester bond for catalysis of hydrolysis t o be significant. 181

Metal ion catalysis of the formation of 2-hydroxyethyl benzoate8 has been studied under various conditions : with terephthalic acid, ester formation (and oligomer formation) in boiling ethylene glycol is catalysed by Co or Mn(I1) acetate :1*2 ester exchange of dimethyl terephthalate in ethylene glycol is catalysed by a large range of metal acetates ;Ie3 and the ethylene glycolysis of substituted benzamides is catalysed by PbO ( p = 0.48)lS4 and a large number of metal salts, with an efficiency correlated with the acid ionization constants of the aquo-complexes.185 These reactions are characterized by very low negative entropies of activation, near -50 e.u.

Pd( 11) salts promote alkyl-oxygen cleavage of the /I-lactones (104).186

I I R (104) R

The hydrolysis of thiobenzamidesl87 is catalysed by the tetrachloroaurate ion. A stopped-flow study of the initial fast formation of the fairly stable complex (106) shows evidence for an intermediate in the reaction of the unsubstituted and N-cyclohexyl compounds (105; R = H or CsH11). A five-co-ordinate structure is suggested for this species.188

4s +AuC14 . INT. - Fast C13AuS=C 'Ph +c1- Faster

\NHR Ph-C

\NHR

(105) (106)

Slow Ha0 I PhCONHR

The hydrolysis of thioimidate esters in dilute acid is catalysed by IIg(I1) but not by silver ions.189 The ester conjugate acids are unreactive towards Hg(I1) ions, so the rate of reaction falls as the hydrogen ion concentration is increased. A similar pattern is found with tetrachlorogold( 111).190

Enzymic Catalysis

Serine Proteinases

Blow has reviewed recent thinking on the structure and m_echanism of action of chymotrypsin.lgl An improved three-dimensional structure of the zymogen at 2.5 A


resolution shows a normal H-bond from the imidazole-N of histidine-57 to the hydroxyl group of serine-195,lQz in contrast to the corresponding long, "bent" H-bond in the active enzyme. A MO study of the charge-relay system of the serine proteinases yields energy profiles for the acylation and deacylation steps with ester and amide substrates, and sensible agreement with structural and kinetic data.1Qs

Rapid-reaction techniques have identified four preacylation steps in the reaction of chymotrypsin with N-acetylphenylalanine p-nitroanilide in 65% aqueous dimethyl- sulphoxide a t -90";lQ4 the first corresponds to substrate binding, to form the Michaelis complex; two pH-independent steps, ascribed to substrate-induced conformation changes, follow; the fourth step depends on an ionization (pK = 5.9) involving the imidazole group of histidine-57, but this is not the formation of the tetrahedral intermediate (which does not appear to accumulate even under these conditions, in agreement with other results); a final lining up of the charge relay system and substrate carbonyl group prior to bond formation are probably also involved.

The kinetics of dimerization of a-chymotrypsin have been measured by monitoring the loss of active sites.lQ5

Rate constants for the deacylation of 5-n-alkylfuroyl- a-chymotrypsins depend on the 5-substituent. The reaction of the 5-n-propyl derivative is fastest, and the Arrhenius plots for this compound (and the 5-Et and unsubstituted compounds) are biphasic, consistent with (a minimum of) two steps for the reaction.196 Linear Hammett plots are found for R = Me, Bun and n-pentyl.

The volumes of activation for the deacylation of di- and tri-methylacetyl- a-chymotrypsin in TRIS buffer (-5, -3 cms mol-1, respectively) are equal to the values found for the overall reaction.197 The effect of pressure on the same reaction of indoleacryloyl-a- chymotrypsin in phosphate buffer is dominated by the effect on the pH of the buffer, and the rate actually decreases with increasing pressure ; this effect can be allowed for, to give a value of -7 om3 mol-1 for the volume of activation for deacylation, in agreement with the figure obtained in TRIS buffer.

Octyl isocyanate specifically inactivates a-chymotrypsin, and the X-ray structure of the resulting octylcarbamoyl-enzyme shows why : the alkyl chain is bqund exclusively in the hydrophobic pocket, without distorting a planar urethane bond to the oxygen atom of serine-195.lQs

Specific reaction in the hydrophobic pocket was the mission of the reagent (107), designed in the hope that the aromatic group would fit into the aromatic binding site. But this reagent also reacts at serine-195, as well as with amino-terminus of the enzyme.19Q

(107) (108)

Phenacyl bromide reacts with chymotrypsin at low pH to alkylate the sulphide-S of methionine-192. The sulphonium salt can be dealkylated with SH reagents, but this is not possible if the alkylated enzyme is allowed to stand, or is formed, at neutral pH. The irreversible reaction is probably a dealkylation of the sulphonium salt (transfer of CHs or phenacyl) by a second nucleophilic group on the protein.200


Reaction of a-chymotrypsin with glycine ethyl ester a t low pH in the presence of a water-soluble carbodi-imide leads to the conversion of 15 of the enzyme’s 17 carboxyl groups into neutral amides. Compared with native enzyme, activity is sharply reduced towards N-acylamino-esters, so that non-acylated, ionizable substrate esters are preferred. But reaction apparently depends on the same ionizations as control the reactivity of the native enzyme, so modification has not blocked the carboxyl ionizing near pH 5. The mechanism proposed201 includes a role for the protonated imidazole of histidine-57 (since the rate does not fall so sharply a t low pH for the modified enzyme), and uses water rather than asp-102 as the proton sink; it raises many more problems than it solves.

More probes have been inserted into the active site of chymotrypsin. The spin-labelled acyl-enzyme (108 ; R = enzyme-serine-195) is formed by acylation with the corresponding p-nitrophenyl ester ;202 and p-bromocinnamic acid binding has been followed by pulsed79~ 81 Br-NMR spectroscopy. Rapid Br exchange is observed, suggesting that the aromatic ring is not bound in the hydrophobic pocket.203

Substrate specificity continues to attract interest, particularly comparisons between different serine proteases. The tripeptide Lys-Phe-Leu has been varied by replacing the Phe by a-aza-phenylalanine or D-Phe. With the D-amino-acid the peptide is stable to aminopeptidase, carboxypeptidase, thermolysin and trypsin, but trypsin cleaves the Lys-azaPhe bond of the first modifieation.204 The hydrolysis of a series of substrates HOOCCH2CH2CO-Ala-Ala-Y-NHC6H4NO2-p with 14 varieties of Y, by elastase, a-chymotrypsin and trypsin, is fastest for elastase with Y = Ala. Chymotrypsin prefers Y = Phe, and trypsin only cleaves two of the 14, where Y = Phe and Tyr.205 Other studies include an examination of N-methylamides as substrates,206 and a study with co-polymers formed from CH-CH CONH(CH2),COPhe aNHCsH4N02-p with a large excess of acrylamide or acylic acid. Susceptibility to cleavage by chymotrypsin falls sharply as the length of the chain separating the anilide group from the polymer backbone is reduced, from a value of Vmax with n = 5 which is equal t o the rate with monomer.207

The binding of a series of both D- and L-N-acyl-a-amino-amides (109) to a-chymotrypsin is correlated with the molar refractivity of the groups Rand R‘CONH. The group with the larger molar refractivity is thought to bind in the hydrophobic pocket, the other in p l space.208 Km values for the natural amino-acid derivatives and Ki values for the D-amino-amides are found t o be correlated by a single equation.

+ R’CONHCHRCONHg (RNMe&H&eH4N=)z

(109) (110)

A study of the effects on binding and catalysis of the oxidation to sulphoxide of the sulphide group of methionine-192 of 8-chymotrypsin is consistent with only a small effect of this side-chain on the binding of polypeptide substrates.209

Ester substrates of chymotrypsin with the same acyl group give values of ko/Km which vary with leaving group in the order methyl < benzyl < cyclohexylmethyl, which is consistent with previous suggestions of a hydrophobic binding site for the leaving group.210

The binding of certain substrates of chymotrypsin can cause a perturbation of the pK of a group on the enzyme, which is not observed with non-substrates of similar structure ; but this effect cannot be identified conclusively as an interaction between the leaving group and the charge-relay system.211

Allosteric effects of azobenzenes (110) with two quaternary ammonium centres on the


hydrolysis of specific substrates by a-chymotrypsin act selectively to increase kcat, under conditions where acylation is rate-determining. Km is unchanged.212

A series of oligopeptides based on the sequence of amino-acids round the active-site serine of chymotrypsin, containing histidine and in some cases aspartic acid residues, have been synthesized and shown to be up to half as reactive as imidazole towards p-nitrophenyl acetate.213

A new 49-amino-acid protein inhibitor of a-chymotrypsin from Russell-Burbank potatoes has three disulphide bonds in its short chain.214 A larger inhibitor from chick peas (66 amino-acids) which also inhibits trypsin, is of the double-headed type, with separate reactive sites for the two enzymes.216

The second-order rate constants for the reaction of a-chymotrypsin with a large series of esters (111) of a thiophosphonic acid show an interesting and characteristic dependence on the hydrophobicity of the alkyl sidechain R. A correlation with the Hansch partition coefficient substituent constant T is linear but biphasic, with a break a t T = 3 corresponding to a limiting length of the alkyl chain.216 Similar previous results with peptide substrates (where R is an amino-acid side-chain) give a similar dependence of log(kcat/Km) upon T.

Difference IR spectroscopy has been used to measure the pK’s of the carboxyl groups remaining when bovine trypsin is modified with semicarbazide in the presence of carbodi- imide. A pK of 6.8 is assigned to aspartic acid-102, and an average value of 2.9 is identified with the sidechain of aspartic acid-104 and the C-terminus.217

The exchange of tritium with the C-2 hydrogen of the imidazole of histidine-57 of trypsin is 25 times slower than the reaction of free histidine, which has a half-life of 2.8 days at pK 8.2. The reactions of His-40 and His-91 are slower still, with half-lives of over lo00 days.218 These reactions are slower than similar reaction observed for histidine residues of ribonuclease and lysozyme, and must mean that the imidazole groups are highly inaccessible to solvent.

The integrity of the disulphide bridge between cysteines 179 and 203 of trypsin is important for substrate binding. When the bond is cleaved and alkylated (by the reaction of borohydride and then the alkylating agent on trypsinogen) the modified trypsin produced reacts normally with non-specific substrates, but Km is increased by 2 to 3 orders of magnitude for specific substrates.219 kcat is not much changed. The binding of specific inhibitors, especially the (protein) soyabean inhibitor, is also much weaker. The disulphide bond concerned is known from X-ray studies to be situated to one side of the opening of the substrate specificity pocket.

The acylation of a-trypsin by substituted phenyl hippurate substrates shows a low sensitivity to leaving group (using u- and k,,/Km, p N 1.0),220 as also observed with chymotrypsin. This in interpreted in terms of electrophilic participation in the acylation step.

The reactions of the lysyl ester analogue (112) with thrombin, trypsin and plasmin, have been compared with those of the arginine analogue (113). Enzyme substrate- binding changes little as enzyme or substrate is varied, and the rate constants for deacylation of the acyl-enzyme from (112) are similar for all three enzymes. The low efficiency of cleavage of lysyl ester bonds by thrombin is a result of kinetic specificity


(112) (llj)

in the acylation step. This step is over 10s faster for the reaction of (112) with trypsin than with thrombin, and 60 times faster for the reaction of thrombin with the arginine analogue (118) than with (112).221

The acylation of subtilisin Carlsberg by substituted phenyl esters of N-acetyl-L- phenylalanine, hippuric acid and dihydrocinnamic acid gives p-values of about zero, 0.4 and 1.0, respectively, depends on a base of pK 2 7.4, and is 2-3 times slower in DzO than in H20.222 An electrophilic component in the acylation mechanism is a possible explanation of the low p-value found for the phenylalanine esters, but it is reasonable that the transition state should be earliest for the best substrates.

The X-ray structure of subtilisin inactivated with the irreversible specific inhibitor Phe-Ala-Lys-CHzC1 has been determined from a 2.5-A electron-density difference map.223 The group alkylated is the nitrogen atom of the catalytic imidazole residue of histidine-64, but the catalytic serine also forms a covalent bond to the inhibitor carbonyl group, forming a hemiacetal analogue of the tetrahedral intermediate of the catalytic reaction as in (114). It is suggested that addition to the carbonyl group must precede the alkylation of the imidazole, since chloro-ketone reagents will not alkylate the iGdazole of anhydrochymotrypsin.

(114)

“Superactivation”, observed earlier with thermolysin, has now been demonstrated with other neutral proteases. Acylation of enzymes from B. subtilis and B. mqccterium with N-hydroxysuccinimide esters of N-acylated amino-acids gives acylated enzymes that are more reactive than the native enzymes. For example, acylation with the N- acetyltryptophan esters leads to increases in kcat/Km of more than an order of magnitude.224 Presumably the activation involves acylation of residues near the active site, which can interact with substrate as i t binds.

The apparent pK of kcat/Kapp for the reaction of acetylcholinesterase with neutral


substrates varies between 5.3 and 6.3, and involves non-linear inhibition by H30+. Deuterium solvent isotope effects also vary with substrate, from k ~ / k ~ = 1.9 for p-nitrophenyl acetate to 1.1 for acetylcholine. The picture, based on the chymotrypsin mechanism, of a rate-determining general base-catalysed step, appears to be too simple to account for these variations, and a rate limiting induced-fit step is proposed to account for the apparent pK values and low values of k H / k D observed with, e.g., phenyl and isopentyl acetate ( k ~ / k ~ = 1.26).225

2-Substituted 1,3,2-dioxaphosphorinane 2-oxides (115; X = F or p-nitrophenyl, etc.) have low anti-cholinesterase activity, not because the acylated enzyme is rapidly dephosphorylated, but because both association and phosphorylation steps are slow with these compounds.226 The more specifically directed compound (116) is a competitive inhibitor for acetylcholinesterase and does not phosphorylate the enzyme.

0 0

Thiol Proteinuses227

Drenth et al. report difference-Fourier studies of the structures of three chloromethyl ketone derivatives of papain.22* The oligopeptide tail (the largest inhibitor used was Ac-Ala-Ala-Phe-AlaCOCH2Cl) fits into the (slightly expanded) active-site groove on the enzyme, much as expected. Starting from these structures, an extrapolation through acyl-enzyme (removal of the CH2 group) to the tetrahedral intermediate gives reasonable pictures of these intermediates. The negatively charged oxygen of the tetrahedral intermediate is placed in a region where it can be stabilized by hydrogen-bonding to two amide groups, the peptide bond of cysteine-21, and the amide group of glutamine-19. There is an obvious similarity here to the situation in chymotrypsin. The nitrogen atom of the leaving group is close enough to the imidazole ring of histidine-159 to make a general-acid role for this group geometrically reasonable.

The fluorine NMR signal from papain irreversibly inactivated with CF3COCH2Br depends on pH. Fluorescence-intensity studies show a dependence on two groups whose ionisation is co-operative. This is very likely a phenomenon induced by the probe : the trifluoroacetyl groupis almost certainlyhydrated, CF&(OH)2-CH2-, andcan thus form hydrogen bonds to the protein. It is suggested that aspartic acid-158 and histidine-159 are together responsible for the acid limb of the dependence on pH of substrate hydrolysis.220 The spectroscopic data allow estimates of chemical exchange rates for the ionization of aspartic acid-158/histidine-l59 and thus also approximate calculations of rate constants for proton transfer; The latter come out rather low, perhaps because of the hydrophobic microenvironment.230

Stopped-flow studies of the fluorescence of two mansyl-peptide substrates of papain show that association with the enzyme is a biphasic process: a very fast initial increase in fluorescence is followed by a slower, first-order enhancement (a similar picture is observed with mercuripapain, but there the second stage is much slower). The rate ofthe second stage shows saturation as enzyme concentration is increased, which is consistent with its being the conversion of ES to a fluorescent species ES*. The hydrolysis of the (Glu-Leu) peptide bond of the oligopeptide is associated with a further first-order

2 Reactiom of Acids a d their Derivatives 61

decrease in fluorescence and is about 140 times faster for a mansylhexapeptide than for a corresponding pentapeptide ; this is further evidence for the importance of secondary enzyme- substrate interactions for catalytic efficiency. The breakdown of ES* is probably the rate-determining step of the overall reaction for these substrates.231

Arguments from resonance Raman spectra of the acyl-papain formed from the substrate (117) indicate a tautomeric change, perhaps with formation of a covalent bond from the carbonyl-oxygen atom (to an unidentified group X), giving a structure

Me2N f J y H - e ph MezN m;:hEn= 0 ‘x NOz 0

(117) (118)

of the type (118).232The technique can be useddirectlytofollowacylationanddeacylation kinetics.

Results with two reactivity probes, 2,2’-dipyridyl disulphide (1 19)233 and 4-chloro-7-nitro-2,1,3-benzoxadiazole (120), and highly purified ficin and bromelain234 a t different pH’s show important differences from the reactions with papain ; so these two enzymes probably lack a carboxyl group conformationally equivalent to aspartic acid-158 of papain.

NO2

(119) (1fLO)

The hydrolysis of substituted phenyl hippurates and methanesulphonylglycinates by bromelain is rather insensitive to the leaving group, consistently with an electrophilic enzyme-substrate interaction.220 Results with these two types of substrates, analysed by Hansch’s quantitative structure-activity relationship technique, show that K , values are correlated by the inductive effect and polarizability of the substituents on the phenyl group.235

Acid Proteinases236

Transpeptidation of both acyl-transfer and amino-transfer types is catalysed by pig pepsin237 and is selectively stimulated by a number of small peptides, as much as 45-fo1d.Z3* Thus Leu-Leu-Leu is the major product from the substrates Leu-Trp-Met-Arg and Leu-Trp-Met a t pH 3.4, and also from Z-Phe-Leu. The acyl-transfer reactions probably also involve a covalent intermediate, since labelled leucine is not incorporated into transpeptidation products and does not exchange with enzyme-bound leucine in the presence of acceptors. Results with oligopeptides suggest a binding site extending over eight or nine amino-acid residues. There is still no direct evidence that acyl- and amino- enzyme intermediates are involved in the hydrolysis of polypeptide substrates, but the high yields of various transpeptidation products obtained-under some conditions the reaction is faster than hydrolysis-suggest strongly that they are.


The acyl-enzyme intermediate is presumed to be an anhydride236 and evidence consistent with a mixed anhydride intermediate has been obtained by a trapping experiment with hydroxylainine.23g Incubation of pepsin with a phenyl sulphite in the presence of hydroxylamine causes progressive deactivation of the enzyme and the formation of up to 3 4 enzyme-bound hydroxamate groups. Lossen rearrangement followed by acid hydrolysis gives only 2,3-diaminopropionic acid, as expected if only aspartic acid residues are involved. These include aspartic acids-32 and 215, which are not essential for enzyme activity towards sulphite ester or protein substrates. Something of a mystery surrounds the possible roles of these and a t least one other pepsin carboxyl group, though i t is possible that hydroxamic acid groups can themselves act catalytically.

The conversion of pepsinogen into the active enzyme probably involves a t least two steps, since a hexadecapeptide fragment can be identified from incubation a t pH 2.5 in the presence of pepstatin.240

A series of elegant experiments in which the Pi-isoleucine residue of soyabean trypsin inhibitor Kunitz is replaced chemically by alanine, leucine and glycine demonstrate that the enzyme tolerates changes in the sidechain of amino-acid-64. About one-third of the natural activity is retained in the modified inhibitor. Full activity is regained if the replacement is reversed;241 but adding an extra isoleucine or alanine a t this position converts the inhibitor into a substrate for the enzyme.242

Metallo-proteinasea

Carboxypeptidase catalyses the exchange of 180 from N-benzoylglycine-carboxyl-ls0 into solvent water. This sort of evidence has been presented before in support of an acyl-enzyme mechanism (anhydride with the carboxyl group of glutamic acid-270) ; but Breslow and Wernick243 have shown that with N-acetylglycine, and especially with N-benzoylglycine, exchange takes place only in the presence of added amino-acids, particularly L-phenylalanine. The preferred explanation of these results is that the exchange reaction is a result of enzyme-catalysed peptide synthesis, the acyl-enzyme being no longer an essential intermediate in the exchange mechanism. Indeed the nucleophilic catalysis mechanism is only consistent with the new evidence if several restrictions-albeit not entirely unreasonable restrictions-are built in; it is suggested that a general-base catalysis mechanism (below) is now more likely. Replacement of the active-

[ O* cH' 'H

COa- I

*o\H

COOH I

Glu-270 61u-270

2 Reactiotis of Acids and their Derivatives 63

site Zn(r1) by CU(II) results in complete loss of the activity of carboxypeptidase towards peptide and ester substrates. The first substrate found for the modified enzyme is the thiolester (121a) which is hydrolysed almost half as efficiently as by the native enzyme ;Z44 the modified enzyme has also been shown to catalyse the oxidation of ascorbic acid, apparently at the same active site.245

The intense chromophore of the intramolecular co-ordination complex formed between the active-site Zn( 11) atom of carboxypeptidase A and arsanilazo-tyrosine-248 allows stopped-flow and T-jump studies of the disruption of the complex by substrate. The modified enzyme is catalytically active, and the reversible formation of the complex is much faster than the catalytic reaction. The chromophore is thus a powerful probe of events in the active site and has given important new results concerning the proposed role of tyrosine-248 in catalysis. In particular i t seems likely that the large movement of this residue towards the active-site Zn atom is not an essential part of the catalytic reaction.246 Other results with this modified enzyme, and with the nitrotyrosine derivative, also lead to the conclusion247 that the ionization of Tyr-248 has little or no effect on the catalytic reaction, when the substrate is the ester (121b).

Ph

Further studies of substrate activation and inhibition in the carboxypeptidase- catalysed hydrolysis of esters have involved a series of hippuric acid esters.248

Bovine pro-carboxypeptidase A catalyses the hydrolysis of N-(haloacy1)amino-acids, and the differences in substrate binding sites between enzyme and zymogen have been explored by using a series of N-acylphenylalanine derivatives.249

Crystalline human carboxypeptidase A has been isolated by an affinity chromato- graphy technique250 and shown to have many similarities to the bovine enzyme. The relative reactivities of various metal-substituted derivatives are also similar, except for the sequence Mn = Zn > Co = Cd > Ni for esterase activity.

The effects of alkyl substitution (Me, Et, Pr, Pr*, But) a t the €-amino group on the hydrolysis of benzoylglycyl-lysine by carboxypeptidase B are consistent with increasing steric interactions between the.amino-group and amino-acid residues of the cleft binding the lysyl sidechain. The N-formyl and N,N-dimethyl derivatives are particularly unreactive.251

Thermolysin and other Zn neutral proteases have been shown t o possess esterase activity also252 and to be activated (“superactivation”, see above, p. 31) by acylation with N-hydroxysuccinimide esters of acylamino-acids.253

Other Enzymes

Citramalate lyase from C1. tetanomorphum catalyses the cleavage of citramalate t,o pyruvate, using a series of bound thiolester derivatives of 4’-phosphopantetheine. The active enzyme complex is the SCOCH3 form. The SH form is inactive but can be reactiva- ted by treatment with acetic anhydride. This reaction exhibits competitive inhibition


with citramalate and appears to involve an enzyme-acetic anhydride complex, perhaps with the anhydride binding a t a site specific for citramalic acetic anhydride (122). It is suggested that this is an intermediate in the conversion of S-citramalab into the thiolester (123).254 This process is similar in principle, though the intermediate anhydride is

S-Citramalete Ed3.COCHs CH&OCOO-

CHsCOO- 1 E n z B W "%..OH

coo- (128)

OH ( coo-

not in this case between enzyme-carboxyl groups, to recent proposals for the mechanism of formation of the amino-enzyme in pepsin-catalysed reactions.236 A model suggested for the thiolester conversion is the formation of the 6-citryl thiolester of acetyl-coenzyme A from the 1-citryl ester, via the cyclic anhydride (124).254

coo- -0oc -0oc

HO <- / O = H e O + - S B - H F s R coo-

0

(124)

An alternative mechanism for the thiolester conversion which is difficult to assess is a four-centre concerted process, e.g. (125). An approach to this problem is described by

SR

i i Enz-S - - - C R '

I: b (125)

White and Jencks.255 A feature of transition states such as (125) is a low sensitivity to electronic effects; but the reactivity of a series of substituted acetates as substrates for succinyl-CoA :3-ketoacid coenzyme A transferase increases strongly with increasing basicity: a plot of log kcat (or log kcat/Km) against the pK of the acid has unit slope. This is

2 Reactions of Acids am1 their Derivatives 65

consistent with a stepwise mechanism involving an intermediate anhydride. I n this case, also, an enzyme thiolester is involved, but here the enzyme provides the carboxyl group : i t is known that reaction proceeds by an enzyme CoA intermediate in which the coenzyme A is bound to the enzyme as the thiol ester of a glutamate- y-carboxyl.

Under the reaction conditions the half-reaction of the enzyme with acetoacetyl-Cod is much faster than the subsequent reaction of the Enz-SCoA produced with substituted acetate, and the two half-reactions (both presumably going by the anhydride mechanism) are :

Enz + AcAcSCoA F== Enz-SCoA + AcAc

Enz-SCoA +RCOO- Enz + RCOSCoA

En2 RCOO- + AcAcSCoA RCOSCoA + AcAc

kcnt/K, is 2 x 105 times greater for acetoacetate (AcAc) than for acetate in the second half reaction, so that the binding of the extra acetyl group effects a decrease in AG' of 7.2 kcal mol-l. The binding of the coenzyme A contributes a similar factor: experimentally CoA is found not to exchange from the medium into the enzyme-substrate complex. When formed as an intermediate in the anhydride mechanism i t must be tightly bound. A conformational change on formation of the enzyme-CoA intermediate can be detected by its effect on the rate of inactivation by thiol reagents and is explained in terms of an alligator-type model.256

The overall error rate in protein biosynthesis is much lower than expected on the basis of binding specificities with the relevant amino-acid-tRNA synthetases. Fersht and Kaethner257 have now provided evidence relevant to the mechanism by which this hyperspecificity is achieved. Valyl-tRNA synthetase from B. stearothermophilus activates the isosteric amino-acid threonine and forms a 1 : 1 complex with threonyladenylate, but does not catalyse net formation of threonyl-tRNAVa1. This can be isolated by quenched- flow experiments and shown to be rapidly hydrolysed by the enzyme. Studies of inhibition indicate a distinct second site for the hydrolysis. So i t appears that hyperspecificity is achieved by using a normal type of specificity response twice, once in the acylation step, and then again in a subsequent hydrolysis step if mis-acylation does in fact occur.

Phenylglyoxal reacts with an essential arginine a t the active site of aspartate transcarbamylase, thus destroying catalytic activity. This arginine can be protected by N-phosphonoacetyl-L-aspartate. Under these conditions phenylglyoxal reacts (more slowly) with one or more other arginines, causing the loss of activation by ATP and inhibition by CTP. A single nucleotide-binding site is probably involved.258

The binding of 90~o-enriched[~3C]-carbamoyl phosphate t o the active site of aspartate transcarbamylase has been studied by CMR. There is only a slight change (2 Hz upfield a t pH 7.0) in chemical shift on binding, but when a substrate analogue (succinate) is added there is a large downfield shift (18 Hz) as the ternary complex is formed. A similar large downfield shift is observed for the 13C peak from N-(phosphonoacety1)-L-aspartate, enriched at the amide-carbonyl group, but an upfield shift of ca. 6 Hz is observed for [ 1-~3C]-phosphonoacetarnide on binding. The large downfield shift appears to be associated with an isomerized ternary complex, induced specifically by phosphonoacetyl aspartate, as suggested previously to explain T-jump and proton-NMR results; i t could be due to partial protonation of the substrate amide group.259

The activated form of C02 suggested as an intermediate in the enzymic synthesis of carbamoy1 phosphate from ATP, hydrogen carbonate and ammonia (or glutamate) has


been identified by two independent methods as carbonic phosphoric anhydride (126). 1%-Labelled compound is reduced by borohydride to formate ; and the trimethyl ester

(126) (127)

(127) is the probable product when reaction mixtures of enzyme [y-32P]-ATP and H14COi are methylated with diazomethane.260

The action of pyruvate carboxylase on CDsCOCOO- shows an isotope effect, k H / k D = 2.1, on VmaX/Km, but none on V- itself. The intramolecular tritium isotope effect (relative rate of loss of H us. T, by product studies) is 4.8. The results are interpreted in terms of a rate-determining step in the part of the reaction involving carboxylation of enzyme-bound biotin rather than in the half-reaction in which pyruvate is carboxyl- ated.261 .

Catalytic aspects of enzyme-catalysed racemization have been reviewed.262

Decarbox yletion

The decarboxylation of benzisoxazole-3-carboxylic acids (128) (pKa of acid when X=H = 1.95) to salicylonitriles (129) a t pH 2 in tritiated water produces no detectable [3-3H]-benzisoxazole and is thought to be a one-step concerted process, as illustrated.263 This is consistent with the lack of reactivity of the neutral compound since even the zwitterion (128; NH+) is not decarboxylated to nn ylide. Full details have now been published for the large effects of solvent on the reaction, spanning a range of 108 in rate

(lf8) (139) (180)

over 30 solvents and solvent mixtures for the reaction catalysed by tetramethyl- guanidine.264 The Hammett reaction constant p varies from 1.37 in water to 2.40 in hexamethylphosphoramide, solvents in which (128) shows the lowest and highest reactivity, respectively. Ground-state stabilization by hydrogen-bonding in protic solvents, and transition-state stabilization in aprotic solvents are clearly dominant factors.265 Thus (130) is stabilizedrelative to the 5-hydroxy-compound even in water, and its rate of decarboxylation is almost independent of solvent.

The CU( 11) oxaloacetate complex (131) undergoes competing decarboxylation and spontaneous and acid-catalysed enolization a t similar rates. COz formation is consequent- ly biphasic, a rapid reaction of [ 131) being followed by decarboxylation of the substrate

67 2 Reactaons of Acids and their Derivatives

which has enolized; the rate of this process is limited by the reketonization step.266 The rate of protonation of the Cu(1r)-pyruvate enolate product (132) is pH-independent, and it is suggested that Cu-0 bond-breaking is rate-limiting for this reaction.

The rates of decarboxylation of 2-methyl-3-0x0 succinic acid (133) and of ketonization of the enol produced are conveniently studied by proton-NMR spectroscopy.267

Me\N\

A N COOH I

HO q COOH 0

0 Me

(188) (134)

1,3-Dimethylorotic acid (134) is decarboxylated in sulpholane a t 180-220" by way of the zwitterion in neutral solvents and through the anion in the presence of an excess of base. The rate of reaction via the zwitterion (135) is estimated by studying the reaction of

0 OMe

Me Me

(135) (188) (137)

(136). This compound is some 1010 times more reactive than (134) a t 35", so the function of the enzyme orotidine 5'-phosphate decarboxylase is probably to displace the corresponding equilibrium for the nucleotide to favour the zwitterionic form.268

The acid-catalysed decarboxylation of a large series of carbamic acid derivatives (137; X = CH2,0,S,NH or NMe; Y = 0,s or Se) has been studied in water. The relative

phYo S I ph70 s

Ph


second-order rate constants obtained are about 1 : 105: 1010 for the diseleno-, dithio- and dioxo-carbamates, respectively.260

Biotin-dependent carboxylation continues to attract attention. The methoxycarbonyl group of (138) is transferred to the neighbouring carbon atom, but the neutral compound (139) simply cyclizes to (140). Methylation, and therefore also protonation, evidently favour breakdown of the initial tetrahedral intermediate with C-N cleavage. Methoxide is eliminated if the alternative is N-. It is suggested that COZ-transfer to an acceptor group in biotin-dspendent reactions is similarly facilitated by initial protonation of the CO2-biotin intermediate.270

A possible model for the carboxylation of biotin itself is discussed below (page 72).271

NON-CARBOXYLIC ACIDS

Phosphorus-containing Acids

A major event is the publication of a new single-volume Chemistry of Phosphorus, which takes a notably broad view of the subject.272

Non-enzymic Reactions

Results have begun to appear of a major investigation by Westheimer’s group of the mechanism of acid-catalysed hydrolysis of phosphate and phosphonate esters. It is known that the rate of hydrolysis of esters such as triphenyl phosphate and p-nitrophenyl phosphate reaches a maximum in the region of 2 M-acid. A similar phenomenon is observed for (carboxylic) amide hydrolysis and is explained satisfactorily by the decrease in the activity of water in strongly acidic solutions, where an amide would be fully protonated. Phosphate esters are much less basic than amides (pK’s in the region -15-6) have been estimated), so a simple explanation of this sort cannot apply. Since acid-catalysis is apparent, a t least in dilute acid, it is assumed that the protonated phosphate ester e.g. (141), is involved. Since (141) is normally present only in low concentrations, a stable phosphonium compound (142) is used as a model, and the first results concern the properties and hydrolysis of two of these phosphonium salts (prepared as the trifluoromethanesulphonates by reaction of the triaryl phosphite with CF3S020Me).

The rate of hydrolysis of (Ph0)3P+CH3 is too large to measure, even by the stopped- flow technique, in pure water, so the sterically hindered phosphonium compound (143), which is conveniently some 100 times less reactive, is used in solvents containing high concentrations of water.

The two phosphonium salts (142) and (143) show parallel behaviour in solvents of low water content (< 8% of water in acetonitrile) : log khyd is a linear function of log HzO, with a high slope, between 3 and 4. Above 8% of water, in the region accessible for the hindered salt only, the rate levels off to become independent of water concentration; a


rate of hydrolysis for (142) in pure water can thus be estimated if it is assumed that its behaviour continues to parallel that of (143) in the water-rich region.273 These results are explained in terms of a reaction scheme involving both the conjugate acid and the conjugate base (146) of the phosphorane (145) ; kl is rate-determining in dilute acid, but

(PhO)aP+Me + HzO

(PhO)zP, + PhO- Me

at high acid concentrations the concentration of the key intermediate (146) is reduced, so that k2 becomes rate-determining, and the rate thus becomes inversely proportional to the square of the acidity function; in fact the hydrolysis of both (142) and (143) does show marked acid-inhibition below IM-acid (CFaS03H).

These results provide a qualitative explanation of the maximum in the pH-rate profile for hydrolysis of triphenyl phosphate, if it is assumed that (Ph0)3P+OH (141) reacts as (142) does. But triphenyl phosphate is actually hydrolysed about lo00 times more slowly then predicted on this basis, and the model is clearly inadequate at this stage.273 More recent work suggests that a large part of the observed acid-inhibition of the hydrolysis of the phosphonium salts may be accounted for by salt effects, which are unexpectedly large in the mixed solvents used. For example, 1 .SM-lithiurn trifluoro- methanesulphonate depresses the rate of hydrolysis of the hindered phosphonium salt (143) in 34% aqueous acetonitrile by a factor of 30.274

As part of this broad investigation, the association of phosphonium compounds with oxyanions has been measured by the conductivity method in acetonitrile as a model for the attack of nucleophiles at a phosphonium centre. Under the conditions oxyphos- phoranes are found to be only weakly dissociated. Thus the 31P-NMR spectrum of an equimolar solution of pentaphenoxyphosphorane and sodium phenoxide (in 4 : 1 DMF- CD3CN) shows tetraphenoxyphosphonium cation to be absent ; the predominant signal is that of the hexaco-ordinate anion, (PhO)aP-, which accounts for over 80% of the starting materia1.275 Equilibrium constants for the two dissociations [R = Me or Ph; n = 1, 2

K: RnP(0Ph)i-n R,P(OPh)5-n & RnP(OPh)+4-, (4

ka + PhO- + PhO-

or 3); see equilibrations (a)] are in the region 10-9-10-10 for K1, 10-3-10-4 for K2. Combined with rate constants, measured by NMR spectroscopy, for the dissociation of the phosphorane, these results allow the calculation of rate constants ( ka) for association. In two cases these are close to the diffusion limit.276

The hydrolysis of tris-(2,4-dichlorophenyl) phosphate in aqueous dioxan a t 98" shows a rate maximum a t 2.5na-HC1, and a negative salt effect on the acid reaction which may account for the reduced rate in stronger acid. At higher pH the rate falls, reaching a


low pH-independent value near pH 7.277 The neutral hydrolysis of the monoester (2,4-dichlorophenyl dihydrogen phosphate) under the same conditions278 is described in terms of SN2(P) attack by water, on the basis of an entropy of activation near -20 e.u. If this is correct, it is out of line with results for a large number of similar esters, which generally have entropies of activation near zero and are thought to hydrolyse by the SNI(P) (metaphosphate) mechanism.

Pyridine-2-carbaldoxime phosphate (147) is activated towards hydrolysis and nucleophilic attack when it binds Zn(n) ions as, e.g. in (148). The Zn(n) complex behaves as a

WL? . , . , O V / N \ O p 0 3 H - H

2+% . , ... : /o- (147) o-p\()-

typical phosphate monoester with leaving-group activation. The pK of the conjugate acid of the leaving group is reduced from 10.0 to 6.0 by co-ordination of the Znz+ ion. This is stillnot low enough to produce a large increase in the rate of hydrolysis, because this will be dominated by the monoanion reaction for a leaving group of this pK, and will thus be rather insensitive to the pK of the leaving group. SN2( P ) reactions with amines, on the other hand, show a high sensitivity to leaving group for both mono- and &-anions, and the hydrolysis of (148), but not of (147), shows strong nucleophilic catalysis by substituted pyridines (@ = 0.15).27@

The reverse of this reaction [also occurring by way of transitionstate (148)], asexempli- fied by phosphate transfer from N-phosphorylimidazole to pyridine-2-carbaldoxime, involves a ternary complex of oxime, Znz+ and phosphorylimidazole. Recent results with Niz+ give a clearer picture of this reaction, which is consistent with the conclusions reached from the work described above. The role of the metal cation is two-fold : a tem- plate effect brings the reactants together, and charge neutralization overcomes the electrostatic barrier to attack of an oxyanion on a dianionic phosphoryl group (l49).280

,+q, ----* \

The sN2(P) reaction of hydroxylamine with N-(4-~hlorophenyl)phosphoramidate (150) shows saturation kinetics. The second-order rate constant falls with increasing hydroxylamine concentration, and curvature is apparent well below laa-amine. Hydroxylamine gives linear second-order plots with other substrates, so that activity coefficient effects can be ruled out (with the possible exception of a specific salt effect on


this particular reaction). The simplest explanation of this result is that the hydroxyl- aminolysis of (150) is a two-stage reaction ; attack on the intermediate, which is rate- determining a t low nucleophile concentrations, becomes faster than its reversion to starting materials as the concentration of hydroxylamine is increased (formation of the intermediate rate-determining).281

kr NHiOH I p-CICaH4NH2 + HONHPOaH-

The values obtained for kzlk-1 and kslk-1 are 0.05 and 0.65 1 mol-l, respectively, at 40°, indicating that the intermediate shows little selectivity between water and the highly nucleophilic hydroxylamine, but a strong preference for reaction with the minute amount of p-chloroaniline present. This suggests that the aniline has not become entirely free in the intermediate and leads the authors to propose the solvated induced-dipole pair structure (151).

H I

Ar- N : P :0, / +*., H 0 0- H’

(151)

The low selectivity for the nucleophile suggests that reaction with other nucleophiles should show similar kinetics. Certainly more evidence of this sort would be very helpful in assessing the suggestion that a mechanism involving an intermediate such as (151) is common to phosphoramidate and phosphate monoester monoanion hydrolysis.

The high (electrophilic) reactivity of the metaphosphate anion [central to (El), and to discussions of S N ~ (P) mechanisms generally] is ascribed to two diffuse low-lying unoc- cupied antibonding orbitals, one ?r* and one u*, centred on phosphorus, which both interact with the HOMO of the nucleophile.282

Intramolecular nucleophilic displacement of o-chlorophenoxide from dinucleotide derivatives such as (152) is much more efficient than attack by external hydroxide, leading to the unwanted 5’ --f 5’-iuomers.283

+ ArO-


Details have appeared of the acid-catalysed hydrolysis of the diethyl phosphonate (153), which involves participation by the neighbouring amide gr0up.284 Rate-determin-

0

(158)

ing nucleophilic attack of the amide-oxygen on the protonated phosphoryl group accounts nicely for the observed kinetics, and the authors point out that this phosphorylation of an amide might be relevant to biochemical phosphate transfer.285

A similar reaction occurs between the oxygen atom of urea and the phosphoryl group of (154). The heterocycle (155) is obtained when the dimethyl ester of (154) is treated

Me

(154) (155)

with 1 equivalent of p-toluenesulphonic acid in CDCla, supporting the nucleophilic catalysis mechanism. It is suggested that this reaction (156) is a possible model for the cleavage of ATP catalysed by biotin csrboxylase, as in (157).271

Q-

L

Biotin

(156) (157)

Cyclic N , N-diphenylethylenephosphorodiamidates (158) react with formate in aqueous solution to give the N-formyldiamines (159). Results with oxygen-18 show that an oxygen atom is transferred from formate to inorganic phosphate in the course of the reaction, and a mechanism involving initial nucleophilic attack by formate on phosphorus is indicated.286 The formic phosphoric anhydride (160) formed will readily acylate the amine in a second, intramolecular reaction. There are intriguing similarities between t h s reaction and the enzyme-catalysed formylation of tetrahydrofolate, which requires ATP.

The high nucleophilicity of fluoride ion towards phosphorus is apparent in reactions of phosphate esters with good leaving groups. CsF, and the tetra-n-butylammonium salt, catalyse the transesterification of triphenyl phosphate to a trialkyl ester in the alcohol as solvent.287 If a dialkyl phenyl phosphate is used, only the aryl group is exchanged.

2 Reactions of Acids and their Derivatives

Ar

N-POsa- I

H + - (N-CHO I

Ar I

Ar

73

Hi01 fa t I (158) (160)

CNHh N-CHO

I

(159)

Ar

Other leaving groups selectively displaced are cyanoethyl and trichloroethyl as alcohols. When the reaction is carried out in THF the (presumed) intermediate phosphoro- fluoridates are hydrolysed on work up, and the diester is obtained (from a dialkyl trichloroethyl ester, etc.).zss The mild conditions used (30 min a t room temperature in THF) suggest that the reaction will find use for removing phosphate protecting groups.

Another striking instance of the effects of cations on the S&(P) reaction is in the phosphorylation of p-methoxyphenol by the cyclic phosphorochloridate (161). In acetonitrile in the absence of added salt, or in the presence of KC104 or Mg(ClO&, over 90% of the inversion product (163) is obtained. But if 1 mol of LiC104 is added, 9694 of the

product of retention (162) is found. It is suggested that bonding between the phosphoryl- oxygen and Lif develops, to stabilizing a pentacovalent intermediate in which the OLi group is much more apicophilic than 0- would be.289

Studies have been reported concerning the metal-catalysed hydrolysis of uridine


diphosphate glucose and several related compounds, which yield the carbohydrate cyclic phosphate and the mononucleotide ;290 also concerning the hydrolysis of ATP in micellar and reversed micelle systems.291

Functional micelles of cetyl-2-(hydroxyethyl)dimethylammonium bromide are better catalysts for the alkaline hydrolysis of dialkyl p-nitrophenyl phosphates than is cetyltrimethylammonium bromide ;zQ2 the solvent deuterium isotope effect is similar to that for uncatalysed alkaline hydrolysis for the diethyl ester k ~ / k ~ = 0.87) ; a larger, inverse, isotope effect is observed for the di-n-hexyl ester, but this disappears a t high hydroxide concentrations. This is explained if reaction involves the N+CH2CH20- group of the surfactant, since this will be fully ionized at high pH.

Micellization of the substrate has little effect on the rate of hydrolysis of the monoanion of decyl phosphate, but the reaction of the undissociated acid is favoured a t the expense of the acid-catalysed and chloride-promoted reactions in dilute HCl.zs3

The hydrolysis of ethylene phosphates and their applications in synthesis have been reviewed.294 Ramirez et al. report2951 296 just such an application, namely a versatile one-flask synthesis of unsymmetrical phosphate diesters, in which the cyclic enediol N-phosphorylimidazole (165) used is generated in situ from the pyrophosphate (164). The product (165) phosphorylates alcohols rapidly, to produce triesters (166) which are themselves reactive phosphorylating agents, The final product (167) is readily hydrolysed

ROE, (166) x > P f ) R ,P-OR'

'OR

to the required diester, ROP(02H)OR'. The second phosphorylation step (166 + 167) is catalysed by acetate salts and tertiary amines in chloroform or acetonitrile. A nucleophilic role for the catalyst is indicated, and an interesting mechanism involving a hexaco-ordinate intermediate (168) is suggested.296


A theoretical investigation (CND0/2) of the conformations of dimethyl phosphate monoanion and 5- and 6-ring cyclic phosphates indicates a strong coupling between RO-P-OR bond angles and preferred torsional conformations. The restrictions of 5-ring geometry impose torsional strain and the relief of this strain accounts largely for the high heat of hydrolysis of ethylene phosphates. A similar coupling is found for triesters, but the source of the high heats of hydrolysis of some 6-ring cyclic phosphates has not been identified.zg7

Similar considerations must apply to cage compounds such as (169), where structural comparisons (X-ray structural data) show that the POC bond angles are considerably less

(169) (170)

than those in acyclic esters. Comparison with (170) suggests that this cage phosphite is essentially unstrained ; some POC angle strain does, however, appear in the corresponding phosphate.29*

The same sort of strain accounts for the high reactivity towards hydrolysis of 5- membered cyclic phosphoramidates: in certain cases P-N cleavage can be observed even in alkali, and even when P-0 cleavage is an available alternative. A careful study of a series of such compounds by Brown and Hudson2gg shows that the cyclic phosphoramidates (172; R = Me or Ph) react some 104 times faster than the open-chain analogues (171). And whereas the acyclic compounds are hydrolysed exclusively with the

s R I

I N MeN\ 40 p o

[d \OAr P

MeO’ ‘OAr

(171) (173)

loss of the best leaving group, ArO-, the cyclic compounds (172) give significant amounts of ring cleavage; in the case of (172; R = Me) this includes 5% of P-N cleavage.2g9 The results are explained in terms of the pentacovalent intermediates (173) and (174) ; the intermediate (173) first formed cannot eliminate phenoxide without apseudorotation

\ -OH

I

(172) - ‘Me

I I

-0 c OH

(173) (174)

(173 + 174). If the NMe group of (174) is protonated to a small extent [either by addition of a further proton, or by a prototropic shift of (174)], then P-N cleavage can compete with loss of phenoxide. Consistent with this picture is the observed lack of P-N bond cleavage for (172; R = Ph), where the nitrogen would be a better leaving group under other circumstances. A different transition state for loss of aryl oxide from the cyclic phosphoramidates is also indicated by the Hammett p values for the leaving group: p = 1.56 for the acyclic compounds (171), but for the hydrolysis of the cyclic compounds (172) p is about 0.8.299

76 Organic Reactifin Mechanisms 1976

An unusual inversion of stereochemistry is observed in the BF3-catalysed methanolysis of the four-membered ring cyclic phosphinamidate (175; R = benzyl). The explanation seems to be that co-ordination of BFs to phosphoryl-oxygen increases the apicophilicity of this group, so that pseudorotation (176 + 177) becomes possible. Methanolysis is not observed with protic acids.300

lJNHR 2 l--tNHR pb06F3

0 OMe

(175) (176)

11

(177)

The conversion of fully esterified phosphoramidates~o~ and phosphonamidates302 into the thiolester anions, by NaH followed by CS2, has been shown to proceed with retention of configuration a t phosphorus. The reaction as developed originally was a synthesis of i~oth iocyanates ;~~~ it has now been used to introduce sulphur stereospecifically into a mononueleotide (selenium can also be introduced by a similar reaction).302

Ph

+ PhN-

A similar intramolecular displacement a t phosphorus, this time via a five-membered ring, affords a convenient synthesis of aziridines (178);304 This is described as the

0 II ,R

0 (Et0)zP--N II NaH,

R I

+ (EtO)zPO-a A d \ (Et0)tPNHR 7

R P -O R’

\ Jy R’ (178) A - Ph3P

PhsP=NR +

(IS@) (179) R

2 Reactiotis of Acids and their Derivatives 77

“Homer modification” of the similar reaction of an iminophosphorane (179), in which the intermediate oxazaphospholidine (180) can be isolated.305

The hydrolysis of N‘-phosphorocreatine (181) occurs by two distinct pathways. The compound is stable above pH about 5 , and hydrolysis is catalysed by acid in a reaction (dominant below pH 1.0) leading to creatinine (182) and inorganic phosphate, possibly by way of phosphorocreatine. In weakly acidic solutions, hydrolysis occurs by a second pathway that shows a bell-shaped pH-dependence, and the product is creatine (183).306

Strong acid

0 I

H 3 m 4 + H 2 N < y N

Me I

(182)

The reaction in dilute acid is kinetically that expected for an SN~(P) metaphosphate mechanism (AS* -2 e.u., k H / k D = 0.86) ; there is no evidence that the carboxyl group is involved. The results are used to sketch the probable requirements for the active site of creatine kinase, which uses phosphorocreatine to phosphorylate ADP, if a similar mechanism is involved.

pH-rate profiles for the hydrolysis of diethyl, di-n-propyl and di-isopropyl phosphoramidate show minima near pH 6, shifted to pH 7 for the diethyl-N-methyl compound.307

Three papers provide more evidence for an A-2 associative mechanism for the solvolysis of phosphinamides.308 The methanolysis of N-(cyclohexy1)methylphenylphos- phinamidate (184 ; R = cyclohexyl) proceeds with inversion of configuration over a wide

(184) (185)

range of sulphuric acid concentrations.309 So too do the same reactions of the unsubstituted N-phenyl and N-p-nitrophenyl compounds310 in 4.15na-HCl in methanol. In 1.5~-hydrochloric acid the picture is less clear-cut, since the optical activity of the product is reduced, but more so for the anilide (184; G P h ) than for the p-nitroanilide ; the latter might have been expected to favour a dissociative mechanism, having a better leaving group. So these results do not unequivocally support a change to an A-1 mechanism in strong acid; nor do the effects of varying the Ar group in a series of di(substituted-pheny1)phosphinamidates (185) ; for three different leaving groups (R = H, Ph orp-nitrophenyl) in (185) the replacement by one and then two ortho-methyl groups on the aromatic rings (Ar = phenyl, o-tolyl, mesityl) produces large decreases


in the rate of hydrolysis (in 9:l v/v aqueous dioxan containing 1.36wHC104). These results also are consistent with associative mechanisms for all the compounds used, and the role of the A-1 mechanism in the hydrolysis of phosphinamidates is now in some considerable doubt.311

N,N-Di-isopropyl alkyiphosphoramidothioic chlorides Prz*NPfS)ClR show non- equivalent isopropyl groups in the low-temperature proton-NMR spectrum, attributed to barriers to P-N bond rotation in the region of 9-13 kcal mol-1.312

The addition of cations capable of co-ordinating at sulphur (Ag+, Hgzf, not MgZ+) changes the stereochemistry of methanolysis of the thiolate ester (186) to predominant inversion, although retention is observed in the absence of metals.313

SPh OMe 0 I I

The thiono-thiolo-rearrangement of phosphorothionates (187 -+ 188) is catalysed by protic acids.314 The rearrangement of the corresponding Se compounds is faster,

Oxidation of thiophosphates, e.g. (187), with hydrogen peroxide produces the phosphates with net retention of configuration.315 Phosphine sulphides, on the other hand, are converted into phosphine oxides with inversion. The mechanism suggested involves initial oxidation a t 5, followed by formation of a pentacovalent intermediate (189), which loses SOH directly where a, b and c are alkyl or aryl groups. But if one of the

a

b-P--A ,..* \ + ...*

e b*P=fSOH

C .

I HO-P-SOH

4 ".. b c

a I

HO-P-OR 4 +%.

b SOH

groups is alkoxy (more apicophilic than SOH), (190) is formed instead ; loss of SOH then needs a pseudorotation, and retention can be accounted for.

X-ray crystallographic analysis of the spirophosphorane (191) shows that the four endocyclic 0-P bonds are almost identical in length, with the P-OPh bond significantly shorter. Bond lengths (and angles also) are thus not consistent with a structure based on the trigonal bipyramid; nor are the bond angles those expected for even a distorted tetragonal pyramid. It is suggested that the structure is best represented by a configuration 15" into turnstile rotation.316

Pseudorotation of five phosphoranes related to (192) has been studied by 13C-NMR,317

2 Reactions o j Acids and their Derivatives 79

(191) (192)

and that of tetrakis-(2,6-dimethylphenoxy)methylphosphorane by 'H-NMR spectroscopy. Ligand reorganization of the last-mentioned compound is slow enough t o be studied at temperatures between -65" and room temperature, presumably because of steric crowding.318

Evidence that a phosphorane is an intermediate in the conversion of the catechol phosphate (193) into the diphenyl ester (194) is provided by a trapping experiment.

(w (194)

When the trimethylsilyl group is actually on the molecule, as in (195), it is transferred to the phosphoryl-oxygen rather than to the displaced catechol-oxygen, and the product is a stable spirodicatechol-phosphorane (196).310


Hexaco-ordinate compounds, e.g. (197), had been made earlier by the addition of pyridine to a spiropentaoxyphosphorane,320 and indeed earlier, in 1927, by Anschutz. His compound, obtained from PC15 and pyrocatechol, has now been identified as ( 198),3Z1 which appears to exist, a t least in solution, as the free acid, since it gives known salts of the

H+ H&Eta

hexaco-ordinate anion on neutralization with amines. Epimerization a t the (chiral) phosphorus centre can be studied by using a single optical isomer (199) separated from the diastereoisomeric pair formed from the reaction of (+)-mandelic acid with N,N- diethylamino-spirodicatechol-phosphorane. The free energy of activation for the epimerization is similar to that found for similar pentaoxyspirophosphoranes and suggests a mechanism involving initial dissociation to a P( v) intermediate.322

Hexaco-ordinate intermediates are involved in the &2( P) reactions of aryloxy- cyclotriphosphazenes (200) with nucleophiles (CPsCH20-, PhO- or PhS-). The reactions are relatively slow, and when better leaving groups (AI = 0- or p-nitrophenyl) are used attack on the aromatic ring competes (PhO-, PhS-). This aromatic nucleophilic substitution predominates with nitrogen nucleophiles.323

Several cage polycyclic phosphoranes, e.g. (201), have been prepared by the reaction of cyclic phosphites with compounds (202), (203) and hexafluorobiacetyl, and shown to have equivalent CF3 (or CH3) groups, unless the intramolecular motion responsible is slowed because a five-membered ring is fused to phosphorus.324

Phosphoranes also result on reaction of benzil with the diazadiphosphetidine (204) (a bisphosphorane in this case),325 on addition of a phosphinite (205) to acrylic acid or acrylamide,326 and even (206) from the reaction of pyrocatechol with phosphorous acid and dicyclohexylcarbodi-imide.~27

Lautenco and Burgada describe several interesting reactions of the spiran (206),


I i Php/ 1 I t

,P-NPh EtO

including an oxidation-reduction with an enamine that gives a bisphosphorane linked by an OCHzCHzO bridge.328 The similar spirophosphoranes (207 ; R = H) react with - - phenyl azide, clearly by way of the PIIII) form, to give the phosphoranes (207; R = NHPh) .329

Hydrogenolysis of the trimethyl phosphite-biacetyl adduct (208) gives ethyl methyl ketone and trimethyl phosphate, possibly by way of (209).330

Two cases are reported of C-0 cleavage in the acid-catalysed hydrolysis of phosphin- ate esters. SNl-like kinetics are observed for the hydrolysis of several ally], propargyl and tertiary alkyl diallylphosphinates ;331 and hydrolysis of three methyl arylmethyl- phosphinates in ~M-HCIOI a t 67” proceeds with some 10% of C-0 bond cleavage,332 much the same as the figure found for the hydrolysis of trimethyl phosphate under similar conditions. Catalysis of the hydrolysis of aryl dimethylphosphinates by a series of amine and oxyanion bases involves the nucleophilic mechanism333 (although general base catalysis by imidazole is observed for the diarylphosphinates334). Presumably st.eric hindrance to the approach of the nucleophile is reduced in the dimethylphosphinate. Hsmmett p- values are 0.93 for hydroxide and 1.60 for imidazole.

Enzymic Readio~s

Several notable NMR studies of alkaline phosphatase complexes have appeared. Using 3lP-NMR and the Fourier transform technique (up to 250,000 transients per sample), two groups have identified signals from the phosphate group of the non-covalent enzyme phosphate complex E . P, and from the covalent phosphoryl enzyme E-P.335-337 Coupling between phosphorus and the methylene-protons of the active-site serine is observed335 (coupling constant,s 13.1 Hz for the apophosphoryl enzyme, with the metal removed). The phosphoryl group of the apoenzyme has considerable independent mobility, much of which i t loses when a metal [ C ~ ( I I \ ] is present a t the active site.

This experiment is possible because dephosphorylation, which is rapid for the Zn( 11)

enzyme, is slow for the Cd(I1) enzyme, and stops with the apoenzyme (between pH 2 and 9).336 The 31P chemical shifts are 6 and 8 ppm downfield (from inorganic phosphate) for phosphoryl apo- and Cd( 11)-enzymes, respectively, suggesting strain (presumably angle strain) at the phosphoryl centre, largely by the protein microenvironment.335 Strain might account for the high reactivity of the serine phosphate in the metalloenzymes, but it is hard to see how (at any rate angle) strain could be the cause of the

82 Organic Reaction Mechanisms I976

downfield shift of the phosphate signal from the apoenzyme, if the group is indeed relatively mobile.

These papers are packed with interesting information about the enzyme-phosphate complexes, but puzzling features remain. What is clear is that the phosphorylserine a t the active site of alkaline phosphatase is a remarkable phosphate monoester.

18F-NMR spectroscopy is possible with enzyme prepared by in vivo incorporation of m-fluorotyrosine, which contains eleven fluorotyrosine groups distributed through the protein. The fluorine signals are sensitive to the local conformation, and titration with inorganic phosphate indicates that the binding of two molecules per enzyme dimer cause a deep-seated conformational change in the protein.338 Remarkably there is no evidence from this work to link this conformation change to the strong negative co-operativity observed by others with this enzyme system.

The first results of NMR experiments with 1 W d in the active site of alkaline phosphatase are consistent with two metal-binding sites per protein dimer that are identical in the absence of external ligands.338

A careful investigation of the metal content of the native E . coli enzyme shows 4.0 Zn(n) ions and 1.3 f 0.3 Mg(n) ions per dimer of 89,000 molecular weight. By using the spectroscopic properties of the CO(II) enzyme it can be shown that Mg2+ regulates the binding of Co2+ (and Zn2+) and the activity of the resulting metalloenzymes.~0

VOi is a potent competitive inhibitor of alkaline phosphatase, binding in place of inorganic phosphate at the specific binding site.341

The progesterone-induced porcine uterine purple phosphatase is activated by thiols to a pink high-spin Fe(rr1) enzyme, which contains an essential SH group.342

The hydrolysis of pyrophosphate catalysed by yeast inorganic pyrophosphatase shows a solvent deuterium isotope effect, k H / k D = 1.90 (to be compared with 1.45 for the Mg2+-catalysed reaction), and no isotope effect for uncatalysed hydrolysis.343

Potassium ferrate, K2FeO4, is a phosphate analogue which is also a powerful oxidizing agent. It reacts with rabbit muscle phosphorylase b by binding a t the AMP site, then oxidizing a cysteine residue and a t least one tyrosine, and so eliminating the binding of AMP and inactivating the enzyme.3'4

A detailed NMR investigation of the binding of substrates to rabbit muscle pyruvate kinase has produced the best picture yet available of the geometry of the very busy active site of this enzyme.345-347 By means of enzyme-bound Mn(11)~45 [and CO(II)] as paramagnetic probes, relaxation rates have been studied, and thus approximate (+. 10%) distances from the probe obtained, for the phosphorus atoms of ATP (and PEP), protons of the adenine ring of ATP and pyruvate, and carbon atoms of carboxyl- and carbonyl- W-enriched pyruvate. Measurements with Cr(1Ir) ATP provide a second set of data based on a second reference point.346

The unique active-site model (210) obtained from these results goes a long way towards explaining many properties of the enzyme.%' ATP appears to be bound in an extended conformation, with no inner-sphere complexing to enzyme-bound M(n), and with the y-phosphate group overlapping the position of the phosphate group of phosphoenol pyruvate, which it becomes as reaction proceeds, thus accounting for the competitive binding of ATP and PEP.345 Cr(I11) ATP replaces ATP in promoting the enolization of pyruvate, only in the presence of Mg or Mn(I1) and K+, confirming the requirement for a bivalent cation near, but not at, the nucleotide binding site.346 Further, the close approach (3.0 0.5 d) of the pyruvate carbonyl-oxygen to the y-P of ATP is consistent with direct phosphoryl-transfer to substrate, with no phosphoryl enzyme intermediate. The mechanism of the reaction from pyruvate is rather clearly defined by these results (ZlO), with


the fairly minor exception of the geometry of the displacement a t P, which is unlikely to be anything but in-line in the circumstances.

The “phosphoryl” enzyme obtained from phosphoglycerate kinsse ATP in the presence of ADP is probably a phosphoryl (enzyme-phosphoglyceric acid complex), with the extra phosphate group present as tightly bound 1,3-diphosphoglyceric acid.348

Rabbit muscle phosphofructokinase has one very reactive SH group which can be modified, spin-labelled, etc., but turns out not to be an essential active-site functional group.349 The enzyme will accept D-tagatose-&P, but not the psicose or sorbose derivative, indicating specificity for the L-configuration at C-3 and D a t C-5, but tolerance for an epimeric centre a t C-4.350

Creatine kinase forms stable abortive complexes with suitable substrates lacking the transferable phosphate group, allowing the introduction of a paramagnetic probe, such as Mn(n) as MnADP. (The second reference point essential for building up a three- dimensional picture is an organic spin-label, attached to the essential SH group of the

84 Organic Reaction Mechanism I976

active site.) Planar oxyanions, such as formate, and especially nitrate, dramatically increase the binding of creatine to the abortive complexes, perhaps fitting in the place of the transferable PO; group, and thus turning the complex into a transition-state analogue. The best picture of the phosphate-transfer step, built up from the evidence from several complexes, is consistent with direct transfer between ADP and the guanidino-nitrogen of creatine (211).351

An elegant isotope scrambling method allows the detection of reversible y-phosphate- transfer from ATP.

l80-ATP, labelled a t the 8-bridge oxygen, when incubated with glutamine synthetase, shows exchange of labelled oxygen into the non-bridge oxygens of the 8-phosphate group (212) in the presence of glutamate. The results are consistent with a two-step mechanism for the reaction, involving phosphoryl transfer from ATP to glutamate, followed by acylation of ammonia.352

0 0 0 0 0 I1 I1 * I 1 I1 II

I I 1 I I Ad-0-P-O-P-O--P-O- Ado-P-0-P-0' + XPOs-

0- 0- 0- 0- 0-

I I 0 0' 0 0 O* II II II II \I

I 1 I I I Ad-0-P-0-P-0-P-0- Ado-P-0-P-0-

0- 0- 0- 0- 0- (212)

Phosphoglycerate mutase (2,3-diphosphoglycerate-dependent) is rapidly and irreversibly inactivated, probably a t the substrate-binding site, by N-(bromoscety1)ethanol- amine phosphate. The reagent alkylates an essential SH group, though i t is not clear whether this group is directly involved in catalysis.353 The phosphoryl enzymes bisphosphoglycerate mutase and bisphosphoglycerate synthase have been shown to have kinetic properties consistent with their involvement as intermediates in the catalytic reactions.354 So too has a phosphoryl (serine) enzyme in the synthesis of glucose 1,6-diphosphate from glucose 1 -phosphate and glycerate 1,3-diphosphate, catalysed by glucose 1,6-diphosphate synthase from beef brain.355

Phosphoryl-transfer from the Mg( 11) form of rabbit muscle phosphoglucomutase (phosphoryl enzyme) to the 6-hydroxyl group of glucose 1-phosphate is faster than transfer to water by a factor of over 3 x 10'0. This large factor cannot be attributed to any single effect,3se and a study of the effects on the rate of dephosphorylation of the binding of a number of simple substrate analogues provides evidence as to the relative importance of several factors. The binding of a simple phosphate derivative increases the rate of hydrolysis 1000-fold ; an alcohol acceptor is several thousand-fold better and depends (factor of several hundred-fold) on its pK. When the organic phosphate and the acceptor alcohol are bridged, as in 1,4-butanedioI monophosphate, a further rate increase of over 200-fold is observed.357 The equilibrium constant for phosphate transfer to bound glucose monophosphate varies by a factor of 65 as the metal-ion activator is varied from Zn(ir) to C d ( 1 1 ) . ~ ~ ~


The original assignments of the 1H-NMR signals from the C-2 protons of the imidazoles of histidine-12 and -119 of ribonuclease have been reversed.359. 360 A modified ribonuclease S', composed of S-protein and a synthetic tetradecapeptide analogue of S- peptide, with histidine replaced by homohistidine, is formed less readily than natural RNase S' but has practically full enzymic activity. This is the first modification of t.his active-site histidine that does not have sharply decreased activity and it suggests a surprising flexibility in the active-site arrangements.361

The 3lP chemical shifts of the three cytidine monophosphates, and of UMP, depend on the ionization state of the nucleotide and are little affected by binding to RNase, although the phosphate group presumably has important interactions with positively charged centres on the enzyme.362

The binding of guanosine to ribonuclease T1 is different from that of deoxyguanosine but the difference disappears for y-methoxycarbonyl-glutamate-58 enzyme : an interaction between the ribose 2'-OH group and the carboxyl group of glutamate-58 in the enzyme substrate complex is a suggested explanation,363 which would have interesting consequences for the catalytic mechanism.

A 5'-nucleotide phosphodiesterase present in calf intestinal alkaline phosphatase is an efficient catalyst for the hydrolysis of aryl phenylphosphonate esters, but not of phosphate monoesters.364

Sulphur-containing Acids

The acid-catalysed methanolysis of p-nitrophenyl sulphate has been used to estimate the pK's of weak acids in methanol. Catalytic constants (and derived pK's) for the conjugate acids of a handful of weak nitrogen bases have been reported.365

Rate and equilibrium constants for the esterification of propan-2-01 and butan-1- and -2-01 by sulphuric acid have been measured over a range of temperature.366

The maximum in the H,-rate profile for the hydrolysisof N-methyl-N-phenylsulpham- ate salts367 is shifted to lower acidities (by 1.5-2 units) compared with the reaction of derivatives of (the less basic) primary anilines.3'38 The catalytic order of HC1> > HC104 is that generally observed for A-2 reaction, and salt and solvent effects, deuterium isotope effects, and Bunnett-Olson parameters are consistent with this mechanism. The entropy of activation is, however, small and negative, usually considered diagnostic for the A-1 mechanism. Since the balance of the evidence is against this mechanism it is suggested that changes in solvation on formation of the transition state are atypical for hydrolysis of sulphamic acid. A borderline A-2 mechanism is considered the best explanation consistent with the evidence. On the other hand, substituent effects on the hydrolysis of a series of N-(substituted pheny1)sulphamic acids in HC104, have been interpreted in terms of the A-1 mechanism.369 When rate constants are corrected for ionization the rate also appears to be independent of UH%O a t low acid concentrations. The lower rate in strong acid is accounted for by the protonation of thereactive zwitterion.

Nucleophilic catalysis of the hydrolysis of diphenyl sulphite in 91% aqueous acetonitrile by a series of oxyanions is characterized by a Brensted 6 near 0.7 and shows a significant a-effect.370

A sulphene (213) hits been generated by the [3,3]sulpho-Cope rearrangement of ally1 vinyl ~ u l p h o n e . ~ ~ ~ When this is heated in the presence of pyridine in ethanol (170"), N-ethylpyridinium pent-4-ene-1-sulphonate is produced in 70% yield, clearly by way of (214).

86 Organic Reaction Mechanism 1976

(214)

2-Hydroxy-5-nitrotoluene-a-sulphonic acid sultone (215) is hydrolysed with both nucleophilic and general base catalysis. Hindered and tertiary amine nucleophiles act as general bases ( k H / k D = 4.2 for imidazole), whilst oxyanions and primary amines generally act as nucleophiles ( k H / k D = 1 .O for acetate, / 3 ~ = 0.66 for RNHz) ; a significant a-effect is observed. The ElcB mechanism established for similar acyclic compounds is not observed, presumably for stereoelectronic reasons.372

o a N ~ ~ ~ a

\ o (215)

The hydrolysis of benzenesulphonyl chloride in 8&100~o and fuming HzS04 is thought to involve rate-determining S-C1 cleavage.373 A detailed study with 12 substituted derivatives in 99.8% HzSO4 a t 25" ( p = -4.4) leads to the suggestion that S-C1 cleavage is general acid catalysed by HsSO:.~~~ A cyclic transition state avoids the generation of a sulphene intermediate in this case. A similar study of benzenesulphonyl fluorides (p = -4.3) leads to rather similar conclusions.375

The hydrolysis of a series of (substituted-pheny1)methanesulphonyl chlorides in aqueous dioxan (30-50") follows the Hammett equation, with p = 1.3-1.4.376 But no good Taft correlation is found for the reaction of RSOzC1, where solvent effects are greater than sdbstituent effects.377

The acylation of a large range of alcohols and phenols by alkanesulphonyl chlorides in various solvents is catalysed by tertiary amines by either nucleophilic or general base mechanisms.378

Steric and electronic effects on the acylation of ortho-substituted anilines by thiophene-2-sulphonyl chloride are characterized by Taft parameters p * - - - 1.2 and

The alkylation of dimethylaniline and pyridine by benzyl esters of substituted benzenesulphonic acids in acetone a t 30" show similar sensitivities to the leaving gr0up.380 Hydrolysis of the allyl and propyl esters of substituted toluene- a-sulphonic acids in 70% aqueous dioxan at 40-60" follows the Hammett equation, with very similar p-values (40") of 0.69 for the n-propyl esters (&@) and 0.78 for the reaction of allyl esters.381 The entropies of activation' are also very similar and the (60-fold) higher reactivity of the allyl esters is due mainly to a more favourable enthalpy term. Since hydroxide catalyses the hydrolysis of the propyl but not that of the allyl esters, one a t least of the criteria for

6 = 0.89.370

2 Remlions of Acids and their Derivatives 87

an S N ~ mechanism is fulfilled; but i t is difficult to avoid the conclusion that solvent must be involved to a considerable extent in the transition state.381

The equivalent conductivity of phenyl toluene-p-sulphonates carrying ortho-CO0- and 0-, and para-SO- and 0- substituents appears to fall to zero a t high NaOH concentrations (0.5 M), wherefore ion-pair formation has been suggested.%z? 383

Arylmethanesulphonates react with a variety of arene anion radicals in THF to give almost quantitative yields of aryl oxide and methanesulphonate ; two moles of anion radical are consumed. The mechanism involves a rapid electron-transfer from donor to sulphonate, followed by a slow cleavage step. Stopped-flow measurements on three substituted compounds allow separate determinations of p for the electron-transfer step xaoso2Me +ArH' ki X-@oso2Me + ArH

1 Am' FMt

+ArH - Ar'O'+MeSOr-

to form (216) (kl, p = 6 ) and for product formation ( p = 3 ) . With donors of higher reducing power C-0 cleavage appears, which could be a reaction of the dianion.384

Nucleophilic reactivity towards sulphinyl-sulphur (217) parallels reactivity towards sulphonyl-sulphur (218) although the two centres presumably differ significantly in hardness; the marked change in the pattern of reactivity found previously for four nucleophiles (W-, Cl-, Br- and AcO-) is now believed not to be typical.385 Soft-base nucleophiles (CN-, Buns-) show only small deviations, in contrast to their greatly

enhanced reactivity towards the suiphenyl-sulphur of thiolsulphonates.3~~ The data are correlated by Ritchie's N+ parameter less well than those for attack a t SO2 but better than for attack on sulphenyl-S. The a-effect is also less marked than for attack a t the suiphonyl centre, especially for HOO-.

Sulphinate esters are produced by acid-catalysed alcoholysis of sulphinamides (219 -+ 220) in a reaction that involves inversion of stereochemistry a t sulphur. The


competing racemization of the starting material is a problem where the nucleophilicity of the alcohol is reduced by steric hindrance.387

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( 1975).


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119 L. M. Litvinenko, A. S. Savohenko and L. Ye. Galushko, Zh. Org. Khim., 11, 1899 (1976); Chem.

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133 S. V. Bogatkov, Z. P. Golovina and E. M. Cherkasova, Dokl. Akad. Nauk SSSR, ZZB, 98 (1976); Chem. Aba., 86, 142267 (1976).

194 V. N. Sapunov, 0. M. Lemann and N. N. Lebedev, Kratk. Teziay-Vaea. Sweahch. Probl. Mekh. Ueteroliticheakikh Reakta., 1874. 141; Chem. Aba., 85.122917 (1976).

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Timisoara, Ser. Chim., 18.37 (1974); Chcm. A h . , 84,73278 (1976).

Chim., 18,27 (1974); Chem. A h . . 84,73430 (1976).

84,30189 (1976).

(1976).

Zh., 41,018 (1976); Chem. Ale., 84,4060 (1976).

(1976); Chem. Aba., 84,120736 (1976).

Ueterditicheakikh Reakte., 1974, 146; Chem. Aba., 86.77422 (1976).

kikh Reah . , 1874,177; Chem. A h . , 85,77149 (1976).

Probl. Yekh. Beterdificheukikh Reakla., 1874, 182; C h . A h . , 85,77434 (1976).

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A h . , 84,4066 (1976).

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2 Reactions of Acih and their Derivatives 91

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157 J. H. Fendler, Aeeovnta Chem. Red. 9, 163 (1976). 158 D. Piszkiewicz, J. Am. CLm. Soc., 98,3063 (1976). 158 J. P. Guthrie and Y. Uda. can. J . Chem., 54,2746 (1976). 180 Org. Reaction Yech., 1974,41. 1.s1 D. J. Cram, R. C. Helgeeon, L. R. Soues, J. M. Timko, M. Newcomb, P. Moreau, F. ddong, 0. W.

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171 K. Konno, T. M8bUyam8, H. Mizuno and A. Kitahara, N i p p n Kagaku Kaiahi, 1975, 1867;

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Cmnm., 1976,843.

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[Organic Reaction Mechanisms Series] Organic Reaction Mechanisms || Reactions of Acids and their Derivatives