Catalysis by oxyanions

The hydrolysis of the more reactive carboxylic esters is catalyzed by a wide range of oxyanions. The mechanism proposed for the neutral hydrolysis of esters on p. 158 involves two molecules of water, one as a nucleophile and one as a general base. In principle an oxyanion or other nucleophile can replace either of these molecules, and both general base and nucleophilic catalysis of ester hydrolysis are well-known. The detailed mechanism of nucleophilic catalysis depends, to some extent, on the type of anion concerned, but the differences occur at a relatively late stage in the reaction, and the similarities are sufficient to allow generalizations about oxyanion reactions as a class. Some of the differences are not normally kinetically significant, and are best mentioned briefly at this point. 

It is not strictly accurate to describe alkaline hydrolysis as hydroxide-catalyzed, because hydroxide is consumed during ester hydrolysis, viz. 

Most nucleophiles are both more basic and more reactive towards sp -hybridized carbon than is water. Thus catalysis is readily observed wilh those esters which undergo hydrolysis at a significant rate in neutral solution, that is, with esters activated by electron withdrawing substituents in either the acyl or the leaving group. Hydroxide ion, however, is basic enough to attack any ester, and since the mechanism of alkaline hydrolysis appears to be relatively simple this reaction will be discussed first. 

The broad outline of the mechanism of catalysis of ester hydrolysis by hydroxide ion is not in doubt. The reaction is well known to involve acyl-oxygen cleavage, and seems invariably to be of the second order, being first order in both ester and hydroxide anion. General base catalysis in the usual sense is not a possibility, the partial removal of a proton from water cannot generate a species more reactive than hydroxide ion, so direct nucleophilic attack must be involved. (However, if it is accepted that the high ionic nobility of the hydroxide ion in water is explained by a Grotthus-type mechanism 

Although the observation of the exchange reaction proves beyond reasonable doubt that the tetrahedral addition compound is formed during alkaline hydrolysis, it does not prove that it is actually an intermediate in the hydrolysis reaction. In other words, it remains to be shown that it is not a blind-alley intermediate. 

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General base catalysis of Sn 2-type reactions of ordinary aliphatic alcohols by oxy-anions is observed in the cydization of 4-chlorobutanol [26], and of the sulfonium cation 2.6 [27] (Scheme 2.12) at 50 °C and 40 °C, respectively. (Amine buffers prefer to demethylate 2.6.) In all cases (including the reaction of 2.5 discussed above) catalysis by oxyanions shows a low solvent deuterium isotope effect and a Bronsted coefficient p of 0.26 + 0.1. This reaction may be something of a curiosity, but there is little doubt that it has been properly identified. 

Oxyanions of sufficient basicity will catalyze the hydrolysis of all but the least reactive esters but since the latter include the esters of the common aliphatic alcohols, early attempts to detect the reaction were negative or inconclusive. Dawson and Lowson274, claimed to have detected catalysis by acetate ion of the hydrolysis of ethyl acetate as early as 1927, but the extent of catalysis observed was too small to rule out the possibility that salt or solvent effects were, in fact, responsible. 

These principles appear to hold for nucleophilic catalysis of hydrolysis by other species also. The evidence for catalysis by amino-compounds is discussed below. Catalysis by a wide variety of oxyanions (and other anions) has been measured by several authors, particularly of the hydrolysis of p-nitrophenyl acetate. This is a convenient substrate kinetically, since the release of / -nitrophenoxide is easily followed spectrophotometrically at 400 nm, but perhaps not ideal mechanistically, since, as described above, at least some of its reactions involve a mixture of mechanisms. A selection of data, obtained under the same conditions in one laboratory283, is given in Tables 37 and 38. Some of these data are plotted logarithmically (in Fig. 17) against the... 

Sim6n, L. Goodman, J. M. Enzyme catalysis by hydrogen bonds The balance between transition state binding and substrate binding in oxyanion holes, J. Org. Chem. 2009, 75,1831-1840. 

Because the oxyanion hole of subtilisin includes a side-chain NH group in addition to backbone NH groups, it is possible to probe the importance of the oxyanion hole for catalysis by site-directed mutagenesis. The mutation of asparagine 155 to glycine reduced the value of k to 0.2% of its wild-type value but increased the value of. ST by only a factor of two. 

Serine proteinases such as chymotrypsin and subtilisin catalyze the cleavage of peptide bonds. Four features essential for catalysis are present in the three-dimensional structures of all serine proteinases a catalytic triad, an oxyanion binding site, a substrate specificity pocket, and a nonspecific binding site for polypeptide substrates. These four features, in a very similar arrangement, are present in both chymotrypsin and subtilisin even though they are achieved in the two enzymes in completely different ways by quite different three-dimensional structures. Chymotrypsin is built up from two p-barrel domains, whereas the subtilisin structure is of the a/p type. These two enzymes provide an example of convergent evolution where completely different loop regions, attached to different framework structures, form similar active sites. 

For many serine and cysteine peptidases catalysis first involves formation of a complex known as an acyl intermediate. An essential residue is required to stabilize this intermediate by helping to form the oxyanion hole. In cathepsin B a glutamine performs this role and sometimes a catalytic tetrad (Gin, Cys, His, Asn) is referred too. In chymotrypsin, a glycine is essential for stabilizing the oxyanion hole. 

The mechanism for the lipase-catalyzed reaction of an acid derivative with a nucleophile (alcohol, amine, or thiol) is known as a serine hydrolase mechanism (Scheme 7.2). The active site of the enzyme is constituted by a catalytic triad (serine, aspartic, and histidine residues). The serine residue accepts the acyl group of the ester, leading to an acyl-enzyme activated intermediate. This acyl-enzyme intermediate reacts with the nucleophile, an amine or ammonia in this case, to yield the final amide product and leading to the free biocatalyst, which can enter again into the catalytic cycle. A histidine residue, activated by an aspartate side chain, is responsible for the proton transference necessary for the catalysis. Another important factor is that the oxyanion hole, formed by different residues, is able to stabilize the negatively charged oxygen present in both the transition state and the tetrahedral intermediate. 

Tosylate is displaced by weak oxyanions with little elimination in aprotic solvents, providing alternative routes to polymer-bound esters and aryl ethers. Alkoxides, unfortunately, give significant functional yields of (vinyl)polystyrene under the same conditions. Phosphines and sulfides can also be prepared from the appropriate anions (57), the latter lipophilic enough for phase-transfer catalysis free from poisonning by released tosylate. 

Clearly, the oxyanion hole is now as significant a feature of the binding site of such acyl transfer abzymes as it is already for esterases and peptidases — and not without good reason. Knossow has analysed the structures of three esterase-like catalytic antibodies, each elicited in response to the same phosphonate TSA hapten (Charbonnier et al., 1997). Catalysis for all three is accounted for by transition state stabilization and in each case there is an... 

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