Ester hydrolysis isotopic labels

In an extension of the work described m the preceding section Bender showed that basic ester hydrolysis was not concerted and like acid hydrolysis took place by way of a tetrahedral intermediate The nature of the experiment was the same and the results were similar to those observed m the acid catalyzed reaction Ethyl benzoate enriched m 0 at the carbonyl oxygen was subjected to hydrolysis m base and samples were isolated before saponification was complete The recovered ethyl benzoate was found to have lost a por tion of Its isotopic label consistent with the formation of a tetrahedral intermediate... 

We said in Section 21.6 that mechanistic studies on ester hydrolysis have been carried out using ethyl propanoate labeled with lsO in the etherlike oxygen. Assume that Odabeled acetic acid is your only source of isotopic oxygen, and then propose a synthesis of the labeled ethyl propanoate. 

Many mechanistic aspects of the hydrolysis of phosphate esters in protic media remain uncertain. In spite of predictions that racemization at phosphorus should be the final outcome if indeed the (hypothetical) metaphosphate intermediate is involved in the solvolysis of monoesters, the results of several studies on the methanolysis of appropriately O-isotopically labelled compounds are consistent with reactions proceeding with inversion of configuration, as observed for all enzymic and non-enzymic systems so far examined this has resulted in the suggestion that if metaphosphate is actually formed, then it must be in a masked form. 

A kinetic isotope effect 160/180 of 2% in the spontaneous hydrolysis of the 2,4-dinitrophenyl phosphate dianion, whose ester oxygen is labeled, suggests a P/O bond cleavage in the transition state of the reaction, and thus also constitutes compelling evidence for formation of the metaphosphate 66,67). The hydrolysis behavior of some phosphoro-thioates (110) is entirely analogous 68). 

A reaction that has been much investigated is the hydrolysis of esters, e.g. (164), by aqueous base, i.e. eOH. It is found to be kinetically second order, and lsO isotopic labelling experiments on (164) have... 

More recently, isotopic labeling experiments have assumed a major role in establishing the detailed mechanism of enzymic action. It was shown that alkaline phosphatase possesses transferase activity whereby a phos-phoryl residue is transferred directly from a phosphate ester to an acceptor alcohol (18). Later it was found that the enzyme could be specifically labeled at a serine residue with 32P-Pi (19) and that 32P-phosphoserine could also be isolated after incubation with 32P-glucose 6-phosphate (20), providing strong evidence that a phosphoryl enzyme is an intermediate in the hydrolysis of phosphomonoesters. The metal-ion status of alkaline phosphatase is now reasonably well resolved (21-23). Like E. coli phosphatase it is a zinc metalloenzyme with 2-3 g-atom of Zn2+ per mole of enzyme. The metal is essential for catalytic activity and possibly also for maintenance of native enzyme structure. 

Studies of reaction kinetics, and use of isotopic labelling (180) [3], have established the general mechanism for base-catalysed hydrolysis of esters (i) of most primary and secondary alcohols (Bac2 mechanism [3]) shown in Fig. 7. 

Metabolic studies carried out with isotopically labeled dehydroepi-androsterone sulfate [312] showed that this conjugated steroid may follow an indirect metabolic pathway initiated by the hydrolysis of the sulfate group. In the course of the metabolism the conjugated steroid thus becomes a free steroid first and the free steroid may undergo further metabolism. On the other hand, dehydroepiandrosterone sulfate may follow a direct metabolic pathway without a break of the ester group. 

It may often be obvious which bonds are broken and formed in a reaction. However, in some cases it is not so obvious. In the acid-catalysed hydrolysis of esters (reaction 1.6), it is not obvious whether the alkyl-oxygen bond is broken (1.6a) or the acyl-oxygen bond (1.6b). This question can be resolved by use of an lsO isotopic label in the ester. 

Because transition states may have lifetimes of only several nanoseconds, in most cases, it is impossible to observe them directly. However, there are numerous lines of evidence for the existence of a tetrahedral-like transition state for non-enzymatic ester hydrolysis a) substitution at a carbonyl group (as is the case of the hydrolysis of esters) most often proceeds by a tetrahedral mechanism, a second-order addition-elimination (for a review of this mechanism, see (23)) b) the kinetics are pseudo-first order either in the substrate or in the nucleophile, as predicted by the mechanism c) for the 180 labeled esters, the 180 isotope is detectable in both products (in a "normal" Sjj2 reaction all the 180 isotopes should remain in the acid functionality)(24) d) in a few cases tetrahedral intermediates have been isolated or detected spectrally (25). 

The most important species in the mechanism for ester hydrolysis is the tetrahedral intermediate. Evidence in support of the existence of the tetrahedral intermediate was developed by Professor Myron Bender on the basis of isotopic labeling experiments he carried out at the University of Chicago. Bender prepared ethyl benzoate, labeled with the mass-18 isotope of oxygen at the carbonyl oxygen, then subjected it to acid-catalyzed hydrolysis in ordinary (unlabeled) water. He found that ethyl benzoate, recovered from the reaction before hydrolysis was complete, had lost a portion of its isotopic label. This observation is consistent only with the reversible formation of a tetrahedral intermediate under the reaction conditions ... 

Gorenstein and co-workers have used primary 0 isotope effects to study the structure of the transition state in the hydrolysis of 2,4-dinitrophenyl phosphate in which the bridging ester oxygen is labeled with 0 (47). The results of 32 determinations were averaged to reveal an isotope effect of 1.0204 + 0.0044. 

This seemingly small isotope effect must be compared to the isotope effect measured for the hydrolysis of the dibenzyl ester precursor to the isotopically labeled monoester [phosphate triesters can only hydrolyze by an Sn2(P) mechanism], 1.0070 -I- 0.0038, for proper interpretation. The fact that the hydrolysis of the monoester has a larger isotope effect was interpreted as demonstrating that substantial cleavage of the phosphate ester bond occurs in the transition state this is in accord with the expectation based on an Sn 1(P) mechanism. 

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