Hydrolysis formates, nucleophile isotope

In a review of nucleophile isotope effects in chemistry, the hydrolysis of formates was discussed.10 The effect of dioxane on the acid-catalysed hydrolysis of ethyl formate was studied by carrying out the reaction in 0-80% (v/v) dioxane at different temperatures ranging from 20 to 40 °C. It was proposed that up to 1.5 mol of water are associated with the activated complex.11 Kinetic studies of the alkaline hydrolysis of ethyl decanoate12 in DMF-H2O solutions and of ethyl isovalerate13 in aqueous acetone were reported. 

The haloalkane dehalogenase DhlA mechanism takes place in two consecutive Sn2 steps. In the first, the carboxylate moiety of the aspartate Aspl24, acting as a nucleophile on the carbon atom of DCE, displaces chloride anion which leads to formation of the enzyme-substrate intermediate (Equation 11.86). That intermediate is hydrolyzed by water in the subsequent step. The experimentally determined chlorine kinetic isotope effect for 1-chlorobutane, the slow substrate, is k(35Cl)/k(37Cl) = 1.0066 0.0004 and should correspond to the intrinsic isotope effect for the dehalogenation step. While the reported experimental value for DCE hydrolysis is smaller, it becomes practically the same when corrected for the intramolecular chlorine kinetic isotope effect (a consequence of the two identical chlorine labels in DCE). 

The reaction mechanism catalysed by sEH has been recently elucidated from experiments using heavy isotopes, protein, mass spectrometry, site-directed mutagenesis, and has been supported by the recent crystal structure determination at 2.8-A resolution (Fig. 31.28). This two-step reaction mechanism involves a catalytic nucleophile (aspartic acid 333) which can attack the polarized epoxide ring by two tyrosyl residues (tyrosines 381 and 465) leading to the ring opening and the formation of an acyl-enzyme intermediate. The second step corresponds to hydrolysis of this intermediate by a water molecule activated by a histidine 523-aspartic acid 495 pair." ... 

In the first one, the termolecular problem (i.e. the fact that in GAC and GBC three molecules have to come together in the transition state) is avoided by making a reaction intramolecular. Normally, ester formation and hydrolysis are specrfic-acid-catalysed only, but here there is catalysis by a weak acid acetic acid. A normal kinetic isotope effect k(HOAc)/k(DOAc) = 2.3 shows that proton transfer occurs in the rate-determining step and there is a large negative AS = -156 J mol" K". This is GAC of nucleophilic attack on a carbonyl group, admittedly in a rather special molecule. 

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