18O Isotope Effect in 13C Nuclear Magnetic Resonance Spectroscopy. 7. Hydrolysis of 2,2-Dimethyloxirane in Dilute Acid and by Microsomal Epoxide Hydratase

Article abstract of DOI:10.1021/ja00344a038

The 18O isotope effect in 13C NMR spectroscopy affords an analytical technique that may be used to ascertain directly and simultaneously, in a continuous-assay mode, the position of bond cleavage, the rate of hydrolysis, and the extent of any accompanying oxygen exchange during the hydrolysis of oxiranes.The synthesis of 2,2-dimethyl<13C,18O>oxirane (<1-13C,18O>isobutylene oxide) is described.The hydrolysis of this compound in dilute acid, by mouse liver microsomes (epoxide hydratase), and by homogeneous rat liver epoxide hydratase (EC 3.3.2.3) was studied by 13C NMR spectroscopy.The 18O isotope-induced shifts of the 13C resonances of the primary carbon atom and of the tertiary carbon atom in the oxirane are 0.031 and 0.042 ppm upfield, respectively.The corresponding isotope shifts in the product diol (2-methylpropane-1,2-diol) are 0.019 and 0.033 ppm respectively.Dilute-acid-catalyzed hydrolysis of the oxirane results in retention of the 18O label on the primary carbon atom with no accompanying oxygen exchange.The rates of nonenzymatic hydrolysis measured with this technique closely approximate the rates of hydrolysis measured previously by tedious mass spectral analysis.In contrast, when either mouse liver microsomes or homogeneous rat liver microsomal epoxide hydratase was incubated at pH 8 and 35 deg C with 2,2-dimethyl<3-13C,18O>oxirane, the hydrolysis product was 2-methyl<1-13C,2-18O>propane-1,2-diol, consistent with enzyme-catalyzed hydrolytic attack at the primary carbon.Competing base-catalyzed hydrolysis of the oxirane under identical experimental conditions was much slower than the enzyme-catalyzed reactions.Although specific numerical values of k_cat could not be obtained due to inability to saturate the enzymes, k_cat for 2,2-dimethyloxirane must be significantly greater than that for styrene oxide.This study further illustrates the applicability of the 18O isotope effect in 13C NMR spectroscopy in simplifying the analysis of a variety of kinetic and stereochemical problems.