Kinetics of C(2 alpha)-proton abstraction from 2-benzylthiazolium salts leading to enamines relevant to catalysis by thiamin-dependent enzymes

The kinetics of proton transfer from the C(2 alpha) position of 2-(1-methoxybenzyl)thiazolium salts was studied for the pH and p-N(CH3)(3)(+) derivatives as models for the protonation of the enamine/C(2 alpha)-carbanion, a key intermediate in many thiamin diphosphate-dependent enzymatic reactions. The reactions were studied by rapid mixing of the salts with sodium hydroxide in a stopped-flow instrument while monitoring the progress of enamine formation and decomposition in the visible region of the spectrum. It was demonstrated that under the conditions of the experiments, the thiazolium ring opens to give a product with a characteristic H-1-NMR spectrum consistent with both cis and trans stereochemistry about the amide bond generated in the ring opening. Analysis of the progress curves for the p-trimethylammonium-substituted compound (1) gave the following rate constants (25 degrees C): 21 M(-1) s(-1) for deprotonation at C(2 alpha), 300-540 s(-1) for reprotonation, and 3.4 M(-1) s(-1) for thiazolium ring opening. For the unsubstituted compound (2), the respective rate constants were 0.019 M(-1) s(-1), 1 s(-1), and 0.5 M(-1) s(-1). Through comparisons with results for the C(2 alpha)-deuterated form of 1, the primary hydrogen isotope effect on deprotonation of 1 was found to be 4-6. The C(2 alpha)-H pK(2) values were estimated to be 15.0-15.5 for I and 15.7 for 2. When compared with the turnover number for benzoylformate decarboxylase catalysis, the rate constant for reprotonation of the enamine derived from 2 leads to an estimate of 4500 M as a minimum effective molarity for the enzyme.