The reactivity of lactyl-oxythiamin implies the role of the amino-pyrimidine in thiamin catalyzed decarboxylation

It has previously been established that the deprotonated amino substituent of the pyrimidine of thiamin diphosphate (ThDP) acts as an internal base to accept the C2H of the thiazolium in ThDP-dependent enzymes. The amino group has also been implicated in assisting the departure of the aldehydic product formed after loss of CO2 from ketoacid substrates. However, the potential role for the pyrimidine amino group in the key decarboxylation step has not been assessed. Oxythiamin contains a hydroxyl group in place of the pyrimidine amino group in thiamin, providing a basis for comparison of reactivity. Lactyl-oxythiamin (LOTh), the conjugate of pyruvic acid and oxythiamin was prepared by condensation of ethyl pyruvate and hydroxyl- protected oxythiamin followed by deprotection and acidic hydrolysis of the ethyl ester. The rate constants observed for the decarboxylation of LOTh in neutral and acidic solutions are about four times smaller than those for the corresponding compound that contains the amino group, lactylthiamin. The difference in reactivity is consistent with the amino group's participation in facilitating the decarboxylation step by allowing a competitive addition pathway that produces bicarbonate and has implications for the corresponding enzymic reaction. (C) 2016 Elsevier Inc. All rights reserved.