The natural occurrence, enzymatic formation, and biochemical significance of a hydroxyethyl derivative of thiamine pyrophosphate.

Thiamine pyrophosphate is required as a coenzyme for several enzymatic reactions which result in the decarboxylation of cu-keto acids with the concomitant formation of corresponding aldehydes or their condensation products (a-keto alcohols). A number of investigators (l-3) have found that thiamine will catalyze the same reactions under mildly alkaline conditions in the absence of enzymes. This work has stimulated various workers to formulate mechanisms which might account for the action of thiamine in these systems. Theories have been proposed which suggest that the active portion of the thiamine molecule is the amino group on the pyrimidine ring (4), the sulfhydryl group resulting from the opening of the thiazole ring (5)) and the methylene bridge carbon which connects the pyrimidine and thiazole portions of the compound (6). However, all of these theories have been discarded because none was supported by experimental evidence. In 1957, Breslow (7) discovered that deuterium ion rapidly exchanged with the hydrogen atom on position 2 of the thiazolium ring and, as a result of this finding, formulated (8) a mechanism for the action of thiamine in model systems which depends on the formation of a carbanion (due to the ionization of the hydrogen) at position 2 of the thiazole ring. This carbanion would then react with carbonyl carbonium ions to form hydroxyalkyl derivatives of thiamine, which upon decarboxylation would yield aldehyde adducts of thiamine. These adducts might be rendered stable by resonance and be capable of ketol condensation reactions. In support of his formulation, Breslow prepared 2-(l-hydroxybenzyl)-3,4-dimethylthiazolium iodide and showed that it could be decomposed readily into benzaldehyde and 3,4-dimethylthiazolium iodide. The hydroxyalkyl compound which would be formed in the manner described above by the decarboxylation of pyruvate would be 3-[(2-methyl-4-amino-5-pyrimidyl)methyl]-2-(l-hydroxyethyl)-4-methyl-5-(2-hydroxyethyl) thiazole (shown in Fig. 1). In 1958, Krampitz et al. (9) reported that this compound had been synthesized and was about 80% as active as thiamine