Thiol addition to quinones: model reactions for the inactivation of thymidylate synthase by 5-p-benzoquinonyl-2'-deoxyuridine 5'-phosphate.

The reaction of methyl mercaptoacetate (5) with phenyl-p-benzoquinone (6) or 5-p-benzoquinonyl-3',5'-di-O-acetyl-2'-deoxyuridine (10) resulted in the formation of the three possible adducts to the quinone rings of 6 and 10; an additional product in the reaction with 10 was the unsubstituted hydroquinone (14). Both reactions were found to be solvent dependent; in buffered aqueous acetonitrile the meta and para adducts of 10 were formed in the ratio of 2:1. In ethyl acetate the ortho adduct and the reduction product of 10 were isolated in a ratio of 2:3. The second-order rate constant for the reaction of 5 with 10 in acetonitrile was 0.53 M-1 s-1; the reaction was accelerated by the addition of water. Although the initially proposed mechanism-based enzyme inactivation cannot be excluded, the results of the model reactions support the alternative mechanism, active-site thiol addition to the quinone ring. If this is true the title compound would be classed as an affinity label, not a mechanism-based inhibitor.