Interaction of thymidylate synthase with the 5 '-thiophosphates, 5 '-dithiophosphates, 5 '-H-phosphonates and 5 '-S-thiosulfates of 2 '-deoxyuridine,thymidine and 5-fluoro-2 '-deoxyuridine

New analogs of dUMP, dTMP and 5-fluoro-dUMP, including the corresponding 5' -thiophosphates (dUMPS, dTMPS and FdUMPS), 5'-dithiophosphates (dUMPS(2), d TMPS(2) and FdUMPS(2)), 5'-H-phosphonates (dUMP-H, dTMP-H and FdUMP-H) and 5'-Sthiosulfates (dUSSO(3), dTSSO(3) and FdUSSO(3)), have been synthesized and their interactions studied with highly purified mammalian thymidylate s ynthase. dUMPS and dUMPS2 proved to be good substrates, and dTMPS and dTMPS 2 classic competitive: inhibitors, only slightly weaker than dTMP. Their 5- fluoro congeners behaved as potent, slow-binding inhibitors. By contrast, t he corresponding 5'-H-phosphonates and 5'-S-thiosulfates displayed weak act ivities, only FdUMP-H and FdUSSO(3) exhibiting significant interactions wit h the enzyme, as weak competitive slow-binding inhibitors versus dUMP. The pH-dependence of enzyme time-independent inhibition by FdUMP and FdUMPS was found to correlate with the difference in pK(a) values of the phosphate an d thiophosphate groups, the profile of FdUMPS being shifted (approximate to 1 pH unit) toward lower pH values, so that binding of dUMP and its analogs is limited by the phosphate secondary hydroxyl ionization. Hence, together with the effects of 5'-H-phosphonate and 5'-S-thiosulfate substituents, the much weaker interactions of the nucleotide analogs (3-5 orders of magnitud e lower than for the parent 5'-phosphates) with the enzyme is further evide nce that the enzyme's active center prefers the dianionic phosphate group f or optimum binding.