This paper presents improved synthetic methods for the modification of
2'-deoxyuridine-5'-monophosphate and its 5-fluoro derivative, using
trimethylphosphate in aqueous medium at pH 10. These modifications include methylation of the
pyrimidine ring N(3) and/or esterification of the
phosphate group. The 5'-methyl
ester of dUMP was neither a substrate nor an inhibitor of Ehrlich
ascites carcinoma thymidylate synthetase. By contrast, the corresponding methyl
ester of
FdUMP was a tight-binding inhibitor of the
enzyme from L1210, Ehrlich
ascites carcinoma and CCRF-CEM cells. 3-Methyl-dUMP, fixed in the 4-keto form, exhibited only very weak substrate activity with the Ehrlich
ascites carcinoma enzyme. The dUMP analogues
5-ethyl-dUMP and
5-propyl-dUMP were found to be competitive inhibitors of
thymidylate synthetase from L1210, Ehrlich
ascites carcinoma and HeLa cells, the former being the more potent inhibitor. Both analogues were shown to bind cooperatively to each of the mouse tumour
enzymes. Two molecules of inhibitor interacted with a single
enzyme molecule, reflected by the parabolic character of the replots of the slope versus inhibitor concentration. The parent
dTMP was a stronger inhibitor of the mouse tumour
enzymes than its higher alkyl homologues.