title: Studies on the interaction with thymidylate synthase of analogues of 2'-deoxyuridine-5'-phosphate and 5-fluoro-2'-deoxyuridine-5'-phosphate with modified phosphate groups.
abstract: The role of the phosphate moiety of dUMP, and some analogues, in their interaction with mammalian thymidylate synthase, has been investigated. Substrate and inhibitor activities, and the pH-dependence of these activities, of dUMP and 5-FdUMP, as well as analogues with modified phosphate groups, were compared. The methyl ester of dUMP was neither a substrate nor an inhibitor. By contrast, the methyl ester of 5-FdUMP was a slow-binding inhibitor of the enzyme from L1210, Ehrlich ascites carcinoma and CCRF-CEM cells, with Ki values in the micromolar range. Both 5-FdUrd and the newly synthesized 5'-methylphosphonate of 5-FdUrd were also slow-binding inhibitors of the Ehrlich carcinoma enzyme, but with Ki values in the millimolar range. The interaction of dUMP, 5-FdUMP, and the methyl ester of the latter decreased with increase in pH, whereas that of the 5'-methyl-phosphonate of 5-FdUrd remained unchanged. The results are discussed in relation to the role of the phosphate hydroxyls of dUMP in binding to the enzyme. 5-FdUMP and its analogues exhibited differing interactions with two binding sites on the enzyme molecule, consistent with cooperativity of binding. A convenient procedure is described for the synthesis of 5-fluoro-2'-deoxyuridine-5'-methylphosphonate, applicable also to the preparation of other 5'-methylphosphonate analogues.
mesh_terms: D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology; D002460:Cell Line; D003856:Deoxyuracil Nucleotides / Q000494:pharmacology*; D005468:Fluorodeoxyuridylate / Q000031:analogs & derivatives / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D013329:Structure-Activity Relationship; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*
chemical_list: D003856:Deoxyuracil Nucleotides; D005468:Fluorodeoxyuridylate; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase