5-Iodo-5'-amino-2',5'-dideoxyuridine-5'-N'-triphosphate. Synthesis, chemical properties, and effect on Escherichia coli thymidine kinase activity

Abstract

5-Iodo-5'-amino-2',5'-dideoxyuridine-5'-N'-triphosphate (AIdUTP), a phosphoramidate analog of 5-iodo-2',5'-dideoxyuridine 5'-triphosphate (IdUTP), was synthesized and some of its chemical and biological properties were investigated. Although AIdUTP is stable in alkaline solutions, below pH 8 it undergoes degradation by a novel phosphorylysis reaction which exhibits first order kinetics. Inclusion of magnesium ion in the reaction mixture decreased the rate of degradation. Protonation of a group on AIdUTP which has a pKa of 6.10, presumably the secondary ionized oxygen on the gamma phosphate, precedes phosphorylysis. The only detectable reaction products are the nucleoside, 5-iodo-5'-amino-2',5'-dideoxyuridine (AIdUrd), and trimetaphosphate. A mechanism for the acid catalyzed phosphorylysis of AIdUTP is proposed. AIdUTP, like TTP, converts Escherichia coli thymidine kinase into an inactive dimer with a sedimentation coefficient of 5.78 S. AIdUTP is, however, 60-fold more potent as an allosteric inhibitor than is TTP at pH 7.8. Although the inhibitory effect of TTP is markedly reduced at high pH, the activity of AIdUTP is lowered only slightly. The allosteric effects of AIdUTP also differ from those of IdUTP, which is an inhibitor at low pH but a strong activator above pH 7.4. 5-Iodo-2'-deoxycytidine 5'-triphosphate, a potent enzyme activator, cannot completely reverse the AIdUTP inhibition, even when present at a 150-fold molar excess.