Reaction of 5-trifluoromethyl-2′-deoxyuridine and 1-methyl-5-trifluoromethyluracil with methoxyamine: model studies for the interaction between thymidylate synthetase and 5-trifluoromethyl-2′-deoxyuridine 5′-phosphate

Abstract

The reaction of 5-trifluoromethyl-2′-deoxyuridine (CF₃dUR) and 5-trifluoromethyl-1-methyluracil (1-MCF₃U) with methoxyamine in aqueous solution was studied to provide insight into the mechanism of inhibition of thymidylate synthetase by 5-trifluoromethyl-2′-deoxyuridine 5′-phosphate. 5-(Methoxyiminofluoromethyl)-2′-deoxyuridine (4b) and 5-(methoxyiminomethoxyaminomethyl)-2′-deoxyuridine (8b) were the products of the reactions of CF₃dUR with methoxyamine. No other major product was detected by radiochromatography of [6-³H]CF₃dUR after treatment with methoxyamine. The decrease in CF₃dUR on treatment with methoxyamine followed pseudo-first-order kinetics, and (4b) and (8b) were formed sequentially, in that order. At pH 7.5 and 37 °C in 0.5M methoxyamine, the pseudo-first-order rate constant, k_obs, for the decrease in CF₃dUR (0.4 mM) was 0.355 h^–1 and that for the decrease in (4b)(0.4 mM) was 0.06 h^–1. The k_obs for the decrease in CF₃dUR at 37 °C had an optimum at pH 7.5; it was 0.355 h^–1 with 0.5M methoxyamine. Increasing the concentration of methoxyamine at pH 7.5 resulted in greater increases in the rate of CF₃dUR degradation than expected for a first-order reaction. This suggests that more than one molecule of methoxyamine participates in the degradation of CF₃dUR. 1-MCF₃U Reacted with methoxyamine to give 5-(methoxyiminofluoromethyl)-1-methyluracil (4a) and 5-(methoxyiminomethoxyaminomethyl)-1-methyluracil (8a) as products. Under acidic conditions, (4a) was converted into 5-(N-methoxycarbamoyl)-1-methyluracil. The formation of (4a) and (4b) as products of these reactions provides evidence for the formation of a highly reactive intermediate with an exocyclic difluoromethylene group at the 5 position which subsequently reacts with methoxyamine. An analogous mechanism was proposed for the inhibition of thymidylate synthetase by 5-trifluoromethyl-2′-deoxyuridine 5′-phosphate in which a nucleophilic group of the active site of the enzyme participates in the activation of the trifluoromethyl group.