Theoretical analysis of the addition of hydroxylamine to uracil and 5-fluorouracil as a model for the Thymidylate synthase reaction

In the present paper we report a quantum chemical (PM3) investigation of reagents, transition structures, intermediates and final products of the nucleophilic addition of hydroxylamine to uracil (U) and 5-fluorouracil (FU). This reaction serves as a model for the more complex enzymatic methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) and 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) by thymidylate synthase. From the analysis of the frontier orbitals of the isolated and complexed species, as well as from the calculation of activation barriers, we propose that nucleophilic attack usually proceeds after formation of an initial complex between U (or FU) and one neutral and one protonated molecule of hydroxylamine. Our results give some insight into the mechanism of these reactions and account for the higher rate of addition of hydroxylamine to FU, compared to U. The main connection between the chemical simulation and the biological scheme is that in both reactions hydrogen bonding residues are found to be necessary to assist catalysis.