Structure and conformation of N⁴-hydroxycytosine and N⁴-hydroxy-5-fluorocytosine. A theoretical ab initio study

Optimal molecular geometries and molecular energies were obtained for N⁴-hydroxycytosine and its 5-fluoro congener with the use of the theoretical ab initio quantum mechanical calculations within the Self Consistent Field method corrected for the electron correlation effects by the second-order Many Body Perturbation Theory (SCF + MBPT(2)). The 6-31G Gaussian basis set was employed. Several tautomeric and rotameric forms were considered. For N⁴-hydroxycytosine and N⁴-hydroxy-5-fluorocytosine the imino tautomer (in the conformation syn relatively to the N3-nitrogen atom) appeared to be the most stable form. The imino tautomer of N⁴-hydroxy-cytosine in the anti rotameric form is by 12.8 kJ mol^-1 less stable than the imino-syn form. The 5-fluoro substituent raises the energy difference between the syn and anti rotamers up to 38.5 kJ mol^-1. The potential energy barrier for the syn-anti rotation in the imino form of N⁴-hydroxycytosine is estimated to be about 180 kJ/mol. The results presented in this paper suggest that the syn-imino and anti-imino forms can be treated as two structural isomers that do not interconvert at temperatures relevant to biochemical conditions. The theoretical results also show that the amino tautomeric forms do not compete with the imino forms in the gas-phase and in non-polar and weakly-polar environment. In a polar environment (e.g., in aqueous solutions), however, one may expect an increased population of the amino forms. Qualitatively, the results of the present study agree well with the available experimental and theoretical data for N⁴-hydroxycytosine and some of its derivatives. The implications of the present study are discussed in relation to the molecular mechanisms of mutagenesis caused by NH₂OH and of enzyme (thymidylate synthase) inhibition by N⁴-hydroxydeoxycytidine monophosphate.