Tautomerism, acid-base properties and conformation of methylated analogues of the promutagenic N4-hydroxycytosine

Abstract

UV and NMR spectroscopy were employed to study the tautomerism, acid-base properties and conformation of the exocyclic N 4–OH group in 1-methyl- N 4-hydroxycytosine (1-mOH 4C), and its methyl derivatives, viz. the fixed imino forms (1,3-m 2OH 4C and 1,3,5-m 3OH 4C), the fixed amino form (1, N 4-m 2OH 4C), and analogues sterically constrained to the form syn (1,5-m 2OH 4C) or anti (1,3-m 2OH 4C) with respect to the ring N(3). Relative to 1, N 4-m 2OH 4C, UV spectroscopy showed that the other analogues were predominantly imino and that all analogues formed a structurally common cation in acid medium, with results pointing to ∼90% population of the imino species for 1-mOH 4C and 1,5-m 2OH 4C, further supported by NMR spectroscopy. Both exhibited two sequential dissociations in alkaline medium, the first due to N 4–OH, followed by the N(3)–H. 1H and 13C NMR spectroscopy showed 1-mOH 4C in the conformation syn. With 1,3,5-m 3OH 4C, an `overcrowded' planar molecule with steric constraints to both the syn and anti conformations, a syn– anti equilibrium is observed, with a preference of ∼75% for the anti rotamer, independently of the polarity of the medium. Exchange between the rotamers is slow on the NMR time-scale, with a minimal barrier to exchange exceeding 100 kJ/mol. In low-polar media, the analogues associate as dimers via O 4–H⋯O 2 or O 4–H⋯N 4 hydrogen bonds, with association constants at ambient temperature of 4.6 (1,3-m 2OH 4C), 12.8 ( anti 1,3,5-m 3OH 4C), 36 (1,5-m 2OH 4C), 109 ( syn 1,3,5-m 3OH 4C) M −1. Implications of the overall findings to the promutagenic activities of OH 4C and OMe 4C are examined.