Identification by UV Resonance Raman Spectroscopy of an Imino Tautomer of 5-hydroxy-2′-deoxycytidine, a Powerful Base Analog Transition Mutagen with a Much Higher Unfavored Tautomer Frequency than That of the Natural Residue 2′-deoxycytidine

UV resonance Raman spectroscopy was used to detect and estimate the frequency of the unfavored imino tautomer of the transition mutagen 5-hydroxy-2′-deoxycytidine (HO5dCyt) in its anionic form. In DNA, this 2′-deoxycytidine analog arises from the oxidation of 2′-deoxycytidine and induces C → T transitions with 102greater frequency than such spontaneous transitions. An imino tautomer marker carbonyl band (≈ 1650 cm-1) is enhanced at ≈ 65 degrees C against an otherwise stable spectrum of bands associated with the favored amino tautomer. This band is similarly present in the UV resonance Raman spectra of the imino cytidine analogs N3-methylcytidine at high pH and N4-methoxy-2′-deoxycytidine at pH 7 and displays features attributable to the imino form of C residues and their derivatives. The fact that the imino tautomer of HO5dCyt occurs at a frequency consistent with its high mutagenic enhancement lends strong support to the hypothesis that unfavored base tautomers play important roles in the mispair intermediates of replication leading to substitution mutations.