Keto-iminol tautomerism of protonated cytidine monophosphate characterized by ultraviolet resonance Raman spectroscopy: implications of C+ iminol tautomer for base mispairing

Ultraviolet resonance Raman (UVRR) spectra are reported for cytidine monophosphate in neutral and protonated forms. Aside from shifts in cytosine ring modes, protonation induces the appearance of a new RR band at 1730 cm-1 whose excitation profile and temperature-dependent intensity distinguish it from other cytosine bands. The 1730-cm-1 band shifts down by 15 cm-1 in D2O, and it is assigned to the C2=N3 stretching mode of an iminol tautomer. Its high frequency is due to the electronic effect of the OH substituent as well as vibrational coupling with the OH bending mode, as shown by comparison with imidic acid model compounds. The iminol tautomer is protonated at N4, not N3, as judged by comparison with UVRR spectra of N3-methylcytidine, N4,N4-dimethyl-2'-deoxycytidine, and cyclocytidine. The UVRR bands are assigned in a consistent fashion for major and minor tautomers of all these species. UVRR spectra of polycytidylic acid (poly(C)) show duplex formation upon hemiprotonation, with concomitant inhibition of formation of the iminol tautomer, as expected from the C+.C H-bonding pattern. Additional protonation dissociates the poly(C) strands of the duplex, showing that C+.C+ pairing does not allow an alternative duplex structure. The iminol tautomer of C+ may play a role in the stabilization of A-C oppositions via wobble pairing.