STRUCTURE OF OXIDATIVELY DAMAGED NUCLEIC-ACID ADDUCTS .3. TAUTOMERISM, IONIZATION AND PROTONATION OF 8-HYDROXYADENOSINE STUDIED BY N-15 NMR-SPECTROSCOPY

Natural abundance N-15 NMR spectroscopy and ancillary spectroscopic techniques have been employed to study the solution structure of 8-hydroxyadenosine. 8-Hydroxyadenosine is a naturally occurring oxidized nucleic acid adduct that is generally implied to have an 8-hydroxy tautomeric structure. N-15 NMR chemical shifts and coupling constants, however, indicate that the modified base exists as an 8-keto tautomer. The pH dependence of N-15 NMR and UV spectra showed the presence of two pK(a)'s, at 2.9 and 8.7, corresponding to protonation at N1 and ionization at N7, respectively. The latter results in the formation of an 8-enolate structure. Unusual upfield shifts of the H-1 and N-15 resonances of the NH2 group, and a reduction in the one-bond coupling constant 1J(N6-H6), is indicative of an unfavorable steric or electronic interaction between the NH2 group and the adjacent N7-H proton. This interaction results in a subtle change in the structure of the NH2 group. In addition to being a possible mechanism for alteration of hydrogen bonding in oxidized DNA, this type of interaction gives a better understanding into N7-N9 tautomerism of adenine. Furthermore, the structure of 8-hydroxyadenosine has been related to possible mechanisms for mutations.