Computational Study of the pH-Dependent Competition between Carbonate and Thymine Addition to the Guanine Radical.

When oligonucleotides are oxidized by carbonate radical, thymine and carbonate can add to guanine radical, yielding either a guanine-thymine cross-link product (G∧T) or 8-oxo-7,8-dehydroguanine (8oxoG) and its further oxidation products such as spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). The ratio of thymine addition to carbonate addition depends strongly on the pH. Details of the mechanism have been explored by density functional calculations using the ωB97XD/6-31+G(d,p) level of theory with the SMD implicit solvation method, augmented with a few explicit waters. Free energies of intermediates and transition states in aqueous solution have been calculated along the pathways for addition of thymine, CO/HCO and carbonate radical to guanine radical. The pH dependence was examined by using appropriate explicit proton donors/acceptors as computational models for buffers at pH 2.5, 7, and 10. Deprotonation of thymine is required for nucleophilic addition at C8 of guanine radical, and thus is favored at higher pH. The barrier for carbonate radical addition is lower than for bicarbonate or carbonate dianion addition; however, for low concentrations of carbonate radical, the reaction may proceed by addition of bicarbonate/carbonate dianion to guanine radical. Thymine and bicarbonate/carbonate dianion addition are followed by oxidation by O, loss of a proton from C8 and decarboxylation of the carbonate adduct. At pH 2.5, guanine radical cation can be formed by oxidization with sulfate radical. Water addition to guanine radical cation is the preferred path for forming 8oxoG at pH 2.5.