The Guanine Cation Radical: Investigation of Deprotonation States by ESR and DFT

Abstract

This work reports ESR studies that identify the favored site of deprotonation of the guanine cation radical (G•+) in an aqueous medium at 77 K. Using ESR and UV−visible spectroscopy, one-electron oxidized guanine is investigated in frozen aqueous D2O solutions of 2‘-deoxyguanosine (dGuo) at low temperatures at various pHs at which the guanine cation radical, G•+ (pH 3−5), singly deprotonated species, G(−H)• (pH 7−9), and doubly deprotonated species, G(−2H)•- (pH > 11), are found. C-8-deuteration of dGuo to give 8-D-dGuo removes the major proton hyperfine coupling at C-8. This isolates the anisotropic nitrogen couplings for each of the three species and aids our analyses. These anisotropic nitrogen couplings were assigned to specific nitrogen sites by use of 15N-substituted derivatives at N1, N2, and N3 atoms in dGuo. Both ESR and UV−visible spectra are reported for each of the species: G•+, G(−H)•, and G(−2H)•-. The experimental anisotropic ESR hyperfine couplings are compared to those obtained from DFT calculations for the various tautomers of G(−H)•. Using the B3LYP/6-31G(d) method, the geometries and energies of G•+ and its singly deprotonated state in its two tautomeric forms, G(N1−H)• and G(N2−H)•, were investigated. In a nonhydrated state, G(N2−H)• is found to be more stable than G(N1−H)•, but on hydration with seven water molecules G(N1−H)• is found to be more stable than G(N2−H)•. The theoretically calculated hyperfine coupling constants (HFCCs) of G•+, G(N1−H)•, and G(−2H)•- match the experimentally observed HFCCs best on hydration with seven or more waters. For G(−2H)•-, the hyperfine coupling constant (HFCC) at the exocyclic nitrogen atom (N2) is especially sensitive to the number of hydrating water molecules; good agreement with experiment is not obtained until nine or 10 waters of hydration are included.