π-Radical to σ-Radical Tautomerization in One-Electron-Oxidized 1-Methylcytosine and Its Analogs

Abstract

In this work, iminyl σ-radical formation in several one-electron-oxidized cytosine analogs, including 1-MeC, cidofovir, 2'-deoxycytidine (dCyd), and 2'-deoxycytidine 5'-monophosphate (5'-dCMP), were investigated in homogeneous, aqueous (D2O or H2O) glassy solutions at low temperatures by employing electron spin resonance (ESR) spectroscopy. Upon employing density functional theory (DFT) (DFT/B3LYP/6-31G* method), the calculated hyperfine coupling constant (HFCC) values of iminyl σ-radical agree quite well with the experimentally observed ones, thus confirming its assignment. ESR and DFT studies show that the cytosine iminyl σ-radical is a tautomer of the deprotonated cytosine π-cation radical [cytosine π-aminyl radical, C(N4-H)(•)]. Employing 1-MeC samples at various pHs ranging from ca. 8 to 11, ESR studies show that the tautomeric equilibrium between C(N4-H)(•) and the iminyl σ-radical at low temperature is too slow to be established without added base. ESR and DFT studies agree that, in the iminyl σ-radical, the unpaired spin is localized on the exocyclic nitrogen (N4) in an in-plane pure p-orbital. This gives rise to an anisotropic nitrogen hyperfine coupling (Azz = 40 G) from N4 and a near isotropic β-nitrogen coupling of 9.7 G from the cytosine ring nitrogen at N3. Iminyl σ-radical should exist in its N3-protonated form, as the N3-protonated iminyl σ-radical is stabilized in solution by over 30 kcal/mol (ΔG = -32 kcal/mol) over its conjugate base, the N3-deprotonated form. This is the first observation of an isotropic β-hyperfine ring nitrogen coupling in an N-centered DNA radical. Our theoretical calculations predict that the cytosine iminyl σ-radical can be formed in double-stranded DNA by a radiation-induced ionization-deprotonation process that is only 10 kcal/mol above the lowest energy path.

Figure 1

ESR spectra of iminyl σ-radical formed in γ-irradiated (dose = 1.4 kGy at 77 K) matched glassy samples (2 mg/ml each) of (A) 1-MeC (red), (B) cidofovir (blue), and (C) dCyd (teal) in 7.5 M LiCl/D₂O with electron scavenger K₂S₂O₈ (8 mg/ml in each sample), pH ca. 11 via annealing at 160 – 165 K for 15 min after one-electron oxidation of the cytosine base by Cl₂•⁻. The simulated spectra (black) due to iminyl σ-radical of one-electron oxidized 1-MeC (A) and cidofovir (B) are placed underneath the experimental spectra. For simulation parameters see text. (C) The experimentally recorded spectrum of one-electron oxidized dCyd (teal) is shown in Figure 1(C). The experimentally obtained iminyl σ-radical spectrum from 1-MeC (red, Figure 1(A)) has been superimposed on this spectrum for comparison. All ESR spectra shown in Figures A to C were recorded at 77 K. The three reference markers in this figure and in subsequent figures are Fremy’s salt resonances with central marker is at g = 2.0056 and each of three markers is separated from one another by 13.09 G.

Figure 2

ESR spectra obtained from matched 1-MeC samples [concentration of 1-MeC in each sample = 2 mg/ml in 7.5 M LiCl/D₂O] in the presence of the electron scavenger K₂S₂O₈ (8 mg/ml in each sample). Each sample has been γ-irradiated (absorbed dose = 1.4 kGy at 77 K), subsequently annealed to 160 to 165 K for 15 min in the dark at various pHs (A) pH ca. 8 (pink), (B) pH ca. 9.5 and (C) pH ca. 11 (red, spectrum in Figure 1(C)). The simulated spectra (black) due to the π-aminyl radical (1-MeC(N4-H)•) in (A) as well as owing to the iminyl σ-radical of one-electron oxidized 1-MeC in (C) respectively are placed underneath the experimental spectra. The line components of the red (or, black) spectrum in (C) are visible in (B) (see dotted lines). See text for the details of simulation. All ESR spectra are recorded at 77 K.

Figure 3

Proposed role of the cytosine iminyl σ-radical to produce neutral guanyl radical (G(N1-H)•:C (or, *G(-H)C)) via proton coupled electron transfer in the one-electron oxidized 9-Me-G:1-Me-C (Me group at N9 in G and at N1 in C mimic sugar moieties). The calculations were performed employing ωb97x/PCM/6-31++G(d) method for geometry optimization and spin density distribution plots (also see supporting information Figure S10). TE = total energy in atomic unit (A.U.). The HFCC values of N3 and N4 were shown for the 1-Me-C iminyl σ-radical (see left uppermost panel) in the one-electron oxidized 9-Me-G:1-Me-C and these values agree with those observed in the monomer (see Table 1).

Scheme 1

Formation of cytosine π-cation radical via one-electron oxidation of cytosine monomers. The cytosine π-cation radical, via deprotonation and by subsequent tautomerization, leads to the formation of iminyl radical, which is a σ radical by nature. The atom numbering scheme of the cytosine base is shown. The relative stabilities of C(N4-H)• _syn, C(N4-H)•_anti, and iminyl σ-radical in kcal/mol are provided. Optimization of the geometries of these radicals along with their energy values employing optimized structures were obtained with the aid of DFT/B3LYP/6-31G* method.