Reactions of 5-methylcytosine cation radicals in DNA and model systems: thermal deprotonation from the 5-methyl group vs. excited state deprotonation from sugar

Abstract

Purpose: To study the formation and subsequent reactions of the 5-methyl-2'-deoxycytidine cation radical (5-Me-2'-dC•(+)) in nucleosides and DNA-oligomers and compare to one-electron oxidized thymidine.

Materials and methods: Employing electron spin resonance (ESR), cation radical formation and its reactions were investigated in 5-Me-2'-dC, thymidine (Thd) and their derivatives, in fully double-stranded (ds) d[GC*GC*GC*GC*](2) and in the 5-Me-C/A mismatched, d[GGAC*AAGC:CCTAATCG], where C* = 5-Me-C.

Results: We report 5-Me-2'-dC•(+) production by one-electron oxidation of 5-Me-2'-dC by Cl(2)•- via annealing in the dark at 155 K. Progressive annealing of 5-Me-2'-dC•(+) at 155 K produces the allylic radical (C-CH(2)•). However, photoexcitation of 5-Me-2'-dC•(+) by 405 nm laser or by photoflood lamp leads to only C3'• formation. Photoexcitation of N3-deprotonated thyminyl radical in Thd and its 5'-nucleotides leads to C3'• formation but not in 3'-TMP which resulted in the allylic radical (U-CH(2)•) and C5'• production. For excited 5-Me-2',3'-ddC•(+), absence of the 3'-OH group does not prevent C3'• formation. For d[GC*GC*GC*GC*](2) and d[GGAC*AAGC:CCTAATCG], intra-base paired proton transferred form of G cation radical (G(N1-H)•: C(+ H(+))) is found with no observable 5-Me-2'-dC•(+) formation. Photoexcitation of (G(N1-H)•:C(+ H(+))) in d[GC*GC*GC*GC*](2) produced only C1'• and not the expected photoproducts from 5-Me-2'-dC•(+). However, photoexcitation of (G(N1-H)•:C(+ H(+))) in d[GGAC*AAGC:CCTAATCG] led to C5'• and C1'• formation.

Conclusions: C-CH(2)• formation from 5-Me-2'-dC•(+) occurs via ground state deprotonation from C5-methyl group on the base. In the excited 5-Me-2'-dC•(+) and 5-Me-2',3'-ddC•(+), spin and charge localization at C3' followed by deprotonation leads to C3'• formation. Thus, deprotonation from C3' in the excited cation radical is kinetically controlled and sugar C-H bond energies are not the only controlling factors in these deprotonations.

Keywords: 5-Methylcytosine; DNA-oligomers; ESR; HFCC values; TD-DFT calculations; cation radical; one-electron oxidation; thymidine.

Figure 1

(A) ESR spectrum (black) showing line components of Cl₂•⁻ and SO₄•⁻ thereby providing evidence of their formation at 77 K in γ-irradiated (1.4 kGy) sample of 5-Me-2'-dC (2 mg / ml) in the homogeneous glassy solution of 7.5 M LiCl in D₂O (pD (ca. 5)) in the presence of K₂S₂O₈. (B) Spectrum (black) of the sample in (A) after annealing to ca. 155 K for 5 min. (C) Spectrum (black) obtained after further annealing at ca. 155 K for 30 min (total 35 min). (D) Spectrum (black) obtained after further annealing at ca. 155 K for 120 min (total 155 min). (E) The spectrum (Gray) was extracted by subtraction of 25% spectrum D from spectrum B and is assigned to 5-Me-2′-dC•⁺ (see supplemental Figure S1). (F) Subtraction of 20% of spectrum E from spectrum D, the spectrum in black was obtained. The simulated spectrum (gray) due to C-CH₂• is placed underneath the black spectrum. For simulation parameters see text. All ESR spectra shown in Figures A to D 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

(A) ESR spectrum (black) of a γ-irradiated (1.4 kGy, 77 K) matched sample (for Figure 1) of 5-Me-2′-dC (2 mg/ml) in the presence of excess K₂S₂O₈ at the native pD (ca. 5) in the homogeneous glassy solution of 7.5 M LiCl in D₂O. (B) Spectrum (black) of the sample in (A) after annealing to ca. 155 K for 10 min. (C) Using photoflood lamp, photoexcitation of the sample in (B) at 143 K for 30 min. (D) The spectrum (black) obtained after subtraction of line components of Cl₂•⁻ and C-CH₂• from spectrum C and is assigned to C3′•. The simulated (gray) C3′• spectrum obtained using three isotropic β-proton HFCC is superimposed on the black spectrum for comparison.

Figure 3

Spectrum (A) of T(−H)• produced via annealing at 155 K owing to one-electron oxidation of the N3-deprotonated thymine base in 5′-TMP (3 mg/ml) by Cl₂•⁻ in homogeneous aqueous glass (7.5 M LiCl/D₂O) at pD ca. 10 in the presence of excess K₂S₂O₈ ; (B) after photoexcitation of T(−H)• in 5′-TMP shown in (A) using 405 nm laser at 143 K for 40 min; (C) after photoexcitation of T(−H)• in a matched sample of 5′-TDP (3 mg/ml) using 405 nm laser at 143 K for 40 min; (D) after photoexcitation of T(−H)• -of T(−H)• in a matched sample of 5′-TTP (3 mg/ml) using 405 nm laser at 143 K for 40 min; (E) after visible illumination of T(−H)• formed in a similarly prepared sample of Thd (3 mg/ml) at pD ca. 11 at 143 K. (F) The simulated C3′• spectrum has been obtained using three isotropic β-proton HFCC (text). The line components of the C3′• spectrum is visible in spectra (B) to (E) as indicated by the dotted lines. Similar to the results found in Figure 2 and reaction (5), a small extent of Cl₂•⁻ (ca. 10%) formed via one-electron oxidation of the matrix (LiCl) by (T(−H)•)* has been subtracted from each of the experimentally recorded spectra and the subtracted spectra have been presented in Figures 3(B) to 3(E).

Figure 4

Spectrum (A) after visible illumination by photoflood lamp of T(−H)• formed in a sample of Thd (3 mg/ml) at pD ca. 11 at 143 K for 1 h. This spectrum is already shown in Figure 3 (E). Spectrum (B) obtained after adding 50% of spectrum (C) due to C3′• (see spectrum 3(F)) and 50% of spectrum (D) due to UCH₂• as benchmarks.

Figure 5

Spectrum (A) of T(−H)• produced via annealing at 155 K owing to one-electron oxidation of the N3-deprotonated thymine base in 3′-TMP (3 mg/ml) by Cl₂•⁻ in homogeneous aqueous glass (7.5 M LiCl/D₂O) at pD ca. 9 in the presence of excess K₂S₂O₈. This spectrum also contains residual line components from Cl₂•⁻. Spectrum (B) after photoexcitation of T(−H)• using 405 nm laser at 143 K for 1 h. showing photoproduction of C5′•, UCH₂•, and Cl₂•⁻. All the ESR spectra are recorded at 77 K.

Figure 6

(A) ESR spectrum of a γ-irradiated (1.4 kGy, 77 K) matched sample of 5-Me-2′,3′-ddC (2 mg/ml) in the presence of excess K₂S₂O₈ at the native pD (ca. 5) in the homogeneous glassy solution of 7.5 M LiCl in D₂O. (B) Spectrum (black) of the sample in (A) after annealing to ca. 153 K for 15 min in the dark. The spectrum of 5-Me-2′-dC•⁺ (spectrum 1(E) is superimposed on it for comparison). (C) Spectrum after photoexcitation of the sample in (B) using 405 nm laser at 143 K for 40 min. (D) Spectrum (black) obtained after subtraction of line components of Cl₂•⁻. The outer line components due to C3′•_dephos (reaction (11) are indicated by arrows. C-CH₂• spectrum in gray (spectrum 1F) is superimposed on it. (E) The simulated C3′•_dephos spectrum obtained using HFCC from Becker et al. 2003 (see text).

Figure 7

Left panel: (A) ESR spectrum of a γ-irradiated (3 kGy, 77 K) sample of d[GC*GC*GC*GC*]₂ (C* = 5-Me-C) (1.5 mg/ml) in the presence of electron scavenger K₂S₂O₈ (8 mg/ml) at the native pD (ca. 5) in the homogeneous glassy solution of 7.5 M LiCl in D₂O. (B) Spectrum (black) of the sample in (A) after annealing to ca. 154 K for 25 min in the dark. The reported ESR spectrum (gray) of intra-base pair proton transferred state of guanine cation radical (G(N1-H)•:C(+H⁺)) in d[GCGCGC]₂ (Adhikary et al. 2009) is superimposed on it for comparison. Right panel: (C) spectrum of the sample in (B) after photoexcitation employing 405 nm laser for 40 min at 143 K. (D) spectrum obtained after photoexcitation employing 405 nm laser for 40 min at 143 K from a similarly prepared sample of one-electron oxidized d[GGAC*AAGC:CCTAATCG] (supplemental information Figure S6). The line components due to the formation of the C1´• via photoexcitation are indicated by up arrows. The down arrows indicate the C5´• which overwhelm the central peaks of the C1´•.

Figure 8

TD-B3LYP/6–31G** calculated 11^th transition showing the electronic transition originating from doubly occupied inner core molecular orbital (53β) localized on the sugar moiety to the half filled molecular orbital, SOMO, (64 β) localized on the cytosine base of the 5-Me-2′-dC•⁺.