Hydroxyl ion addition to one-electron oxidized thymine: unimolecular interconversion of C5 to C6 OH-adducts.

In this work, addition of OH(-) to one-electron oxidized thymidine (dThd) and thymine nucleotides in basic aqueous glasses is investigated. At pHs ca. 9-10 where the thymine base is largely deprotonated at N3, one-electron oxidation of the thymine base by Cl(2)(•-) at ca. 155 K results in formation of a neutral thyminyl radical, T(-H)·. Assignment to T(-H)· is confirmed by employing (15)N substituted 5'-TMP. At pH ≥ ca. 11.5, formation of the 5-hydroxythymin-6-yl radical, T(5OH)·, is identified as a metastable intermediate produced by OH(-) addition to T(-H)· at C5 at ca. 155 K. Upon further annealing to ca. 170 K, T(5OH)· readily converts to the 6-hydroxythymin-5-yl radical, T(6OH)·. One-electron oxidation of N3-methyl-thymidine (N3-Me-dThd) by Cl(2)(•-) at ca. 155 K produces the cation radical (N3-Me-dThd(•+)) for which we find a pH dependent competition between deprotonation from the methyl group at C5 and addition of OH(-) to C5. At pH 7, the 5-methyl deprotonated species is found; however, at pH ca. 9, N3-Me-dThd(•+) produces T(5OH)· that on annealing up to 180 K forms T(6OH)·. Through use of deuterium substitution at C5' and on the thymine base, that is, specifically employing [5',5"-D,D]-5'-dThd, [5',5"-D,D]-5'-TMP, [CD(3)]-dThd and [CD(3),6D]-dThd, we find unequivocal evidence for T(5OH)· formation and its conversion to T(6OH)·. The addition of OH(-) to the C5 position in T(-H)· and N3-Me-dThd(•+) is governed by spin and charge localization. DFT calculations predict that the conversion of the "reducing" T(5OH)· to the "oxidizing" T(6OH)· occurs by a unimolecular OH group transfer from C5 to C6 in the thymine base. The T(5OH)· to T(6OH)· conversion is found to occur more readily for deprotonated dThd and its nucleotides than for N3-Me-dThd. In agreement, calculations predict that the deprotonated thymine base has a lower energy barrier (ca. 6 kcal/mol) for OH transfer than its corresponding N3-protonated thymine base (14 kcal/mol).

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

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.

Direct formation of the C5'-radical in the sugar-phosphate backbone of DNA by high-energy radiation.

Neutral sugar radicals formed in DNA sugar-phosphate backbone are well-established as precursors of biologically important damage such as DNA strand scission and cross-linking. In this work, we present electron spin resonance (ESR) evidence showing that the sugar radical at C5' (C5'(•)) is one of the most abundant (ca. 30%) sugar radicals formed by γ- and Ar ion-beam irradiated hydrated DNA samples. Taking dimethyl phosphate as a model of sugar-phosphate backbone, ESR and theoretical (DFT) studies of γ-irradiated dimethyl phosphate were carried out. CH(3)OP(O(2)(-))OCH(2)(•) is formed via deprotonation from the methyl group of directly ionized dimethyl phosphate at 77 K. The formation of CH(3)OP(O(2)(-))OCH(2)(•) is independent of dimethyl phosphate concentration (neat or in aqueous solution) or pH. ESR spectra of C5'(•) found in DNA and of CH(3)OP(O(2)(-))OCH(2)(•) do not show an observable ß-phosphorus hyperfine coupling (HFC). Furthermore, C5'(•) found in DNA does not show a significant C4'-H ß-proton HFC. Applying the DFT/B3LYP/6-31G(d) method, a study of conformational dependence of the phosphorus HFC in CH(3)OP(O(2)(-))OCH(2)(•) shows that in its minimum energy conformation, CH(3)OP(O(2)(-))OCH(2)(•), has a negligible ß-phosphorus HFC. On the basis of these results, the formation of radiation-induced C5'(•) is proposed to occur via a very rapid deprotonation from the directly ionized sugar-phosphate backbone, and the rate of this deprotonation must be faster than that of energetically downhill transfer of the unpaired spin (hole) from ionized sugar-phosphate backbone to the DNA bases. Moreover, C5'(•) in irradiated DNA is found to be in a conformation that does not exhibit ß-proton or ß-phosphorus HFCs.