The reaction of hydroxyl radical (OH•) with DNA accounts for about half of radiation-induced DNA damage in living systems. Previous literature reports point out that the reaction of OH• with DNA proceeds mainly through the addition of OH• to the C=C bond of the DNA bases. However, recently it has been reported that the principal reaction of OH• with dGuo (deoxyguanosine) is the direct hydrogen atom abstraction from its exocyclic amine group rather than addition of OH• to the C=C bond. In the present work, these two reaction pathways of OH• attack on guanine (G) in the presence of water molecules (aqueous environment) are investigated using the density functional theory (DFT) B3LYP method with 6-31G* and 6-31++G** basis sets. The calculations show that the initial addition of the OH• at C4=C5 double bond of guanine is barrier free and the adduct radical (G-OH•) has only a small activation barrier of ca. 1 – 6 kcal/mol leading to the formation of a metastable ion-pair intermediate (G•+---OH−). The formation of ion-pair is a result of the highly oxidizing nature of the OH• in aqueous media. The resulting ion-pair (G•+---OH−) deprotonates to form H2O and neutral G radicals favoring G(N1-H)• with an activation barrier of ca. 5 kcal/mol. The overall process from the G(C4)-OH• (adduct) to G(N1-H)• and water is found to be exothermic in nature by more than 13 kcal/mol. (G-OH•), (G•+---OH−), and G(N1-H)• were further characterized by the CAM-B3LYP calculations of their UV-visible spectra and good agreement between theory and experiment is achieved. Our calculations for the direct hydrogen abstraction pathway from N1 and N2 sites of guanine by the OH• show that this is also a competitive route to produce G(N2-H)•, G(N1-H)• and H2O.

Employing electron spin resonance (ESR) spectroscopy, we have characterized the radicals formed in 3′-Azido-3′-deoxythymidine (3′-AZT) and in its 5′-analog 5′-azido-5′-deoxythymidine (5′-AZT) after electron attachment in γ-irradiated aqueous (H 2 O or D 2 O) glassy (7.5 M LiCl) systems. ESR spectral studies and theoretical calculations show that the predominant site of electron capture in 3′-AZT and in 5′-AZT is at the azide group and not at the thymine moiety. The azide group in AZT is therefore more electron affinic than the most electron affinic DNA base, thymine. Electron attachment to 3′-AZT and 5′-AZT results in an unstable azide anion radical intermediate (RN 3 • − ) that is too short lived to be observed in our work even at 77 K. At 77 K we observe the neutral aminyl radical (RNH•) after loss of N 2 from RN 3 • − followed by protonation of nitrene anion radical (RN• − ) to give RNH•. The expected RN• − intermediate is not observed as protonation from water is complete at 77 K even in under highly basic conditions. Formation of RND• in D 2 O solutions confirms water as the source of the NH proton in the RNH•. Our assignments to these radicals are aided by DFT calculations for hyperfine coupling constants which closely match the experimental values. On annealing to higher temperatures (ca. 160-170 K), RNH• undergoes bimolecular hydrogen abstraction reactions from the thymine methyl group and the sugar moiety resulting in the formation of the thymine allyl radical (UCH 2 •) and two sugar radicals -C3′•, C5′•. RNH• also results in one-electron oxidation of the guanine base in 3′-AZG. This work provides a potential mechanism for the reported radiosensitization effects of AZT.

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 Cl2•− at ca. 155 K results in formation of a neutral thyminyl radical, T(−H)•. Assignment to T(−H)• is confirmed by employing 15N 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 Cl2•− 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, i.e., specifically employing [5',5”-D,D]-5'-dThd, [5',5”-D,D]-5'-TMP, [CD3]-dThd and [CD3,6D]-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).

Nucleobase radicals are the major family of reactive intermediates produced when nucleic acids are exposed to γ-radiolysis. 5,6-Dihydrouridin-5-yl radical (1), the formal product of hydrogen atom addition and a model for hydroxyl radical addition was independently generated from a ketone precursor via Norrish Type I photocleavage in single stranded, and double stranded RNA. Radical 1 produces direct strand breaks at the 5'-adjacent nucleotide and only minor amounts of strand scission are observed at the initial site of radical generation. Strand scission occurs preferentially in double stranded RNA and in the absence of O2. The dependence of strand scission efficiency from 5,6-dihydrouridin-5-yl radical (1) on secondary structure under anaerobic conditions suggests that this reactivity may be useful for extracting additional RNA structural information from hydroxyl radical reactions. Varying the identity of the 5'-adjacent nucleotide has little effect on strand scission. Internucleotidyl strand scission occurs via β-elimination of the 3'-phosphate following C2'-hydrogen atom abstraction by 1. The subsequently formed olefin cation radical yields RNA fragments containing 3'-phosphate or 3'-deoxy-2'-ketonucleotide termini from competing deprotonation pathways. The ketonucleotide end group is favored in the presence of low concentrations of thiol, presumably by reducing the cation radical to the enol. Competition studies with thiol show that strand scission from 5,6-dihydrouridin-5-yl radical (1) is significantly faster than from 5,6-dihydrouridin-6-yl radical (2) and is consistent with computational studies using the G3B3 approach that predict the latter to be more stable than 1 by 2.8 kcal/mol.

One electron oxidation of neutral sugar radicals has recently been suggested to lead to important intermediates in the DNA damage process culminating in DNA strand breaks. In this work, we investigate sugar radicals in a DNA model system to understand the energetics of sugar radical formation and oxidation. The geometries of neutral sugar radicals C1′•, C2′•, C3′•, C4′• and C5′• of 2′-deoxyguanosine (dG) and 2′-deoxythymidine (dT) were optimized in the gas phase and in solution using the B3LYP and ωB97x functionals and 6-31++G(D) basis set. Their corresponding cations (C1′+, C2′+, C3′+, C4′+ and C5′+) were generated by removing an electron (one-electron oxidation) from the neutral sugar radicals and their geometries were also optimized using the same methods and basis set. The calculation predicts the relative stabilities of the neutral sugar radicals in the order C1′• > C4′• > C5′• > C3′• > C2′•, respectively. Of the neutral sugar radicals, C1′• has the lowest vertical ionization potential (IPvert) ca. 6.33 eV in the gas phase and 4.71 eV in solution. C2′• has the highest IPvert ca. 8.02 eV in the gas phase and the resultant C2′ cation is predicted to undergo a barrierless hydride transfer from the C1′ site to produce the C1′ cation. One electron oxidation of C2′• in dG is predicted to result in a low lying triplet state consisting of G+• and C2′•. The 5′,8-cyclo-2′-deoxyguanosin-7-yl radical formed by intramolecular bonding between C5′• and C8 of guanine transfers spin density from C5′ site to guanine and this structure has IPvert 6.25 eV and 5.48 eV in the gas phase and in solution.