Amitava Adhikary

UVA-visible photo-excitation of guanine radical cations produces sugar radicals in DNA and model structures

This work presents evidence that photo-excitation of guanine radical cations results in high yields of deoxyribose sugar radicals in DNA, guanine deoxyribonucleosides and deoxyribonucleotides. In dsDNA at low temperatures, formation of C1′• is observed from photo-excitation of G•+ in the 310–480 nm range with no C1′• formation observed ≥520 nm. Illumination of guanine radical cations in 2′dG, 3′-dGMP and 5′-dGMP in aqueous LiCl glasses at 143 K is found to result in remarkably high yields (∼85–95%) of sugar radicals, namely C1′•, C3′• and C5′•. The amount of each of the sugar radicals formed varies dramatically with compound structure and temperature of illumination. Radical assignments were confirmed using selective deuteration at C5′ or C3′ in 2′-dG and at C8 in all the guanine nucleosides/tides. Studies of the effect of temperature, pH, and wavelength of excitation provide important information about the mechanism of formation of these sugar radicals. Time-dependent density functional theory calculations verify that specific excited states in G•+ show considerable hole delocalization into the sugar structure, in accord with our proposed mechanism of action, namely deprotonation from the sugar moiety of the excited molecular radical cation.

Effect of base stacking on the acid-base properties of the adenine cation radical [A*+] in solution: ESR and DFT studies.

In this study, the acid-base properties of the adenine cation radical are investigated by means of experiment and theory. Adenine cation radical (A*(+)) is produced by one-electron oxidation of dAdo and of the stacked DNA-oligomer (dA)6 by Cl2*(-) in aqueous glass (7.5 M LiCl in H2O and in D2O) and investigated by ESR spectroscopy. Theoretical calculations and deuterium substitution at C8-H and N6-H in dAdo aid in our assignments of structure. We find the pKa value of A*(+) in this system to be ca. 8 at 150 K in seeming contradiction to the accepted value of < or = 1 at ambient temperature. However, upon thermal annealing to > or = 160 K, complete deprotonation of A*(+) occurs in dAdo in these glassy systems even at pH ca. 3. A*(+) found in (dA)6 at 150 K also deprotonates on thermal annealing. The stability of A*(+) at 150 K in these systems is attributed to charge delocalization between stacked bases. Theoretical calculations at various levels (DFT B3LYP/6-31G*, MPWB95, and HF-MP2) predict binding energies for the adenine stacked dimer cation radical of 12 to 16 kcal/mol. Further DFT B3LYP/6-31G* calculations predict that, in aqueous solution, monomeric A*(+) should deprotonate spontaneously (a predicted pKa of ca. -0.3 for A*(+)). However, the charge resonance stabilized dimer AA*(+) is predicted to result in a significant barrier to deprotonation and a calculated pKa of ca. 7 for the AA*(+) dimer which is 7 pH units higher than the monomer. These theoretical and experimental results suggest that A*(+) isolated in solution and A*(+) in adenine stacks have highly differing acid-base properties resulting from the stabilization induced by hole delocalization within adenine stacks.

C5′- and C3′-sugar radicals produced via photo-excitation of one-electron oxidized adenine in 2′-deoxyadenosine and its derivatives

We report that photo-excitation of one-electron-oxidized adenine [A(-H)•] in dAdo and its 2′-deoxyribonucleotides leads to formation of deoxyribose sugar radicals in remarkably high yields. Illumination of A(-H)• in dAdo, 3′-dAMP and 5′-dAMP in aqueous glasses at 143 K leads to 80-100% conversion to sugar radicals at C5′ and C3′. The position of the phosphate in 5′- and 3′-dAMP is observed to deactivate radical formation at the site of substitution. In addition, the pH has a crucial influence on the site of sugar radical formation; e.g. at pH ∼5, photo-excitation of A(-H)• in dAdo at 143 K produces mainly C5′• whereas only C3′• is observed at high pH ∼12. 13C substitution at C5′ in dAdo yields 13C anisotropic couplings of (28, 28, 84) G whose isotropic component 46.7 G identifies formation of the near planar C5′•. A β-13C 16 G isotropic coupling from C3′• is also found. These results are found to be in accord with theoretically calculated 13C couplings at C5′ [DFT, B3LYP, 6-31(G) level] for C5′• and C3′•. Calculations using time-dependent density functional theory [TD-DFT B3LYP, 6-31G(d)] confirm that transitions in the near UV and visible induce hole transfer from the base radical to the sugar group leading to sugar radical formation.

Photo-induced Hole Transfer from Base to Sugar in DNA: Relationship to Primary Radiation Damage

Abstract Adhikary, A., Kumar, A. and Sevilla, M. D. Photo-induced Hole Transfer from Base to Sugar in DNA: Relationship to Primary Radiation Damage. Radiat. Res. 165, 479–484 (2006). This work presents the hypothesis that photo-excitation of G·+ in DNA and model systems results in the same electronic states expected from direct ionization of the sugar phosphate backbone and that these states lead to specific sugar radicals on the DNA sugar phosphate backbone. As evidence we show that visible photo-excitation of guanine cation radicals (G·+) in the dinucleoside phosphate TpdG results in high yields (about 85%) of deoxyribose sugar radicals at the C1′ and C3′ sites. Further, we have calculated transition energies of hole transfer from G·+ in TpdG using time-dependent density functional theory (TD-DFT) at the B3LYP/6–31G(d) level in gas phase as well as in a solvated environment. These calculations clearly predict that visible excitation of G·+ in TpdG causes transitions from only inner-shell filled molecular orbitals (MOs) to the singly occupied molecular orbital (SOMO) that effectively result in hole transfer from guanine either to the sugar phosphate backbone or to the adjacent base, thymine. The hole transfer is followed by rapid deprotonation from the sugar to form C1′ and C3′ radicals. These experimental and theoretical results are in agreement with our previously published experimental and theoretical results that photo-excitation of G·+ results in high yields of deoxyribose sugar radicals in DNA, guanine deoxyribonucleosides and deoxyribonucleotides. Photo-excitation of G·+ therefore provides a convenient method to produce and study sugar radicals that are expected to be formed in γ-irradiated DNA systems unencumbered by the many other pathways involved in direct ionization.

Prototropic equilibria in DNA containing one-electron oxidized GC: intra-duplex vs. duplex to solvent deprotonation

By use of ESR and UV-vis spectral studies, this work identifies the protonation states of one-electron oxidized G:C (viz. G˙+:C, G(N1–H)˙:C(+H+), G(N1–H)˙:C, and G(N2-H)˙:C) in a DNA oligomer d[TGCGCGCA]2. Benchmark ESR and UV-vis spectra from one electron oxidized 1-Me-dGuo are employed to analyze the spectral data obtained in one-electron oxidized d[TGCGCGCA]2 at various pHs. At pH ≥7, the initial site of deprotonation of one-electron oxidized d[TGCGCGCA]2 to the surrounding solvent is found to be at N1 forming G(N1–H)˙:C at 155 K. However, upon annealing to 175 K, the site of deprotonation to the solvent shifts to an equilibrium mixture of G(N1–H)˙:C and G(N2–H)˙:C. For the first time, the presence of G(N2–H)˙:C in a ds DNA-oligomer is shown to be easily distinguished from the other prototropic forms, owing to its readily observable nitrogen hyperfine coupling (Azz(N2) = 16 G). In addition, for the oligomer in H2O, an additional 8 G N2–H proton HFCC is found. This ESR identification is supported by a UV-vis absorption at 630 nm which is characteristic for G(N2–H)˙ in model compounds and oligomers. We find that the extent of photo-conversion to the C1′ sugar radical (C1′˙) in the one-electron oxidized d[TGCGCGCA]2 allows for a clear distinction among the various G:C protonation states which can not be easily distinguished by ESR or UV-vis spectroscopies with this order for the extent of photo-conversion: G˙+:C > G(N1–H)˙:C(+H+) ≫ G(N1–H)˙:C. We propose that it is the G˙+:C form that undergoes deprotonation at the sugar and this requires reprotonation of G within the lifetime of exited state

Highly Oxidizing Excited States of One-Electron-Oxidized Guanine in DNA: Wavelength and pH Dependence

Excited states of one-electron-oxidized guanine in DNA are known to induce hole transfer to the sugar moiety and on deprotonation result in neutral sugar radicals that are precursors of DNA strand breaks. This work carried out in a homogeneous aqueous glass (7.5 M LiCl) at low temperatures (77-175 K) shows the extent of photoconversion of one-electron-oxidized guanine and the associated yields of individual sugar radicals are crucially controlled by the photon energy, protonation state, and strandedness of the oligomer. In addition to sugar radical formation, highly oxidizing excited states of one-electron-oxidized guanine are produced with 405 nm light at pH 5 and below that are able to oxidize chloride ion in the surrounding solution to form Cl(2)(•-) via an excited-state hole transfer process. Among the various DNA model systems studied in this work, the maximum amount of Cl(2)(•-) is produced with ds (double-stranded) DNA, where the one-electron-oxidized guanine exists in its cation radical form (G(•+):C). Thus, via excited-state hole transfer, the dsDNA is apparently able to protect itself from cation radical excited states by transfer of damage to the surrounding environment.

Formation of S-Cl phosphorothioate adduct radicals in dsDNA-S-oligomers: Hole transfer to guanine vs. disulfide anion radical formation

In phosphorothioate-containing dsDNA oligomers (S-oligomers), one of the two nonbridging oxygen atoms in the phosphate moiety of the sugar-phosphate backbone is replaced by sulfur. In this work, electron spin resonance (ESR) studies of one-electron oxidation of several S-oligomers by Cl2(•-) at low temperatures are performed. Electrophilic addition of Cl2(•-) to phosphorothioate with elimination of Cl(-) leads to the formation of a two-center three-electron σ(2)σ*(1)-bonded adduct radical (-P-S-̇Cl). In AT S-oligomers with multiple phosphorothioates, i.e., d[ATATAsTsAsT]2, -P-S-̇Cl reacts with a neighboring phosphorothioate to form the σ(2)σ*(1)-bonded disulfide anion radical ([-P-S-̇S-P-](-)). With AT S-oligomers with a single phosphorothioate, i.e., d[ATTTAsAAT]2, reduced levels of conversion of -P-S-̇Cl to [-P-S-̇S-P-](-) are found. For guanine-containing S-oligomers containing one phosphorothioate, -P-S-̇Cl results in one-electron oxidation of guanine base but not of A, C, or T, thereby leading to selective hole transfer to G. The redox potential of -P-S-̇Cl is thus higher than that of G but is lower than those of A, C, and T. Spectral assignments to -P-S-̇Cl and [-P-S-̇S-P-](-) are based on reaction of Cl2(•-) with the model compound diisopropyl phosphorothioate. The results found for d[TGCGsCsGCGCA]2 suggest that [-P-S-̇S-P-](-) undergoes electron transfer to the one-electron-oxidized G, healing the base but producing a cyclic disulfide-bonded backbone with a substantial bond strength (50 kcal/mol). Formation of -P-S-̇Cl and its conversion to [-P-S-̇S-P-](-) are found to be unaffected by O2, and this is supported by the theoretically calculated electron affinities and reduction potentials of [-P-S-S-P-] and O2.

Kr-86 Ion-Beam Irradiation of Hydrated DNA: Free Radical and Unaltered Base Yields

This work reports an ESR and product analysis investigation of Kr-86 ion-beam irradiation of hydrated DNA at 77 K. The irradiation results in the formation and trapping of both base radicals and sugar phosphate radicals (DNA backbone radicals). The absolute yields (G, μmol/J) of the base radicals are smaller than the yields found in similarly prepared γ-irradiated DNA samples, and the relative yields of backbone radicals relative to base radicals are much higher than that found in γ-irradiated samples. From these results, we have elaborated our radiation chemical model of the track structure for ion-beam irradiated DNA as it applies to krypton ion-beams. The base radicals, which are trapped as ion radicals or reversibly protonated or deprotonated ion radicals, are formed almost entirely in the track penumbra, a region in which radiation chemical effects are similar to those found in γ-irradiated samples. By comparing the yields of base radicals in ion-beam samples to the yields of the same radicals in γ-irradiated samples, the partition of energy between the low-LET region (penumbra) and the core is experimentally determined. The neutral sugar and other backbone radicals, which are not as susceptible to recombination as are ion radicals, are formed largely in the track core. The backbone radicals show a linear dose response up to very high doses. Unaltered base release yields in Kr-86 irradiated hydrated DNA are equal to sugar radical yields within experimental error limits, consistent with radiation-chemical processes in which all base release originates with sugar radicals. Two phosphorus-centered radicals from fragmentation of the DNA backbone are found in low yields.

Do Solvated Electrons (eaq–) Reduce DNA Bases? A Gaussian 4 and Density Functional Theory-Molecular Dynamics Study

The solvated electron (e(aq)⁻) is a primary intermediate after an ionization event that produces reductive DNA damage. Accurate standard redox potentials (E(o)) of nucleobases and of e(aq)⁻ determine the extent of reaction of e(aq)⁻ with nucleobases. In this work, E(o) values of e(aq)⁻ and of nucleobases have been calculated employing the accurate ab initio Gaussian 4 theory including the polarizable continuum model (PCM). The Gaussian 4-calculated E(o) of e(aq)⁻ (-2.86 V) is in excellent agreement with the experimental one (-2.87 V). The Gaussian 4-calculated E(o) of nucleobases in dimethylformamide (DMF) lie in the range (-2.36 V to -2.86 V); they are in reasonable agreement with the experimental E(o) in DMF and have a mean unsigned error (MUE) = 0.22 V. However, inclusion of specific water molecules reduces this error significantly (MUE = 0.07). With the use of a model of e(aq)⁻ nucleobase complex with six water molecules, the reaction of e(aq)⁻ with the adjacent nucleobase is investigated using approximate ab initio molecular dynamics (MD) simulations including PCM. Our MD simulations show that e(aq)⁻ transfers to uracil, thymine, cytosine, and adenine, within 10 to 120 fs and e(aq)⁻ reacts with guanine only when a water molecule forms a hydrogen bond to O6 of guanine which stabilizes the anion radical.

One-electron oxidation of gemcitabine and analogs: mechanism of formation of C3' and C2' sugar radicals.

Gemcitabine is a modified cytidine analog having two fluorine atoms at the 2′-position of the ribose ring. It has been proposed that gemcitabine inhibits RNR activity by producing a C3′• intermediate via direct H3′-atom abstraction followed by loss of HF to yield a C2′• with 3′-keto moiety. Direct detection of C3′• and C2′• during RNR inactivation by gemcitabine still remains elusive. To test the influence of 2′- substitution on radical site formation, electron spin resonance (ESR) studies are carried out on one-electron oxidized gemcitabine and other 2′-modified analogs, i.e., 2′-deoxy-2′-fluoro-2′-C-methylcytidine (MeFdC) and 2′-fluoro-2′-deoxycytidine (2′-FdC). ESR line components from two anisotropic β-2′-F-atom hyperfine couplings identify the C3′• formation in one-electron oxidized gemcitabine, but no further reaction to C2′• is found. One-electron oxidized 2′-FdC is unreactive toward C3′• or C2′• formation. In one-electron oxidized MeFdC, ESR studies show C2′• production presumably from a very unstable C3′• precursor. The experimentally observed hyperfine couplings for C2′• and C3′• match well with the theoretically predicted ones. C3′• to C2′• conversion in one-electron oxidized gemcitabine and MeFdC has theoretically been modeled by first considering the C3′• and H3O+ formation via H3′-proton deprotonation and the subsequent C2′• formation via HF loss induced by this proximate H3O+. Theoretical calculations show that in gemcitabine, C3′• to C2′• conversion in the presence of a proximate H3O+ has a barrier in agreement with the experimentally observed lack of C3′• to C2′• conversion. In contrast, in MeFdC, the loss of HF from C3′• in the presence of a proximate H3O+ is barrierless resulting in C2′• formation which agrees with the experimentally observed rapid C2′• formation.