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Single Strand Break in DNA Coupled to the O—P Bond Cleavage. A Computational Study Janusz Rak, Monika Kobyzecka, and Piotr Storoniak* Department of Chemistry, University of Gda nsk, Sobieskiego 18, 80-952 Gdansk, Poland
bS Supporting Information ABSTRACT:
In the present study, we consider the formation of a single strand break (SSB) in DNA via an alternative mechanism involving O—P bond splitting that was observed as a minor route to DNA damage induced by low-energy electrons (LEEs) or γ radiation. We postulate and characterize, at the B3LYP/6-31þþG** level, a path that starts with LEE attachment to the nucleotide of thymine resulting in a stable valence radical anion localized on pyrimidine. In the next step, a proton is attached to the C5 position of thymine, producing a neutral monohydroradical of this nucleotide. This event triggers the subsequent intramolecular transfer of a sugar hydrogen atom from C30 or C50 to the C6 site of thymine. In the final elemental reaction, O—P bond dissociation takes place, which yields the phosphoryl radical and a cyclic ketone or aldehyde. Identification of the latter species as well as 5,6-dihydropyrimidines in DNA solutions irradiated with ionizing radiation could provide experimental confirmation of the suggested mechanism.
’ INTRODUCTION In 2000, the Canadian group of Leon Sanche published results that unequivocally demonstrated that under an ultrahigh vacuum, low-energy electrons (LEEs) with energies above 4 eV are able to induce single (SSBs) and double (DSBs) strand breaks in the plasmid DNA deposited on a tantalum substrate.1 Later on, with the advent of higher-resolution equipment, the same group demonstrated that electrons with a nominal energy of ∼0 eV gave rise to SSBs when interacting with DNA and that the damage yield peaks around 0.8 eV with values similar to the maximum observed for the higher-energy region of incident electrons, that is, for energies from the 8-10 eV range.2 So far, several proposals explaining this amazing phenomenon have been published, in which electrons of 0 eV trigger the dissociation of the phosphodiester bond of 3 eV. One of these hypotheses, published by Simons et al.,3 assumes that an excess electron that localizes first as a π-shaped resonance anion on a pyrimidine nucleobase is, in turn, transferred to the C30 -O σ* orbital. The latter process leads directly to the dissociation of the phosphodiester bond, that is, to the formation of a single strand break.3 The same authors also examined the phosphate—sugar C—O bond breakage resulting from electron attachment directly to the phosphate PdO π* orbital.4 They found that electrons r 2011 American Chemical Society
with energies of 2-3 eV can attach to the PdO π* orbital and induce C—O cleavage at this site, with metastable π* anions as intermediates. However, as the bond cleavage rates in the PdO π* anions were calculated to be slow, damage involving direct electron capture by phosphate does not seem to be competitive with electron autodetachment and, therefore, is unlikely to be the main path of the DNA backbone damage.4 A similar mechanism based on the assumption that an excess electron localizes on the π* orbital of the PdO bond (forming the corresponding resonance anion) and then moves to the CX0 —O π*orbital was modeled by Sevilla et al.5 The mechanisms published by the Illenberger group6-8 also belong to the category of “resonance mechanisms”; these authors assumed that SSBs are generated in DNA as a result of its interactions with hydrogen atoms released from the resonance anions of nucleobases during the so-called dissociative electron attachment (DEA) process. The resonance nature of SSB formation is suggested by the shape of the curve, reflecting the damage yield versus electron Received: November 19, 2010 Revised: January 5, 2011 Published: February 4, 2011 1911
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The Journal of Physical Chemistry B energy (at certain electron energies, maxima are observed, whereas at other energies minima, are measured).9 It is, however, worth noticing that if resonance anions were directly responsible for the observed strand breaks, the chemical reactions (leading to breaks) would have to compete with the extremely fast electron autodetachment process (k = 10(14-15) s-1).10 Moreover, if the activation barriers associated with the cleavage of the stable anion were relatively low (less than 20-23 kcal/mol), the plot of SSB yield versus incident electron energy should have a shape reflecting the cross section for resonance anion formation because, in this case, the electron attachment efficiency would directly affect the strand break yield. The shape of the curve reflecting the damage yield versus electron kinetic energy does reflect the described above behavior.9 Indeed, the first sharp peak near 1 eV corresponds to the nucleobases LUMO energies,11 while the next feature, near 2-4 eV, corresponds to their LUMOþ1 and LUMOþ2 energies,11 suggesting, thus, that π-shape resonances are responsible for the initial attachment of an electron. Note that if stable anions were eventually formed in the situation described above, the limitation concerning the fast electron autodetachment process would not apply as the lifetimes of electronically bound anions are essentially unlimited (at least at 0 K).12 The first mechanism of LEE-induced SSB formation in DNA based on electronically stable anions was published by Simon’s group.3,4,13 Although in the gas phase the cleavage of the C—O bond begins from the metastable anions of pyrimidines nucleotides, the solvated anions of these nucleotides were found to be adiabatically stable.3,4,13 Another mechanism, involving a stable anion of nucleotide, was published in 2005 by Da)bkowska et al.14 The attachment of an electron to cytosine, leading to the formation of the adiabatically stable anion radical of 30 -monophosphate-20 -deoxycytidine (30 -dCMP), is the very first step in this two-electron mechanistic proposal. Next, the anion radical is protonated at the C5 position of cytosine, which yields a neutral monohydroradical of the nucleotide. Then, a second electron enters the monohydroradical, which triggers low-energy-barrier hydrogen atom transfer from the C30 position of 20 -deoxyribose to the C6 atom of cytosine. The latter process is coupled to the barrier-free dissociation of the C30 —O bond, which ends the reaction sequence. The scenario mentioned above is not a probable mechanism for the bombardment with LEEs carried out within the linear regime of the dose response curve. It may, however, be operative in aqueous solutions of DNA irradiated with ionizing radiation because then, the concentration of solvated electrons in the so-called “spur” is significant.15 A year later, two papers were published by Leszczynski’s group,16,17 who suggested the involvement of the adiabatically stable anions of pyrimidine nucleotides in SSB formation. These researchers demonstrated that both the 30 - and 50 -monophosphates of these nucleosides support radical anions exhibiting a substantial electron adiabatic affinity (0.34-0.56 eV), and once formed, these anions can dissociate into the experimentally observed SSBs, surmounting only a small kinetic barrier of 4.4-12.8 kcal/mol.16,17 To this end, it is worth emphasizing that the existence of adiabatically stable anions of nucleotides has been confirmed in several computational and experimental studies.18-22 Independently of the reports published by Leszczynski and co-workers, the theoretical investigations of Schaefer and co-workers.18 revealed that 30 -dCMP is able to capture near 0 eV electrons, forming a radical anion that has a lower energy than the corresponding neutral species in both the gaseous phase and aqueous solution, and that the excess electron density in this anion is
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localized primarily on the nucleobase. Furthermore, the electronaccepting properties of thymine in the 50 -dTMP 3 3 3 adenine pair were theoretically investigated by the same group.19 The adiabatic electron affinity of the above system was predicted to be positive, with the excess electron localized on the thymine moiety. Similarly, the computational studies by Rak and co-workers on 50 monophosphate-20 -deoxyadenosine (50 -dAMP) in four representative conformations demonstrated the formation of its adiabatically stable valence anions,20 and the existence of these anions in the gaseous phase was confirmed experimentally by Bowen et al. in photoelectron spectroscopy studies.21 Moreover, Schaefer et al. studied the electron capture ability of the 30 ,50 -diphosphate of 20 deoxynucleosides and found that their gaseous-phase adiabatic electron affinities (AEAs) diminish in the following order: (30 ,50 dTDP) > (30 ,50 -dCDP) > (30 ,50 -dGDP) > (30 ,50 -dADP).22 Finally, the inclusion of solvent effects suggests that the radical anions of nucleotides are substantially stabilized in aqueous solution.16,18,19,22 In the above mechanisms of SSB formation, the attachment of an excess electron to the nucleobase or phosphate ends up with the dissociation of the CX—O (where X = 30 or 50 ) bond, leading to a sugar radical and a terminal phosphate anion. Another possible route to SSBs is the splitting of the O—P bond. Indeed, O—P cleavage has been reported in ESR studies on argon ions irradiated and γ-irradiated hydrated DNA.23,24 By the same token, studies on LEE-induced damage in model oligonucleotides25,26 suggested the cleavage of the O—P bond as a minor route to the formation of single strand breaks in DNA. To the best of our knowledge, O—P cleavage leading to SSBs in DNA has not yet been explored using molecular modeling. In order to fill this gap, we postulate, and characterize at the B3LYP level, a mechanism in which SSB formation is coupled to O—P bond splitting. Our proposal, demonstrated for the electroninduced degradation of the 30 -dTMP and 50 -dTMP, starts with LEE attachment to a nucleotide leading to the adiabatically stable radical valence anion localized on thymine. In the next step, a proton is attached to the C5 position of thymine, producing a neutral monohydroradical. The latter process makes the transfer of a sugar hydrogen to the C6 site of thymine possible. Finally, O—P bond dissociation takes place, yielding the phosphoryl radical and a cyclic ketone or an aldehyde sugar derivative of 30 dTMP and 50 -dTMP, respectively. The possible usefulness of these compounds in the identification of O—P dissociative damage, as well as instances where the mechanism postulated here could be a major route to DNA breaks, is briefly discussed in the last section of the paper.
’ METHODS We applied primarily the density functional theory method with Becke’s three-parameter hybrid functional (B3LYP)27-29 and the 6-31þþG** basis set.30,31 The ability of the B3LYP method to reliably predict the stability of valence-type molecular anions32 as well as reaction energetics has been assessed in the past. Moreover, in a series of our own studies employing a combination of anion photoelectron spectroscopy with molecular modeling, we confirmed the usefulness of the B3LYP/631þþG** approach for reproducing the energetics of the anionic nucleotide of adenine as well as the anionic complexes between nucleobases and various proton donors like amino acids, inorganic acids, alcohols, formic acid, and other nucleobases.33 All geometries were fully optimized without geometrical constraints, and the analysis of harmonic frequencies proved 1912
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The Journal of Physical Chemistry B that all of them are either geometrically stable (all force constants positive) or first-order saddle points (all but one force constant positive). In order to verify that the TS geometries obtained connect the correct reactants, the IRC calculations34,35 were performed for every saddle point structure. The energies of stationary geometries are expressed in terms of the electronic energy E. In addition to the electronic energy, we calculated the free energy G by correcting the values of E for zero-point vibrational terms, thermal contributions to energy, the pV term, and the entropy term. These terms were calculated in the rigid rotor-harmonic oscillator approximation for T = 298 K and p = 1 atm. All of the quantum chemical calculations were carried out with the GAUSSIAN0336 code, and the images of molecular orbitals were plotted with the GaussView 5 package.37
’ RESULTS AND DISCUSSION Our mechanistic proposal, which corresponds to a reaction sequence starting from an electron attachment to the nucleotide of thymine and ending with O—P bond breakage, is based, among other premises, on the fact that abstraction of any hydrogen atom from the sugar moiety in DNA results in a strand break. Indeed, it was demonstrated in the excellent review by Pogozelski and Tullius38 that at least one pathway leading to a strand break is associated with a hydrogen atom abstraction event concerning each of the sugar hydrogens. Therefore, the main assumption of the current studies is that electron attachment to a nucleobase makes possible the transfer of a hydrogen atom between the sugar and that nucleobase. Furthermore, we assumed that the strand break is generated within this nucleotide, which initially binds an electron. Consistent with theoretical16,17,22 and experimental25,26 studies demonstrating that nucleobases (especially thymine)39 are likely to be the prime target for incoming electrons, we propose
Figure 1. Singly occupied molecular orbital of the 1-methylthymine radical anion plotted with a contour value of 0.02 bohr-3/2.
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that the adiabatically stable valence anion of thymine is formed at the very first step of the modeled damage. The electron capture ability and, therefore, the tendency of thymine to form stable anionic states in the DNA context is reflected by the positive gasphase adiabatic electron affinities (AEA) of its nucleotides. Indeed, the B3LYP/DZPþþ AEAs of 20 -deoxythymidine monophosphates, 30 -dTMP and 50 -dTMP, were calculated to be 0.56 and 0.44 eV, respectively,16,17 while that of 20 -deoxythymidine30 ,50 -diphosphate22 was 0.52 eV. Moreover, our own computational studies demonstrate that virtually all double-stranded DNA sequences should support the adiabatically stable thymine anions.40-42 The excess charge is localized mainly on the C5 and C6 atoms (see Figure 1) of the thymine valence anion. Taking this fact into account, as well as the geometrical arrangement in thymine nucleotides (of all of the atoms of thymine, C5 and C6 are the closest centers to the sugar), C5 or C6 should play the role of H atom acceptor in this hydrogen atom transfer. At the last stage of the reaction sequence under study, the O—P bond is cleaved at the C30 or C50 end of the nucleotide. Therefore, hydrogen atom transfers that precede the above-mentioned cleavage should be those involving the C30 or C50 centers. The transfer of the H atom from C40 to the C5/C6 center of thymine is not favored for steric reasons. In order to examine the energetics of hydrogen atom transfer between the sugar unit and the negatively charged nucleobase, we first performed calculations for the isolated reagents (see Scheme 1). In order to model the DNA backbone, we modified 20 -deoxyribose with phosphate residues at positions 30 and 50 . We also replaced the N-glycosidic sugar-base bond with the —NH2 group. In turn, the thymine radical anion at N1 was substituted with a methyl group as this atom takes part in the N-glycosidic bond (1-methylthymine is denoted by 1-MT). In addition to the C30 and C50 sugar atoms, we also took C20 into consideration as a potential hydrogen-atom-donating center. From the thermodynamic data that describe H atom transfer from 1-amino-30 ,50 diphosphate-20 -deoxyribose to positions C5 and C6 of 1-MT, it is evident that the investigated processes are improbable. The lowest barrier of 22.7 kcal/mol was found for H atom transfer from the C30 atom of the sugar to position C6 in pyrimidine (see Table 1), and the remaining data span the range from 23.9 to 55.5 kcal/mol on the free-energy scale. The calculated thermodynamic characteristics are so unfavorable that even the deficiencies of our approach, arising out of the simplistic molecular model and approximate computational treatment, cannot be blamed for the above conclusion. However, it is worth emphasizing that in the cell, DNA interacts with a number of molecular components, like proteins (histones, enzymes) or hydrated metal cations. As far as interactions
Scheme 1. Hydrogen Atom Transfer from Position C30 of 1-Amino-30 ,50 -diphosphate-20 -deoxyribose to the C6 Site of the 1-Methylthymine Radical Anion
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between proteins and DNA are concerned, contacts between amino acid side chains and nucleobases seem to be of crucial importance. The most potent proton donors are amino acids which possess protonated side chains under physiological conditions, that is, arginine and lysine. Out of 576 contacts between thymine and side chains of amino acids described in an amino acid-nucleotide database, comprising 1213 crystallographic structures of protein-nucleic acid complexes, 163 and 119 fall to arginine and lysine, respectively.43 The above numbers suggest, thus, a significant probability of proton transfer from the protonated arginine or lysine if the radical anion of thymine is formed in a protein-DNA complex. Finally, protons in the DNA surrounding may appear due to the interaction of ionizing radiation with water present in DNA hydration layer, Table 1. Thermodynamic Data for Hydrogen Atom Transfer from X (X = 20 , 30 , and 50 ) Centers of 1-Amino-30 ,50 -diphosphate-20 -deoxyribose to Positions C5 and C6 of the 1-Methylthymine (1-MT) Radical Anion, Calculated at the B3LYP/6-31þþG** Level (all values in kcal/mol) X
ΔEr
ΔHr
ΔGr
H Atom Transfer to C5(1-MT•-) 0
59.8
58.2
55.5
30
46.3
45.3
47.2
50
49.4
48.2
2
48.3 •-
H Atom Transfer to C6(1-MT ) 20
35.5
34.1
31.1
30
22.0
21.1
22.7
50
25.1
24.0
23.9
Table 2. Thermodynamic Data for Hydrogen Atom Transfer from X (X = 20 , 30 , and 50 ) Centers of 1-Amino-30 ,50 -diphosphate-20 -deoxyribose to Positions C5 and C6 of Monohydroradicals 1-MTHC6 and 1-MTHC5, Calculated at the B3LYP/6-31þþG** Level (all values in kcal/mol) X
ΔEr
ΔHr
ΔGr
H Atom Transfer to C5(1-MTHC6) 0
20.4
19.8
30
6.9
6.9
9.8
50
10.0
9.8
10.9
2
18.1
H Atom Transfer to C6(1-MTHC5) 0
17.3
16.8
14.0
30
3.9
3.8
5.6
50
7.0
6.7
6.8
2
H2O f H2O•þ þ e- f OH• þ Hþ þ e-.44 To indicate the probable sites of protonation, we calculated proton affinities (PA) for all centers in the anionic 1-MT (see Table S1, Supporting Information). It can be seen that the strongest proton-accepting tendency is demonstrated by the C5 and C6 atoms, for which the calculated PA values amount to ∼349 and 352 kcal/mol, respectively. The proton affinities of C5 and C6 are at least 10 kcal/mol higher than those of the other centers; therefore, only the two most stable monohydroradicals, denoted by 1-MTHC5 and 1-MTHC6, are likely to appear following protonation of the anionic 1-MT. In contrast to the 1-MT valence anion, its monohydroradical derivatives readily accept hydrogen atoms derived from the sugar moiety. Table 2 shows the thermodynamic quantities characterizing hydrogen atom transfer from 1-amino-30 ,50 -diphosphate-20 deoxyribose to the 1-MTHC5 and 1-MTHC6 monohydroradicals. The energy barriers for H atom transfer in sugar/monohydroradical systems are substantially lower than the corresponding barriers in sugar/radical anion systems (cf. Table 1 with Table 2). For example, the lowest barrier for sugar/radical anion systems (the 30 -H atom transfer to the C6 position of the 1-MT anion) amounts to 22.7 kcal/mol (see Table 1), while the analogous process for H transfer to 1-MTHC5 involves a barrier of only 5.6 kcal/mol (see Table 2). Inspection of the data in Table 2 shows that the latter process (Scheme 2) is the most probable one among the reactions considered. It will also be noticed that H atom transfer from C30 or C50 is accompanied by a significantly lower barrier than that from C20 . Thus, protonation of the valence anion of thymine seems to facilitate intramolecular hydrogen transfer. Although thermodynamic barriers of ∼6 kcal/mol are still rather high, they may have been overestimated by our simplified molecular model. In order to obtain more realistic data, we assumed two nucleotides, 30 -dTMP and 50 -dTMP (Scheme 3), to be the ultimate molecular model. The former molecule enabled SSB formation at the 30 site and the latter SSB formation at the 50 site to be studied. Because the results obtained at the preliminary stage of modeling (see above) showed that the thymine anion must be protonated before H atom transfer from the sugar moiety is possible, we assumed that SSBs form within the respective nucleotide monohydroradicals. The latter species are formed as a consequence of the protonation of the C5 position of thymine in the anion radical of a nucleotide. An alternative route to such monohydroradicals could be the attachment of hydrogen atoms, produced in DNA by LEE during so-called dissociative attachment (DEA),45 to thymine in the respective nucleotides. Thus, intramolecular transfer of the H atom can occur within a given nucleotide from the C30 or C50 sugar site to the C6 atom of thymine, as depicted by the 30 RI and 50 RI reactions in Scheme 3.
Scheme 2. Hydrogen Atom Transfer from Position C30 of 1-Amino-30 ,50 -diphosphate-20 -deoxyribose to the C6 Site of the 1-Methylthymine Neutral Radical Resulting from the Hydrogenation of the Radical Anion at C5
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Scheme 3. Proposed Mechanism of the Low-Energy Electron-Induced Single Strand Break in the Thymidine Nucleotide Modeled by 30 - and 50 -Monophosphate of 20 -Deoxythymidine Monohydroradicals (pathways labeled as 30 R and 50 R, respectively)
Such processes are driven by the formation of the stable product of hydrogenation of the double C5dC6 bond in thymine. Indeed, 5,6-dihydropyrimidines have been detected as the products of the reaction of ionizing radiation-produced electrons with DNA in an aqueous environment.44 Following the conclusions from the previous calculations (see above) where we made use of isolated compounds, pathways involving H atom transfer from C20 were neglected because of their unfavorably high thermodynamic barriers (see Table 2). Hydrogen atom transfer between the sugar residue and the monohydroradical of thymine may be followed by dissociation of the O—P bond (see Scheme 3, steps 30 RII and 50 RII), which closes the reaction sequence under consideration and produces a strand break. Once a radical is localized at the C30 or C50 sugar sites (due to H transfer from those atoms to the C6 site of the thymine monohydroradical), O—P bond dissociation is accompanied by the formation of a cyclic ketone or aldehyde (see reactions 30 RII and 50 RII in Scheme 3). Here, the question arises why CX—O bond dissociation is not favored instead. We have examined this possibility by elongating the CX—O bond by 1 Å; this led to a monotonic increase in the system’s energy of up to 60 kcal/mol. The explanation of this fact may be as follows: elongation of the CX—O bond is coupled to its homolytic dissociation, which results in a high-energy open-shell singlet localized on the sugar and a doublet localized on the phosphate unit. On the other hand, O—P bond dissociation yields the carbonyl group instead of the above-mentioned high-energy open-shell singlet. Depending on which O—P bond is broken, from the 30 - or 50 -side, the carbonyl group becomes part of the cyclic ketone or aldehyde (see Scheme 3). The B3LYP geometries of the stationary points localized on the 30 R and 50 R reaction pathways (see Scheme 3) are shown in Figure 2. Each sequence comprises two reactions, intramolecular
hydrogen transfer and O—P dissociation. The subscripts S, P, and TS used in the names of the individual structures in Figure 2 refer to the respective geometries of the substrate, product, and transition state. The thermodynamic and kinetic characteristics of the elemental reactions (Scheme 3) are summarized in Table 3. Inspection of these data reveals that H atom transfers from C30 or C50 to the thymine monohydroradical are associated with almost identical thermodynamic stimuli; the free energies for 30 RI and 50 RI amount to 3.7 and 3.6 kcal/mol, respectively. Moreover, these values are lower than the corresponding ΔG's calculated for the isolated reagents (5.6 and 6.8 kcal/mol, respectively; see Table 2). Because a nucleotide makes a better model of DNA than the isolated sugar and nucleobase, one can conclude that the DNA environment probably facilitates intramolecular H atom transfer; nevertheless, the calculated thermodynamic barriers of 3-4 kcal/mol (see Table 3) are still substantial. The accuracy of the B3LYP method is within the same range, however. Besides, a single nucleotide is quite a crude approximation of the whole, complex DNA molecule, and one can expect that inclusion of the effects originating from DNA into the computational model could lower the calculated free energies. The second and last stage of the mechanism, XRII (X = 30 , 50 ), depicted in Scheme 3, is the O—P bond break. For reaction 30 RII, the thermodynamic stimulus was found to be negative (-1.2 kcal/mol; see Table 3). Reaction 50 RII, in turn, is connected with a thermodynamic barrier equal to 4.9 kcal/ mol, which is slightly higher than the one predicted for the preceding process (50 RI). These data suggest, therefore, that SSBs, if they occur via this mechanism, should develop at position 30 rather than 50 . Table 3 lists the kinetic barriers for the four elemental reactions involved in the assumed mechanism (see Scheme 3). Their values, ranging from 17.7 to 26.8 kcal/mol (see Table 3), 1915
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CX0 —O bond splitting in the anionic nucleotide of thymine ranges from only 4 to 12 kcal/mol while the free energy of the dissociation process varies from -23 to -24 kcal/mol.16,17 However, under physiological conditions, where DNA interacts with proteins and other molecules having proton-donor properties, a rapid protonation of the pyrimidine anion may prevent electron transfer to the phosphate group and open the reaction channel proposed here that leads to the dissociation of the O—P bond.
Figure 2. Optimized structures appearing on the 30 R and 50 R pathways. The substrates (20 -deoxythymidine-30 -monophosphate and the 20 -deoxythymidine-50 -monophosphate monohydroradicals formed from the anion radicals as a result of C5 protonation) are labeled with subscripts S. The transition states occurring during intramolecular H atom transfer are labeled with subscripts TS. The products of the corresponding O—P bond cleavage are marked with the subscript P.
Table 3. Thermodynamic (ΔEr, ΔHr, ΔGr) and Kinetic (ΔrE*, ΔHr*, ΔGr*) Characteristics of the Reactions Leading to O—P Bond Dissociation in the Product of Proton Attachment to the C5 Center in the Anion of 30 - or 50 -Monophosphate of 20 -Deoxythymidine (30 R and 50 R pathways; see Scheme 3) reaction
ΔEr
ΔrE*
ΔHr
ΔHr*
ΔGr
ΔGr*
30 R Pathway 0
3 RI
4.7
27.6
4.6
24.1
3.7
26.8
30 RII
1.7
20.1
1.5
18.7
-1.2
17.7
50 R Pathway 50 RI
4.8
23.7
4.6
20.3
3.6
21.5
50 RII
6.1
25.5
6.0
24.5
4.9
24.2
are typical of chemical processes. The lowest barrier of 17.7 kcal/ mol was predicted for the dissociation of the O—P bond through the 30 R sequence, whereas the highest kinetic barrier of 26.8 kcal/ mol was calculated for the H atom transfer from the 30 center of the sugar to the C6 site of thymine (reaction 30 RI). The kinetic and thermodynamic barriers related to the mechanism proposed here disfavor it with respect to the LEEinduced damage pathway involving the CX0 —O bond breakage. Indeed, it was demonstrated that the activation energy for the
’ SUMMARY We report on computational studies of electron-induced single strand cleavage in DNA, which, in contrast to literature proposals, assume the dissociation of the O—P rather than the CX—O bond. In the first step of the postulated mechanism, which is demonstrated for the thymine nucleotide, an excess electron is captured by the nucleobase, which leads to a stable valence anion localized on the pyrimidine. This anion is protonated on C5, the protonation being followed by intramolecular H atom transfer from the sugar moiety to the C6 of thymine, which leads to a carbon-centered radical on the sugar. Finally, O—P bond cleavage occurs at the C30 or C50 site, yielding a neutral cyclic ketone or aldehyde, respectively. Typical activation energies and small thermodynamic barriers have been predicted at the B3LYP/6-31þþG(d,p) level. However, in order to compensate for the weaknesses of our computational model, analogous calculations should be performed for a representative DNA fragment using the hybrid QM/QM approach. Calculations of this type are under way in our laboratory. Reports published so far23-26 indicate that O—P bond dissociation is only a minor route to the SSBs induced in DNA by LEEs. It is worth noticing, however, that those experimental studies concern dry DNA deposited on a metal surface. Under such experimental conditions, there are no proton donors of sufficient acidity to protonate the radical anions forming in DNA. However, the situation is quite different when the biomolecule is in its natural environment, where DNA interacts with proteins and other molecules having proton-donor properties. We therefore suggest that the O—P splitting mechanism may be a dominant route for SSB formation in aqueous, especially acidic, solutions of DNA irradiated with ionizing radiation. The mechanism proposed in this paper could be validated experimentally; identification of the cyclic ketone and aldehyde sugar derivatives together with 5,6-dihydropyrimidines in DNA solution irradiated with ionizing radiation would provide compelling evidence that SSBs were generated according to this mechanism. ’ ASSOCIATED CONTENT
bS
Supporting Information. Proton affinities (PA) of the C, N, and O centers in the 1-methylthymine valence anion calculated at the B3LYP/6-31þþG** level. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
’ ACKNOWLEDGMENT This work was supported by the Polish Ministry of Science and Higher Education (MNiSW), Grants N N204 023135 ( J.R.) 1916
dx.doi.org/10.1021/jp111059q |J. Phys. Chem. B 2011, 115, 1911–1917
The Journal of Physical Chemistry B and DS/8221-4-0140-1 (P.S.). The calculations were performed at the Academic Computer Center in Gda nsk (TASK).
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