Carbonate radical anion as an efficient reactive oxygen species: Its reaction with guanyl radical and formation of 8-oxoguanine

Chemical Physics 405 (2012) 76–88

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Carbonate radical anion as an efficient reactive oxygen species: Its reaction with guanyl radical and formation of 8-oxoguanine Amarjeet Yadav, P.C. Mishra ⇑ Department of Physics, Banaras Hindu University, Varanasi 221 005, India

a r t i c l e

i n f o

Article history: Received 15 April 2012 In final form 27 June 2012 Available online 4 July 2012 Keywords: Carbonate radical anion Guanine radical 8-Oxoguanine Reaction mechanism Cancer Hydration Proton transfer

a b s t r a c t Mechanisms of reactions of four different tautomers (G(-H1)
, G(-H2a)
, G(-H2b)
and G(-H9)
) of guanyl radical with carbonate radical anion (CO3. ) producing the mutagenic product 8-oxoguanine (8-oxoG) have been investigated theoretically using density functional theory and second order Møller–Plesset perturbation (MP2) theory. Geometries of reactant, intermediate and product complexes and those of transition states were optimized in gas phase. Solvent effect of aqueous media was treated by including eight specific water molecules in a hydration cell during optimization and single point energy calculations employing the polarizable continuum model. It is found that the specific water molecules catalyze proton transfer involving the different sites of the guanyl radical during the reaction. Possible roles of aeration, stirring and photoirradiation of reaction media which are employed to facilitate the reaction in experimental studies are explained. The carbonate radical anion is found to be a very effective reactive oxygen species. Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction Reactive oxygen species (ROS) and reactive nitrogen oxide species (RNOS) generated in biological systems by ionizing radiation, UV radiation, metabolic processes and reactions involving pollutants cause several types of modifications of the DNA bases [1–4]. These modifications of the DNA bases may cause alteration in base pairing patterns which may result in mutation and different diseases including inflammation, aging, cancer and neurodegenerative diseases such as the Alzheimer’s and Parkinson’s diseases [4–7]. Among the DNA bases, guanine, due to its lowest ionization potential, is the primary target of attack of the various reactive agents [8,9]. Reactions of ROS and RNOS with guanine produce different mutagenic products e.g. 8-oxoguanine and 8-nitroguanine [10–16]. Formation of guanine radical cation (G.+) can take place due to oxidation of guanine without deprotonation [17,18] while guanyl radical (G
) is formed due to a rapid deprotonation of guanine radical cation, the corresponding rate constant being 107 s 1 at pH = 7 [17,18]. This reaction has been observed in pulse radiolysis, [18] transient absorption spectroscopy, [19] electron spin resonance [20] and X-ray photoelectron spectroscopy [21]. Deprotonation of guanyl radical cation can take place from any of the N1, N2 and N9 sites [22]. Thus four tautomers of guanyl radical can be ⇑ Corresponding author. E-mail address: [email protected] (P.C. Mishra). 0301-0104/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chemphys.2012.06.012

generated which can cause DNA damage though different reactions. Earlier theoretical calculations and ESR measurements in aqueous media have shown that the N1 site of guanyl radical cation is most favourable for deprotonation [23]. The carbonate radical anion (CO3. ), a reactive oxygen species (ROS), plays an important oxidative role in DNA damage [24]. Its formation can occur in different ways in biological media. For example, it can be formed due to one electron oxidation of bicarbonate anion at the active site of copper-zinc dismutase [24,25]. Another mechanism of formation of CO3. is homolytic dissociation of nitrosoperoxycarbonate anion (ONOOCO2 ) which can be formed transiently in the cellular environment due to reaction between peroxynitrite (ONOO ) and carbon dioxide (CO2) [26–28]. Peroxynitrite produced due to reaction between nitric oxide radical (NO
) and superoxide radical anion (O2
) [29,30] combines rapidly with carbon dioxide to yield the unstable product ONOOCO2 [26]. Homolytic dissociation of ONOOCO2 leads to production of two species, i.e., CO3. and nitrogen dioxide radical (NO2
) [26,31]. Also, CO3. can be formed by one electron oxidation of bicarbonate anion (HCO3 ) employing photochemically generated sulphate radical anion (SO4. ). The carbonate radical anion is a strong one electron oxidant that can oxidize the DNA bases [32]. It can abstract electrons from appropriate donors rapidly but it abstracts hydrogen atoms slowly [32]. The guanine base of DNA is oxidized by CO3. producing 8-oxoguanine (8-oxoG) [33–35]. Crean et al. [36] have studied experimentally the reaction of CO3. at the C8 site of guanyl radical deprotonated at the N1 site

A. Yadav, P.C. Mishra / Chemical Physics 405 (2012) 76–88

and this reaction was observed to produce 8-oxoguanine (8-oxoG). Further, the reactions of 8-oxoG with CO3. lead to formation of other mutagenic products [34,36]. 8-OxoG mispairs with adenine during DNA replication which causes GC ? AT transversion mutation and it leads to serious consequences on cell functioning including different diseases [37,38]. Formation of 8-nitroguanine due to reaction between the NO2 radical and the guanyl radical in the presence of six water molecules was studied earlier [39]. In this study [39], important catalytic roles of the specific water molecules in proton transfer facilitating the reaction were found. It is highly desirable to study the reaction between the carbonate radical anion and guanyl radical in view of a high oxidizing power of the former species as it can produce the mutagenic product 8-OxoG. The results obtained from such a study are presented here. 2. Computational details Reactions of four different tautomers of guanyl radical (G(-Hx)
, x = 1, 2a, 2b, 9) (Figs. 1–4) with carbonate radical anion (CO3. ) were studied using density functional [40,41] and second order Møller–Plesset perturbation (MP2) theories [42]. Geometries of all the reactant complexes (RCs), intermediate complexes (ICs), transition states (TSs) and product complexes (PCs) involved in the various reaction schemes were optimized in the presence of eight complexed specific water molecules at the B3LYP/6–31G(d,p) level of density functional theory in gas phase. The eight complexed specific water molecules represent a hydration shell around each of the RCs, ICs, TSs and PCs, and also these molecules are involved in proton transfer involving the different sites of the guanyl radical. Single point energy calculations were performed both in gas phase (including the eight complexed specific water molecules) and aqueous media at the B3LYP/AUG-cc-pVDZ, BHandHLYP/AUG-cc-pVDZ and MP2/AUG-cc-pVDZ levels of theory employing the B3LYP/6– 31G(d,p) level gas phase optimized geometries. Bulk solvent effect of aqueous media was treated by single point energy calculations on each of the RCs, ICs, TSs and PCs including the eight complexed specific water molecules employing the integral equation formalism of the polarizable continuum model (IEFPCM) [43,44]. Gibbs free barrier and released energies involved in the different reaction steps were obtained at all the above mentioned levels of theory in both gas phase and aqueous media. Vibrational frequency analyses for all the optimized molecular geometries were carried out. All the RCs, ICs and PCs were found to be associated with all real vibrational frequencies while all the transition states (TSs) were found to be associated with an imaginary vibrational frequency each. Genuineness of each optimized transition state was ensured by visually examining the vibrational mode corresponding to the imaginary frequency applying the condition that it connected properly the two complexes between which the transition state under consideration was located. As genuineness of all the calculated transition states was obvious, intrinsic reaction coordinate (IRC) calculations were not performed. The thermal energy corrections obtained in geometry optimization calculations at the B3LYP/6–31G(d,p) level of theory in gas phase giving Gibbs free energies were also taken to be valid, as an approximation, for the corresponding single point energy calculations in both gas phase and aqueous media. Surface molecular electrostatic potential (MEP)-fitted net charges located at the atomic sites were obtained at the MP2/AUG-cc-pVDZ level of theory employing the CHelpG algorithm [45]. All the calculations were carried out using the Windows version of the Gaussian 09 (G09 W) (rev. A.1) suite of programs [46] while the GaussView program (rev. 5.0.9) was used for visualization of optimized structures and vibrational modes [47].

77

3. Results and discussion The radicals G(-H1)
and G(-H9)
are obtained by removing the hydrogen atoms attached to the N1 and N9 sites of guanine respectively. When the hydrogen atoms of the amino group of guanine lying towards the N1 and N3 sites are removed, the radicals G(-H2a)
and G(-H2b)
respectively are obtained. Geometries of these four tautomeric forms of guanyl radical were optimized initially at the B3LYP/6–31G(d,p) level of theory in gas phase without including any complexed water molecule. The relative Gibbs free energies of G(-H1)
, G(-H2a)
, G(-H2b)
and G(-H9)
optimized at the B3LYP/6–31G(d,p) level of theory in gas phase were found to be 0.00, 5.15, 0.24 and 1.99 kcal/mol respectively while the relative Gibbs free energies of these radical tautomers obtained by single point energy solvation calculations in aqueous media were found to be 1.36, 2.54, 0.00 and 3.43 kcal/mol. Thus, among the four tautomers of guanyl radical, G(-H2a)
and G(-H9)
are found to be most stable in gas phase and aqueous media respectively. In a previous BHandHLYP/AUG-cc-pVDZ level geometry optimization study in gas phase also, G(-H2a)
was found to most stable radical tautomer of guanine [39]. Further, it is also in agreement with the result obtained by Schaefer and coworkers using four carefully calibrated density functional methods [48]. In the presence of eight complexed specific water molecules forming a hydration cell around each of the four tautomers of guanyl radical, geometry optimization at the B3LYP/6–31G(d,p) level of theory yielded the relative Gibbs free energies of G(-H1)
, G(-H2a)
, G(-H2b)
, G(-H9)
in gas phase as 3.27, 0.00, 4.67, 5.98 kcal/mol while in bulk aqueous media (solvated including the eight complexed water molecules), the relative Gibbs free energies were found to be 4.24, 0.00, 3.08, 6.03 kcal/mol respectively. Thus among the four radical tautomers of guanine, in the presence of eight water molecules, G(-H9)
is found to be most stable in both gas phase and aqueous media. The next most stable radical of guanine in aqueous media after G(-H9)
is found to be G(-H1)
. In guanosine, the N9 site would be bonded to a sugar moiety and therefore, in that case, G(-H9)
would not exist. The above mentioned Gibbs free energies suggest that the most stable guanosine radical in aqueous media including the eight complexed specific water molecules would be G(-H1)
. Adhikary et al. [49] on the basis of an electron spin resonance (ESR) experimental study along with density functional theoretical calculations considering seven water molecules surrounding guanyl radicals have also found that G(-H1)
is most stable among the various radical tautomers of guanyl radical. Thus the present result obtained considering eight complexed water molecules is in agreement with the previous experimental and theoretical results [49]. In a previous study [39], mechanisms of reaction between nitrogen dioxide radical (NO2
) and different tautomers of guanyl radical in the presence of one, two, three and six water molecules leading to formation of the product 8-nitroguanine were investigated using the same methods and basis sets as those employed here. The experimental study of reaction of CO3. with guanyl radical was performed starting with several dissolved chemicals in water e.g. the oligonucleoside, NaHCO3, Na2S2O8 and NaH2PO4 [34]. The experimental material was aerated, stirred and excited by an excimer laser to effect the reaction [34]. The same intended end products were also obtained by another process involving photooxidation of the oligonucleoside employing the dye methylene blue as a photosensitizer and a xenon arc lamp as the photoexcitation source [34]. Due to photoexcitation of the material and occurrence of non-radiative and other photochemical processes [34], there would be a significant amount of energy dumped in it. The photo and thermal energies would help the system overcome barriers enabling the reaction between CO3. and guanyl radical

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Fig. 1. Free reactant (FR1), reactant complex (RC1), intermediate complexes IC1 to IC7, product complex PC1 and transition states TS1 to TS8 involved in the reaction of CO3. at the C8 site of G(-H1)
in the presence of eight water molecules one of which is consumed in the reaction. Gibbs free barrier energies (kcal/mol) obtained at the single point MP2/AUG-cc-pVDZ level of theory in gas phase are given. Some important optimized interatomic distances (Å) are also given. The locations of different species are not to scale.

to occur [34]. Obviously, the experimental material used was quite complex and it is desirable to investigate what roles are likely to played by aeration and stirring, and which reaction steps would require appreciable activation energies in such a situation [34,36]. Stability of the reactant complex RC1 that includes G(-H1)
, CO3. and eight complexed water molecules was found to be significantly affected by dielectric constants (e) of solvent medium. The Gibbs free binding energies of RC1 were found in bulk water (e = 78.36), chlorobenzene (e = 5.67), toluene (e = 2.37), benzene (e = 2.27), argon (e = 1.43) and gas phase (e = 1) to be 82.0, 26.4, 9.0, 7.8, 8.4 and 25.2 kcal/mol respectively. Thus RC1 becomes stabilized when the equivalent dielectric constant of the medium is close to that of argon which is not very different from that of

gas phase. The Gibbs free energies of the reactant complexes RC1, RC2, RC3 and RC4 that involve the guanyl radicals G(-H1)
, G(-H2a)
, G(-H2b)
and G(-H9)
in aqueous media respectively were found to be 46–60 kcal/mol more negative than those in gas phase. These results show that though the aqueous medium helps the reactant complexes stabilize, its components i.e. the reacting species CO3. and guanyl radical would separately polarize the medium and get stabilized in the same to greater extents than each of the reactant complexes. Therefore, it seems that the above mentioned treatments of the experimental medium including aeration and stirring reduce the equivalent dielectric constant to around that of air. Thus it appears that the present gas phase results would corresponds to experimental results better than those obtained in

A. Yadav, P.C. Mishra / Chemical Physics 405 (2012) 76–88

79

Fig. 2. Free reactant (FR2), reactant complex (RC2), intermediate complexes IC8 to IC15, product complex PC2 and transition states TS9 to TS17 involved in the reaction of CO3. at the C8 site of G(-H2a)
in the presence of eight water molecules one of which is consumed in the reaction. Gibbs free barrier energies (kcal/mol) obtained at the single point MP2/AUG-cc-pVDZ level of theory in gas phase are given. Some important optimized interatomic distances (Å) are also given. The locations of different species are not to scale.

aqueous media. In a previous study also, aqueous medium was not found to be appropriate for reactions between guanyl radical cation and carbonate radical anion [50]. It may be argued that a possible reason behind this result is that we optimized all molecular geometries in gas phase and performed only single point energy calculations, instead of geometry optimization, in aqueous media. However, we do not expect that geometry optimization in aqueous media would change the results drastically in view of the following reasons: (i) It has been found that single point energy calculations in aqueous media using the PCM generally yield correct qualitative trend of results that would be obtained by full geometry optimization in aqueous media using the same method [51,52], and (ii) The results obtained using different solvents, as discussed above, show

that there is a regular variation in solvation energy with dielectric constant and those obtained in aqueous media only follow an expected trend. It is to be noted that as eight specific water molecules are considered to be involved in each of the reactant complexes even in gas phase, the results obtained in the present study in gas phase would also include a significant amount of solvent effect (specific solvent effect) of water. In view of the above results and as electron correlation treatment at the MP2 level is better than those at the other levels of theory employed here, we would mainly discuss the results obtained at the MP2/AUG-cc-pVDZ level in gas phase. In all the reaction mechanisms studied here, CO3. is considered to attack the C8 site of the guanyl radical in question while the

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Fig. 3. Free reactant (FR3), reactant complex (RC3), intermediate complexes IC16 to IC21, product complex PC3 and transition states TS18 to TS24 involved in the reaction of CO3. at the C8 site of G(-H2b)
in the presence of eight water molecules one of which is consumed in the reaction. Gibbs free barrier energies (kcal/mol) obtained at the single point MP2/AUG-cc-pVDZ level of theory in gas phase are given. Some interatomic distances (Å) are also given. The locations of different species are not to scale.

surrounding water molecules catalyze proton transfer between its different sites. The calculated Gibbs free barrier and released energies involved in the different reaction steps obtained at the B3LYP/ 6–31G(d,p), B3LYP/AUG-cc-pVDZ, BHandHLYP/AUG-cc-pVDZ and MP2/AUG-cc-pVDZ levels of theory in both gas phase and aqueous media are given in Tables 1–4 while the locations of extrema on the potential energy surfaces obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase in each case are presented in Figs. 1–4. In each of these figures, all the eight water molecules are shown in the reactant complexes RC1-RC4. Although all these water molecules were considered at all the steps of the reactions, only those water molecules are shown at the different reaction steps that are explicitly involved in proton transfer, in order to avoid crowding. The corresponding reaction mechanisms including all the eight water molecules in the various cases are shown in Figs. S1–S4 (Supporting information). In each of the four reaction schemes (Figs. 1–4), one of the eight complexed water molecules is consumed while the other seven water molecules remain

complexed with the system. A separate set of free reactants is involved in each of the four reaction schemes (Figs. 1–4). These sets of free reactants can in general be represented as X + 8H2O + CO3. where X stands for G(-H1)
, G(-H2a)
, G(-H2b)
or G(-H9)
, and may be named as FR1, FR2, FR3 and FR4 respectively. The locations of these four sets of free reactants in terms of their Gibbs free energies are also shown in the different reaction schemes (Fig. 1–4). In gas phase, the reactant complexes RC1, RC2 and RC3 have higher Gibbs free energies than the corresponding sets of free reactants FR1, FR2 and FR3 by 26.0, 23.1 and 11.1 kcal/mol respectively, while in bulk aqueous media, the reactant complexes have much higher Gibbs free energies (by 51–68 kcal/mol) than the corresponding sets of free radicals. In gas phase, the reactant complex RC4 corresponding to G(-H9)
was found to be almost isoenergetic with FR4 as the former was found to be only slightly lower in Gibbs free energy (by 0.1 kcal/mol) than the latter. However, in bulk aqueous media, RC4 was found to be 48.4 kcal/mol higher in Gibbs free energy than FR4.

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Fig. 4. Free reactant (FR4), reactant complex (RC4), intermediate complexes IC22 to IC25, product complex PC4 and transition states TS25 to TS29 involved in the reaction of CO3. at the C8 site of G(-H9)
in the presence of eight water molecules one of which is consumed in the reaction. Gibbs free barrier energies (kcal/mol) obtained at the single point MP2/AUG-cc-pVDZ level of theory in gas phase are given. Some interatomic distances (Å) are also given. The locations of different species are not to scale.

Formation of the appropriate reactant complex is necessary for initiation of a reaction. It appears that for the reactions involving RC1, RC2 and RC3, aeration, stirring and photoirradiation of the experimental medium which are actually practiced as discussed earlier, would serve two very useful purposes: (i) Reducing the Gibbs free energy differences between the corresponding sets of free reactants and reactant complexes e.g. FR1, RC1, and so on, and (ii) Populating the reactant complexes by providing the necessary activation energies to the free reactants as discussed earlier. However, in the case of the fourth reaction scheme involving G(-H9)
, the reactant complex RC4 would be easily formed from FR4 in view of the results discussed above. In all the reactions studied here (Tables 1–4), the first Gibbs barriers (DGib, i = 1, 9, 18, 25) are found to be appreciably different at the various levels of theory. However, it is noted that all the first Gibbs barrier energies are

negative which shows that the corresponding reaction steps would be barrierless. In this sense, the various results for the first Gibbs barriers are in agreement. However, from the quantitative point of view, we would consider the values obtained at the MP2/AUGcc-pVDZ level to be most reliable in view of the fact that electron correlation treatment at this level is better than those at the other levels of theory employed here. 3.1. Reaction between carbonate radical anion and the guanyl radical G(-H1)
The mechanism of reaction between G(-H1)
and CO3. shown in Fig. 1 involves the free reactants FR1 consisting of G(-H1)
, eight water molecules and CO3. , reactant complex RC1, seven intermediate complexes ICn (n = 1–7), eight transition states TSn (n = 1–8)

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A. Yadav, P.C. Mishra / Chemical Physics 405 (2012) 76–88

Table 1 Gibbs free barrier (DGib) and released energies (DGir) (i = 1–8) at 298.15 K (kcal/mol) involved in reaction of carbonate radical anion (CO3. ) with G(-H1)
in the presence of eight water molecules, according to the scheme of Fig. 1 in gas phase and aqueous media.a Gibbs free barrier and released energies

DG1b DG1r

a b c

B3LYP/6-31G(d,p)b 19.8 ( 22.8) 26.6 ( 24.7)

B3LYP/AUG-cc-pVDZc 23.4 ( 23.5) 25.0 ( 23.4)

BHandHLYP/AUG-cc-pVDZc 51.1 ( 42.4) 26.3 ( 24.5)

MP2/AUG-cc-pVDZc 75.5 ( 50.3) 19.9 ( 18.2)

DG2b DG2r

4.4 (5.2) 7.0 ( 3.4)

3.6 (5.1) 6.8 ( 3.7)

5.5 (7.1) 7.7 ( 4.4)

0.7 (2.0) 6.3 ( 3.2)

DG3b DG3r

5.2 (4.1) 8.8 ( 13.2)

5.2 (4.2) 11.6 ( 16.2)

7.8 (6.8) 16.2 ( 21.2)

8.9 (8.4) 9.7 ( 14.4)

DG4b DG4r

14.0 (17.4) 35.3 ( 35.6)

20.2 (23.5) 39.0 ( 42.3)

25.8 (29.3) 45.3 ( 48.8)

15.7 (18.9) 40.8 ( 44.3)

DG5b DG5r

4.7 ( 2.6) 4.0 ( 6.2)

6.9 ( 4.5) 4.8 ( 5.5)

2.1 (0.3) 7.9 ( 8.7)

0.7 (3.1) 6.2 ( 7.2)

DG6b DG6r

13.2 (11.1) 7.2 ( 6.7)

12.2 (9.2) 5.6 ( 5.0)

11.6 (8.6) 5.3 ( 4.9)

10.9 (8.0) 5.2 ( 4.7)

DG7b DG7r

3.5 (5.9) 15.5 ( 14.6)

3.7 (5.7) 9.9 ( 10.0)

6.5 (8.6) 13.1 ( 13.2)

5.1 (7.2) 13.0 ( 12.7)

DG8b DG8r

13.2 (8.4) 18.2 ( 20.8)

9.5 (4.4) 19.3 ( 21.7)

13.3 (8.7) 22.2 ( 25.0)

14.4 (9.4) 15.9 ( 18.6)

Quantities given in parentheses corresponds to aqueous media. Geometry optimization was performed at the B3LYP/6–31G(d,p) level of theory. Single point energy calculations were performed at these levels of theory employing the geometries optimized at the B3LYP/6–31G(d,p) level.

Table 2 Gibbs free barrier (DGib) and released energies (DGir) (i = 9–17) at 298.15 K (kcal/mol) involved in reaction of carbonate radical anion (CO3. ) with G(-H2a)
in the presence of eight water molecules, according to the scheme of Fig. 2 in gas phase and aqueous media.a

a b c

Gibbs free barrier and released energies

B3LYP/6–31G(d,p)b

B3LYP/AUG-cc-pVDZc

BHandHLYP/AUG-cc-pVDZc

MP2/AUG-cc-pVDZc

DG9b DG9r DG10b DG10r DG11b DG11r DG12b DG12r DG13b DG13r DG14b DG14r DG15b DG15r DG16b DG16r DG17b DG17r

19.4 ( 15.3) 24.4 ( 25.0) 2.0 (2.1) 8.8 ( 7.9) 9.3 (9.8) 5.0 ( 8.7) 10.7 (11.3) 36.7 ( 36.6) 15.7 (4.8) 1.6 ( 2.9) 10.0 (6.9) 5.8 ( 6.4) 10.4 (12.9) 33.9 ( 30.9) 11.0 (9.3) 15.5 ( 14.6) 13.3 (8.4) 18.3 ( 20.8)

51.4 ( 11.0) 23.7 ( 24.6) 1.6 ( 0.98) 8.5 ( 7.9) 8.0 (8.5) 6.0 ( 9.9) 15.6 (16.4) 38.9 ( 38.9) 2.1 (1.7) 3.1 ( 4.4) 10.3 (6.8) 4.5 ( 4.5) 9.7 (11.8) 26.9 ( 24.1) 5.2 (3.7) 10.0 ( 9.9) 9.5 (4.4) 19.3 ( 21.7)

45.8 ( 28.9) 26.5 ( 27.6) 2.7 (3.8) 8.8 ( 8.4) 9.4 (10.1) 10.4 ( 14.7) 22.2 (23.1) 44.3 ( 44.3) 5.8 (5.3) 6.0 ( 7.2) 9.8 (6.6) 4.5 ( 4.8) 13.1 (15.2) 31.6 ( 28.6) 8.1 (6.7) 13.1 ( 13.2) 13.3 (8.7) 22.2 ( 25.0)

68.8 ( 38.2) 23.0 ( 23.8) 3.6 (4.1) 9.5 ( 8.8) 13.8 (14.5) 5.6 ( 9.7) 14.8 (15.5) 37.5 ( 37.6) 6.9 (6.5) 3.5 ( 4.8) 9.6 (6.5) 4.6 ( 5.1) 8.2 (10.4) 29.6 ( 26.8) 8.6 (7.3) 13.1 ( 12.7) 14.4 (9.4) 15.9 ( 18.6)

Quantities given in parentheses corresponds to aqueous media. Geometry optimization was performed at the B3LYP/6–31G(d,p) level of theory. Single point energy calculations were performed at these levels of theory employing the geometries optimized at the B3LYP/6–31G(d,p) level.

and the product complex PC1 which is a complex of 8-oxoG with bicarbonate anion (HCO3 ) and seven water molecules. The calculated Gibbs free barrier (DGb) and released (DGr) energies obtained at four different levels of theory (B3LYP/6–31G(d,p), B3LYP/AUGcc-pVDZ, BHandHLYP/AUG-cc-pVDZ and MP2/AUG-cc-pVDZ) in both gas phase and aqueous media are presented in Table 1, and those obtained at the MP2/AUG-cc-pVDZ level in gas phase are also shown in Fig. 1. It is noted that though some of the Gibbs free barrier and released energies obtained at the different levels of theory are appreciably different from the others, qualitatively similar trends are obtained at all the levels (Table 1). At RC1, the distance between the C8 site of G(-H1)
and O8 (nearest oxygen atom of the CO3. group) is 2.78 Å and the CHelpG charges associated with the CO3. and G(-H1)
moieties are 1.59 and 0.75 respectively. We find that although CO3. which is a part of FR1 was already having 1 charge (in the unit of magnitude of

electronic charge), on coming close to the C8 site of G(-H1)
at RC1, it takes up an additional 0.59 charge. Thus an appreciable negative charge transfer occurs from G(-H1)
to CO3. in going from FR1 to RC1 so that G(-H1)
would look like G(-H1)
+ while CO3. would look like CO3.2 , and the binding between the two moieties at RC1 can be ascribed largely to charge transfer. At the first reaction step i.e. in going from RC1 to IC1 through TS1 (Fig 1), the O8 oxygen atom of the CO3. group gets bonded to the C8 site of the guanine radical. At TS1, the total CHelpG charges associated with the CO3. and G(-H1)
moieties are 0.94 and 0.10 respectively. The distance between C8 of G(-H1)
and O8 of CO3. at TS1 is 1.71 Å. Thus though the CO3. moiety has come fairly close to G(-H1)
at TS1, it still retains  1 charge. The Gibbs free barrier energy (DG1b) involved at this step is appreciably negative at all the levels of theory employed here in both gas phase and aqueous media (Table 1). The Gibbs free barrier energy corresponding to

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Table 3 Gibbs free barrier (DGib) and released energies (DGir) (i = 18–24) at 298.15 K (kcal/mol) involved in reaction of carbonate radical anion (CO3. ) with G(-H2b)
in the presence of eight water molecules, according to the scheme of Fig. 3 in gas phase and aqueous media.a

a b c

Gibbs free barrier and released enegies

B3LYP/6–31G(d,p)b

B3LYP/AUG-cc-pVDZc

BHandHLYP/AUG-cc-pVDZc

MP2/AUG-cc-pVDZc

DG18b DG18r DG19b DG19r DG20b DG20r DG21b DG21r DG22b DG22r DG23b DG23r DG24b DG24r

25.4 ( 19.2) 26.9 ( 30.2) 8.5 (8.0) 4.6 ( 2.0) 2.8 (5.7) 4.2 ( 8.0) 32.7 (31.8) 17.1 ( 19.8) 2.7 (1.4) 12.6 ( 15.4) 23.6 (20.2) 75.3 ( 66.6) 25.3 (12.7) 12.7 ( 14.8)

26.6 ( 11.8) 27.0 ( 30.4) 5.7 (6.1) 5.0 ( 2.8) 3.4 (6.1) 5.5 ( 9.5) 31.3 (30.4) 15.1 ( 17.6) 2.6 (6.3) 14.7 ( 18.1) 18.8 (16.4) 71.3 ( 62.4) 18.8 (7.1) 14.0 ( 16.5)

49.7 ( 31.8) 25.7 ( 29.4) 8.0 (8.6) 4.8 ( 2.5) 3.6 (6.3) 10.3 ( 14.3) 33.9 (32.3) 18.9 ( 21.3) 6.3 (10.8) 18.5 ( 21.6) 25.0 (22.2) 78.0 ( 68.8) 24.7 (12.5) 17.9 ( 20.5)

61.5 ( 39.3) 21.5 ( 24.8) 2.7 (3.1) 2.4 ( 0.3) 5.7 (9.0) 5.2 ( 9.5) 27.1 (24.5) 16.4 ( 18.9) 5.5 (10.4) 15.2 ( 18.1) 25.3 (22.5) 69.9 ( 61.2) 19.8 (8.2) 13.7 ( 16.0)

Quantities given in parentheses corresponds to aqueous media. Geometry optimization was performed at the B3LYP/6–31G(d,p) level of theory. Single point energy calculations were performed at these levels of theory employing the geometries optimized at the B3LYP/6–31G(d,p) level.

Table 4 Gibbs free barrier (DGib) and released energies (DGir) (i = 25–29) at 298.15 K (kcal/mol) involved in reaction of carbonate radical anion (CO3. ) with G(-H9)
in the presence of eight water molecules, according to the scheme of Fig. 4 in gas phase and aqueous media.a

a b c

Gibbs free barrier and released enegies

B3LYP/6–31G(d,p)b

B3LYP/AUG-cc-pVDZc

BHandHLYP/AUG-cc-pVDZc

MP2/AUG-cc-pVDZc

DG25b DG25r DG26b DG26r DG27b DG27r DG28b DG28r DG29b DG29r

6.7 ( 3.6) 28.6 ( 30.9) 3.9 (6.7) 2.9 ( 3.7) 6.2 (6.4) 4.1 ( 10.3) 18.5 (23.7) 75.3 ( 66.6) 25.3 (12.7) 12.7 ( 14.8)

5.9 ( 1.5) 27.1 ( 30.2) 1.2 (4.7) 6.0 ( 6.8) 8.7 (8.7) 7.0 ( 13.5) 19.4 (24.8) 71.3 ( 62.4) 18.8 (7.1) 14.0 ( 16.5)

24.7 ( 14.6) 28.9 ( 32.1) 5.4 (9.1) 6.8 ( 7.7) 9.7 (9.9) 11.2 ( 18.2) 23.5 (29.2) 78.0 ( 68.8) 24.7 (12.5) 17.9 ( 20.5)

30.9 ( 22.6) 21.9 ( 25.3) 0.80 (4.08) 4.5 ( 5.4) 10.8 (11.2) 3.2 ( 10.0) 19.7 (25.1) 69.9 ( 61.2) 19.8 (8.2) 13.7 ( 16.0)

Quantities given in parentheses corresponds to aqueous media. Geometry optimization was performed at the B3LYP/6–31G(d,p) level of theory. Single point energy calculations were performed at these levels of theory employing the geometries optimized at the B3LYP/6–31G(d,p) level.

TS1 obtained at the MP2/AUG-cc-pVDZ level in gas phase is 75.5 kcal/mol (Table 1). It implies that the reaction step would occur rapidly and in a barrierless manner. At the second step of the reaction i.e. in going from IC1 to IC2 via TS2, a CO2 group gets dissociated from the CO3. moiety so that the distance between the O8 and C9 atoms which belonged to this moiety is increased from 1.50 Å to 3.95 Å. At TS2, the CHelpG charges associated with the CO2 and G(-H1)
-O8 moieties obtained at the MP2/AUG-cc-pVDZ level in gas phase are 0.12 and 0.64 respectively. The Gibbs free barrier energy corresponding to TS2 in gas phase obtained at the MP2/AUG-cc-pVDZ level is quite low i.e. 0.7 kcal/mol. In IC2, the CO2 group is associated with only a small negative charge i.e. 0.05, while the G(-H1)
-O8 moiety is associated with the net charge 0.63. Thus at both TS2 and IC2, a major portion of the negative charge of the CO3. moiety is transferred to the G(-H1)
-O8 moiety. At TS3, a water molecule dissociates into a proton H24 and an anion O22H23, and these species move towards O8 bonded to C8 and C9 of the CO2 group respectively. Consequently, at IC3, a bicarbonate anion (HCO3 ) carrying a net negative charge of 0.81 is formed. The Gibbs free barrier energy obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase for the reaction step corresponding to TS3 is 8.9 kcal/mol. In going from IC3 to IC4 via TS4, the proton H8 is transferred from C8 to N7 of the imidazole ring and a water molecule located near the N7 site catalyzes this process. IC4 is a tautomer of 8hydoxyguanine (8-OHG) protonated at N7 and deprotonated at N1 complexed with a bicarbonate anion (HCO3 ) and seven water

molecules. The vibrational mode corresponding to the imaginary frequency at TS4 shows that the H8 proton moves from C8 towards O25 i.e. the oxygen atom of the water molecule located near N7, and thus an H3O+-like species is formed transiently. The net CHelpG charge associated with the water molecule placed near N7 including the H8 proton is 0.83 which supports formation of a H3O+-like species at TS4. The C8H8, H8O25 and H8N7 distances at TS4 are 1.35, 1.27 and 1.95 Å respectively. Thus, at TS4, from the point of view of bonding, H8 is shared by both C8 and O25 while it is hydrogen bonded with N7. When the energy is minimized starting with the geometry of TS4, the H8 proton leaves the H3O+-like species and gets bonded to N7. The Gibbs free barrier energy for transfer of the proton H8 from C8 to N7 starting from IC3 in the absence of any water molecule in gas phase at the B3LYP/6–31G(d,p) was found to be 30.7 kcal/mol while the corresponding value in the presence of seven water molecules, one of which is explicitly involved in the proton transfer process, as mentioned above, was found to be 14 kcal/mol (Table 1). Thus the water molecules, particularly that placed near N7 plays a catalytic role in proton transfer from C8 to N7. The Gibbs free barrier energy corresponding to TS4 at the MP2/AUG-cc-pVDZ level of theory in gas phase was found to be 15.7 kcal/mol (Table 1, Fig. 1). The proton attached to N7 in IC4 is transferred to O6 via TS5 involving catalytic action of a water molecule. There is a relay action involving the movement of two protons at TS5. Thus as the H8 proton moves from N7 towards the O19 atom of the water molecule located near the O6 site of the 8-OHG moiety, the H20 proton

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of the water molecule moves towards the O6 site. In IC5 which is formed after TS5, the H8 and H20 protons get fully bonded to the O19 atom of the water molecule and O6 site of the enol form of the 8-OHG moiety respectively. The Gibbs free barrier energy corresponding to TS5 at the MP2/AUG-cc-pVDZ level of theory in gas phase was found to be fairly low i.e. 0.7 kcal/mol. At the transition state TS6, the dihedral angle N1C6O6H20 is 85.0° and the vibrational motion corresponding to the imaginary frequency shows that the proton H20 moves to and fro forming cis and trans conformations with respect to this dihedral angle. At IC6, the conformation of system with respect to the dihedral angle N1C6O6H20 is cis, while at IC5, it was trans. The Gibbs free barrier energy corresponding to the transition state TS6 in gas phase as obtained at the MP2/AUG-cc-pVDZ level theory was found to be 10.9 kcal/mol. At TS7, there is a relay action in operation in which the proton H20 attached to the O6 atom of guanine moves towards the O42 atom of the water molecule located near N1 while the proton H41 of the water molecule moves towards the N1 site of the 8OHG moiety. The Gibbs free barrier energy involved in this process was obtained at the MP2/AUG-cc-pVDZ level theory in gas phase as 5.1 kcal/mol. The intermediate complex IC7 formed after TS7 is the keto form of the 8-OHG where the proton H41 is bonded to the N1 site. At the next step in the reaction scheme shown in Fig. 1, the proton attached at O8 bonded to C8 is transferred to N7 through the transition state TS8. A relay action involving movement of two protons i.e. H24 from O8 towards the O25 atom of a water molecule located near N7, and H26 from O25 towards N7 is in operation at TS8. Consequent to the transfer of these two protons, the product complex PC1 which is 8-oxoG complexed with a bicarbonate radical anion and seven water molecules is formed. The Gibbs barrier energy for the reaction step corresponding to TS8 was found at the MP2/AUG-cc-pVDZ level of theory in gas phase to be 14.4 kcal/mol. The net CHelpG charges associated with the 8-oxoG and bicarbonate moities are 0.01 and 0.88 respectively. Thus the net -1 charge of the initial reactant CO3. is located in the product complex mostly at the bicarbonate moiety, and therefore, it is also a radical anion.

3.2. Reaction between carbonate radical anion and the guanyl radical G(-H2a)
A reaction scheme that involves the free reactants guanyl radical G(-H2a)
, eight water molecules and CO3. (FR2), a complex of these reactants (RC2), intermediate complexes ICn (n = 8–15), transition states TSn (n = 9–17) and product complex PC3 which is a complex of 8-oxoG, bicarbonate anion (HCO3 ) and seven water molecules is shown in Fig. 2. The calculated Gibbs free barrier and released energies involved in this mechanism at four different levels of theory i.e. B3LYP/6–31G(d,p), B3LYP/AUG-cc-pVDZ, BHandHLYP/AUG-cc-pVDZ and MP2/AUG-cc-pVDZ), in both gas phase and aqueous media, are presented in Table 2. The Gibbs free barrier (DGb) and released (DGr) energies obtained at the MP2/ AUG-cc-pVDZ level in gas phase for the various reaction steps in this case are shown in Fig. 2. The reactant complex RC2 required for initiation of the reaction shown in this scheme would be formed when the necessary activation energy i.e. 23.1 kcal/mol is obtained by the free reactants FR2 through the general mechanism discussed earlier. At the reactant complex RC2, the CO3. moiety is located above the C8 site of the imidazole ring of the guanyl radical G(-H2a)
while eight water molecules surround it forming a hydration shell. At the reactant complex RC2, the net CHelpG charges associated with the guanyl radical G(-H2a)
and CO3. obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase were found to be 0.77 and -1.59 respectively. Thus, the CO3. moiety, at RC2, becomes more negatively charged by taking up additional 0.59

charge and would, therefore, look somewhat like CO3.2 . Thus RC2 like RC1 has a strong charge transfer character. At the first step of the reaction i.e. in going from RC2 to IC8 via TS9 (Fig. 2), the CO3. moiety gets bonded to the C8 site of the guanyl radical G(-H2a)
. At TS9, the CHelpG charges associated with G(-H2a)
and CO3. are 0.21 and -1.01 respectively. Thus in going from RC2 to TS9, the CO3. moiety loses about 0.58 charge. The Gibbs free barrier energy involved at this step is large negative at all the levels of theory employed here in both gas phase and aqueous media (Table 2) which shows that the reaction step in question would be barrierless. At the second step of the reaction (Fig. 2) that involves the transition state TS10, an oxygen atom of the CO3. group, being dissociated from it, gets bonded to C8 while a CO2 molecule gets dissociated from CO3. . At TS10, the CHelpG charges associated with the CO2 and G(-H2a)
-O8 moieties, as obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase, are 0.11 and 0.46 respectively. Thus at TS10, the CO2 group is associated with a small amount of negative charge while the G(-H2a)
-O8 moiety is associated with a fairly large amount of negative charge. The Gibbs free barrier energy of this reaction step is 3.6 kcal/mol as obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase. In IC9 formed after TS10, the CHelpG charges associated with the CO2 and G(-H2a)
-O8 moieties as obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase are 0.07 and 0.57 respectively. Thus the G(-H2a)
-O8 moiety continues to be associated with a fairly large amount of negative charge. At the third step of the reaction (Fig. 2) i.e. in going from IC9 to IC10 via TS11, the proton H24 dissociates from a water molecule located near C8 and binds with the oxygen atom bonded to C8 while the O22H23 group of the dissociated water molecule combines with the CO2 group. Thus, at IC10, a bicarbonate group is produced. The Gibbs free barrier energy corresponding to TS11 is 13.8 kcal/mol as obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase. At IC10, the bicarbonate group is associated with a net negative charge of 0.84. Thus the -1 charge which was initially associated with the CO3. moiety is now largely localized at the bicarbonate moiety and so the bicarbonate moiety is in the anionic form. At the fourth step of the reaction, IC11 is formed from IC10 via TS12. IC11 is a tautomeric form of 8-OHG. At this step, the proton H8 is transferred from C8 to the N7 site through a water molecule located near these sites. This step is catalyzed by a water molecule since the H8 proton during its transit from C8 to N7 remains weakly bonded to the oxygen atom (O25) of it. The CHelpG charge associated with the atoms of water molecule and H8 is found to be 0.86 which shows that an H3O+-like species is formed at TS12 transiently. The Gibbs free barrier energy for this step as obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase is 14.8 kcal/mol. At the fifth step of the reaction that involves the transition state TS13, IC12 is formed from IC11. At this step, the H8 proton is transferred from the N7 site to the O6 site of the guanine moiety and this process is catalyzed by a water molecule located near these sites. A relay action involving movement of two protons, H8 and H20, takes place at TS13 such that while one is approaching the O19 atom of water molecule, the other is leaving it and vice versa. At IC12, a tautomeric form of 8-OHG where the guanine moiety is in the enol form is formed. The barrier energy corresponding to TS13 as obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase is 6.9 kcal/mol. At the transition state TS14, the dihedral angle N1C6O6H20 is 85.8and thus the O6H20 group is almost perpendicular the ring plane. The conformation of the system, with respect to the dihedral angle N1C6O6H20, is trans at IC12 while it is cis at IC13. The vibrational mode of the imaginary frequency at TS14 shows a to and fro motion of the O6H20 group between these two conformations. The Gibbs free barrier energy corresponding to TS14 as obtained at the MP2/AUG-cc-pVDZ level of

A. Yadav, P.C. Mishra / Chemical Physics 405 (2012) 76–88

theory in gas phase is 9.6 kcal/mol. At the next transition state i.e. TS15, a relay action involving the motion of two protons, H1 which is attached to N1 in guanine and H18 which belongs to a water molecule located near N1 at IC13, takes place, and thus a proton gets transferred to the N2 atom attached to the C2 atom of guanine. This step leads to formation of the intermediate complex IC14 from IC13. A tautomer of 8-OHG where the guanine moiety is in the enol form is formed at IC14 and it is complexed with seven water molecules and a bicarbonate anion. The Gibbs barrier energy involved at this reaction step as obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase was found to be 8.2 kcal/mol. At the next reaction step that involves the transition states TS16, the enol form of 8-OHG is converted to the keto form of the same. At this transition state, a relay action involving the motion of two protons, H20 and H41, which in IC14 are attached to the O6 atom of guanine and O42 atom of the water molecule located near O6 respectively, takes place. The Gibbs free barrier energy as obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase for this reaction step was found to be 8.6 kcal/mol. After TS16, the intermediate complex IC15 which is a complex of 8-OHG, a bicarbonate anion and seven water molecules is formed. The last step of the reaction shown in Fig. 2 involves the transition state TS17. At this step also, a relay action involving the motion of two protons, H24 which at IC15 belongs to the OH group bonded to C8, and H26 which at IC15 belongs to a water molecule located near N7, takes place. Thus, at TS17, as the H24 proton moves to get bonded to the O25 atom of the water molecule located near N7, the H26 proton bonded to O25 in IC15 moves to get bonded to N7. The product complex PC2 formed at this reaction step is 8-oxoG complexed with seven water molecules and a bicarbonate anion. The Gibbs barrier energy involved at this reaction step as obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase was found to be 14.4 kcal/mol. In PC2, the CHelpG charges associated with 8-oxoG and bicarbonate are 0.01 and 0.88 respectively. Thus, the -1 charge that was associated with the carbonate moiety in FR2, is mostly associated with the bicarbonate moiety in PC2. 3.3. Reaction between carbonate radical anion and the guanyl radical G(-H2b)
A reaction scheme that involves G(-H2b)
, eight water molecules and CO3. as free reactants (FR3), reactant complex RC3, intermediate complexes ICn (n = 16–21), transition states TSn (n = 18–24) and product complex PC3 which is a complex of 8oxo-G, bicarbonate anion and seven water molecules is shown in Fig. 3. The Gibbs free barrier and released energies involved in this reaction scheme are presented in Table 3. At RC3, the net CHelpG charges associated with G(-H2b)
and CO3. are 0.69 and -1.50 respectively. Thus, RC3 has acquired an additional negative charge 0.50 mainly from the guanyl radical than what was initially associated with the carbonate radical anion. At the first step of the reaction, IC16 is formed from RC3 via TS18 (Fig. 3). In IC16, an oxygen atom of the CO3. group has become bonded to the C8 site of G(-H2b)
. The barrier energy of this step is large negative at all the levels of theory in both gas phase and aqueous media (Fig. 3, Table 3) which shows that the reaction step is barrierless. At TS18, the net CHelpG charges associated with the G(-H2b)
and CO3. moieties are 0.09 and 0.85 respectively. Thus in going from RC3 to TS18, G(-H2b)
gains 0.60 charge mainly from the CO3. group. At the second step of the reaction i.e. in going from IC16 to IC17 via TS19, a CO2 molecule has become detached from the CO3. group while an oxygen atom of this group has become bonded to C8 producing a species that may be denoted by G(-H2b)
-O8. At TS19, the CHelpG charges associated with the CO2 component and G(-H2b)
-O8 moiety are 0.12 and 0.65 while at IC17, the corresponding charges associated with these

85

groups are 0.11 and 0.64 respectively. Thus the G(-H2b)
-O8 moiety is associated with a fairly large amount of negative charge at both TS19 and IC17. The Gibbs free barrier energy corresponding to TS19 as obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase was found to be 2.7 kcal/mol. At the third step of the reaction scheme shown in Fig. 3, IC18 is formed from IC17 via the transition state TS20. At this step, a water molecule located near C8 is dissociated into the O22H23 group and the proton H24. The proton H24 moves to become bonded to O8 attached at C8 while the O22H23 group binds with CO2 forming a bicarbonate group. At IC18, the bicarbonate group carries a negative CHelpG charge of 0.84 which shows that it is in the anionic state. Thus we find that the first three reaction steps are similar in the schemes discussed so for (Figs. 1–3). The Gibbs free barrier energy corresponding to TS20 as obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase is 5.7 kcal/mol. At the fourth step of the reaction shown in Fig. 3, IC19 is formed from IC18 via the transition state TS21. At this step, a relay action involving the movement of two protons i.e. H9 attached to N9 in IC18 and H31 of a water molecule located near N3 is in operation. Consequent to this relay action, a proton is transferred to the N3 site of the guanine moiety while N9 is deprotonated. The Gibbs free barrier energy for this reaction step as obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase was found to be 27.1 kcal/mol. The intermediate complex IC20 is formed from IC19 via the transition state TS22 at the fifth step of the reaction scheme shown in Fig. 3. At the transition state TS22 also, a relay action involving the motion of two protons i.e. H31 attached to N3 in IC19 and H28 of a water molecule located near N3 takes place. This relay action transfers a proton to the deprotonated N2 atom of the amino group of the guanine moiety. The Gibbs free barrier energy for this reaction step as obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase is 5.5 kcal/mol. At the sixth step of the reaction that involves the transition state TS23, IC21 is formed from IC20 by transfer of the proton H8 from the C8 site to the N7 site. The Gibbs free barrier energy for this step as obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase is fairly high i.e. 25.3 kcal/mol. This proton transfer is catalyzed by a water molecule located near N7. At the seventh and last step of the reaction scheme shown Fig. 3 that involves the transition state TS24, the product complex PC3 is formed from the intermediate complex IC21. At this step, a relay action involving movement of two protons is in operation. Thus, at TS24, as the proton H24 bonded to O8 in IC21 moves towards the O35 atom of the water molecule located near N9, the proton H34 which was attached to the water molecule in IC21 moves towards N9, and vice versa. The Gibbs free barrier energy for this reaction step as obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase was found to be 19.8 kcal/mol. The product complex PC3 is a complex of 8-oxoG with bicarbonate and seven water molecules. The total CHelpG charges associated with 8-oxoG and bicarbonate are 0.00 and 0.89 respectively which shows that the latter species is in the anionic state. Thus the -1 charge which was located in FR3 on the carbonate moiety is mostly associated with the bicarbonate moiety in PC3. 3.4. Reaction between carbonate radical anion and guanyl radical G(-H9)
The components of the free reactant FR4 involved in this reaction scheme are G(-H9)
, eight water molecules and CO3. (Fig. 4). This reaction scheme involves the intermediate complexes IC22 to IC25, transition states TS25 to TS29 and leads to formation of the product complex PC4. In this case, RC4 lies slightly below FR4 (by 0.1 kcal/mol). This situation is different from those of Figs. 1–3 where the reactant complexes RC1-RC3 were found to lie appreciably above the free reactants FR1-FR3 respectively.

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A. Yadav, P.C. Mishra / Chemical Physics 405 (2012) 76–88

However, all the calculated barrier energies in this case were also found to be negative at the different levels of theory employed in both gas phase and aqueous media (Fig. 4, Table 4), as found in the other cases (Figs. 1–3, Tables 1–3). Thus the first reaction step is barrierless in this case also (Fig. 4, Table 4). At the reactant complex RC4, the distance between the C8 site of G(-H9)
and O8 (nearest oxygen atom of the CO3. group) is 2.56 Å and the CHelpG charges associated with the CO3. group and G(-H9)
are -1.31 and 0.45 respectively. It shows that in going from FR4 to RC4, the CO3. group has withdrawn 0.31 charge from the other components of FR4. At IC22, an oxygen atom of the CO3. group is bonded to the C8 site of G(-H9)
. At TS25, the CHelpG charges associated with G(-H9)
and the CO3. group were found to be 0.02 and 0.90 respectively. Thus at this transition state, the CO3. group retains most of its initial -1 charge.

At the second step of the reaction that involves the transition stateTS26, a CO2 group is detached from the CO3. moiety and an oxygen atom of this group remains bonded with the C8 site of the guanine moiety (Fig. 4). At TS26, the CHelpG charges associated with the CO2 component and G(-H9)
-O8 moiety, as obtained at the MP2/AUG-cc-pVDZ level in gas phase, are 0.16 and 0.52 respectively. Thus, at the transition state TS26, the G(-H9)
-O8 moiety is associated with about 52% of the initial -1 charge of the carbonate radical anion. The Gibbs free barrier energy corresponding to TS26 as obtained at the MP2/AUG-cc-pVDZ level in gas phase is quite small i.e. 0.8 kcal/mol. One of the complexed water molecules become consumed in the reaction at the third step that involves the transition state TS27 (Fig. 4). Thus, at TS27, the proton H24 of a water molecule moves near the oxygen atom O8 bonded to C8 while the hydroxyl group of same water molecule moves near

Fig. 5. Summary of reaction schemes given in Figs. 1–4 obtained at the single point MP2/AUG-cc-pVDZ level of theory in gas phase. Gibbs free energies (kcal/mol) of the different reactant complexes, intermediate complexes, transition states and product complexes are given with respect to that of RC1. Relative locations of the different sets of free radicals, complexes and transition states are qualitatively in order, but not exactly to scale.

A. Yadav, P.C. Mishra / Chemical Physics 405 (2012) 76–88

the carbon atom C9 of the CO2 group located nearby. At IC24 that is formed after TS27, the proton H24 is bonded to O8 which is attached to C8 while the hydroxyl group becomes bonded to CO2 forming a bicarbonate group. The CHelpG charge associated with the bicarbonate group is 0.89. Thus the bicarbonate group at IC24 is in the anionic state (HCO3 ). The Gibbs free barrier energy corresponding to TS27 as obtained at the MP2/AUG-cc-pVDZ level in gas phase is 10.8 kcal/mol. IC25 is formed from IC24 via the transition state TS28 at the fourth step of the reaction. At this step, the H8 proton is transferred from the C8 site to the N7 site. AT TS28, the H8O25 interatomic distance where O25 belongs to the water molecule located near N7, is 3.53 Å. Thus the water molecule located near N7 would not have a significant role in the transfer of the proton H8 from C8 to N7. The Gibbs free barrier energy corresponding to TS28 was obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase to be 19.7 kcal/mol. At the fifth and final step of the reaction (Fig. 4), the product PC4 is formed from IC25 via the transition state TS29. At this step, a relay action involving the motion of two protons i.e. H24 and H34 is involved. Thus, at TS29, as the proton H24 moves away from the oxygen atom O8 bonded to C8 towards the oxygen atom O35 of a water molecule located near N9, the proton H34 of the same water molecule moves towards N9 and vice versa. The Gibbs free barrier energy for this reaction step was obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase as 19.8 kcal/mol. The product complex PC4 is a complex of 8-oxoG, bicarbonate and seven water molecules. In PC4, the CHelpG charges associated with the 8-oxoG and bicarbonate moieties are 0.00 and 0.89 respectively. Thus the bicarbonate group is in the anionic state in PC4. 3.5. Summary and comparison A summary of all the reaction schemes given in Figs. 1–4 obtained at the MP2/AUG-cc-pVDZ level of theory in gas phase is presented in Fig. 5. In this figure, locations of the various free reactants, transition states, intermediate complexes and product complexes are shown considering their relative Gibbs free energies qualitatively and treating the Gibbs free energy of RC1 as the reference. The following information is revealed by this figure: (i) The relative stabilities of some of the intermediate and product complexes follow the following order (relative Gibbs free energies are given in parentheses): IC21 ( 138.1) > IC25 ( 137.8) > PC1 ( 136.1) > PC2 ( 136.0) > IC7 ( 134.6) > IC15 ( 134.5) > IC5 ( 132.4) > PC3 ( 132.0) > PC4 ( 131.7) > IC14 ( 130.0). Among these complexes, IC21, IC25, IC7 and IC15 have an 8-OHG component each where guanine is in the keto (C6O6) form, and IC5 and IC14 have an 8-OHG component each where guanine is in the enol (C6O6H) form. The product complexes PC1 to PC4 include 8-oxoG where guanine is in the keto (C6O6) form. In those cases where 8-OHG is more stable than 8-oxoG, the former species would be formed preferentially over the latter, and so the reaction steps corresponding to conversion of 8OHG to 8-oxoG would not occur as long as the hydrogen bonds involved in the complexes are maintained. The relative Gibbs free energies of the product complexes show that when the guanyl radical is obtained by deprotonating the N1 or N2a site, the most stable oxidized guanine product would be 8-oxoG but when the guanyl radical is obtained by deprotonating the N2b or N9 site, the most stable oxidized guanine product would be 8-OHG. (ii) There is a good deal of similarity among the reactions involving the four guanyl radicals. The only notable difference found in scheme 4 is that while RC4 lies slightly below

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FR4, RC1, RC2 and RC3 lie appreciably above FR1, FR2 and FR3 respectively. (iii) The results obtained from the present study of reactions between any of the four different guanine radicals and CO3. may be compared in terms of Gibbs free energy with those obtained from the study of corresponding reactions involving NO2
[39]. A significant difference between the reactions of the two radicals with guanyl radical is that all the reactant complexes lie appreciably below the corresponding first transition states (by 12.5–22.4 kcal/mol) in the different reaction schemes involving NO2
[39] while all the reactant complexes (as well as the free reactants) are located significantly above the corresponding first transition states in the different reaction schemes involving CO3. (Figs. 1–5). The unusual situation found in the case of CO3. suggests that it would be a highly efficient reactive oxygen species would oxidize guanyl radical to 8-oxoG. 4. Conclusion We arrive at the following conclusions from the present study: 1. The reactions between CO3. and guanyl radical in aerated aqueous media are catalyzed by specific water molecules present in the hydration shell though their involvement in proton transfer between the different sites of the guanyl radical. One of the complexed water molecules in the hydration shell is consumed and it is involved in conversion of the carbonate radical anion to bicarbonate radical anion. The various proton transfer steps are important for oxidation of guanyl radicals to 8-oxoG. 2. The present study helps explain the roles of aeration, stirring and photoirradation that are employed to effect the reaction in the experimental medium. 3. All the transition states, from the point of view of Gibbs free energy, are located below the corresponding free reactants or the reactant complexes. In this situation, the thermal energy present in the medium would suffice to enable the system to overcome all the reaction barriers. Therefore, except for the energy required for initial activation to populate the reactants RC1, RC2 and RC3 from the corresponding free reactants (no activation is required in the case of RC4), all the reaction steps would effectively be barrierless. It appears that CO3. would be a highly efficient reactive oxygen species to oxidize guanyl radical to 8-oxoG.

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