J. Phys. Chem. B 2009, 113, 5645–5652
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Effect of N7-Protonated Purine Nucleosides on Formation of C8 Adducts in Carcinogenic Reactions of Arylnitrenium Ions with Purine Nucleosides: A Quantum Chemistry Study Shi-Fei Qi,*,† Xiao-Nan Wang,† Zhong-Zhi Yang,‡ and Xiao-Hong Xu† School of Chemistry and Materials Science, Shanxi Normal UniVersity, Linfen 041004, People’s Republic of China, and Chemistry and Chemical Engineering Faculty, Liaoning Normal UniVersity, Dalian 116029, People’s Republic of China ReceiVed: December 19, 2008; ReVised Manuscript ReceiVed: February 28, 2009
A theoretical analysis of the reactions of four N7-protonated purine bases with phenylnitrenium and 4-biphenylylnitrenium ions, both in the gas phase and in aqueous solution, is studied to elucidate the effect of protonated purines on the formation of C8 adducts in a series of complicated carcinogenic reactions. Based on this analysis, four important conclusions are drawn. (i) The mechanism of C8 adduct formation by the addition of arylnitrenium ions directly to C8 sites of nucleotide bases in DNA is still supported. (ii) The N7 protonation of purine bases will lower the rate constants of these carcinogenic reactions, which agrees with observations and proves the experimental presumption. (iii) More complicated arylnitrenium ions have more of an effect on the reduction in the rate constants of these reactions involving N7-protonated purine bases. (iv) The rate constant of the C8 deprotonation process becomes larger when N7-protonated purine bases are involved in these carcinogenic reactions. Introduction Arylamines and heterocyclic amines constitute a large class of chemical carcinogens.1 A common feature of this class is their ability to transfer the arylamine group to DNA. The predominant site of attachment of this group to DNA is on the C8 position of guanine. Although first structurally characterized over 30 years ago, mechanistic details for forming these C8 adducts are still unclear. Recent experiments by the Novak group have now conclusively established the nitrenium pathway and show classic evidence for a nucleophile reacting with an intermediate, which is not the rate-limiting step of the reaction.4 The groups of McClelland and Falvey have also corroborated this conclusion through laser flash photolysis (LFP) experiments involving the same nitrenium ions.5,6 It is now apparent that ground-state arylnitrenium ions (Figure 1) are formed during the ground-state hydrolysis of ester precursors, and it is these electrophiles that react with guanine derivatives to form the C8 adducts. However, there is a problem with this mechanism. According to some reports, C8 does not appear to be the normal position of electrophilic addition in purine bases.7,8 As is shown in Scheme 1, three experimental mechanisms of the reaction between arylnitrenium ion and guanosine or deoxyguanosine (dG) have been proposed: the Humphreys scheme,8 the Navak scheme,9 and the McClelland scheme.10 Shortly after the McClelland et al. findings were published, Guengerich and his co-workers11 studied the formation and reactions of N7 aminoguanosine and concluded that the Humphreys and Navak schemes cannot be excluded. For this reason, the actual detailed mechanism of C8 adduct formation by the reaction of nitrenium ions with purine nucleosides remains unclear at present. Recently, we have performed theoretical studies on the direct transfer mechanism12 between the N7 adduct and the C8 adduct * Corresponding author. E-mail address:
[email protected]. † Shanxi Normal University. ‡ Liaoning Normal University.
Figure 1. Arylnitrenium ion and purine bases concerned in this article. The atomic numbering is also shown.
(the key steps involved in the Humphreys and Navak schemes), on the formation mechanism13 of an unusual imine N6 adduct, as well as on the direct formation mechanism14 of the C8 adduct (the McClelland scheme) in the reactions of arylnitrenium ions with nucleosides. These theoretical studies provide detailed theoretical insights into the formation of these important C8 adducts and have confirmed that their formation proceeds directly by the addition of arylnitrenium ions to the C8 position of nucleoside bases in DNA. On the other hand, it is also well-known that the pKa values of DNA nucleobases are in the range of pKa < 4 or pKa > 9, which permits Watson-Crick pairing between the neutral forms of the complementary bases. However, in some cases, the pKa values of nucleobases are shifted into the near-neutral pH range.15-20 Structural studies have revealed that, in a large number of RNAs and DNAs, adenine and cytosine pKa values arequitedifferentfromthevaluesoftheirfreemononucleobases.21-27 For example, NMR has revealed an unusual pKa of 6.5 at 25 °C for an adenine that is close to the cleavage site in a W/lSNlabeled lead-dependent ribozyme.17,28 There can also be a preferential strengthening of base pairing in triplex formation when the participating modified nucleobase has an increased pKa (6.8) relative to dC (4.3), which gives better pKa matching.29 This results in more effective protonation at physiological pH, leading to an improved hydrogen bonding. Heptameric isosequential ssDNA and ssRNA molecules studied using pH
10.1021/jp811262x CCC: $40.75 2009 American Chemical Society Published on Web 03/30/2009
5646 J. Phys. Chem. B, Vol. 113, No. 16, 2009
Qi et al.
SCHEME 1
titration and NMR methods have indicated significant pKa perturbation of nucleobases as an intrinsic property of the sequence context in DNA and RNA. The perturbation of the pKa of a particular nucleobase appears to be due to a sequencedependent nearest-neighbor electrostatic effect; consequently, protonated nucleobases can exist at physiological pH in some RNAs and DNAs.30 As mentioned above, the pKa values of nucleobases can be shifted into the near-neutral pH range in some cases. The purpose of our present study is to shed light on the complicated carcinogenic reactions of arylnitrenium ions with DNA. If the pKa values of a site in some nucleobases of DNA can be perturbed at the near-neutral pH range, or if these nucleobases are in acidic solutions at low pH value, then protonations could occur in these nucleobases. In fact, some experiments on the reactions of arlynitrenium ions and nucleosides in acidic solutions9,10 have shown that the rate constants of these reactions become smaller compared with those obtained at physiological pH. However, these experiments did not present a clear explanation of this phenomenon but only presumed that nucleosides changed into their protonated forms. In contrast, some relevant studies have revealed that the N7 site of four purine bases could first be protonated; thus, N7 may be the most important site for the formation of C8 adducts, in agreement with our previous theoretical studies.12-14 Therefore, in this article, we will theoretically discuss in detail the effect of N7protonated nucleobases on the formation of C8 adducts in these carcinogenic reactions. This article is organized as follows: First, the pKa values of N7 sites in the four purine bases are evaluated. Then, the reactions of these N7-protonated purine bases with phenylnitrenium ions are theoretically explored. Third, the solvent effect on these reactions is presented by the polarizable continuum dielectric method (PCM). Finally, the effect of N7-protonated
purine bases on the C8 deprotonation process in these carcinogenic reactions is also revealed. 2. Computational Details The following ab initio and DFT calculations were performed using the GAUSSIAN 03 suite of programs.31 pKa Value. The estimation of the theoretical pKa value usually involves a thermodynamic cycle, such as is shown in Figure 2. In this cycle, the free energy of the dissociation in solution, ∆G°, s is related to the pKa value,
pKa ) ∆G°s /2.303RT
(1)
This is defined as indicated in Figure 2.
∆G°s ) ∆G°[A-(aq)] + ∆G°[H+(aq)] - ∆G°[HA(aq)] + ∆G1corr atm(g)f1 M(g)(2) ∆Gcorr 1 atm(g)f1 M(g) is the free energy correction term corresponding to the free energy change accompanied by the reversible state change of 1 mol of gas from 1 atm (24.47 L mol-1) to 1 M (1 mol L-1). The ideal-gas law yields
∆G1corr atm(g)f1 M(g) ) RT ln(24.46)
(3)
At 298 K, this has a value of 1.89 kcal/mol. The standard free energy ∆G° of each species in water can be broken down into those of the corresponding reactions in the gas phase, ∆Gg, plus the free energy of solvation, ∆Gs:
∆G°[HA(aq)] ) ∆Gg[HA(g)] + ∆Gs(HA) ∆G°[A-(aq)] ) ∆Gg[A-(g)] + ∆Gs(A-)
(4)
∆G°[H+(aq)] ) ∆Gg[H+(g) + ∆Gs(H+)
Figure 2. Thermodynamic cycle used to evaluate the pKa value by the absolute method.
The free energy ∆Gg in the gas phase can be obtained from quantum mechanical calculations. The free energy of solvation, ∆Gs, can be calculated by discrete solvent models or by
Reactions of Arylnitrenium Ions with Purine Nucleosides
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continuum models. In this article, we have adopted the IEFPCM model to calculate the variation employing the corresponding gas geometry. Taken from the literature,32,33 the Gibbs free energy of a proton is ∆Gg[H+(g)] ) -6.28 kcal/mol. The free energy of solvation of a proton in water, ∆Gs(H+), is very important for obtaining an accurate value of pKa. We have used Tissandier et al.’s value of -265.9 kcal/mol.34 In this article, the pKa values of N7 sites in the following four purine bases: guanine, hypoxanthine, xanthine, and adenine, were estimated by the computational approach described above. Reaction Mechanism. According to previous studies of similar reaction systems,12-14 the geometries of reactants, transition states (TSs), and C8 intermediates considered in the present study, have been optimized at the B3LYP35/6-31G(d,p) and the B3LYP/6-31+G(d,p) theoretical levels. All stationary points have been characterized as minima or transition states by vibrational frequency calculations at the same level of theory as that of the geometry optimizations. In addition, all transition states have also been analyzed, either by calculating an intrinsic reaction coordinate (IRC) 36,37 or by visual inspection of the transition vector with the correct vibration.38 Frequency calculations at 298 K and 101.325 kPa, using the same level of geometry optimization, gave the zero-point, thermal, and Gibbs free energy corrections. The solvent effect on this reaction mechanism is also important. Considering the convergence problems associated with optimization in solution, the solvent phase calculations were completed at the optimized geometries of the gas phase, using the self-consistent reaction field (SCRF)39,40 method. Tomasi found that the free energy of solvation could be accurately computed using the optimized geometry in Vacuo at the same level of theory.41 In addition, utilizing the geometries obtained at the B3LYP/6-31+G(d,p) level in the gas phase, single-point energies were calculated at the MP242/6-311+G(2d,p) level in aqueous solution. All solution phase calculations were completed with the IEF-PCM43 model, as implemented in GAUSSIAN 03. Rate Constant. In conventional transition state theory (TST), the activation rate constant is expressed as
k ) (kBT/h)(QTS* /QRC) exp(-V0 /RT)
(5)
where T is temperature, kB is the Boltzmann constant, h is the Planck constant, R is the gas constant, QTS* is a partition function of the TS, QRC is the partition function of the reactant (RC) state, and V0 ) E0TS - E0RC. An equivalent form of eq 5 is
k ) (kBT/h) exp(-∆G*/RT)
(6)
where ∆G* is the free energy of activation and ∆G* ) GTS GRC. In this article, eq 6 was used to obtain the rate constants of the four reactions. 3. Results and Discussion 3.1. pKa Value of the N7 Site in Four Purine Bases. Accurate estimates of the acidities and basicities of nucleic bases are essential for a more complete understanding of fundamental biological issues. It is now recognized that DNA and RNA bases are modulated by hydrogen bonding and that hydrogen bonding, in turn, is dependent on the intrinsic acidity and basicity of acceptor and donor groups on the nucleic bases.44 Furthermore, elucidating the intrinsic reactivity of nucleic bases can improve the understanding of key biosynthetic mechanisms for which nucleobases are substrates.45
TABLE 1: Theoretical and Experimental pKa Values of N7 Sites for Four Nucleic Bases pKa for nucleic bases base guanine hypoxanthine xanthine adenine
site N7 N7 N7 N7
theory b
2.9 2.2b -1.5b (1.2)c 0.1b
experimenta 3.3 2.0 0.8
