Insight into Mechanism of Formation of C8 Adducts in Carcinogenic

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J. Phys. Chem. B 2009, 113, 254–259

Insight into Mechanism of Formation of C8 Adducts in Carcinogenic Reactions of Arylnitrenium Ions with Purine Nucleosides Zhong-Zhi Yang,* Shi-Fei Qi, Dong-Xia Zhao, and Li-Dong Gong Chemistry and Chemical Engineering Faculty, Liaoning Normal UniVersity, Dalian 116029, P. R. China ReceiVed: May 9, 2008; ReVised Manuscript ReceiVed: September 15, 2008

For the most important arylnitrenium ion-guanosine C8 adducts in the reactions involving arylamine-initiated carcinogenesis, a detailed mechanism of their formation still remains unclear. In this paper, we employ quantum chemistry methods to explore this issue. Our study indicates that formation of these C8 adducts proceeds directly by additions of arylnitrenium ions to C8 position of nucleoside bases in DNA. The good agreements of theoretical rate constants, pKa value, and NMR chemical shifts of C8 intermediate with experimental data support this theoretical finding. Excitingly, predictions of what adducts can be observed in reactions of arylnitrenium ions with guanine and hypoxanthine are in fair agreement with experimental observations. This study answers an important question, in carcinogenesis researches, of what is the mechanism for formation of C8 adducts. 1. Introduction Arylamines and heterocyclic amines constitute a large class of chemical carcinogens.1-3 A common feature of this class is attack of the arylamine group to DNA. The predominant site of attachment to DNA is the C8 position of guanine. Although these C8 adducts were first structurally characterized over 30 years ago, mechanistic details for forming them have still been uncertain. But it is generally accepted that bioactivation through oxidation and O-esterification is required. The esters so formed have been generally assumed to react via a SN1 pathway with an electrophilic intermediate, the arylnitrenium ion (Figure 1), when the species actually react with guanine.1,4,5 Suggestions, however, have also been presented about a direct interaction at the ester stage, which is essentially a SN2 reaction of guanine at the ester nitrogen.6-8 Only recently have experiments from Novak group conclusively established the nitrenium pathway. They find classic evidence for nucleophile reacting with an intermediate which is not in the rate-limiting step of the reaction.9,10 McClelland and Falvey groups have also corroborated this conclusion through laser flash photolysis (LFP) experiments involving the same nitrenium ions.11-17 As a result, arylnitrenium ions are formed in the hydrolysis of N-acetoxy compound precursors, and these electrophiles do react with guanine derivatives to form the C8 adducts. In some studies, C8 of purine bases is not regarded as the normal position of electrophilic addition.18,19 But direct addition of radicals to the C8 position of guanine is also common.20-22 At present, three mechanisms (shown in Figure 2) of the reaction between arylnitrenium ion and guanosine or deoxyguanosine (dG) have been proposed. Humphreys’ group claimed (HKG Scheme) that they had found the experimental evidence for initial adduct formation at N7 of guanosine. In their experiment, 2-fluorenylnitrenium ion in the presence of 8-methylguanine derivatives generated an unstable species, and its reduction product was obtained and characterized as N7 adduct 1 (Figure 2).19 However, in a similar experiment, Kennedy et al. performed reactions of 4-biphenylylnitrenium and N-acetyl-4-biphenylylni* Corresponding author: e-mail [email protected], Tel (86) 41182159607, Fax (86) 411-82158977.

Figure 1. Arylnitrenium ion and purine bases concerned in this study. The atomic numbering is shown.

trenium ions with 8-methyldeoxyguanine (8-MedG). They also observed an unstable species and a reduction product, but they assigned it as C8 structure 5 (Figure 2). On the basis of a correlation of reactivity with N7 basicity of purine nucleosides, they still suggested N7 as the initial site of attack (KNK Scheme).10 Later, McClelland’s group also studied the irradiation of 2-azidofluorene in an aqueous solution containing 2-deoxyguanosine. They obtained a C8 adduct with good yields which was implicated in the carcinogenicity of 2-aminofluorene. In the process of their experiment, they observed two intermediates. One was the 2-fluorenylnitrenium ion, and another subsequent longer-lived species was assigned the initial C8 adduct 3 (Figure 2). As to the intermediate, they thought that the reaction of the nitrenium ion and dG proceeded directly by an addition at C8.23 Shortly after the McClelland et al. finding, Guengerich and coworkers24 studied formation and reactions of N7 aminoguanosine. Though they merely provided the evidence that N7 adduct to C8 adduct transfer was feasible in the case of N7NH2guanosine, they concluded that the mechanism of N7 to C8 transfer might be viable for arylamines. The above introduction indicates that the question as to initial site of attachment for arylnitrenium ions is uncertain. In investigation of the nitrenium ions, theoretical computations have also played a particularly crucial role. For example, theoretical predictions25-40 mainly by Ford and Cramer et al. helped guide the experiments leading to the first characterization of a substituted arylnitrenium ion possessing a triplet ground

10.1021/jp804128s CCC: $40.75  2009 American Chemical Society Published on Web 12/15/2008

Reactions of Arylnitrenium Ions with Purine Nucleosides

J. Phys. Chem. B, Vol. 113, No. 1, 2009 255

Figure 2. Three experimental schemes on the formation of C8 adduct in the carcinogenic reactions of guanosine or deoxyguanosine with arylnitrenium ions.

state.41,42 The relationships between nitrenium ion properties and their mutagenicity have also been explored by Ford.43 To our knowledge, only Cramer et al.44 first investigated the reaction of guanine with the phenylnitrenium ion theoretically. They studied the conversion of N7 cation adduct to C8 cation adduct in detail and thought that the direct conversion was impossible besides dissociation/recombination of reactants. In order to explain the general failure of observing N7 products experimentally, they suggested an alternative rearrangement of converting a 2-N7 cation adduct to a C8 cation adduct. This work from Cramer et al. establishes a foundation for theoretical study of the carcinogenic reactions between arylnitrenium ions and purine nucleosides. In view of great importance and interest of these carcinogenic biochemical reactions and their controversy mechanisms about arylamines and heterocyclic amines suggested by some experiments, more theoretical investigations of the mechanism of these reactions are needed. In two recent publications, we have successfully provided the direct transfer mechanism between N7 adduct and C8 adduct45 and the formation mechanism46 of the unusual imine N6 adduct in these reactions of arylnitrenium ions with nucleosides. In this paper, in order to more closely mimic the actual carcinogenic reactions, we present a theoretical model for these reactions: the reactions of 4-biphenylylnitrenium ion with four purine bases (shown in Figure 1). In addition, an actual reaction of guanosine with 4-biphenylylnitrenium ion is also studied. 2. Computational Methods The following ab initio and DFT calculations were performed using the GAUSSIAN 03 suite of programs47 on the SGI-3700 Server with 64 CPUs. 2.1. Mechanism on Direct Formation of C8 Adducts. In view of the two configurational character of singlet nitrenium ion, various theoretical levels were used to estimate relative accuracy of these methods (see Table S2 in Supporting Information). In another two papers,45,46 we investigated the similar formation mechanisms of other adducts in the reactions of arylnitrenium ions with purine bases. It is found that MP2 energy calculations based on the geometry optimized by DFT methods are feasible to our studied system. At last, the geometries of reactants, transition states (TSs), and intermediates

considered in the model were optimized using the B3LYP48,49/ 6-31G(d), B3LYP/6-31G(d,p), B3LYP/6-31+G(d,p), and B3LYP/ cc-pVDZ50 methods. The solvent effect on reaction mechanism was also evaluated at the optimized geometries of gas phase. Utilizing the geometries obtained at the B3LYP/6-31+G(d,p) level in gas phase, the aqueous single-point energies have been obtained at MP251/6-311+G(2d,p) level. For the actual reaction of guanosine with 4-biphenylylnitrenium ion, the mechanism of formation of C8 adduct was explored at the B3LYP/6-31G(d) level. All species concerned in this study were characterized as minima or transition states by vibrational frequency calculations at the same level of theory as the geometry optimizations were done. In addition, every transition state was analyzed by calculating an intrinsic reaction coordinate (IRC).52,53 Frequency calculations at 298 K and 101.325 kPa using the same level of optimized geometry gave the zero-point, thermal, and Gibbs free energy corrections. All single-point energies were corrected by zero-point energies (ZPEs) which were unscaled. All above solution phase calculations were completed with the IEF-PCM54 model as implemented in GAUSSIAN 03. 2.2. Rate Constant. In conventional transition state theory (TST), the activation rate constant is expressed as

k ) (kBT/h)(QTS* /QRC) exp(-V0 /RT)

(1)

where T is temperature, kB is the Boltzmann constant, h is the Planck constant, R is the gas constant, QTS* is the partition function of the TS state, QRC is the partition function of the reactants (RC) state, and V0 ) E0TS - E0RC. An equivalent form of eq 1 is

k ) (kBT/h) exp(-∆G*/RT)

(2)

where ∆G* is the free energy of activation and ∆G* ) GTS GRC. In this article, eq 2 was used to obtain the rate constants of four reactions in the theoretical model. The computational details of N1 pKa value and NMR chemical shifts for C8 intermediate in the reaction of guanine with 4-biphenylylnitrenium ion can be found in the Supporting Information.

256 J. Phys. Chem. B, Vol. 113, No. 1, 2009 3. Results and Discussion 3.1. Mechanism of Direct Formation of C8 Adduct. 3.1.1. Mechanism. In our model, we explore the reactions of 4-biphenylylnitrenium ion with four purine basessguanine (reaction 1), hypoxanthine (reaction 2), xanthine (reaction 3), and adenine (reaction 4)sin gas phase and aqueous solution. An important result for these model reactions is that four transition states (TSs) related to the formation of C8 adducts have been properly located. It is noted that such transition states cannot be found in simpler model reactions of these four purine bases with phenylnitrenium ion when DFT and MP2 methods are used (see Table S1 in Supporting Information). Hence, the reactions of purine bases with phenylnitrenium ion are not an appropriate model to explore the formations of the C8 adducts. The most relevant geometrical parameters of the four TSs located in our model are summarized in Figure 3a and Figure S2 (three TSs in model reactions of 4-biphenylylnitrenium ion with hypoxanthine, xanthine, and adenine) and Table S4 of the Supporting Information. The key parameters of distances between N10 and C8 for the four transition states are respectively 2.204, 2.014, 2.099, and 2.054 Å at the B3LYP/cc-pVDZ level and 2.106, 1.971, 2.033, and 2.005 Å at the B3LYP/631+G(d,p) level in gas phase. It is apparent that the largest distance between C8 and N10 appears in the TS for reaction 1 and then in the TS for reaction 3 at all employed computational levels. Another important geometrical parameter for the four TSs is the distance between H atom of nitrenium ion and N7 atom in purine base, as is shown in Figure S2, which are longer compared with the distances between C8 and N10 atoms in four transition states at all employed computational levels, such as B3LYP/6-31+G(d,p) and B3LYP/cc-pVDZ levels. For the four TSs, the corresponding IRC calculations indicate that they do connect with four C8 intermediates in one direction, but we cannot confirm whether they connect with the separated reactants in another direction. We think that the electrostatic interaction between H atoms of 4-biphenylylnitrenium ion and N7 atoms, O6 atoms of purine bases is strong, which causes that the complete reaction paths of the four reactions in the theoretical model cannot be passed through. In actual biological reactions, arylnitrenium ions often attack purine or deoxypurine nucleosides. Thereby, we also explore an actual reaction of 4-biphenylylnitrenium ion and guanosine. At the B3LYP/6-31G(d) level, the transition state (Figure 3c) has been located with careful search, and the analysis of the IRC calculations clearly indicates that the TSs do connect the separated reactants through an ion-molecule complex (Figure 3b). As a consequence, the mechanism (shown in Figure 4) of direct formation of C8 adducts is established; that is, the reacting molecules initially form an ion-molecule complex, then the complex crosses a transition state to arrive at C8 intermediate, and then it converts to the final C8 adduct through a C8 deprotonation process. Distinctly, it is noted that the C8 intermediate corresponds to the intermediate 3 in Figure 2 indicated by the McClelland experiment.23 3.1.2. Thermodynamics. For the sake of time-savings, we adopt the four model reactions to compute their thermodynamical quantities at different levels of theory. The relative energy and relative Gibbs free energy for the TS mentioned in the following refer to those of separated reactants in each reaction. As indicated in Table 1, calculations at several levels of theory were performed in order to check the relative accuracies of the methods in both gas phase and aqueous solution. Different correlation calculations, such as B3LYP, B3PW91, and MP2 calculations, have an effect on the calculated values of

Yang et al.

Figure 3. Geometry of TS (a) for the reaction of 4-biphenylylnitrenium ion with guanine at B3LYP/6-31+G(d,p) and (B3LYP/cc-pVDZ) levels of theor, and geometries of ion-molecule complex (b) and TS (c) in the actual reaction of guanosine with 4-biphenylylnitrenium ion at the B3LYP/6-31G(d) level.

thermodynamical quantities of the reaction system. Table 1 lists the relative energies (relative to reactants) and relative Gibbs free energies (relative to reactants) of the four transition states in the four reactions of the theoretical model at several levels of theory in both gas and aqueous phases. The gas-phase relative energies of TSs are -11.1, -2.2, -2.3, and -1.6 kcal mol-1 at the B3LYP/6-31+G(d,p)//B3LYP/6-31+G(d,p) level of theory and -16.0, -7.3, -7.5, and -7.0 kcal/mol at the

Reactions of Arylnitrenium Ions with Purine Nucleosides

Figure 4. Gas (solid lines) and aqueous (dashed lines) schematic potential energy profiles for the actual reactions of guanosine with 4-biphenylylnitrenium ion.

B3PW91/cc-pVDZ//B3LYP/cc-pVDZ level of theory. It indicates that B3PW91 and B3LYP methods give negative energy of TS relative to reactants in every reaction of the theoretical model, and the increasing and decreasing sequence of relative energies of these reactions is similar in the two methods. But the relative sequence of energies for TSs between these reactions obtained at MP2/6-31+G(d,p)//B3LYP/6-31G(d) level is not in agreement with the results of MP2/6-311+G(2d,p)//B3LYP/631+G(d,p) calculations. For instance, the relative energy for TS in reaction 2 in gas is second low at the MP2/6-311+G(2d,p) level. However, it is third low according to the results of MP2/ 6-31+G(d,p) calculations. The differences of geometries obtained using two methods and the basis sets may all have effects on increasing and decreasing order of these four relative energies. In general, the values of relative energies for TSs obtained from MP2/6-311+G(2d,p)//B3LYP/6-31+G(d,p) calculations are lower than those from other methods. According to above results, we can obviously see that MP2 and B3PW91 methods provide comparable results to each other, and the B3LYP method overestimates energies of TSs compared with those mentioned above. The quantity of interest is the free energy since it controls the rate of reaction. In the following, the activation energies of TSs will be discussed. Table 1 also lists the activation energies of the four transition states in gas phase and aqueous solution at several levels of theory. In gas phase, the activation energies of four TSs are all negative using all correlation methods of computation. Clearly, the activation energies of TSs obtained using B3LYP methods are overestimated compared with those obtained above from MP2 and B3PW91 calculations. All methods show that the activation energy of TS in the reaction of guanine with 4-biphenylylnitrenium ion is the lowest in the four gas reactions. As indicated in Table 1, interestingly, inclusion of bulk solvent effect increases the activation energies for the four transition states compared with that obtained in gas phase. The aqueous activation energies for all TSs increase by about 14 kcal/mol compared with corresponding gas values and become higher than the values of reactants in all methods. Additionally, the charges of C8 site in four purine bases (given in Table 2) were obtained by using the ABEEM method55-60 and the HF/6-31G(d) method, and the results are analogous to each other for two methods. For four electrophilic reactions of the model, the charges at C8 positions of four bases relate to their aqueous activation energies of transition states. For instance, the C8 charge, 0.504e (ABEEM), for guanine corresponds to aqueous activation energy, 7.2 kcal/mol, of its reaction with 4-biphenylylnitrenium ion. However, these two quantities

J. Phys. Chem. B, Vol. 113, No. 1, 2009 257 are 0.525e (ABEEM) and 12.6 kcal/mol in the case of adenine, respectively. Such results coincide with general understanding on electrophilic reaction. In the four reactions of the model, the reaction of 4-biphenylylnitrenium ion with guanine (reaction 1) in aqueous solution gives, especially using the IEFPCMMP2/6-311+G(2d,p)//B3LYP/6-31+G(d,p) method, the lowest activation energy (7.2 kcal mol-1). Correspondingly, the rate constant for the reaction of guanosine with 4-biphenylylnitrenium ion is also largest in the four experimental reactions.10 The agreement between theory and experiment hints that the theoretical models and mechanism given in this article are reasonable. 3.2. Rate Constant. Using the relative Gibbs free energies in Table 1 obtained at the IEFPCM-MP2/6-311+G(2d,p) level in aqueous solution, the rate constants (summarized in Table 2) of the four reactions in the model were calculated. Generally, the relative values of four reactions agree with the experimental data though our results are lower than the experimental data. For the reaction of adenine with 4-biphenylylnitrenium ion, our computations show that the rate constant is comparable with reactions 2. However, Kennedy et al.10 did not obtain a C8 adduct; they found an unusual benzene imine adduct at N6 of adenosine. But they provided an upper limit of the rate constant of the formed C8 adduct according to the inability to detect the C8 adduct. They thought that the rate constant was quite low compared with other three reactions in their experiment. Also, on the basis of a correlation of reactivity with N7 basicity of purine nucleosides, Kennedy et al. still suggested N7 as the initial site of attack. But later, the correlation broke down when imidazoles were included by McClelland et al.13 In the next part, we will provide a reasonable interpretation on the experimental observation in reaction of arylnitrenium ion with adenosine. Considering the agreement between theoretical rate constants and Novak experiment, the mechanism of direct formation on C8 adduct suggested in this work should be reasonable. 3.3. NMR Chemical Shift and N1 pKa Value of C8 Intermediate. The 13C NMR chemical shifts of C8 intermediate for the reaction guanine and 4-biphenylylnitrenium ion were obtained at the HF/6-31G(d)//B3LYP/6-31G(d) level and partly listed in Table 3. These data have been compared with the experimental 13C NMR chemical shifts of C8 adduct for the reaction of N-acetyl-4-aminobiphenylnitrenium ion with 8-methylguanosine.10 Theoretical chemical shifts are in good agreement with the corresponding experimental values except for C6 atom. As calculated using the HF/6-31G(d)//B3LYP/6-31G(d) method, the chemical shift of C6 atom is 150.1 ppm. However, the experimental value is 170.3 ppm, and it is the maximum value among these carbon atoms. The charges of bonds and lone-pair electrons are again partitioned into the corresponding atoms by the ABEEM scheme. These data are listed in Table 3. We can see that global ABEEM charges of different carbons can well reflect the changes of NMR chemical shifts at corresponding positions. Especially for the C6 atom, ABEEM charge, 0.502e, is also maximum in all these carbon atoms. Compared with the values of chemical shifts at the positions, there is a good agreement between our calculation and experiment. In conclusion, ABEEM charges can correctly reflect the changes of NMR chemical shifts, at least for the carbon atoms. In an experiment,23 McClelland et al. obtained the pKa value of N1 position in the C8 intermediate for the reaction of 2-fluorenylnitrenium ion with 2′-deoxyguanosine, and they thought that the C8 deprotonated process was a rate-limiting step. Here, we compute the pKa value of the N1 position in the

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TABLE 1: Calculated Relative Energies (in kcal/mol) and Relative Gibbs Free Energies (in kcal/mol) for Transition States in the Reactions of Four Purine Bases with 4-Biphenylylnitrenium Ion in Vacuo and Aqueous Phasea method

guanine b

vacuo

B3LYP/6-31G(d)//A B3LYP/6-31G(d,p)//Be B3LYP/6-31+G(d,p)//Cf B3LYP/cc-pVDZ//Dg B3PW91/cc-pVDZ//D MP2/6-31+G(d,p)//A MP2/6-311+G(2d,p)//C B3LYP/6-31G(d)//A B3LYP/6-31G(d,p)//B B3LYP/6-31+G(d,p)//C B3LYP/cc-pVDZ//D B3PW91/cc-pVDZ//D MP2/6-311+G(2d,p)//C

aqueous

-14.7 (-3.0) -14.7 (-2.9) -11.1 (0.9) -15.4 (-3.6) -16.0 (-4.3) -17.2 (-5.5) -20.0 (-8.1) -0.3 (12.1) -0.5 (12.1) 3.3 (16.0) -1.5 (11.0) -2.1 (10.1) -5.3 (7.2) c

d

hypoxanthine

xanthine

adenine

-5.7 (6.3) -5.5 (6.4) -2.2 (9.8) -6.2 (5.8) -7.3 (4.7) -11.8 (0.2) -13.1 (-1.1) 6.2 (18.8) 6.2 (18.8) 10.0 (22.7) 5.3 (17.9) 4.2 (16.8) -1.6 (11.1)

-6.0 (6.0) -5.8 (6.2) -2.3 (9.8) -6.9 (5.5) -7.5 (4.6) -11.1 (0.9) -13.4 (-1.3) 3.6 (16.7) 3.6 (16.6) 7.4 (20.6) 2.6 (15.6) 1.7 (14.8) -4.3 (8.7)

-5.4 (6.5) -5.2 (6.9) -1.6 (11.6) -6.0 (6.0) -7.0 (5.0) -10.2 (1.7) -11.9 (1.3) 5.4 (18.2) 5.4 (18.5) 8.9 (23.3) 4.3 (17.2) 3.4 (16.3) -1.6 (12.6)

a All energies and Gibbs free energies are relative to those of separated reactants in the reactions. b “A” denotes geometry optimized at the B3LYP/6-31G(d) level in gas. c Relative energy. d Relative Gibbs free energy. e “B” denotes geometry optimized at B3LYP/6-31G(d,p) level in gas. f “C” denotes geometry optimized at B3LYP/6-31+G(d,p) level in gas. g “D” denotes geometry optimized at B3LYP/cc-pVDZ level in gas.

TABLE 2: Summary of Experimental and Present Theoretical Values of Rate Constants (M-1 dm3 s-1) Evaluated from the Reactions of Four Purine Bases with 4-Biphenylylnitrenium Ion in the Model Using the IEFPCM-MP2/6-311+G(2d,p)//B3LYP/6-31+G(d,p) Method charge (e) C8 site base guanine hypoxanthine xanthine adenine

theoretical Knuc

experimental Knuca

guanine

ABEEM HFb 4-biphenylyl nucleoside 4-biphenylyl 0.504 0.506 0.503 0.525

0.517 0.523 0.510 0.544

guanosine 1.2 × 10 inosine 5.9 × 107 xanthosine 3.0 × 107 adenosine