Nucleic bases alkylation with acrylonitrile and cyanoethylene oxide: A

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Nucleic bases alkylation with acrylonitrile and cyanoethylene oxide: A computational study Martin Gladovi#, Eva Španinger, and Urban Bren Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.7b00268 • Publication Date (Web): 22 Dec 2017 Downloaded from http://pubs.acs.org on December 24, 2017

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Chemical Research in Toxicology

Nucleic bases alkylation with acrylonitrile and cyanoethylene oxide: A computational study Martin Gladovic,†,‡ Eva Spaninger,† and Urban Bren∗,†,¶ †Faculty of Chemistry and Chemical Technology, University of Maribor, Smetanova 17, SI-2000 Maribor, Slovenia ‡Faculty of Chemistry and Chemical Technology, University of Ljubljana, Večna pot 113, SI-1000 Ljubljana, Slovenia ¶National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia E-mail: [email protected] Phone: +386 2 2294 421

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Abstract Acrylonitrile (AN) is widely used in the manufacture of resins, plastics and polymers, where workers are exposed to it during its production, transportation and application. After intake a portion of AN is converted to cyanoethylene oxide (CEO) by cytochrome P450 2E1. Both AN and CEO represent possible chemical carcinogens leading to DNA damage mainly in the form of the major 7-(2-oxoethyl)deoxyguanosine adduct. A kinetic model for its formation was devised and a corresponding second-order rate constant obtained from the experimental data on the reaction with CEO. Then a series of ab initio, density functional theory and semiempirical calculations of activation free energies was performed on the alkylation of nucleic bases with both CEO and AN. The combination of Hartree-Fock level of theory with the flexible 6-311++G(d,p) basis set and Langevin dipoles implicit solvation model gave the best agreement with the experimental activation barrier. It also predicted relative reactivities of all four nucleobases that are in agreement with the experimentally reported adduct yields. Moreover, this combination predicted higher reactivity of CEO than AN with all four nucleobases corroborating the experimental hypothesis that SN 2 substitution of CEO rather than direct Michael addition of AN is responsible for the genotoxic properties of AN. Last but not least, in a broader context this paper points to the applicability of quantum-chemical methods to the studies of carcinogenesis.

Introduction Acrylonitrile (AN) is a colorless volatile liquid used in the manufacture of resins, plastics and polymers, where humans can be exposed to it via air or water during its production, transportation and application. 1 An analysis performed in 2014 by Merchant Research & Consulting Ltd. reported a yearly production of 5.7 million tonnes of AN worldwide with a predicted global production to reach 7 million tonnes by 2017. 2 Acrylonitrile AN is carcinogenic in rats 3,4 and suspectedly carcinogenic in humans. 5–8 The human carcinogenicity of AN is somewhat controversial, with some studies reporting its role in the development of 3



lung, colon and stomach cancers, 9,10 while others report no significant association between AN and human carcinogenicity. 11–15 After intake the major route of AN metabolism involves direct conjugation via Michael addition to glutathione. 16 However, in the presence of oxygen and NADPH a portion of AN is converted to cyanoethylene oxide (CEO) via the epoxidation by cytochrome P450 2E1. 17–19 CEO is a mutagenic and suspectedly carcinogenic compound. In this article we present the calculation of the second-order rate constant and the corresponding activation free energy for alkylation of guanine with CEO from the available experimental data. Moreover, we also calculated the activation barriers for the reactions of CEO and AN with all four DNA bases leading to the most common covalent adducts observed in vitro and in vivo at several ab initio, density functional theory (DFT) and semi-empirical molecular-orbital (MO) theory levels. Solvation effects were modeled using the self-consistent reaction field (SCRF) method of Miertuš et al. 20 and with the Langevin dipoles (LD) model of Florian and Warshel. 21 For semi-empirical MO methods, the AM1SM1 and PM3-SM3 models were applied. 22–24 The experimental activation free energy was used as a measure to determine the most reliable combinations of QM method, basis set and solvation model. Using these theory levels, we finally compared the calculated activation barriers for direct alkylation of nucleobases with AN via Michael addition versus the indirect alkylation with its metabolite CEO via substitution, which was experimentally determined to be much more reactive experimentally. 25 Similar computational studies of DNA alkylation with small carcinogenic compounds have been carried out previously. Kranjc and Mavri performed a study of guanine alkylation with ethylene oxide. 26 Bren and coworkers studied the alkylation of guanine with chloroethylene oxide 27 and aflatoxin B1 . 28 Kržan and Mavri performed a QM study of the alkylation of guanine by styrene. 29 Galeša et al. examined the alkylations with acrylamide. 30 Mavri simulated DNA alkylation with propylene oxide. 31 Lajovic et al. finally performed an extensive computational and experimental investigation of urethane-induced carcinogenesis. 32 4


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The proposed mechanism for the formation of the main 7-(2-oxoethyl)deoxyguanosine adduct is depicted in Scheme 1. The reaction proceeds via SN 2 substitution. The achiral terminal epoxide carbon of CEO attacks the nucleophilic endocyclic nitrogen N7 of guanine. An unstable zwitterionic intermediate is formed, which quickly decomposes through the elimination of the cyano group. The SN 2 substitution represents the rate-limiting step of the reaction. Similarly, the SN 2 substitutions occur at nucleophilic nitrogens of other nucleic bases as well; they are depicted in Schemes 2 - 4. It was also experimentally determined that in the Michael additions of AN the cyano group does not leave during the reaction, and instead remains as a part of the final adduct. See Supporting Material Schemes S1 - S4 for the proposed mechanisms for the reactions with AN.

Methods Calculation of the rate constant for CEO alkylation of guanine from experimental data Due to the intrinsic reactivity of CEO in aqueous solutions, the rate constant for the reaction between CEO and guanine leading to the main adduct 7-(2-oxoethyl)guanine has not yet been directly measured. However, the concentration of this adduct as a function of time was determined and reported by Guengerich and coworkers. 25 We devised a kinetic model for CEO alkylation of guanine and fitted it on these experimental data to obtain the corresponding reactionrate constant. The adduct concentrations for guanine alkylation with AN are also available, 25 but could not be further analyzed due to insufficient data resolution.

Computational methods All calculations were performed at the National Institute of Chemistry in Ljubljana on the CROW cluster. 33,34 To obtain the Born-Oppenheimer hypersurfaces and the activation

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energies for the reactions between AN or CEO and nucleobases, we performed a series of ab initio, DFT, and semi-empirical MO simulations using the Gaussian 09 program package. 35 The ab initio calculations were executed at the Hartree-Fock (HF) and the Møller-Plesset perturbation theory of the second order (MP2) levels of theory in combination with flexible 6-31G(d), 6-31+G(d,p) and 6-311++G(d,p) basis sets. In addition, we considered the DFT method B3LYP consisting of Becke’s exchange functional 36 combined with the correlation functional of Lee, Yang and Parr. 37 We also considered the M06-2X global hybrid functional of Zhao and Truhlar 38 and MPW1K, a modified version of the Perdew-Wang gradientcorrected exchange functional developed by the Truhlar research group. 39 Again, the flexible 6-31G(d), 6-31+G(d,p) and 6-311++G(d,p) basis sets were used also in combination with the DFT methods. Finally, two semi-empirical methods, AM1 and PM3, were applied. These are favorable for mixed quantum-mechanical/molecular mechanical (QM/MM) description and thermal averaging due to their low computational cost. To accurately calculate the activation energy, we first had to find a structure close to the transition state of the reaction using a relaxed potential-energy surface scan, as described previously by Lajovic et al. 32 The structure with the highest energy was used as a starting point for the Berny algorithm 40 which produced the optimized transition state (TS) structure. Vibrational analysis in the harmonic approximation was performed on the TS structure to ensure that there was only one imaginary frequency present and that its vibrational mode coincided with the breaking of the existing epoxidic C-O bond and the formation of the new C-N bond between CEO and the corresponding nucleobase. The reactant state was obtained by increasing the TS distance between the reactive centers by approximately 0.2 Å followed by the energy minimization procedure, during which the reacting fragments moved further apart. Again, vibrational analysis on the resulting structure was performed to ensure that only real frequencies were present, thus indicating that a true minimum was located. Finally, the activation energy of the SN 2 reaction in vacuo was calculated from the energy 6


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difference between the TS and the reactant state. Analogous procedure was used for the Michael addition of AN on the corresponding nucleobases. The hydrogen atom at the N3 position in the most stable (keto) thymine tautomer acts as a steric barrier for the studied reactions and at the same time reduces the nucleophilic character of this nitrogen atom. Therefore, the second-most stable (enol) tautomeric form of thymine 41,42 was chosen for obtaining the TS and reactant state geometries (Scheme 4). The calculated activation energies, solvation and activation free energies were corrected for this tautomerization with respect to the most stable thymine tautomer. Solvation free energies of reactants and TS were calculated with the SCRF method of Miertuš et al. 20 and with the LD model of Florian and Warshel. 21 The SCRF method included in the Gaussian 09 program package was applied to all ab initio and DFT levels using both the standard variational approach and the perturbational approach with external iteration. Merz-Kollman partial atomic charges served as an input for the LD model included in the ChemSol program. 43 For the semi-empirical MO methods, the AM1-SM1 and PM3-SM3 solvation models were used. 24 Corresponding calculations were performed with the AMSOL 5.4.1 program of Truhlar and coworkers. 24

Results and discussion Determination of the experimental reaction rate constant. The reaction between CEO and guanine is competitive with CEO hydrolysis, which leads to the formation of glycoaldehyde (GA). The main adduct 7-(2-oxoethyl)guanine is unstable and is further transformed through reactions like depurination leading to the final product (FP). We can thus summarize the reaction mechanism with the following scheme: CEO

+ H2 O 7

kh

GA



CEO

+ guanine

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ka

7-(2-oxoethyl)guanine kd FP, 7-(2-oxoethyl)guanine

and write the corresponding system of differential equations:

d[GA] = kh [CEO] dt d[guanine] = −ka [guanine][CEO] dt d[FP] = kd ([guanine]0 − [guanine] − [FP]) dt

(1)

where [ ] and [ ]0 indicate current and initial molar concentrations, respectively, kh is the pseudo-first-order rate constant for CEO hydrolysis, ka is the second-order rate constant for 7-(2-oxoethyl)guanine adduct formation, and kd is the first-order rate constant for its decomposition. The current CEO concentration can be calculated with the following massbalance equation

[CEO] = [CEO]0 − [GA] − [guanine]0 + [guanine]

(2)

and the current concentration of 7-(2-oxoethyl)guanine adduct can thus be obtained using relation

[7-(2-oxoethyl)guanine] = [guanine]0 − [guanine] − [FP]

(3)

The experimental half-life for CEO hydrolysis was reported to be about 2 hours at the temperature of 310.15 K, 25 indicating a pseudo-first-order rate constant kh = 9.6 × 10−5 s−1 . The rate constant for the adduct formation ka was obtained through a best fit to the experimental data from equations (2) and (3) and a numerical solution of the system of differential equations (1) using the Levenberg-Marquardt optimization algorithm in the Wolfram Mathematica 10 environment (see Supporting Material for the corresponding code). Experimental 8


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data and the best-fit solution are presented in Figure 1. The second-order rate constant for guanine alkylation with CEO was determined as ka = 0.05±0.02 M −1 s−1 . The rate constant for the decomposition of the 7-(2-oxoethyl)guanine adduct was also optimized as a fitting parameter and was determined as kd = 6 × 10−4 s−1 . As a proof of model validity, when all three rate constants ka , kh and kd were used as fitting parameters, we calculated the kh value of 3 × 10−5 s−1 , which is close to the experimental value. The rate constant is related to the corresponding activation free energy via Eyring’s transition state theory, ∆G‡a kB T exp − ka = h kB T

(4)

where kB is the Boltzmann constant, h the Planck constant and T the temperature. Using equation 4, the experimental activation free energy was calculated as ∆G‡a = 19.2±0.2 kcal/mol. This value was used to determine the best combination of theory level, basis set and solvation model for theoretical calculations of activation barriers.

QM calculations of free energy barriers. Out of many ab initio, DFT and semiempirical MO methods used (see Supporting Material Tables S1-S32) the Hartree-Fock level of theory in combination with the flexible 6311++G(d,p) basis set and the LD solvation model was found to reproduce the experimental activation free energy of the reaction between CEO and guanine in the most accurate way. Consequently, this combination set was selected as a default for comparison of activation free energies for the reactions between the nucleic bases and CEO or AN. The corresponding activation energies in vacuo, imaginary vibrational frequencies of the transition state, lowest frequencies of the reactant states, and distances between the reacting atoms are collected in Table 1. We were able to obtain viable transition states for the reactions of all nucleic bases, because all vibrational modes with imaginary frequencies coincide with the desired creation of a new 9



C-N bond between the reacting molecules as well as with the cleavage of the adjacent epoxidic C-O bond in the case of CEO. The corresponding distances between the reactive centers are very similar for all four nucleic bases, with a maximum spread of 0.01 Å and 0.02 Å for the reactions with CEO and AN, respectively. The reacting molecules in the transition state are on average 0.16 Å closer for the reactions with AN than with CEO. The energy barriers for the reactions in vacuo fail to predict the experimental order of reactivity. For all four bases the reaction with AN appears to be thermodynamically more favorable than with CEO. On the contrary, reaction rates calculated using our model based on the experimental data reported by Guengerich et. al 25,44 for the reactions with CEO are on the order of hours, while the rates for the reactions with AN reported by Solomon and Segal 45 are on the order of weeks. To obtain the correct prediction of the relative reactivities of nucleic bases, it is necessary to employ a biologically relevant environment using a water solvent. The hydration free energies of the transition state and the reactants, their differences and the free energy barriers of the corresponding reactions calculated using the Langevin dipoles implicit solvation model are summarized in Table 2. Activation free energies using this level of theory give a much more accurate prediction of relative reactivities. The barriers are now lower for the reactions with CEO in all cases. The solvent stabilizes the transition state more than the reactants, resulting in a negative difference in the hydration free energies. This reduction in the activation free energy ranges from −4.8 kcal/mol to −22.2 kcal/mol. The observed solvent acceleration is in line with the more polar nature of the transition state close to the one of the unstable zwitterionic intermediates. The transition states with the same base are better hydrated in the reactions with CEO, while the reactants are better hydrated in the reactions with AN. In addition to the presented level of theory, alternative combinations of quantum mechanical methods, basis sets and solvation models were also evaluated (see Supporting Material Tables S1-S32). B3LYP, MP2 and MPW1K all gave significantly lower activation free en10


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ergies using the same basis set and solvation model; true to its nature, the M0626 method gave results in between the pure HF method and the B3LYP functional. On the contrary, the semi-empirical methods resulted in too high activation free energies, with PM3 being closer to the ab initio HF calculations. Formation of guanine adducts. The structures of the transition states for the reactions with CEO and AN are shown in Figure 2. The tables with the corresponding numerical results can be found in the Supporting Material (Tables S1, S5, S9, S13, S17, S21, S25, S29). On the basis of the calculations at the HF/6-311++G(d,p) level of theory in conjunction with the implicit LD solvation model, the activation free energy for the reaction with CEO is 19.0 kcal/mol, which is very close to its experimental value (19.2 ± 0.2 kcal/mol), while the activation barrier for the reaction with AN amounts to 24.4 kcal/mol (Table 2). This translates to pseudo-first order reaction half-times of 3.4 minutes and 44 days, respectively. Guanine is the main adduct experimentally determined for the reactions with both CEO and AN, 44,45 and it indeed also has the lowest calculated activation free energies for both cases. For comparison, the activation free energies obtained with other QM methods and solvent models range from 8.3 kcal/mol (MP2/LD2) to 35.5 kcal/mol (AM1/SM1) for the reaction with CEO and from 5.4 kcal/mol (MPW1K/LD2) to 29.9 kcal/mol (HF/SCRF) for the reaction with AN. Out of all the other tested combinations B3LYP/SCRF (19.5 kcal/mol) and M062X/LD1 (18.9 kcal/mol) also yield activation barriers close to the experimental value for the reaction with CEO. Formation of adenine adducts. The structures of the transition states for the reactions with CEO and AN are depicted in Figure 3. The tables with the corresponding numerical results can be found in the Supporting Material (Tables S2, S6, S10, S14, S18, S22, S26, S30). On the basis of the 11



calculations at the HF/6-311++G(d,p) level of theory in conjunction with the implicit LD solvation model, the activation free energy is 26.4 and 32.2 kcal/mol for the reaction with CEO and AN, respectively (Table 2). The calculated activation barriers are second highest, which is in agreement with the second lowest experimental reaction yields of the alkylation with CEO. 44 On the other hand, the alkylation with AN experimentally yields the second largest amount of adenine adducts. 45 For comparison, the activation free energies obtained with other QM methods and solvent models range from 10.4 kcal/mol (B3LYP/LD2) to 40.4 kcal/mol (AM1/SM1) for the reaction with CEO and from 9.9 kcal/mol (M062X/LD2) to 35.0 kcal/mol (HF/SCRF) for the reaction with AN. Formation of cytosine adducts. The structures of the transition states for the reactions with CEO and AN are shown in Figure 4. The tables with the corresponding numerical results can be found in the Supporting Material (Tables S3, S7, S11, S15, S19, S23, S27, S31). On the basis of the calculations at the HF/6-311++G(d,p) level of theory in conjunction with the implicit LD solvation model, the activation free energy is 20.9 and 32.2 kcal/mol for the reaction with CEO and AN, respectively (Table 2). Cytosine indeed gives the second-largest yield in the reaction with CEO, but gives the lowest yield in the reaction with AN. 44,45 Its calculated activation free energy is, however, the second lowest in both cases. For comparison, the activation free energies obtained with other QM methods and solvent models range from 5.4 kcal/mol (MP2/LD2) to 32.9 kcal/mol (AM1/SM1) for the reaction with CEO and from 7.9 kcal/mol (M062X/LD2) to 34.0 kcal/mol (HF/SCRF) for the reaction with AN. Formation of thymine adducts. The structures of the transition states for the reactions with CEO and AN are depicted in Figure 5. The tables with the corresponding numerical results can be found in the Supporting Material (Tables S4, S8, S12, S16, S20, S24, S28, S32). On the basis of the calculations at the 12


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HF/6-311++G(d,p) level of theory in conjunction with the implicit LD solvation model, the activation free energy barrier is 38.0 and 42.6 kcal/mol for the reaction with CEO and AN, respectively (Table 2). This is in agreement with experimental results of CEO alkylations, where the yield of thymine adducts yield is the lowest among all nucleic bases. 44 For the reaction with AN, however, the computationally predicted relative reactivity seems to be too low. 45 For comparison, the activation free energies obtained with other QM methods and solvent models range from 18.9 kcal/mol (MP2/LD2) to 49.0 kcal/mol (AM1/SM1) for the reaction with CEO and from 21.3 kcal/mol (MPW1K/LD2) to 46.7 kcal/mol (HF/SCRF) for the reaction with AN. Reactivity of CEO. In addition to the analyis of the regioselectivity, we can also establish the relative reactivity of CEO in comparison with the other previously studied ultimate carcinogens of the epoxy type. Focusing on the main reaction with guanine, it appears that the free energy barrier of CEO of around 19 kcal/mol is among the lowest of all studied carcinogens, which indicates that CEO represents one of the most genotoxic chemicals. The calculated free energy barriers for vinyl carbamate epoxide, chloroethylene oxide, glycidamide, ethylene oxide, propylene oxide and styrene oxide range from 19.1 to 27.7 kcal/mol, 26,27,29–32 and the corresponding experimentally determined values lie between 19.5 and 26.5 kcal/mol. 26,27,29–32 On the contrary, the well-known aflatoxin B1 possesses an even lower free energy barrier, calculated at 14.3 kcal/mol and experimentally determined at 15.1 kcal/mol, due to its ability to form favourable stacking interactions that lead to the intercalation of the toxin inbetween the Watson-Crick base pairs. 28

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Conclusions We performed a quantum mechanical evaluation of biologically relevant chemical interactions between nucleic bases and possible human carcinogens CEO and AN. We considered the alkylations from a kinetic standpoint by assessing the ab initio calculated activation free energies and comparing them to the experimentally determined reaction rate constants. 25 With the selection of the right combination of the quantum theory level (Hartree-Fock), basis set (6-311++G(d,p)) and appropriate solvation model (LD), we could obtain accurate predictions of absolute guanine reactivity and of relative reactivities of remaining nucleic bases towards the alkylation with CEO. 44 This holds also in the case of the Michael addition of AN, with cytosine being the only exception. The relative reactivity for cytosine was overestimated, placing it right after guanine in the reactivity series, while experimentally the yield of cytosine adducts was the lowest of all nucleic bases. 45 An alternative combination of B3LYP/6-311++G(d,p) level of theory with the standard solvent reaction field (SCRF) method giving accurate predictions of experimental activation barriers for guanine alkylation with CEO made a somewhat better placement of cytosine reactivity with AN behind guanine and adenine. The quantum mechanical calculations also correctly predicted a significantly higher reactivity of all nucleobases towards CEO than AN. 25 Finally, it has to be emphasized that in real biological system, the nucleic bases are confined in a relatively rigid and sterically much more crowded environment, whereas our model systems consisted of the free methylated nucleobase floating in implicit solvent. Steric hindrance of the nascent Watson-Crick base pairs could indeed explain some discrepancies between the experimental and theoretical values. This was checked by superposing the transition state of the reaction between cytosine and AN onto a DNA model from X-ray crystallography. Visual inspection of the superposed geometry reveals a clear overlap of AN and the complementary guanine base (Supporting Information Figure S1). For future studies it would therefore be prudent to consider these reactions using a QM/MM model system with 14


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the alkylation taking place in a biologically more accurate environment. In addition, the epoxidation of AN by cytochrome P450 2E1 and the interaction of the obtained DNA adducts with DNA polymerases should be computationally and experimentally further investigated to obtain a full understanding of the early AN-related carcinogenesis. 46–51 Nevertheless, our quantum mechanical study of the alkylation of nucleobases provided us with valuable insights in the reaction mechanisms and the geometries of the transition states. Alkylated bases represent genetic mutations, which are removed by the base excision repair mechanism, where endonucleases play a vital role. 52,53 Consequently, computational studies of the interactions between damaged nucleobases and endonucleases are envisioned as future perspectives. The results of the study can also be used for the design of novel molecular scavengers that could prevent DNA alkylation damage by covalently binding to the ultimate carcinogens via a lower activation barrier. 54

Funding Sources Financial support through Slovenian Research Agency project grant J1-6736 and Slovenian Ministry of Education, Science and Sports program grant F4F is gratefully acknowledged.

Supporting Information Available • Schemes for proposed mechanisms of the reactions between nucleic-bases and AN; tables with activation energies, activation free energies using all combinations of implicit solvent models, imaginary frequencies of the transition state, lowest frequencies of the reactant state, and distances between the reacting atoms for all combinations of nucleic bases, reactive species, quantum mechanical methods and basis sets; transition state of the reaction between cytosine and AN superposed onto a DNA model; the code numerically solving the set of differential equations (1). This material is available free of charge via the Internet at http://pubs.acs.org. 15



Abbreviations AN

acrylonitrile

CEO

cyanoethylene oxide

DFT

density functional theory

DNA

deoxyribonucleic acid

FP

final product

GA

glycoaldehyde

HF

Hartree-Fock

LD

Langevin dipoles

MO

molecular orbital

MP2

Møller-Plesset perturbation theory of the second order

QM/MM quantum-mechanical/molecular mechanical SCRF

self-consistent reaction field

TS

transition state

References (1) Baus, D. K., Hsu, R. S., Neal, M. W., Santodonato, J., Sugatt, R. H., Bayard, S., Bayliss, D. L., Hiremath, C. B., and Vaughn- Dellarco, V. Health Assessment Document for Acrylonitrile. Technical Report EPA-600/8-82-007F. 1983. (2) Merchant Research & Consulting Ltd., Acrylonitrile (ACN): 2014 World Market Outlook and Forecast up to 2018. 2014. (3) Maltoni, C., Cilberti, A., and Di Maio, V. (1971) Carcinogenicity bioas- says on rats of acrylonitrile administered by inhalation and by ingestion. Med. Lavoro 68, 401–411. (4) Quast, J. F. (2002) Two-year toxicity and oncogenicity study with acrylonitrile incorporated in the drinking water of rats. Toxicol. Lett. 132, 153–196.

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of workers exposed to acrylonitrile in fiber production. Scandinavian Journal of Work, Environment & Health 54–62. (14) Marsh, G. M., Youk, A. O., and Collins, J. J. (2001) Reevaluation of lung cancer risk in the acrylonitrile cohort study of the National Cancer Institute and the National Institute for Occupational Safety and Health. Scandinavian Journal of Work, Environment & Health 5–13. (15) Schulz, M. R., Hertz-Picciotto, I., Todd, L., and Ball, L. M. (2001) Reconciling animal and human data in a cancer risk assessment of acrylonitrile. Scandinavian Journal of Work, Environment & Health 14–20. (16) Lambotte-Vandepaer, M., and Duverger-van Bogaert, M. (1984) Genotoxic properties of acrylonitrile. Mutat. Res. Genet. Toxicol. 134, 49–59. (17) Kedderis, G. L., Batra, R., and Koop, D. R. (1993) Epoxidation of acrylonitrile by rat and human cytochromes P450. Chem. Res. Toxicol. 6, 866–871. (18) Sumner, S. C. J., Fennell, T. R., Moore, T. A., Chanas, B., Gonzalez, F., and Ghanayem, B. I. (1999) Role of Cytochrome P450 2E1 in the Metabolism of Acrylamide and Acrylonitrile in Mice. Chem. Res. Toxicol. 12, 1110–1116. (19) Wang, H., Chanas, B., and Ghanayem, B. I. (2002) Cytochrome P450 2E1 (CYP2E1) is Essential for Acrylonitrile Metabolism to Cyanide: Comparative Studies Using CYP2E1-Null and Wild-Type Mice. Drug Metabolism and Disposition 30, 911–917. (20) Miertuš, S., Scrocco, E., and Tomasi, J. (1981) Electrostatic interaction of a solute with a continuum. A direct utilizaion of AB initio molecular potentials for the prevision of solvent effects. Chem. Phys. 55, 117–129. (21) Florián, J., and Warshel, A. (1997) Langevin Dipoles Model for ab Initio Calculations of Chemical Processes in Solution: Parametrization and Application to Hydration Free 18


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Tables Table 1: Activation energies for the reactions of nucleic bases with cyanoethylene oxide or acrylonitrile HF/6-311++G(d,p) guanine+CEO adenine+CEO cytosine+CEO thymine+CEO guanine+AN adenine+AN cytosine+AN thymine+AN

∆E‡ + ∆ZPE [kcal/mol]a

ω TS [i cm−1 ]b

ωR [cm−1 ]c

dTS [Å]d

dR [Å]e

40.6 42.5 43.1 61.3 37.5 39.1 41.9 49.5

574 617 584 568 424 439 454 470

15.4 12.3 26.2 21.5 13.7 10.6 17.3 10.1

1.95 1.96 1.96 1.95 1.79 1.79 1.80 1.81

3.81 3.70 3.47 3.57 3.77 7.02 3.92 4.56

Gas phase activation energy including zero point vibrational energy correction. b Imaginary vibrational frequency corresponding to the transition state. c Lowest vibrational frequency corresponding to the reactant state. d Distance between the reacting N atom of guanine and the C atom of cyanoethylene oxide or acrylonitrile in the transition state. e Distance between the reacting N atom of nucleobase and the C atom of cyanoethylene oxide or acrylonitrile in the reactant state. a

Table 2: Activation free energies for the reactions of nucleic bases with cyanoethylene oxide or acrylonitrile HF/6-311++G(d,p)/LD guanine+CEO adenine+CEO cytosine+CEO thymine+CEO guanine+AN adenine+AN cytosine+AN thymine+AN

∆GLD hydr TS [kcal/mol]a −48.2 −34.4 −42.6 −41.5 −43.0 −26.9 −35.2 −32.9

∆GLD hydr R [kcal/mol]b −26.6 −18.3 −20.4 −19.9 −29.9 −20.0 −22.3 −28.1

∆∆GLD hydr [kcal/mol]c −21.6 −16.2 −22.2 −21.6 −13.2 −6.9 −12.9 −4.8

∆G‡LD [kcal/mol]d 19.0 (19.2 ± 0.2)e 26.4 20.9 38.0 24.4 32.2 29.0 42.6

Hydration free energy of the transition state, obtained with the LD method. b Hydration free energy of the reactants, obtained with the LD method. c Hydration free energy of the transition state minus hydration free energy of the reactants. d Activation free energy. e Experimental value. a

23



Figure Legends Figure 1: Fitting of the solution of differential equations for the reaction kinetics between guanine and CEO to the experimental data. Best fit with the second-order rate constant ka = 0.05 M −1 s−1 is shown as a blue curve, while experimental data points are depicted in red.

Figure 2: Structure of the transition state for the reaction of the N7 atom of guanine with cyanoethylene oxide (A) and acrylonitrile (B) as predicted at the HF/6-311++G(d,p) level of theory. Carbon is depicted in gray, oxygen in red, nitrogen in blue and hydrogen in white. The newly forming and breaking bonds are represented with gray dashed lines. The highlighted bond lengths are given in Å.

Figure 3: Structure of the transition state for the reaction of the N1 atom of adenine with cyanoethylene oxide (A) and acrylonitrile (B) as predicted by the HF/6-311++G(d,p) level of theory. Carbon is depicted in gray, oxygen in red, nitrogen in blue and hydrogen in white. The newly forming and breaking bonds are represented with gray dashed lines and hydrogen bonds are represented with yellow dashed lines. The highlighted bond lengths are given in Å.

Figure 4: Structure of the transition state for the reaction of the N3 atom of cytosine with cyanoethylene oxide (A) and acrylonitrile (B) as predicted at the HF/6-311++G(d,p) level of theory. Carbon is depicted in gray, oxygen in red, nitrogen in blue and hydrogen in white. The newly forming and breaking bonds are represented with gray dashed lines and hydrogen bonds are represented with yellow dashed lines. The highlighted bond lengths are given in Å.

Figure 5: Structure of the transition state for the reaction of the N3 atom of thymine with cyanoethylene oxide (A) and acrylonitrile (B) as predicted at the HF/6-311++G(d,p) level of theory. Carbon is depicted in gray, oxygen in red, nitrogen in blue and hydrogen in white. The newly forming and breaking bonds are represented with gray dashed lines and hydrogen bonds are represented with yellow dashed lines. The highlighted bond lengths are given in Å.

24


Page 24 of 29

Page 25 of 29

Figures 25 ●

[7 -(2-oxoethyl)guanine] [nmol/mL]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


best fit exp. data

20

15

10

5

0 0

1

2 time [h]

3

4

Figure 1: Fitting of the solution of differential equations for the reaction kinetics between guanine and CEO to the experimental data. Best fit with the second-order rate constant ka = 0.05 M −1 s−1 is shown as a blue curve, while experimental data points are depicted in red.

25



Figure 2: Structure of the transition state for the reaction of the N7 atom of guanine with cyanoethylene oxide (A) and acrylonitrile (B) as predicted at the HF/6-311++G(d,p) level of theory. Carbon is depicted in gray, oxygen in red, nitrogen in blue and hydrogen in white. The newly forming and breaking bonds are represented with gray dashed lines. The highlighted bond lengths are given in Å.

Figure 3: Structure of the transition state for the reaction of the N1 atom of adenine with cyanoethylene oxide (A) and acrylonitrile (B) as predicted by the HF/6-311++G(d,p) level of theory. Carbon is depicted in gray, oxygen in red, nitrogen in blue and hydrogen in white. The newly forming and breaking bonds are represented with gray dashed lines and hydrogen bonds are represented with yellow dashed lines. The highlighted bond lengths are given in Å.

26


Page 26 of 29

Page 27 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4: Structure of the transition state for the reaction of the N3 atom of cytosine with cyanoethylene oxide (A) and acrylonitrile (B) as predicted at the HF/6-311++G(d,p) level of theory. Carbon is depicted in gray, oxygen in red, nitrogen in blue and hydrogen in white. The newly forming and breaking bonds are represented with gray dashed lines and hydrogen bonds are represented with yellow dashed lines. The highlighted bond lengths are given in Å.

Figure 5: Structure of the transition state for the reaction of the N3 atom of thymine with cyanoethylene oxide (A) and acrylonitrile (B) as predicted at the HF/6-311++G(d,p) level of theory. Carbon is depicted in gray, oxygen in red, nitrogen in blue and hydrogen in white. The newly forming and breaking bonds are represented with gray dashed lines and hydrogen bonds are represented with yellow dashed lines. The highlighted bond lengths are given in Å.

27



Page 28 of 29

Scheme Labels Scheme 1: Proposed mechanism of the reaction between guanine and cyanoethylene oxide.

Scheme 2: Proposed mechanism of the reaction between adenine and cyanoethylene oxide.

Scheme 3: Proposed mechanism of the reaction between cytosine and cyanoethylene oxide.

Scheme 4: Proposed mechanism of the reaction between thymine and cyanoethylene oxide.

Schemes

O 6

1

HN 2

H2 N

–

O

H H 5

N7

4

N9

O

H ∗

C

∗

O N HN

N

+

3

N

HN

8

N

O

O C

N H

−CN

N

N

+

H2 N

N

N

R

R

O

N

HN

+

–

H2 N

O −R

H2N

N

N

R 1

Scheme 1: Proposed mechanism of the reaction between guanine and cyanoethylene oxide.

N N

N N

O

C H

∗

H

NH2

H 1

6

N 2

–

5

N7

4

N9

8

N 3

O ∗ CH N

HH

O

NH2

+

+

N

N

NH2 N

N

–

N

N

−CN

N R

R

R

N

on izati cycl 2O H −

N

N R

Di rear mroth rang eme HOOC nt

NH OH

N

N N

N R

1

Scheme 2: Proposed mechanism of the reaction between adenine and cyanoethylene oxide.

28


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N

O

C H

H

∗

NH2

H 3

O

5

N 2

N1

HH

–

O ∗

4

CH

6

N

NH2

NH2 O

+

N

N

O

−CN

N

O

R

N

oxidation

–

R

NH H OO C

+

OH

N

O

N

R

R

1

Scheme 3: Proposed mechanism of the reaction between cytosine and cyanoethylene oxide.

N

O

C H

∗

H H

3

4

HN O

N

O

2

CH3

5

O

C H

H

∗

OH

H

CH3

N N1 R

N

OH

OH CH3

+

N

CH

6

O

HH

–

O ∗

O

N

–

O

N

N R

R

−CN

O CH3

+

O

O

N

−H

O

R

1

Scheme 4: Proposed mechanism of the reaction between thymine and cyanoethylene oxide.

29


CH3

N

+

N R

Nucleic bases alkylation with acrylonitrile and cyanoethylene oxide: A

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