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Ab Initio GB Study of Methylation Reaction of Adenine, Cytosine, Guanine, and Thymine by Methanediazonium Ion
J. theor. Biol. (2002) 215, 13}22 doi:10.1006/jtbi.2001.2500, available online at http://www.idealibrary.com on
Ab Initio GB Study of Methylation Reaction of Adenine, Cytosine, Guanine, and Thymine by Methanediazonium Ion NAOFUMI NAKAYAMA, SYUNSUKE TANAKA AND OSAMU KIKUCHI Department of Chemistry, ;niversity of ¹sukuba, ¹sukuba 305-8571 Japan (Received on 16 March 2001, Accepted in revised form on 22 June 2001)
Methylation reaction at ten nucleophilic sites in four DNA base molecules by methanediazonium ion (N Me>) was examined by ab initio MO/GB calculation which includes the solvent e!ect with the continuum model using the generalized Born formula. The stabilization energy of the ion}dipole complex as well as the energy of the transition state are roughly consistent with the experimental fraction of methylation by N-methyl-N-nitrosourea. For the guanine N7 site, which is the principal site of methylation, the stabilization energy is the largest and the energy of the transition state is low. The reactions at the guanine O and N7 sites were analysed by drawing the potential energy surface with respect to two parameters, the O}C(Me) distance and the N7}C(Me) distance. The methylation reactions of the guanine O and N7 sites begin from the common geometry, the global minimum in the two-dimensional potential energy surface. 2002 Elsevier Science Ltd.
Introduction N-nitroso compounds are typical mutagenic and carcinogenic compounds (Loeppky & Michejda, 1994). They are believed to owe their activity to conversion to highly reactive diazonium ions which are strong alkylation agents to the nucleophilic sites in DNA bases (Scheme 1). Singer and Grunberger summarized the relative fractions of methylation of single and double-stranded nucleic acids by some methylating reagents (Table 1, Singer & Grunberger, 1983). It is indicated that the principal site of methylation is guanine N7 site. However, the alkylation of this site is innocuous because it is not the hydrogen-bonding site. Although dimethylsulfate (Me SO ) and methyl methanesulfonate (MeMS) alkylate many nucleophilic sites simultaneously, they have weak carcinogenicity. Mutagenic and carcinogenic activity are attributed not to the fraction of methyl0022}5193/02/050013#10 $35.00/0
ation of guanine N7 but to the fraction of guanine O, because O-alkylguanine gives rise to transcriptional errors in both DNA and RNA (Singer & Grunberger, 1983). Recently, molecular orbital calculations are employed for the analyses of the bond formation between the chemical carcinogens (including N-nitrosamines) and DNA bases (Lea o & Pava o, 1997). These analyses are based on the simple frontier orbital theory, and are not responsible for the quantitative analysis of the DNA alkylation by N-nitrosamines. Most N-nitroso compounds have carcinogenicity due to the alkylation of DNA base molecules by alkanediazonium ions. There are several previous reports associated with diazonium ions themselves (Vincent & Radom, 1978; Glaser & Choy, 1993) and diazohydroxide as a precursor of diazonium ion (Reynolds & Thomson, 1986a, b; 1987). 2002 Elsevier Science Ltd.
14
N. NAKAYAMA E¹ A¸.
TABLE 1 Percentage of alkylation of single and double-stranded nucleic acids in vitro* (Singer & Grunberger, 1983) Base
Adenine Cytosine Guanine Thymine
Site
N1 N3 N7 O N3 N3 O N7 O O
Single stranded-
Double stranded?
Me SO
MeMSA
MeNU
Me SO
MeMSA
MeNU
13.2 2.6 3.1
18 1.4 3.8
2.8 2.6 1.8
9.5 :0.1 (0.2 62
10 :1 nd 69
2.3 0.4 3 69
1.9 18 1.9 (nd) ((2) 1.1 0.2 74
3.8 10.4 (1.8) (nd) ((1) (0.6) (0.3) 83
1.3 9 1.7 0.1 0.6 0.8 6.3 67 0.11 0.4
*The absolute amount of alkylation varied greatly but the proportion of derivatives was not noticeably a!ected. &&nd'' indicates that the derivative was not detected. Parentheses indicate either a single value or the average of two very di!erent values and thus less reliability than other data shown. -Analyses are from experiments using DNA from M13 phage and RNA from TMV, yeast, HeLa cells, animal ribosomes, and 2 phage. ?Analyses are from experiments using DNA from salmon sperm, calf thymus, salmon testes, rat liver and brain, human "bloblasts, and HeLa and V79 cells. AMethylmethanesulfonate. N-Methyl-N-nitrosourea.
There are also several theoretical investigations on the alkylation of DNA bases and the model compounds of DNA bases by alkanediazonium ions (Mohammad & Hop"nger, 1980; Ford & Scribner, 1983, 1990; Kim & LeBreton, 1996). Of these studies, two representative investigations dealt with DNA base molecules. Ford & Scribner (1990) examined the methylation, ethylation, and propylation (only for the guanine O and N7) using the MNDO method. They showed that the alkylation site shifts from N7 to O as the bulkiness of the alkyl group increases and suggested that the shift is due to the increase of the S 1 character in the , transition state of their S 2 reactions (in other , words, the transition state structure for ethylation is looser than that for methylation). Their results of calculation roughly reproduced the
experimental trend between methylation and ethylation (Singer & Grunberger, 1983). Kim & LeBreton (1996) examined the methylation by RHF/4-31G level of theory using MNDO optimized molecular structures. They also investigated the ionization potentials of nucleotides by both UV photoelectron and ab initio study, and indicated that polarization e!ects in the nucleotide base play an important role in determining DNA methylation patterns. These works were based on semi-empirical levels of calculations and were examined for speci"c alkylation sites. Further, they did not discuss the reason as to why the alkylation fraction of guanine O site by N-nitroso compounds is larger than other alkylating reagents (Table 1). Theoretical study of the preferences of the cations was reported only for cytosine N3 and O
AB INI¹IO GB STUDY OF METHYLATION REACTION
(Pullman & Armbruster, 1977). Also, no environmental e!ects were taken into account in these studies. It is well known that a cation like the methanediazonium ion is very unstable in the gas phase and MO calculations for the ionic species give the potential energy curves which are far from those expected in the real system. Recently, ab initio calculations including the solvent e!ects have become available (Tomasi & Persico, 1994; Cramer & Truhlar, 1999). It is recommended that the reaction of the diazonium ion is analysed by ab initio MO method including solvent e!ect. Figure 1 shows the schematic energy pro"le of nucleophilic addition reaction of methanediazonium ion with DNA bases. In the investigation of reaction mechanism of the cation species with nucleophilic sites, the stabilization energy of the ion}dipole complex (E ) as well AMKN as the energy of the transition state (E ) is 21 important. E represents the ability of elecAMKN trostatically capturing the cation by the nucleophile. Therefore, E may be an e!ective AMKN descriptor for the experimental fractions of total methylation (Table 1). In this study, the energy pro"les of methylation of DNA base molecules by methanediazonium
15
ion (Scheme 2) are examined by using ab initio method including solvent e!ect. The analysis rationalizes the reason why the fraction of DNA base alkylation by N-nitroso compounds is different from other alkylating reagents. In this paper, nucleophilic addition reactions of methanediazonium ion for ten selected sites of four DNA base molecules (N1, N3, and N7 sites of adenine, O and N3 sites of cytosine, N3, O, and N7 sites of guanine, and O and O of thymine, see Fig. 2) are investigated. In addition, the reactions of guanine O and N7 sites are investigated in detail by drawing the potential energy surface with respect to two parameters, the O}C(Me) distance (R ) and the N7}C(Me) distance (R ) (Fig. 3). Method Ab initio SCF calculations with generalized Born (GB) formula have been described in previous articles (Kikuchi et al., 1994; Takahashi et al., 1997). In the continuum model using the GB formula, the solvation free energy of a molecule is expressed as
1 1 G "! 1! Q Q , QMJ 2
(1)
where is the dielectric constant of the solvent, A and B are atoms in the molecule, and Q and Q are the partial charges on the atoms A and B, respectively. The partial charges are calculated from the nuclear charge and LoK wdin (1950) population. represents the interaction be tween A and B atoms. The energy of a molecule in solution is expressed as the sum of the energy of the molecule in its isolated state, E, and the solvation free energy, G : QMJ EQMJ"E#G . QMJ FIG. 1. Energy pro"le for the reaction between methanediazonium ion and the nucleophilic site in DNA bases.
(2)
By applying the variational theorem to eqn (2), the Fock matrix elements including the solvent
16
N. NAKAYAMA E¹ A¸.
NH2
O
O
NH2 N
N 7
N1
3 2 N H
O
3 N
N H Adenine
HN H2N
Cytosine
6 3 N Guanine
N 7 N H
4
HN
Me
2 O
N H Thymine
FIG. 2. Nucleophilic sites in DNA base molecules. N N C
R1 R2
O H
N 6
N 7
H2N
N
N
H H
FIG. 3. Two parameters for the potential energy surface for the guanine O and N7 reactions.
e!ect for a closed-shell molecule are F "F #FQMJ , IJ IJ IJ
(3)
G QMJ (S ) (S ) , FQMJ" IH JH IJ Q H H
(4)
where F is the Fock matrix element for an IJ isolated molecule, and FQMJ describes the contriIJ bution of the solvent. Q is the charge of the atom H to which the basis function belongs. The partial derivatives in eqn (4) are expressed as
G 1 QMJ"! 1! Q
1 Q # Q 2 Q
. # Q Q Q BOA
(5)
The ab initio GB method has been applied to many chemical reactions including ionic species. It reproduces well the relative solvation energy of ionic species. This was con"rmed by comparing the solvation energies calculated by ab initio GB method with those calculated by Monte Carlo
simulation in which many solvent molecules were considered explicitly (Kikuchi et al., 1997). In this study, the dielectric constant "78.5 was used to represent the aqueous solution. Geometry optimizations and calculations for searching the TS were carried out using ABINIT/GB program package (Kikuchi et al., 1999) on the DEC 500/500 workstation. The transition state (TS) structures were determined by minimizing the norm of energy gradients. The RHF method was employed with the 3-21G basis set. We calculated the possible energy-minimum structures of methylated products in each compound. Figure 4 shows the most stable structure of each methylated DNA base molecules in solution (all structures have C symmetry). For the Q guanine O adduct, the proximal conformation in which the Me group is on the N7 side is more stable than the distal conformation in which the Me group is the opposite to the N7 site. This contradicts the previous result (Pedersen et al., 1988) which was obtained by partial geometry optimization. In the calculation of ion}dipole complex and the TS, the orientation of the methyl group was postulated to be the same as the direction of the methyl group in each methylated DNA base. Therefore, the calculations were carried out under the C symmetry. Q Results and Discussion ENERGY PROFILE AND STRUCTURE FOR EACH REACTION SITE
Figure 5 shows the ion}dipole and the TS structures for guanine N7 and O sites. Table 2 shows the total energies of the methylated DNA base cation, the ion}dipole complex and the TS structure in each alkylation reaction. In the
AB INI¹IO GB STUDY OF METHYLATION REACTION
17
FIG. 4. The most stable structures of methylated DNA base cations.
calculations of the TS structure, only the selected structural parameters were optimized: methanediazonium ion (N Me>) moiety, distance be tween C(Me) and X (R, X"N or O atom of base site), and C(Me)}X}C (base) angle (, see Fig. 6). The parameters associated with the base framework were adopted from those in the ion}dipole
complex, and were "xed in the calculations of the TS searching. Table 3 shows the relative energies of methylation reactions shown in the energy pro"le in Fig. 1. A large stabilization, E, is obtained by the methylation at each nucleophilic site even in an aqueous solution where methanediazonium
18
N. NAKAYAMA E¹ A¸.
cation is largely stabilized. It is worth pointing out that E becomes larger if the solvent e!ect is not included. Although the E values at nucleophilic sites appear to have a strong correlation with the values of molecular electrostatic potential (MEP) calculated by both ab initio (Politzer
& Truhlar, 1981) and DFT method (Santamaria et al., 1998), three energies, E , E , and E, AMKN 21 have little correlation to one another. The possibility for the alkylating reagent to access each nucleophilic site can be the reactivity index. Thus, the calculated E value of AMKN guanine N7 site is the largest of all sites, which is consistent with the experiment that the most reactive site of guanine is N7 (Singer & Grunberger, 1983). However, the calculated E enerAMKN gies of cytosine are close to that of guanine N7, although the experimental fractions of methylation in cytosine are much smaller than those in guanine. In the case of thymine and adenine, the E values are larger in thymine, while the AMKN E values are larger in adenine. The E 21 21 values correlate well with the experiment. In adenine, three sites, N1, N3, and N7, are pyridinetype nitrogen atoms and were predicted to have similar reactivity. The N3 site has the smallest activation energy, which is parallel to the experiment. Excluding cytosine, the E values in 21 aqueous solution re#ect well the site of alkylation
FIG. 5. Structures of the ion}dipole complex and the TS for the methylation of guanine N7 and O sites.
FIG. 6. Structural parameters optimized in the TS calculation.
TABLE 2 ¹otal energies (hartree) of the methylated product, the ion}dipole complex and the ¹S on each site of DNA base molecules* Base
Site
Methylated product
Complex
TS
Adenine
N1 N3 N7 O N3 N3 O N7 O O
!501.189567 !501.188700 !501.186298 !429.708579 !429.727406 !575.649971 !575.644849 !575.674172 !488.236425 !488.238571
!609.405935 !609.405118 !609.404939 !537.938895 !537.940354 !683.871148 !683.891080 !683.895002 !596.495136 !596.495822
!609.399644 !609.399228 !609.398461 !537.936610 !537.934859 !683.862914 !683.886676 !683.890792 !596.488213 !596.488508
Cytosine Guanine Thymine
*RHF/3-21G calculation in aqueous solution ("78.5).
AB INI¹IO GB STUDY OF METHYLATION REACTION
N site, and 2.6}2.7 As for the O site (guanine N3 and thymine O are exceptional). In the TS, both distances for the N site alkylation are also the same except for guanine N3. However, the distances for the O site are diverse. Table 5 shows the N}C(Me)}Site and N}N}C(Me) bond angles in the ion}dipole complex and the TS structure for each reaction site. They indicate that both in complex and TS, N}N}C(Me)}Site is approximately linear for all sites, except for the complex of cytosine N3. Table 6 shows the total charge of methanediazonium ion in the ion}dipole complex and the TS structure. These values exhibit the extent of electron transfer from the DNA molecule to the cation. They indicated that the extent of transfer from the cytosine N3 and guanine N7 sites are larger than others in the complex, and this may correspond to the magnitude of E . AMKN
by methanediazonium ion. In cytosine and thymine, the steric repulsion due to the sugar and phosphate groups may be an important factor to determine the sites of methylation. The steric interaction should be included for a more quantitative discussion. Table 4 shows the N}C(Me)} and C(Me)}Site distances in the ion}dipole complex and the TS structure for each reaction site. In the complex, the N}C(Me) distance is almost constant in all sites, while C(Me)}Site distances are roughly divided into two categories: 2.9}3.1 As for the TABLE 3 Relative energies (kcal mol\) of ion}dipole complex, ¹S, and product on each site of DNA base molecules (see Fig. 1)* Base
Site
E AMKN
E 21
E
Adenine
N1 N3 N7 O N3 N3 O N7 O O
!5.1 !4.6 !4.5 !13.0 !13.9 !0.7 !13.2 !15.7 !8.3 !8.7
3.9 3.7 4.1 1.4 3.4 5.2 2.8 2.6 4.3 4.6
!58.2 !57.7 !56.2 !57.3 !69.1 !50.8 !47.5 !65.9 !34.8 !36.2
Cytosine Guanine Thymine
POTENTIAL ENERGY SURFACE FOR THE METHYLATION REACTIONS OF GUANINE N7 AND O SITES
In guanine, the O and N7 sites have much stable ion}dipole complexes and small activation energies. This distinctly re#ects the experimental fact that these two sites are easily alkylated. However, E values for their sites are close to 21 each other, although there are large di!erences between their experimental fractions of methylation. In order to survey the potential energy surface for guanine N7 and O, two parameters (R and
*These values are obtained from total energies of each species (in Table 2), DNA base molecules, N , and N Me>. Total energies (hartree) in aqueous solution of adenine, cytosine, guanine, thymine, N , and N Me> are !461.917052, !390.437458, !536.389323, !449.001191, !108.300954, and !147.480710, respectively.
TABLE 4 ¹he distances (As ) in the ion}dipole complex and ¹S N}C(Me) and C(Me)}Site Base
Adenine Cytosine Guanine Thymine
Site
N1 N3 N7 O N3 N3 O N7 O O
19
Complex
TS
N}C(Me)
C(Me)}Site
N}C(Me)
C(Me)}Site
1.513 1.513 1.512 1.536 1.516 1.518 1.517 1.506 1.526 1.523
3.005 2.989 2.987 2.646 3.013 2.673 2.669 3.091 2.720 2.906
1.808 1.805 1.809 1.762 1.802 1.831 1.825 1.795 1.900 1.894
2.458 2.450 2.433 2.350 2.466 2.441 2.319 2.447 2.228 2.227
20
N. NAKAYAMA E¹ A¸.
TABLE 5 ¹he bond angles (deg) in the ion}dipole complex and ¹S N}C(Me)}site and N}N}C(Me) Base
Adenine Cytosine Guanine Thymine
Site
Complex N}C(Me)}Site
N}N}C(Me)
N}C(Me)}Site
N}N}C(Me)
178.1 178.3 177.9 177.0 168.6 178.4 179.8 173.3 176.0 176.7
180.0 180.0 179.9 180.0 178.9 179.9 179.9 179.9 180.0 179.2
179.1 178.9 179.5 177.7 175.2 178.3 177.9 177.3 177.8 177.2
179.8 179.9 180.0 179.8 177.7 180.0 178.9 178.1 179.9 179.7
N1 N3 N7 O N3 N3 O N7 O O
TABLE 6 ¹otal charge (¸oK wdin, 1950) of methanediazonium ion moiety in the ion}dipole complex and ¹S Base
Site
Complex
TS
Adenine
N1 N3 N7 O N3 N3 O N7 O O
0.976 0.976 0.976 0.955 0.933 0.980 0.958 0.933 0.971 0.971
0.896 0.898 0.896 0.900 0.862 0.890 0.890 0.864 0.900 0.895
Cytosine Guanine Thymine
TS
R , see Fig. 3) were varied in steps of 0.25 As in the range of 2.25}3.50 As . Other parameters were fully optimized in C symmetry. Q Figure 7 shows the potential energy surface of the methylation reactions of both guanine N7 and O sites. In this map, both the minimum of ion}dipole complex and the TS are included: O}C(Me)"3.173 As and N7}C(Me)"2.907 As for the ion}dipole complex for the N7 site, O}C(Me)"2.720 As and N7}C(Me)"3.326 As for the ion}dipole complex for the O site, O}C(Me)"3.157 As and N7}C(Me)"2.455 As for the TS for the N7 site, and O}C(Me)" 2.315 As and N7}C(Me)"3.258 As for the TS for the O site. The energy di!erence between the ion}dipole complexes for guanine N7 and O is 2.5 kcal mol\ (see Table 3).
Figure 7 indicates that the minimum of the ion}dipole complex of guanine N7 is the global minimum is this surface, and that the minimum of guanine O is very shallow. It may be reasonable to assume that the diazonium ion is trapped at the global minimum to form the ion}dipole complex and the alkylation to N7 and O starts from the common complex. The energy of the TS of O site relative to the global minimum is expected to be less than 4 kcal mol\. It is lower than the energy of TS in Table 4 (5.2 kcal mol\) because the structure of guanine framework is "xed in the calculation of TS searching. We conclude that the di!erence of experimental fraction between O and N7 of guanine is due to the energy di!erence of the TS between O and N7. The reaction mechanisms for guanine O and N7 are expected to be such that the methylation reaction of guanine O site begins from the N7 minimum which is the global minimum in the two-dimensional energy surface. Conclusion The energy pro"le of the nucleophilic addition reactions of methanediazonium ion to nucleophilic sites of DNA bases were clari"ed by ab initio MO calculation including solvent e!ects. The stabilization energy for the formation of the ion}dipole complex in guanine N7 site is the largest in all calculated sites. It is consistent with the experiment that the most reactive site is guanine N7. The methylation fractions of other
AB INI¹IO GB STUDY OF METHYLATION REACTION
FIG. 7. Two-dimensional potential energy surface of the methylation reactions of both guanine N7 and O sites. Energies cited are in kcal mol\ and are relative to the guanine N7 ion}dipole complex. R1 and R2 indicate the distances of O C(Me) and N7 C(Me), respectively (see Fig. 3). 䊊 and 䊉 are the ion}dipole minima, and £ and 䉲 are TS for N7 and O, respectively.
sites correspond roughly to the energy barrier of the TS except for cytosine. From the potential energy surface, the reaction for guanine O site may begin from the minimum of ion}dipole complex for guanine N7. Therefore, the experimental alkylation fractions of guanine O by N-nitroso compounds, which is larger than those of other methylation compounds, is responsible for this energy surface. REFERENCES CRAMER, C. J. & TRUHLAR, D. G. (1999). Implicit solvation model: equilibria, structure, spectra, and dynamics. Chem. Rev. 99, 2161}2200. FORD, G. P. & SCRIBNER, J. D. (1983). Theoretical study of gas-phase methylation and ethylation by diazonium ions and rationalization of some aspects of DNA reactivity. J. Am. Chem. Soc. 105, 349}354. FORD, G. P. & SCRIBNER, J. D. (1990). Prediction of nucleoside-carcinogen reactivity. Alkylation of adenine, cytosine, guanine, and thymine and their deoxynucleosides by alkanediazonium ions. Chem. Res. ¹oxicol. 3, 219}230. GLASER, R. & CHOY, G. S.-C. (1993). Importance of the anisotropy of atoms in molecules for the representation of electron density distributions with Lewis structures. A case
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study of aliphatic diazonium ions. J. Am. Chem. Soc. 115, 2340}2347. KIKUCHI, O., MATSUOKA, T., SAWAHATA, H. & TAKAHASHI, O. (1994). Ab initio molecular orbital calculations including solvent e!ects by generalized Born formula. Conformation of zwitterionic forms of glicyne, alanine and serine in water. J. Mol. Struct. (¹HEOCHEM) 305, 79}87. KIKUCHI, O., WATANABE, T., OGAWA, Y., TAKASE, H. & TAKAHASHI, O. (1997). Ab initio MO and Monte Carlo simulation study on the conformation of L-alanine zwitterion in aqueous solution. J. Phys. Org. Chem. 10, 145}151. KIKUCHI, O., MORIHASHI, K., TAKAHASHI, O., NAKANO, T., OGAWA, Y. & INADOMI, Y. (1999). ABINI¹/GB, Ab initio MO/GB program package. University of Tsukuba. KIM, N. S. & LEBRETON, P. R. (1996). UV photoelectron and ab initio quantum mechanical evaluation of nucleotide ionization potentials in water-counterion environments: polarization e!ects on DNA alkylation by carcinogenic methylating agents. J. Am. Chem. Soc. 118, 3694}3707. LEA O, M. B. C. & PAVA O, A. C. (1997). Molecular orbital analysis of chemical carcinogens. Int. J. Quant. Chem. 62, 323}328. LOEPPKY, R. N. & MICHEJDA, C. J. (1994). Nitosamines and Related N-Nitroso Compounds, ACS Symposium Series, Vol. 553. Washington, DC: American Chemical Society. LOG WDIN, P.-O. (1950). On the non-orthogonality problem connected with the use of atomic wave functions in the theory of molecules and crystals. J. Chem. Phys. 18, 365}375. MOHAMMAD, S. N. & HOPFINGER, A. J. (1980). Chemical reactivity of a methyldiazonium ion with nucleophilic centers of DNA bases. J. theor. Biol. 87, 401}419. PEDERSEN, L. G., DARDEN, T. A., II, D. W. D., ANDERSON, M. W. & HOEL, D. G. (1988). A theoretical study of the e!ect of methylation or ethylation at O-guanine in the structure and energy of DNA double strands. Carcinogenesis 9, 1553}1562. POLITZER, P. & TRUHLAR, D. G. (eds) (1981). Chemical Applications of Atomic and Molecular Electrostatic Potential. New York: Plenum Press. PULLMAN, A. & ARMBRUSTER, A. M. (1977). On the a$nity of cytosine towards electrophiles. ¹heor. Chim. Acta 45, 249}256. REYNOLDS, C. & THOMSON, C. (1986a). Ab initio calculations relevant to the mechanism of chemical carcinogenesis by nitrosamines. Part 5. The role of diazometane. J Chem. Soc. Perkin ¹rans. II, 1927}1931. REYNOLDS, C. & THOMSON, C. (1986b). A theoretical study of N-nitrosamine metabolites: possible alkylating species
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in carcinogenesis by N,N -dimethyl nitrosamine. Int. J. Quant. Chem. 30, 751}762. REYNOLDS, C. & THOMSON, C. (1987). Ab initio calculations relevant to the mechanisms of chemical carcinogenesis by N-nitrosamines. J. Chem. Soc., Faraday ¹rans. II 83, 485}502. SANTAMARIA, R., COCHO, G., CORONA, L. & GONZAD LEZ, E. (1998). Molecular electrostatic potentials and Mulliken charge populations of DNA mini-sequences. Chem. Phys. 227, 317}329. SINGER, B. & GRUNBERGER, D. (1983). Molecular Biology of Mutagens and Carcinogens. New York: Plenum Press.
TAKAHASHI, O., SAWAHATA, H., OGAWA, Y. & KIKUCHI, O. (1997). Incorporation of solvent e!ects into ab initio molecular orbital calculations by the generalized Born formula. Formulation, parametrization, and applications. J. Mol. Struct. (¹HEOCHEM) 393, 141}150. TOMASI, J. & PERSICO, M. (1994). Molecular interaction in solution: an overview of methods based on continuum distributions of the solvent. Chem. Rev. 94, 2027}2094. VINCENT, M. A. & RADOM, L. (1978). Prototypes for aliphatic and aromatic diazonium ions. An ab initio study of the methane- and benzenediazonium ions. J. Am. Chem. Soc. 100, 3306}3311.
Ab Initio GB Study of Methylation Reaction of Adenine, Cytosine, Guanine, and Thymine by Methanediazonium Ion NAOFUMI NAKAYAMA, SYUNSUKE TANAKA AND OSAMU KIKUCHI Department of Chemistry, ;niversity of ¹sukuba, ¹sukuba 305-8571 Japan (Received on 16 March 2001, Accepted in revised form on 22 June 2001)
Methylation reaction at ten nucleophilic sites in four DNA base molecules by methanediazonium ion (N Me>) was examined by ab initio MO/GB calculation which includes the solvent e!ect with the continuum model using the generalized Born formula. The stabilization energy of the ion}dipole complex as well as the energy of the transition state are roughly consistent with the experimental fraction of methylation by N-methyl-N-nitrosourea. For the guanine N7 site, which is the principal site of methylation, the stabilization energy is the largest and the energy of the transition state is low. The reactions at the guanine O and N7 sites were analysed by drawing the potential energy surface with respect to two parameters, the O}C(Me) distance and the N7}C(Me) distance. The methylation reactions of the guanine O and N7 sites begin from the common geometry, the global minimum in the two-dimensional potential energy surface. 2002 Elsevier Science Ltd.
Introduction N-nitroso compounds are typical mutagenic and carcinogenic compounds (Loeppky & Michejda, 1994). They are believed to owe their activity to conversion to highly reactive diazonium ions which are strong alkylation agents to the nucleophilic sites in DNA bases (Scheme 1). Singer and Grunberger summarized the relative fractions of methylation of single and double-stranded nucleic acids by some methylating reagents (Table 1, Singer & Grunberger, 1983). It is indicated that the principal site of methylation is guanine N7 site. However, the alkylation of this site is innocuous because it is not the hydrogen-bonding site. Although dimethylsulfate (Me SO ) and methyl methanesulfonate (MeMS) alkylate many nucleophilic sites simultaneously, they have weak carcinogenicity. Mutagenic and carcinogenic activity are attributed not to the fraction of methyl0022}5193/02/050013#10 $35.00/0
ation of guanine N7 but to the fraction of guanine O, because O-alkylguanine gives rise to transcriptional errors in both DNA and RNA (Singer & Grunberger, 1983). Recently, molecular orbital calculations are employed for the analyses of the bond formation between the chemical carcinogens (including N-nitrosamines) and DNA bases (Lea o & Pava o, 1997). These analyses are based on the simple frontier orbital theory, and are not responsible for the quantitative analysis of the DNA alkylation by N-nitrosamines. Most N-nitroso compounds have carcinogenicity due to the alkylation of DNA base molecules by alkanediazonium ions. There are several previous reports associated with diazonium ions themselves (Vincent & Radom, 1978; Glaser & Choy, 1993) and diazohydroxide as a precursor of diazonium ion (Reynolds & Thomson, 1986a, b; 1987). 2002 Elsevier Science Ltd.
14
N. NAKAYAMA E¹ A¸.
TABLE 1 Percentage of alkylation of single and double-stranded nucleic acids in vitro* (Singer & Grunberger, 1983) Base
Adenine Cytosine Guanine Thymine
Site
N1 N3 N7 O N3 N3 O N7 O O
Single stranded-
Double stranded?
Me SO
MeMSA
MeNU
Me SO
MeMSA
MeNU
13.2 2.6 3.1
18 1.4 3.8
2.8 2.6 1.8
9.5 :0.1 (0.2 62
10 :1 nd 69
2.3 0.4 3 69
1.9 18 1.9 (nd) ((2) 1.1 0.2 74
3.8 10.4 (1.8) (nd) ((1) (0.6) (0.3) 83
1.3 9 1.7 0.1 0.6 0.8 6.3 67 0.11 0.4
*The absolute amount of alkylation varied greatly but the proportion of derivatives was not noticeably a!ected. &&nd'' indicates that the derivative was not detected. Parentheses indicate either a single value or the average of two very di!erent values and thus less reliability than other data shown. -Analyses are from experiments using DNA from M13 phage and RNA from TMV, yeast, HeLa cells, animal ribosomes, and 2 phage. ?Analyses are from experiments using DNA from salmon sperm, calf thymus, salmon testes, rat liver and brain, human "bloblasts, and HeLa and V79 cells. AMethylmethanesulfonate. N-Methyl-N-nitrosourea.
There are also several theoretical investigations on the alkylation of DNA bases and the model compounds of DNA bases by alkanediazonium ions (Mohammad & Hop"nger, 1980; Ford & Scribner, 1983, 1990; Kim & LeBreton, 1996). Of these studies, two representative investigations dealt with DNA base molecules. Ford & Scribner (1990) examined the methylation, ethylation, and propylation (only for the guanine O and N7) using the MNDO method. They showed that the alkylation site shifts from N7 to O as the bulkiness of the alkyl group increases and suggested that the shift is due to the increase of the S 1 character in the , transition state of their S 2 reactions (in other , words, the transition state structure for ethylation is looser than that for methylation). Their results of calculation roughly reproduced the
experimental trend between methylation and ethylation (Singer & Grunberger, 1983). Kim & LeBreton (1996) examined the methylation by RHF/4-31G level of theory using MNDO optimized molecular structures. They also investigated the ionization potentials of nucleotides by both UV photoelectron and ab initio study, and indicated that polarization e!ects in the nucleotide base play an important role in determining DNA methylation patterns. These works were based on semi-empirical levels of calculations and were examined for speci"c alkylation sites. Further, they did not discuss the reason as to why the alkylation fraction of guanine O site by N-nitroso compounds is larger than other alkylating reagents (Table 1). Theoretical study of the preferences of the cations was reported only for cytosine N3 and O
AB INI¹IO GB STUDY OF METHYLATION REACTION
(Pullman & Armbruster, 1977). Also, no environmental e!ects were taken into account in these studies. It is well known that a cation like the methanediazonium ion is very unstable in the gas phase and MO calculations for the ionic species give the potential energy curves which are far from those expected in the real system. Recently, ab initio calculations including the solvent e!ects have become available (Tomasi & Persico, 1994; Cramer & Truhlar, 1999). It is recommended that the reaction of the diazonium ion is analysed by ab initio MO method including solvent e!ect. Figure 1 shows the schematic energy pro"le of nucleophilic addition reaction of methanediazonium ion with DNA bases. In the investigation of reaction mechanism of the cation species with nucleophilic sites, the stabilization energy of the ion}dipole complex (E ) as well AMKN as the energy of the transition state (E ) is 21 important. E represents the ability of elecAMKN trostatically capturing the cation by the nucleophile. Therefore, E may be an e!ective AMKN descriptor for the experimental fractions of total methylation (Table 1). In this study, the energy pro"les of methylation of DNA base molecules by methanediazonium
15
ion (Scheme 2) are examined by using ab initio method including solvent e!ect. The analysis rationalizes the reason why the fraction of DNA base alkylation by N-nitroso compounds is different from other alkylating reagents. In this paper, nucleophilic addition reactions of methanediazonium ion for ten selected sites of four DNA base molecules (N1, N3, and N7 sites of adenine, O and N3 sites of cytosine, N3, O, and N7 sites of guanine, and O and O of thymine, see Fig. 2) are investigated. In addition, the reactions of guanine O and N7 sites are investigated in detail by drawing the potential energy surface with respect to two parameters, the O}C(Me) distance (R ) and the N7}C(Me) distance (R ) (Fig. 3). Method Ab initio SCF calculations with generalized Born (GB) formula have been described in previous articles (Kikuchi et al., 1994; Takahashi et al., 1997). In the continuum model using the GB formula, the solvation free energy of a molecule is expressed as
1 1 G "! 1! Q Q , QMJ 2
(1)
where is the dielectric constant of the solvent, A and B are atoms in the molecule, and Q and Q are the partial charges on the atoms A and B, respectively. The partial charges are calculated from the nuclear charge and LoK wdin (1950) population. represents the interaction be tween A and B atoms. The energy of a molecule in solution is expressed as the sum of the energy of the molecule in its isolated state, E, and the solvation free energy, G : QMJ EQMJ"E#G . QMJ FIG. 1. Energy pro"le for the reaction between methanediazonium ion and the nucleophilic site in DNA bases.
(2)
By applying the variational theorem to eqn (2), the Fock matrix elements including the solvent
16
N. NAKAYAMA E¹ A¸.
NH2
O
O
NH2 N
N 7
N1
3 2 N H
O
3 N
N H Adenine
HN H2N
Cytosine
6 3 N Guanine
N 7 N H
4
HN
Me
2 O
N H Thymine
FIG. 2. Nucleophilic sites in DNA base molecules. N N C
R1 R2
O H
N 6
N 7
H2N
N
N
H H
FIG. 3. Two parameters for the potential energy surface for the guanine O and N7 reactions.
e!ect for a closed-shell molecule are F "F #FQMJ , IJ IJ IJ
(3)
G QMJ (S ) (S ) , FQMJ" IH JH IJ Q H H
(4)
where F is the Fock matrix element for an IJ isolated molecule, and FQMJ describes the contriIJ bution of the solvent. Q is the charge of the atom H to which the basis function belongs. The partial derivatives in eqn (4) are expressed as
G 1 QMJ"! 1! Q
1 Q # Q 2 Q
. # Q Q Q BOA
(5)
The ab initio GB method has been applied to many chemical reactions including ionic species. It reproduces well the relative solvation energy of ionic species. This was con"rmed by comparing the solvation energies calculated by ab initio GB method with those calculated by Monte Carlo
simulation in which many solvent molecules were considered explicitly (Kikuchi et al., 1997). In this study, the dielectric constant "78.5 was used to represent the aqueous solution. Geometry optimizations and calculations for searching the TS were carried out using ABINIT/GB program package (Kikuchi et al., 1999) on the DEC 500/500 workstation. The transition state (TS) structures were determined by minimizing the norm of energy gradients. The RHF method was employed with the 3-21G basis set. We calculated the possible energy-minimum structures of methylated products in each compound. Figure 4 shows the most stable structure of each methylated DNA base molecules in solution (all structures have C symmetry). For the Q guanine O adduct, the proximal conformation in which the Me group is on the N7 side is more stable than the distal conformation in which the Me group is the opposite to the N7 site. This contradicts the previous result (Pedersen et al., 1988) which was obtained by partial geometry optimization. In the calculation of ion}dipole complex and the TS, the orientation of the methyl group was postulated to be the same as the direction of the methyl group in each methylated DNA base. Therefore, the calculations were carried out under the C symmetry. Q Results and Discussion ENERGY PROFILE AND STRUCTURE FOR EACH REACTION SITE
Figure 5 shows the ion}dipole and the TS structures for guanine N7 and O sites. Table 2 shows the total energies of the methylated DNA base cation, the ion}dipole complex and the TS structure in each alkylation reaction. In the
AB INI¹IO GB STUDY OF METHYLATION REACTION
17
FIG. 4. The most stable structures of methylated DNA base cations.
calculations of the TS structure, only the selected structural parameters were optimized: methanediazonium ion (N Me>) moiety, distance be tween C(Me) and X (R, X"N or O atom of base site), and C(Me)}X}C (base) angle (, see Fig. 6). The parameters associated with the base framework were adopted from those in the ion}dipole
complex, and were "xed in the calculations of the TS searching. Table 3 shows the relative energies of methylation reactions shown in the energy pro"le in Fig. 1. A large stabilization, E, is obtained by the methylation at each nucleophilic site even in an aqueous solution where methanediazonium
18
N. NAKAYAMA E¹ A¸.
cation is largely stabilized. It is worth pointing out that E becomes larger if the solvent e!ect is not included. Although the E values at nucleophilic sites appear to have a strong correlation with the values of molecular electrostatic potential (MEP) calculated by both ab initio (Politzer
& Truhlar, 1981) and DFT method (Santamaria et al., 1998), three energies, E , E , and E, AMKN 21 have little correlation to one another. The possibility for the alkylating reagent to access each nucleophilic site can be the reactivity index. Thus, the calculated E value of AMKN guanine N7 site is the largest of all sites, which is consistent with the experiment that the most reactive site of guanine is N7 (Singer & Grunberger, 1983). However, the calculated E enerAMKN gies of cytosine are close to that of guanine N7, although the experimental fractions of methylation in cytosine are much smaller than those in guanine. In the case of thymine and adenine, the E values are larger in thymine, while the AMKN E values are larger in adenine. The E 21 21 values correlate well with the experiment. In adenine, three sites, N1, N3, and N7, are pyridinetype nitrogen atoms and were predicted to have similar reactivity. The N3 site has the smallest activation energy, which is parallel to the experiment. Excluding cytosine, the E values in 21 aqueous solution re#ect well the site of alkylation
FIG. 5. Structures of the ion}dipole complex and the TS for the methylation of guanine N7 and O sites.
FIG. 6. Structural parameters optimized in the TS calculation.
TABLE 2 ¹otal energies (hartree) of the methylated product, the ion}dipole complex and the ¹S on each site of DNA base molecules* Base
Site
Methylated product
Complex
TS
Adenine
N1 N3 N7 O N3 N3 O N7 O O
!501.189567 !501.188700 !501.186298 !429.708579 !429.727406 !575.649971 !575.644849 !575.674172 !488.236425 !488.238571
!609.405935 !609.405118 !609.404939 !537.938895 !537.940354 !683.871148 !683.891080 !683.895002 !596.495136 !596.495822
!609.399644 !609.399228 !609.398461 !537.936610 !537.934859 !683.862914 !683.886676 !683.890792 !596.488213 !596.488508
Cytosine Guanine Thymine
*RHF/3-21G calculation in aqueous solution ("78.5).
AB INI¹IO GB STUDY OF METHYLATION REACTION
N site, and 2.6}2.7 As for the O site (guanine N3 and thymine O are exceptional). In the TS, both distances for the N site alkylation are also the same except for guanine N3. However, the distances for the O site are diverse. Table 5 shows the N}C(Me)}Site and N}N}C(Me) bond angles in the ion}dipole complex and the TS structure for each reaction site. They indicate that both in complex and TS, N}N}C(Me)}Site is approximately linear for all sites, except for the complex of cytosine N3. Table 6 shows the total charge of methanediazonium ion in the ion}dipole complex and the TS structure. These values exhibit the extent of electron transfer from the DNA molecule to the cation. They indicated that the extent of transfer from the cytosine N3 and guanine N7 sites are larger than others in the complex, and this may correspond to the magnitude of E . AMKN
by methanediazonium ion. In cytosine and thymine, the steric repulsion due to the sugar and phosphate groups may be an important factor to determine the sites of methylation. The steric interaction should be included for a more quantitative discussion. Table 4 shows the N}C(Me)} and C(Me)}Site distances in the ion}dipole complex and the TS structure for each reaction site. In the complex, the N}C(Me) distance is almost constant in all sites, while C(Me)}Site distances are roughly divided into two categories: 2.9}3.1 As for the TABLE 3 Relative energies (kcal mol\) of ion}dipole complex, ¹S, and product on each site of DNA base molecules (see Fig. 1)* Base
Site
E AMKN
E 21
E
Adenine
N1 N3 N7 O N3 N3 O N7 O O
!5.1 !4.6 !4.5 !13.0 !13.9 !0.7 !13.2 !15.7 !8.3 !8.7
3.9 3.7 4.1 1.4 3.4 5.2 2.8 2.6 4.3 4.6
!58.2 !57.7 !56.2 !57.3 !69.1 !50.8 !47.5 !65.9 !34.8 !36.2
Cytosine Guanine Thymine
POTENTIAL ENERGY SURFACE FOR THE METHYLATION REACTIONS OF GUANINE N7 AND O SITES
In guanine, the O and N7 sites have much stable ion}dipole complexes and small activation energies. This distinctly re#ects the experimental fact that these two sites are easily alkylated. However, E values for their sites are close to 21 each other, although there are large di!erences between their experimental fractions of methylation. In order to survey the potential energy surface for guanine N7 and O, two parameters (R and
*These values are obtained from total energies of each species (in Table 2), DNA base molecules, N , and N Me>. Total energies (hartree) in aqueous solution of adenine, cytosine, guanine, thymine, N , and N Me> are !461.917052, !390.437458, !536.389323, !449.001191, !108.300954, and !147.480710, respectively.
TABLE 4 ¹he distances (As ) in the ion}dipole complex and ¹S N}C(Me) and C(Me)}Site Base
Adenine Cytosine Guanine Thymine
Site
N1 N3 N7 O N3 N3 O N7 O O
19
Complex
TS
N}C(Me)
C(Me)}Site
N}C(Me)
C(Me)}Site
1.513 1.513 1.512 1.536 1.516 1.518 1.517 1.506 1.526 1.523
3.005 2.989 2.987 2.646 3.013 2.673 2.669 3.091 2.720 2.906
1.808 1.805 1.809 1.762 1.802 1.831 1.825 1.795 1.900 1.894
2.458 2.450 2.433 2.350 2.466 2.441 2.319 2.447 2.228 2.227
20
N. NAKAYAMA E¹ A¸.
TABLE 5 ¹he bond angles (deg) in the ion}dipole complex and ¹S N}C(Me)}site and N}N}C(Me) Base
Adenine Cytosine Guanine Thymine
Site
Complex N}C(Me)}Site
N}N}C(Me)
N}C(Me)}Site
N}N}C(Me)
178.1 178.3 177.9 177.0 168.6 178.4 179.8 173.3 176.0 176.7
180.0 180.0 179.9 180.0 178.9 179.9 179.9 179.9 180.0 179.2
179.1 178.9 179.5 177.7 175.2 178.3 177.9 177.3 177.8 177.2
179.8 179.9 180.0 179.8 177.7 180.0 178.9 178.1 179.9 179.7
N1 N3 N7 O N3 N3 O N7 O O
TABLE 6 ¹otal charge (¸oK wdin, 1950) of methanediazonium ion moiety in the ion}dipole complex and ¹S Base
Site
Complex
TS
Adenine
N1 N3 N7 O N3 N3 O N7 O O
0.976 0.976 0.976 0.955 0.933 0.980 0.958 0.933 0.971 0.971
0.896 0.898 0.896 0.900 0.862 0.890 0.890 0.864 0.900 0.895
Cytosine Guanine Thymine
TS
R , see Fig. 3) were varied in steps of 0.25 As in the range of 2.25}3.50 As . Other parameters were fully optimized in C symmetry. Q Figure 7 shows the potential energy surface of the methylation reactions of both guanine N7 and O sites. In this map, both the minimum of ion}dipole complex and the TS are included: O}C(Me)"3.173 As and N7}C(Me)"2.907 As for the ion}dipole complex for the N7 site, O}C(Me)"2.720 As and N7}C(Me)"3.326 As for the ion}dipole complex for the O site, O}C(Me)"3.157 As and N7}C(Me)"2.455 As for the TS for the N7 site, and O}C(Me)" 2.315 As and N7}C(Me)"3.258 As for the TS for the O site. The energy di!erence between the ion}dipole complexes for guanine N7 and O is 2.5 kcal mol\ (see Table 3).
Figure 7 indicates that the minimum of the ion}dipole complex of guanine N7 is the global minimum is this surface, and that the minimum of guanine O is very shallow. It may be reasonable to assume that the diazonium ion is trapped at the global minimum to form the ion}dipole complex and the alkylation to N7 and O starts from the common complex. The energy of the TS of O site relative to the global minimum is expected to be less than 4 kcal mol\. It is lower than the energy of TS in Table 4 (5.2 kcal mol\) because the structure of guanine framework is "xed in the calculation of TS searching. We conclude that the di!erence of experimental fraction between O and N7 of guanine is due to the energy di!erence of the TS between O and N7. The reaction mechanisms for guanine O and N7 are expected to be such that the methylation reaction of guanine O site begins from the N7 minimum which is the global minimum in the two-dimensional energy surface. Conclusion The energy pro"le of the nucleophilic addition reactions of methanediazonium ion to nucleophilic sites of DNA bases were clari"ed by ab initio MO calculation including solvent e!ects. The stabilization energy for the formation of the ion}dipole complex in guanine N7 site is the largest in all calculated sites. It is consistent with the experiment that the most reactive site is guanine N7. The methylation fractions of other
AB INI¹IO GB STUDY OF METHYLATION REACTION
FIG. 7. Two-dimensional potential energy surface of the methylation reactions of both guanine N7 and O sites. Energies cited are in kcal mol\ and are relative to the guanine N7 ion}dipole complex. R1 and R2 indicate the distances of O C(Me) and N7 C(Me), respectively (see Fig. 3). 䊊 and 䊉 are the ion}dipole minima, and £ and 䉲 are TS for N7 and O, respectively.
sites correspond roughly to the energy barrier of the TS except for cytosine. From the potential energy surface, the reaction for guanine O site may begin from the minimum of ion}dipole complex for guanine N7. Therefore, the experimental alkylation fractions of guanine O by N-nitroso compounds, which is larger than those of other methylation compounds, is responsible for this energy surface. REFERENCES CRAMER, C. J. & TRUHLAR, D. G. (1999). Implicit solvation model: equilibria, structure, spectra, and dynamics. Chem. Rev. 99, 2161}2200. FORD, G. P. & SCRIBNER, J. D. (1983). Theoretical study of gas-phase methylation and ethylation by diazonium ions and rationalization of some aspects of DNA reactivity. J. Am. Chem. Soc. 105, 349}354. FORD, G. P. & SCRIBNER, J. D. (1990). Prediction of nucleoside-carcinogen reactivity. Alkylation of adenine, cytosine, guanine, and thymine and their deoxynucleosides by alkanediazonium ions. Chem. Res. ¹oxicol. 3, 219}230. GLASER, R. & CHOY, G. S.-C. (1993). Importance of the anisotropy of atoms in molecules for the representation of electron density distributions with Lewis structures. A case
21
study of aliphatic diazonium ions. J. Am. Chem. Soc. 115, 2340}2347. KIKUCHI, O., MATSUOKA, T., SAWAHATA, H. & TAKAHASHI, O. (1994). Ab initio molecular orbital calculations including solvent e!ects by generalized Born formula. Conformation of zwitterionic forms of glicyne, alanine and serine in water. J. Mol. Struct. (¹HEOCHEM) 305, 79}87. KIKUCHI, O., WATANABE, T., OGAWA, Y., TAKASE, H. & TAKAHASHI, O. (1997). Ab initio MO and Monte Carlo simulation study on the conformation of L-alanine zwitterion in aqueous solution. J. Phys. Org. Chem. 10, 145}151. KIKUCHI, O., MORIHASHI, K., TAKAHASHI, O., NAKANO, T., OGAWA, Y. & INADOMI, Y. (1999). ABINI¹/GB, Ab initio MO/GB program package. University of Tsukuba. KIM, N. S. & LEBRETON, P. R. (1996). UV photoelectron and ab initio quantum mechanical evaluation of nucleotide ionization potentials in water-counterion environments: polarization e!ects on DNA alkylation by carcinogenic methylating agents. J. Am. Chem. Soc. 118, 3694}3707. LEA O, M. B. C. & PAVA O, A. C. (1997). Molecular orbital analysis of chemical carcinogens. Int. J. Quant. Chem. 62, 323}328. LOEPPKY, R. N. & MICHEJDA, C. J. (1994). Nitosamines and Related N-Nitroso Compounds, ACS Symposium Series, Vol. 553. Washington, DC: American Chemical Society. LOG WDIN, P.-O. (1950). On the non-orthogonality problem connected with the use of atomic wave functions in the theory of molecules and crystals. J. Chem. Phys. 18, 365}375. MOHAMMAD, S. N. & HOPFINGER, A. J. (1980). Chemical reactivity of a methyldiazonium ion with nucleophilic centers of DNA bases. J. theor. Biol. 87, 401}419. PEDERSEN, L. G., DARDEN, T. A., II, D. W. D., ANDERSON, M. W. & HOEL, D. G. (1988). A theoretical study of the e!ect of methylation or ethylation at O-guanine in the structure and energy of DNA double strands. Carcinogenesis 9, 1553}1562. POLITZER, P. & TRUHLAR, D. G. (eds) (1981). Chemical Applications of Atomic and Molecular Electrostatic Potential. New York: Plenum Press. PULLMAN, A. & ARMBRUSTER, A. M. (1977). On the a$nity of cytosine towards electrophiles. ¹heor. Chim. Acta 45, 249}256. REYNOLDS, C. & THOMSON, C. (1986a). Ab initio calculations relevant to the mechanism of chemical carcinogenesis by nitrosamines. Part 5. The role of diazometane. J Chem. Soc. Perkin ¹rans. II, 1927}1931. REYNOLDS, C. & THOMSON, C. (1986b). A theoretical study of N-nitrosamine metabolites: possible alkylating species
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N. NAKAYAMA E¹ A¸.
in carcinogenesis by N,N -dimethyl nitrosamine. Int. J. Quant. Chem. 30, 751}762. REYNOLDS, C. & THOMSON, C. (1987). Ab initio calculations relevant to the mechanisms of chemical carcinogenesis by N-nitrosamines. J. Chem. Soc., Faraday ¹rans. II 83, 485}502. SANTAMARIA, R., COCHO, G., CORONA, L. & GONZAD LEZ, E. (1998). Molecular electrostatic potentials and Mulliken charge populations of DNA mini-sequences. Chem. Phys. 227, 317}329. SINGER, B. & GRUNBERGER, D. (1983). Molecular Biology of Mutagens and Carcinogens. New York: Plenum Press.
TAKAHASHI, O., SAWAHATA, H., OGAWA, Y. & KIKUCHI, O. (1997). Incorporation of solvent e!ects into ab initio molecular orbital calculations by the generalized Born formula. Formulation, parametrization, and applications. J. Mol. Struct. (¹HEOCHEM) 393, 141}150. TOMASI, J. & PERSICO, M. (1994). Molecular interaction in solution: an overview of methods based on continuum distributions of the solvent. Chem. Rev. 94, 2027}2094. VINCENT, M. A. & RADOM, L. (1978). Prototypes for aliphatic and aromatic diazonium ions. An ab initio study of the methane- and benzenediazonium ions. J. Am. Chem. Soc. 100, 3306}3311.