Chem. Res. Toxicol. 1992,5, 756-759
756
Communications Distinct Conformers of Alkylchrysene Diol Epoxide-Deoxyguanosine Adducts Detected by Proton NMR Bijaya Misra, Jyh-Ming Lin, Shantu Amin,* and Stephen S. Hecht Division of Chemical Carcinogenesis, American Health Foundation, Valhalla, New York 10595 Received July 2, 1992 Proton NMR spectra, obtained in MeOH-d4; of the major DNA adduct of 5,7-dimethylchrysene1,2-diol 3,4-epoxide, identified as 1(R),2(S),3(S)-trihydroxy-4(S)-(Rn-deoxyguanosyl)-l,2,3,4tetrahydro-5,7-dimethylchrysene,showed the presence of two distinct conformers. One conformer, similar to those observed previously in spectra of peracetates of related DNA adducts of anti-diol epoxides of polynuclear aromatic hydrocarbons, had a chair-like conformation of the tetrahydrobenzo ring. The other conformer, which has not been previously observed, had a boat-like conformation of the tetrahydrobenzo ring. This conformer was converted to the chair-like conformer upon addition of DzO or trifluoroacetic acid to the MeOH-d4 solutions of the adduct. The new conformers were also observed in proton NMR spectra of major DNA adducts of 5-methylchrysene- and 5,6-dimethylchrysene-1,2-diol 3,4-epoxides.
Introduction PAHl are a well-established class of potent carcinogens formed in the incomplete combustion of organic matter. They are ubiquitous in the environment and are probably important in the etiology of human cancers (1). Angular ring diol epoxides such as 1a-c, in which the epoxide ring is in the bay region, are major ultimate carcinogens of many PAH (2). The adducts produced by covalent binding
an anti-diol epoxide, the chair-like conformation,C2,has been observed. In this communication, we describe the first evidence for an additional major conformer, C1,of anti-diol epoxide-dG adducts. We investigated the DNA H.
R\ /H
H4*H1
OH
I
OH
c1
c2
adducts of la-c as part of our ongoing studies on the mechanisms underlying structure-tumorigenicity relationships among carcinogenic methylchrysenes (4,5,14, 15). The new conformers were observed in proton NMR spectra run in MeOH-d4. of these diol epoxides to DNA are critical in the initiation of carcinogenesis. These adducts have been extensively characterized (3-13). The major adducts formed from the generally more carcinogenic anti-diol epoxides such as la-c arise by trans addition of the exocyclic amino group of dG,and in some cases dA,to the benzylic carbon of the epoxide ring. The conformations of the tetrahydrobenzo rings in these adducts have been deduced from the coupling constants in the proton NMR spectra of the corresponding peracetates and in some instances from the spectra of the underivatized adducts. In the adducts resulting from trans addition of the exocyclic amino group of dG or dA to the benzylic carbon of the epoxide ring of
Experimental Procedures
Racemic diol epoxides la-c were synthesized (16-18). Each diol epoxide (1 mg in 1 mL of dry THF) was reacted with calf thymus DNA (10 mg in 10 mL 0.05 M Tris buffer, pH 7.0) at 37 "C for 18 h. The resulting mixtures were extracted with 3 X 10-mL portions of EtOAc, and the DNA was precipitated from the aqueous layer by addition of cold EtOH. The DNA waa hydrolyzed to deoxyribonucleosidesas previously described (5). The hydrolysates were analyzed by HPLC using a 4.6 X 250 mm Beckman Ultrasphere C-18 reverse-phase column (5 pm), eluted with a MeOH/H20 gradient as follows: 15-45% MeOH in HzO in 25 min, then 45-55% MeOH in HzO in 5 min, followed by 55-70% MeOH in 25 min, and finally to 100% MeOH in 5 min. The peracetate of NZ-dG-5,7-diMeCDE was prepared from a sample which had been dissolved in MeOH-dr for NMR. The 'Abbreviations: PAH, polynuclear aromatic hydrocarbons; N2-dG5,7-diMeCDE,1~~),2(~,3(s)-trihydroxy-4(s)-(~-deoxy~~yl)-l,2,3,4- MeOH-d4 was evaporated, and the residue was dissolved in a tetrahydro-5,7-dimethylchrysene;NZ-dG-5-MeCDE, 1(R),2(S),3(S)- mixture of 3 mL of pyridine and 2 mL of Ac2O. The resulting trihydroxy-4~S~-(Nz-deoxyguanosyl)-1,2,3,4-tetrahydro-5-methmixture was stirred overnight at 37 "C. After concentration to ylchrysene; NZ-dG-5,6-diMeCDE,1(R),2(S),3(S)-trihydrox~4(S)-(~deoxyguanosyl~-1,2,3,4-tetrahydro-5,6-dimethylc~sene. dryness, the residue was redissolved in T H F and the peracetate
0 1992 American Chemical Society
Communications
Chem. Res. Toricol., Vol. 5, No. 6,1992 757
A
3
9.0 8.8 8.6 8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2
PPM I
0
10
20
30
40
50
60
Retention Time (min)
Figure 1. HPLC trace of an enzymatic hydrolysate of calf thymus DNA that had been reacted with racemic antid,7-dimethylchrysene-l,2-diol 3,4-epoxide (la).
Figure 3. Downfield portions of the 360-MHz proton NMR spectra of N2-dG-5,7-diMeCDE,0.64 mM, in (A) MeOH-dd and (B) 1:l MeOH-d$DzO.
reported for the peracetate of the major dG adduct of lb, which is farmed by trans ring opening of its l(R),2(S)-diol 3(S),4(R)-epoxideenantiomer by the exocyclicamino group of dG. The large coupling constant, J 1 , 2 = 8.82 Hz, and small coupling constants, J2,3 = 2.42 Hz and J3,4 = 4.12 Hz, are consistent with the chair-like conformation, C2, of the peracetate, and with previously reported data on related adducts. These data demonstrate that peak 3, the major adduct formed by reaction of la with DNA, has the structure illustrated in Figure 2 (N2-dG-5,7-diMeCDE). The downfield portion of the proton NMR spectrum of underivatized peak 3,0.64 mM in MeOH-d4, is illustrated in Figure 3A. It was remarkably different from that shown in Figure 2. Several of the proton resonances appeared as pairs of peaks; this was clearly evident for H11 and 9.0 8.0 7.0 6.0 guanine Hg. All assignments were confiimed by decoupling PPM experiments. In other spectra of peak 3 obtained at Figure 2. Downfield portion of the 360-MHz proton NMR concentrations of 0.3-1.0 mM in MeOH-d4, we have spectrum of the peracetate of N2-dG-5,7-diMeCDE. observed varying amounts of each paired resonance. The spectrum of peak 3, 0.64 mM in 1:l MeOH-ddID20, is was purified by HPLC on the Ultrasphere column with elution by linear MeOH in HzO gradients as follows: 1545% MeOH in illustrated in Figure 3B; the paired resonances have 15min, and then 65-100% in 20 min. The retention time of the virtually disappeared. This was also accomplished by peracetate was 32 min. adding one drop of trifluoroacetic acid to the MeOH-d4 Proton NMR spectra were obtained on a Bruker AM 360 WB solutions. These data can be explained by the presence NMR spectrometerin MeOH-d4at 22 OC, unlessnoted otherwise. of two distinct conformers, C1 and C2, whose interconversion in MeOH-d4 is slow on the NMR time scale. The Results and Discussion chemical shifts and coupling constants of the two conformers are summarized in Table I. The two conformers Reaction of racemic la with DNA, followedby enzymatic were not observed in spectra run in DMSO or acetone-& hydrolysis to deoxyribonucleosides and HPLC analysis, nor were they seen in the peracetate spectrum (Figure 2). gave the chromatogram illustrated in Figure 1. In this The coupling constants are of interest. Some of study, we focused on the major adduct, peak 3. Comparison of its UV spectrum and retention time to those of the appropriate resonances were obscured by HzO in the spectra of peak 3 run a t 22 OC. However, in spectra run a standard prepared by reaction of la with poly(deoxyat 35 "C, the H2O peak shifted upfield, allowingassignment guanylic acid) indicated that it was a dG adduct. Its CD of the peaks and measurement of the coupling constants. spectrum had a positive sign, consistent with trans ring Conformer C2, which increases in concentration upon opening of the l(R),2(S)-diol3(5'),4(R)-epoxideenantiomer of la by the exocyclic amino group of dG (5,11,13,19). addition of D20, has a large coupling constant J1,2 = 8.82 The downfield portion of the proton NMR spectrum of Hz and smaller coupling constants J2,3 = 2.48 Hz and J3,4 = 3.38 Hz. These data are consistent with the chair-like the peracetate derivative of peak 3 is illustrated in Figure conformation C2 in which HI and H2 are pseudodiaxial, 2. Assignments were made by decoupling experiments while HSand H4 are pseudodiequatorial. This chair-like and by reference to published spectra (11, 13). The conformer is the predominant one observed to date in the chemical shifts of H1-H4 were virtually identical to those
758 Chem. Res. Toxicol., Vol. 5, No. 6,1992 Table I. Chemical Shifts and Coupling Constants in the Proton NMR Spectrum, R u n in MeOH-dd of NZ-dG-S,7-diMeCDE
Communications Table 11. Chemical Shifts and Coupling Constants in the Downfield Portions of the Proton NMR Spectra, R u n in MeOH-d4, of W-dG-6-MeCDE and W-dG-S,6-diMeCDE chemical shift, 6 (couDlhtz constant. Hz)
chemical shift, 6 (coupling constant, Hz) Droton
conformer C1
conformer C2
4.98, d ( J 1 , 2 = 1.48) 4.65, dd (J1,2 = 1.79; J z ,=~ 7.20) 4.92, dd (Jz,~ = 7.14; J 3 , 4 = 2.36) 6.51, d (J3,4 = 2.28) 7.90, s
5.09, d (J1,2 = 8.82) 4.03, dd (J1,z = 9.00, J z ,=~ 2.48) = 2.48, 4.70, dd (Jz,~ 5 3 4 = 3.38) 6.40 (J3,4 = 4.30) 7.91, s
proton
conformer C1
conformer C2
~~
7.48, m
7.5, m
8.60, d ( J ~ o 8.11) J~ 8.85, d (5113 = 8.67) 7.70, d ( 5 1 1 ~ 2 8.44) 3.17,s 2.74, s 7.96, s 6.41, dd (Ji,,z,= 6.17; J1,,2, 6.78) 2.55,3.09, m 4.87, m 3.98, m 3.78,3.88, m
8.62, d (Jio,ii = 8.86) 8.98, d (Ji1,iz = 9.16) 8.07, d ( 5 1 1 ~ 2 8.98) 3.06,s 2.72, s 8-08,s 6.48, dd (51,~ = 5.46; J1,,y = 6.31) 2.56,2.80, m 4.76, m 4.06, m 3.65,3.82, m
proton NMR spectra of virtually all PAH anti-diol epoxide deoxyribonucleosideadducts formed by trans ring opening (6-12). The bulky deoxyguanosineresidue is pseudoaxial to minimize steric interactions in the bay region. The coupling constants of H1-H4 in conformer C1 are distinctly different from those in C2. In this conformer, Cl,J1,2 = 1.48 Hz and J3,4 = 2.36 Hz are small, while J2,3 = 7.20 Hz is large. These data can be explained by the boat-like conformation illustrated for C1. The dihedral angle of Oo between HZand H3 is consistent with the large coupling constant while the dihedral angles between HI and Ha, as well as Hs and Hq, are approximately 70°, consistent with the small coupling constants. This conformer appears to be stabilized by hydrogen bonding between the adjacent hydroxyl groups on carbons 2 and 3 of the tetrahydrobenzo ring. Addition of D20 or trifluoroaceticacid could disrupt the hydrogen bond, resulting in conversion of C1 to C2. Hydrogen bonding of this type would not be possible in peracetate derivatives. NMR data from the downfield portions of spectra of the major DNA adducts of diol epoxides l b and IC are summarized in Table 11. These adducts are also formed by trans ring opening of the l(R),a(S)-diol 3(S),4(R)epoxide enantiomer by the exocyclic amino group of deoxyguanosine. While these spectra have not been analyzed in as much detail as those discussed above, it is clear that the two conformers are present. We are not certain whether the presence of a methyl group in the bay regions of adducts of la-lc is necessary for observation of the two adduct conformers. This requires further investigation. Most NMR spectral data on PAH diol epoxide deoxyribonucleoside adducts have been obtained on the corresponding peracetates, in which conformer C1 is not observed. The facile conversion of C1 to C2 by D20, by trace amounts of acid, or perhaps by minor sample impurities may explain why it has not been detected in previous NMR studies of underivatized adducts. The conformationsof PAH diol epoxideadducts in DNA appear to play a role in determining their carcinogenic effects (20). Studies on the conformationsof these adducts at the monomer level may provide insights on their steric
(A) NZ-dG-5-MeCDE 7.7,s NAa
Hs H7
7.7,s 7.8, m
Hio Hi1 Hi2 Gua-Ha
8.75, m 8.72, m 8.86, d (Jii,iz = 9.47) 8.98, d ( 5 1 1 ~ 2= 9.20) 7.69, d ( 5 1 1 ~ 2= 9.47) 8-09,d ( 5 1 1 ~ 2= 9.20) 7.95, s 8.08, s (B)N2-dG-5,6-diMeCDE 8.15, m 8.07, m
7.6, m
7.6, m
HI Ha He Hio Hi1
Hiz Gua-Ha
7.62, m
7.62, m
8.75, m 8.77, d ( 5 1 1 ~ 2 8.82) 7.62, m 8.0,s
8.75, m 8.85, d ( 5 1 1 ~ 2 9.19) NA NA
NA = not assigned.
features in DNA and possibly on the resulting biological effects. Further research is required to assessthe potential significance in carcinogenesis of the distinct conformers observed in this study.
Acknowledgment. We thank Professor Nicholas E. Geacintov, Chemistry Department, New York University, for obtaining the CD spectrum. This study was supported by Grant CA-44377 from the National Cancer Institute. This is paper 146 in the series "A Study of Chemical Carcinogenesis". References International Agency for Research on Cancer (1983)ZARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Volume 32. Polynuclear Aromatic Compounde, Part 1,Chemical, Environmental, andExperimentalData, IARC, Lyon, France. Conney, A. H. (1982) Induction of microsomal enzymes by foreign chemicalsand carcinogenesisby polycyclic aromatic hydrocarbons: G. H. A. Clowes Memorial Lecture. Cancer Res. 42,48754917. Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Weinatein, I. B., Beland, F. A., Harvey, R. G., Kaeai, H., Miura, I., and Nakanishi, K. (1976) Benzo[alpyrene-nucleic acid derivative found in vivo: structure of a benzo[alpyrenetetr&ydrodiol e p o x i d m o e i n e adduct. J. Am. Chem. SOC.98,5714-5715. Melikian,A. A., Ami, S., Hecht, S. S., Hoffmann,D., Pataki, J.,and Harvey, R. G. (1984) Identification of the major adducts formed by reaction of 5-methylchryseneanti-dihydrodiolepoxides with DNA in vitro. Cancer Res. 44, 2524-2529. Melikian, A. A., Amin, S., Huie, K., Hecht, S. S., and Harvey, R. G. (1988) Reactivity with DNA bawa and mutagenicity toward Salmonella typhimurium of methylchrysenediol epoxideenantiomers. Cancer Res. 48, 1781-1787. Agarwal, 5. K., Sayer, J. M., Yeh, H. J., Pannell, L. K., Hilton, B. D., Pigott, M. A., Dipple, A., Yagi, H., and Jerina, D. M. (1987) Chemical characterization of DNA adducta derived from the configurationally isomeric benzo[clphenanthrene3,4-diol1,2-epoxides. J. Am. Chem. SOC.109, 2497-2604. Cheng, S. C., Prakaah, A. S., Pigott, M.A., Hilton, B. D., Roman, J. M., Lee, H., Harvey, R. G., and Dipple, A. (1988)Characterization of 7,12-dimethylbenz[alanthracene-adenine nucleoside adducts. Chem. Res. Toxicol. 1,216-221. Peltonen, K., Cheng, S. C., Hilton,B. D., Lee,H., Cortez, C., Harvey, R. G., and Dipple, A. (1991)Effect of bay region methyl group on reactions of anti benz[a]anthracene-3,4-dihydrodiol 1,2-epoxides with DNA. J. Org. Chem. 66,4181-4188.
Chadha,A.,Sayer,J.M.,Yeh,H.J.C.,Yagi,H.,Cheh,A.M.,Pannell, L. K., and Jerina, D. M. (1989) Structures of covalent nucleoside adducts formed from adenine, guanine,and cytoeine bases of DNA and the optically active bay-region 3,4-dioll,2-epoxidesof dibenz[ajlanthracene. J. Am. Chem. SOC.111,5456-5463.
Communications (10)Nair, R. V.,Gill, R. D., Cortez, C., Harvey, R. G.,and DiGiovanni, J. (1989)Characterization of DNA adducts derived from (*)-tram3,4-dihydroxy-anti-l,2-epox~l,2,3,4-tetr~ydrodibenz[a~lanthracene and (f)-7-methyl-tram-3,4-dihydroxy-anti-l,2-epoxy1,2,3,4-tetrahydrodibeenz[oj]anthracene. Chem. Res. Toricol. 2, 341-348. (11) Reardon, D. B., Prakash, A. S.,Hilton, B. D., Roman, J. M., Pataki, J., Harvey, R. G., and Dipple, A. (1987) Characterization of 5-methvlchene1.2-dihvdrodiol-3.4-e~oxid~DNA adducts. Car. cinogenesk k, 1317-1322: (12) . . Cheng. S.C., Hilton, B. D., Roman, J. M., and Diode, A. (1989) D N A i d d u k from ckinogenic and noncarcinoge4; enantiomers of benzo[a]pyrene dihydrodiol epoxide. Chem.Res. Toxicol. 2,334340. (13) Peltonen, K.,Hilton, B. D., Pataki, J., Lee, H., Harvey, R. G.,and Dipple, A. (1991)Spectroscopic characterization of syn-bmethyladducts. chrysene 1,2-dihydrcdiol-3,4-epoxide-deoxyribonucleoside Chem. Res. Toricol. 4,305-310. (14) Hecht, S.S.,Melikian, A. A., and Amin, S. (1986)Methylchrysenes as probes for the mechanism of metabolic activation of carcinogenic methylated polynuclear aromatic hydrocarbons. Acc. Chem. Res. 19,174-180. (15) Hecht, S. S., Amin, S., Huie, K., Melikian, A. A., and Harvey, R. G. (1987)Enhancing effect of a bay region methyl group on tumori-
Chem. Res. Toricol., Vol. 5, No. 6, 1992 769 genicity in newborn mice and mouse skin of enantiomeric bay region diol epoxides formed stereoselectively from methylchrysenea in mouse epidermis. Cancer Res. 47,5310-5315. (16) Harvey, R. G.,Pataki, J., and Lee, H. (1986) Synthesis of the dihydrodiol and diol epoxide metabolites of chrysene and S-methylchrysene. J. Org. Chem. 51, 1407-1412. (17) Amin, S.,Balanikas, G.,Huie, K., and Hecht, S. S. (1988)Synthesis and tumor initiating activities of dimethylchrysenes. Chem. Res. Toricol. 1, 349-355. (18) Misra, B., Amin, S., and Hecht, S. S. (1992)Dimethylchrysene diol epoxides: mutagenicity in S. typhimurium, tumorigenicity in newborn mice, and reactivity with deoxyadenosine in DNA. Chem. Res. Toricol. 5, 248-254. (19) Sayer, J. M.,Chandha, A., Agarwal, S. K., Yeh, H. J. C., Yagi, H., and Jerina, D. M. (1991)Covalent nucleoside adducts of benzo[o]pyrene 7,8-diol9,lO-epoxides: structural reinvestigation and characterization of a novel adenosine adduct on the ribose moiety. J. Org. Chem. 56,2+29. (20)Singh,S.B.,Hinger&y,B.E.,Singh,U. C., Greenberg,J. P.,Geacintov, N. E., and Broyde, S. (1991)Structures of the (+)- and (-)-tram7 , g d i h y d r o x y - a n t i - 9 , l ~ e p o ~ 7 , 8 , 9 , 1 9 - t e ~ ~ y ~ad~~(a)p~ne ducts to guanine-N2in a duplex dodecamer. Cancer Res. 51, 34823492.
