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Chem. Res. Toxicol. 1997, 10, 378-385
Characterization of DNA Adducts Formed by the Four Configurationally Isomeric 5,6-Dimethylchrysene 1,2-Dihydrodiol 3,4-Epoxides Jan Szeliga,† Bruce D. Hilton,‡ Gwendolyn N. Chmurny,‡ Jacek Krzeminski,§ Shantu Amin,§ and Anthony Dipple*,† Chemistry of Carcinogenesis Laboratory, ABL-Basic Research Program, and Chemical Synthesis and Analysis Laboratory, SAIC Frederick, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, and Naylor Dana Institute for Disease Prevention, American Health Foundation, Dana Road, Valhalla, New York 10595 Received October 21, 1996X
The DNA adducts formed from the racemic syn and anti dihydrodiol epoxides of 5,6dimethylchrysene were characterized through various spectroscopic methods. Substantial reaction with the amino groups of both deoxyadenosine and deoxyguanosine residues were detected with both the syn and anti derivatives. The chemical shifts and coupling constants for the cis and trans opened adducts from the syn dihydrodiol epoxide were distinctly different, whereas for the anti dihydrodiol epoxide these properties were fairly similar for cis and trans adducts. In the latter case, assignment of trans and cis configurations was less obvious, and the finding that trans adducts have always predominated over cis adducts for all dihydrodiol epoxides studied to date was helpful in making these assignments. The preferential formation of cis adducts in DNA by the syn dihydrodiol epoxide is more like the chemistry of the dihydrodiol epoxide of benzo[c]phenanthrene than of benzo[g]chrysene, although both of these, like 5,6-dimethylchrysene, are non-planar compounds.
Introduction Studies of DNA adduct formation by polycyclic aromatic hydrocarbons have revealed that the dihydrodiol epoxide metabolites, which are thought to be responsible for initiating carcinogenic effects (1), react with DNA in a fashion similar to that of the 7-bromomethylbenz[a]anthracenes (2, 3), i.e., they primarily react with the amino groups of the DNA bases (4, 5). However, the reactions of the anti diastereomer of benzo[a]pyrene dihydrodiol epoxide with DNA yielded deoxyguanosine adducts almost exclusively (5-7), whereas reactions of the anti dihydrodiol epoxide derivative of 7,12-dimethylbenz[a]anthracene with DNA yielded both deoxyguanosine and deoxyadenosine adducts in comparable amounts (8-10). Further studies of other dihydrodiol epoxide-DNA reactions have led to the generalization that dihydrodiol epoxides derived from planar polycyclic aromatic hydrocarbons react preferentially with deoxyguanosine residues in DNA, whereas dihydrodiol epoxides from non-planar hydrocarbons react to more similar extents with both deoxyguanosine and deoxyadenosine residues in DNA (11). To investigate the chemical basis for this generalization, we wish to compare the reactions with DNA of dihydrodiol epoxides (both syn and anti) from a planar and from a closely related non-planar hydrocarbon. The hydrocarbons selected for study were the largely planar 5-methylchrysene and the non-planar 5,6-dimethylchrysene (12). The DNA adducts formed from the syn- and anti-dihydrodiol epoxides of 5-methylchrysene have been previously characterized (13-15). Though several stud-
ies of 5,6-dimethylchrysene dihydrodiol epoxide-DNA reactions have been completed (16-18), full characterization of all the adducts has not yet been achieved. Therefore, in this paper, we describe the characterization of deoxyadenosine and deoxyguanosine adducts from all four configurationally isomeric 1,2-dihydrodiol 3,4-epoxides of 5,6-dimethylchrysene.
Experimental Section Calf thymus DNA, deoxyribonucleotides, and enzymes were all obtained commercially. Racemic syn- and anti-5,6-dimethylchrysene 1,2-dihydrodiol 3,4-epoxides were synthesized as described previously (18). Circular dichroism (CD)1 spectra in methanol were measured on a Jasco Model J500A spectropolarimeter equipped with a data processing system for signal averaging. The spectra of nucleoside adducts were normalized to 1.0 absorbance unit at λmax. UV absorption spectra were recorded with a Milton Roy Spectronic 3000 diode array spectrophotometer. NMR analyses were on samples (these ranged from 3 to 120 A262 units) dissolved in ca. 130 µL of acetone-d6, 99.96% D, placed in a 3-mm o.d. Wilmad tube. A Nalorac MID-500-3 microprobe was used with a Varian VXR-500S spectrometer (proton frequency 599.884 MHz), equipped with a Varian variable temperature unit. Temperature was controlled for all experiments; most were performed at 27 °C; some proton experiments were performed at -10 °C to improve resolution; other experiments were performed from -50 to 45 °C to examine the rotational isomerization occurring at the dihydrodiol epoxide C4-NH purine bond. All compounds were analyzed using 1D proton and either COSY or TOCSY 2D NMR. The trans tetraol derived from the anti dihydrodiol epoxide was analyzed in addition with 1D 13C and 2D heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond connectivity (HMBC)
†
Chemistry of Carcinogenesis Laboratory. Chemical Synthesis and Analysis Laboratory. Naylor Dana Institute for Disease Prevention. X Abstract published in Advance ACS Abstracts, March 15, 1997. ‡ §
S0893-228x(96)00178-6 CCC: $14.00
1 Abbreviations: CD, circular dichroism; HMQC, heteronuclear multiple quantum coherence; HMBC, heteronuclear multiple bond connectivity.
© 1997 American Chemical Society
5,6-Dimethylchrysene-DNA Adducts spectra. Where sufficient material was available, TrRoesy 2D Overhauser spectra were obtained. Homodecoupling and D2O exchange experiments were used if necessary to assist in analysis. Analysis of the 1D spectra was carried out with the use of in-house Zero Field Decoupling software. Stability checks using 1D 1H spectra were performed frequently to assure reproducibility of spectra and to ascertain reversiblity after temperature studies. Mass spectral analyses on the 16 nucleoside adducts shown in Figure 1 were undertaken by J. Ding and P. Vouros, Northeastern University, and all were found by VG Quattro electrospray mass spectrometry to have the anticipated molecular ion. Resolution of trans-1,2-Dihydroxy-1,2-dihydro-5,6-dimethylchrysene (18). The racemic dihydrodiol (100 mg) was esterified with four equivalents of (-)-menthoxyacetyl chloride and, after solvent removal and flash chromatography on silica gel, the diastereomeric bis-menthyloxy esters [hexane: EtOAc (10:1) fraction] were separated by preparative HPLC on an Econosil SI 10 OU, 250 × 22 mm column (Alltech) using 2% diethyl ether in cyclohexane at 24 mL/min. The faster eluting diester yielded trans-(1R,2R)-dihydroxy-1,2-dihydro-5,6-dimethylchrysene and the slower eluting diester yielded trans(1S,2S)-dihydroxy-1,2-dihydro-5,6-dimethylchrysene upon hydrolysis in THF/methanol containing sodium methoxide. 1,2-Dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-5,6-dimethylchrysenes. Two enantiomeric pairs of the anti and syn dihydrodiol epoxides were obtained by separately converting each optically active dihydrodiol to the anti dihydrodiol epoxide with m-chloroperbenzoic acid (18) and to the syn dihydrodiol epoxide by sequential treatment with N-bromoacetamide and Amberlite IRA-400(OH) ion exchange resin (19). DNA Adduct Formation. DNA (1 mg/mL) in Tris/HCl buffer (pH 7) was treated with 0.05 vol of the racemic dihydrodiol epoxides (2 mg/mL) in tetrahydrofuran, enzymically digested to nucleosides using a venom phosphodiesterase/DNA ratio that was twice that previously found necessary to digest 7,12-dimethylbenz[a]anthracene-modified DNA (20), and subjected to HPLC (16). Adduct Formation with Deoxyribonucleotides. These reactions were similar to analogous reactions described before (21). For preparative purposes, 40 mg/mL solutions of deoxyribonucleotides were treated at pH 7 with 0.1 vol of dihydrodiol epoxide (1 mg/mL) in acetone. These solutions were extracted with ethyl acetate and ether, and the aqueous phase was passed through a Sep-Pak (Waters) that was washed with water to remove most unreacted nucleotide. The adducted nucleotide was then eluted in methanol, dried, and redissolved in buffer prior to digestion with Escherichia coli alkaline phosphatase to convert the nucleotide to nucleoside. These products were then separated by HPLC (see below). Substantial amounts of each adduct were collected by repetitive chromatography and were acetylated with acetic anhydride in pyridine overnight (22) and purified by normal-phase HPLC for NMR analyses. Tetraols were recovered from the organic solvent extractions above and were collected after chromatography on an ODS column. For syn dihydrodiol epoxide-derived tetraols, isocratic elution for 35 min with 25% acetonitrile, followed by a linear increase to 30% over the next 10 min, and isocratic elution thereafter gave the trans tetraol at 34 min and the cis tetraol at 48 min. For the anti dihydrodiol epoxide-derived tetraols, the trans isomer eluted at 45 min in 22% acetonitrile and the cis isomer, prepared by osmium tetroxide oxidation of the 1,2-dihydrodiol, eluted at 13.5 min in 60% methanol. Reactions with optically active dihydrodiol epoxides were essentially similar to those with racemic compounds except that only 10 mg/mL solutions of nucleotides were used, and these solutions were analyzed by HPLC directly without organic solvent extraction or conversion to nucleosides. Adduct Separations. Unacetylated Adducts: Analytical HPLC separations were achieved using Beckman Ultrasphere ODS columns (5 µm, 0.46 × 25 cm) eluted at 1 mL/min. Whether derived from DNA or deoxyribonucleotide reactions,
Chem. Res. Toxicol., Vol. 10, No. 4, 1997 379
Figure 1. Structures of the enantiomeric pair of syn 5,6dimethylchrysene dihydrodiol epoxides and their possible reaction products with DNA or purine nucleotides (a) and of the enantiomeric pair of anti 5,6-dimethylchrysene dihydrodiol epoxides and their possible reaction products with DNA or purine nucleotides (b). Each adduct is labeled with the configuration at C4 (R or S), the nucleoside involved, i.e., deoxyguanosine (G) or deoxyadenosine (A), and the cis (c) or trans (t) opening of the epoxide. deoxyribonucleoside adducts from the syn dihydrodiol epoxide were eluted isocratically for 10 min with 23% acetonitrile followed by a gradient that decreased acetonitrile to 0% and increased methanol to 50% over the next 50 min. This was followed by a further increase to 65% methanol over the next
380 Chem. Res. Toxicol., Vol. 10, No. 4, 1997
Szeliga et al.
40 min. Those adducts derived from the anti dihydrodiol epoxide were eluted isocratically with 40% methanol for 10 min, followed by a linear increase to 56% methanol over 80 min, and isocratic elution for a further 10 min. For preparative separations of nucleoside adducts, a larger column (5 µm, 1 × 25 cm) and increased flow rate (4 mL/min) were used. HPLC separations of adducts as nucleotides used 0.05 M K2HPO4/KH2PO4 (pH 7) buffer in combination with organic solvents. Separation of the syn dihydrodiol epoxide-deoxyguanylic acid adducts used isocratic elution with 17% acetonitrile in buffer for 45 min, followed by a linear increase to 30% acetonitrile over the next 45 min. Separation of the syn dihydrodiol epoxide-deoxyadenylic acid adducts used elution with 18% acetonitrile in buffer for 5 min, followed by a linear increase to 22% acetonitrile over the next 55 min, and then to 30% acetonitrile over the following 17.5 min. The anti dihydrodiol epoxide-deoxyguanylic acid adducts were eluted isocratically with 35% methanol for 15 min, followed by a linear gradient to 45% methanol over the next 65 min, and then to 50% methanol over the next 20 min. The anti dihydrodiol epoxide-deoxyadenylic acid adducts were separated using isocratic elution with 15% acetonitrile in buffer for 5 min, followed by a linear increase to 25% over the next 55 min, and to 30% over the following 17.5 min. Acetylated Adducts: All adducts were separated on a DuPont Zorbax SIL column (5 µm, 0.46 × 25 cm) eluted at 1 mL/min. For all acetylated nucleoside adducts, elution was with dichloromethane/ethyl acetate (5.7/1) containing the following percentages of 90% methanol: for syn dihydrodiol epoxide adducts, (S)At and (R)At, 0.5%; (S)Gt, 0.8%; (R)Gt, 0.9%; (S)Ac and (R)Ac, 1%; (R)Gc and (S)Gc, 2.5%; for anti dihydrodiol epoxide adducts, all A adducts, 0.5%; (S)Gc and (S)Gt, 1.5%; (R)Gc and (R)Gt, 2%.
Results and Discussion On the basis of studies with other dihydrodiol epoxides (14, 22-26), the anticipated major products of reaction of the racemic syn and anti dihydrodiol epoxides of 5,6dimethylchrysene with DNA would be the trans and cis opened adducts with deoxyadenosine and deoxyguanosine residues shown in Figure 1. In this figure, each adduct is assigned a notation based upon the configuration at carbon-4, the site of attachment to the nucleoside (R or S), the nucleoside from which it was derived, deoxyguanosine (G) or deoxyadenosine (A), and the direction in which the epoxide was opened, cis (c) or trans (t). For clarity in the description, chromatographic peaks in the figures have been labeled with the structures eventually assigned to them. (Proof for these assignments is presented later in the text.) Calf thymus DNA and the purine deoxyribonucleotide components of DNA were reacted with the racemic syn and anti dihydrodiol epoxides; the reaction products were enzymically digested to deoxyribonucleosides; and the products obtained were chromatographically separated on a reverse-phase HPLC column. Comparison of the nucleoside adducts obtained from the nucleotide reactions with those obtained from DNA reactions allowed the nucleotide of origin of each DNA-derived adduct to be assigned (16), as summarized in Figure 2. In concert with studies of several other dihydrodiol epoxides (8, 14, 15, 22-28), all of the deoxyguanosine adducts eluted earlier than the deoxyadenosine adducts; adducts with the purine nucleotides accounted for most of the DNA adducts found. From reactions with large quantities of purine deoxyribonucleotides, multiple chromatographic runs allowed substantial amounts of each of the adducts (labeled in Figure 2) to be collected. These larger samples of each
Figure 2. HPLC separations of nucleoside products from reaction of racemic syn (a) or anti (b) 5,6-dimethylchrysene dihydrodiol epoxide with DNA, deoxyguanylic acid and deoxyadenylic acid. The early eluting unmarked peak is tetraol in which the epoxide was opened trans according to retention time and UV absorption spectrum. The abbreviations used to mark individual peaks were defined in Figure 1.
5,6-Dimethylchrysene-DNA Adducts
Chem. Res. Toxicol., Vol. 10, No. 4, 1997 381 Table 1. Carbon and Proton Assignments for trans Tetraol from anti Dihydrodiol Epoxide
Figure 3. Circular dichroism spectra in methanol for syn (a) and anti (b) 5,6-dimethylchrysene dihydrodiol epoxide nucleoside adducts. The assignments of absolute configuration (S) or (R) at C4 in hydrocarbon-deoxyribonucleoside adducts were made from separate reactions with the optically active dihydrodiol epoxides (Figure 4).
nucleoside adduct were used to collect UV absorbance spectra, CD spectra, and after acetylation, NMR spectra for the adducts derived from both the syn and anti dihydrodiol epoxides. Ultraviolet absorbance spectra in methanol for all adducts were fairly similar with λmax 262 nm for deoxyguanosine adducts and closer to 263 nm for deoxyadenosine adducts. Shoulders that were more pronounced in some cases than in others were seen at 278 and 310 nm. The ratio of absorbance 278/262-3 nm was greater for deoxyadenosine adducts than for deoxyguanosine adducts. The CD spectra obtained for the adducts are summarized in Figure 3. As shown, the spectra for the syn (Figure 3a) and anti (Figure 3b) dihydrodiol epoxidedeoxyribonucleoside adducts could all be grouped into pairs for which the two spectra exhibited a mirror image
assignment
HMBC (8 Hz)
1AcCO 2(3)AcCO 3(2)AcCO 4AcCO C12a C10a(5a) C5a(10a) C6a C11a C6 C5 C8 C4a C9 C11 C12 C7 C10 C1 C2 C4 C3 5Me 1AcMe 4AcMe 2(3)AcMe 3(2)AcMe 6Me
6.55, 2.23 2.03 2.00 7.02, 1.98 8.91, 7.02, 6.55sm (8.91, 8.17, 7.02, 2.72, 2.69) (8.91, 8.17, 7.02, 2.72, 2.69) 8.79, 2.69 8.79sm, 7.52 8.91, 8.17sm, 7.67vvs 2.72, 2.69 8.79 7.52, 7.02, 6.54 8.17 7.67 7.67 7.51 7.01, 6.54 7.01
HMQC
7.68 7.67 8.91 7.52 8.17 8.79 6.54 5.60 7.02 5.81 2.72 2.23 1.99 2.04 2.01 2.69
δ(ppm) 171.30 170.67 170.30 170.05 136.75 134.33 134.29 132.59 131.54 129.94 129.58 128.20 128.19 127.03 125.46 125.25 125.06 124.00 71.09 70.67 69.88 69.72 21.89 21.00 20.80 20.74 20.62 16.44
relationship. This pairing of the spectra indicates that each member of the pair is identical except that the hydrocarbon residues for each member are enantiomeric, e.g., compare (R)Gt and (S)Gt in Figure 1. It is anticipated that one pair of spectra for each nucleoside will be cis adducts and one pair will be trans adducts, but it is not possible to determine which is which from the CD spectra. NMR Studies. The most extensive analysis of primary structure was undertaken for the trans tetraol derived from the anti dihydrodiol epoxide (Figure 1b). This tetraol was available in sufficient quantity to permit a complete assignment of the proton and carbon spectra (Table 1) through 2D HMBC and HMQC experiments (29). The other tetraols could then be assigned on the basis of their COSY spectra. Adduct proton spectra were assigned largely on the basis of the COSY or TOCSY 2D spectra (Table 2) (30). The chemical shifts of the aromatic protons were very similar in all adducts, and long-range coupling from these protons to C1-H and C4-H in the partially saturated ring (detected in COSY and TOCSY experiments) confirmed the assignments of the C1-H and C4-H protons in several of the adducts. In favorable cases, the coupling between the proton at position 2 in the adenine residue and the NH proton was observed as further confirmation of the manner of attachment of the nucleoside to the hydrocarbon. A connection between the C4-H proton and the NH proton was observed in most of the adducts; generally a COSY cross peak was present and/or an NH-C4-H coupling could be observed on the C4-H proton multiplet. Sharpening of the C4-H signal on D2O exchange or decoupling the NH proton in some cases provided evidence of the NH-C4-H connectivity. The appearance of the C1-H proton (a simple doublet coupled to C2-H) facilitated its assignment. The connection C1-H-C2-HC3-H-C4-H-NH was clear from the 2D experiments. Further support for the assignments made was derived from the similarity between the chemical shifts and
382 Chem. Res. Toxicol., Vol. 10, No. 4, 1997 Table 2.
1H
Szeliga et al.
NMR Data for Acetates of Deoxyribonucleoside Adducts and of Tetraols from 5,6-Dimethylchrysene 1,2-Dihydrodiol 3,4-Epoxides
adduct (as the acetates)
C1-H
C2-H
(S)Gt (R)Gt (R)Gc (S)Gc (S)At (R)At (S)Ac (R)Ac trans-tetraol cis-tetraol
6.65 6.65 6.35 6.35 6.85 6.85 6.16 6.17 6.62 6.46
J1,2 ) 8.3 J1,2 ) 8.0 J1,2 ) 5.7 J1,2 ) 5.7 J1,2 ) 8.3 J1,2 ) 8.3 J1,2 ) 2.5 J1,2 ) 2.6 J1,2 ) 8.4 J1,2 ) 6.0
5.2 5.16 5.84 5.82 5.23 5.25 5.8 5.8 5.11 5.82
syn J2,3 ) 3.5 J2,3 ) 2.7 J2,3 )10.3 J2,3 )10.1 J2,3 ) 3.4 J2,3 ) 2.4 J2,3 ) 8.6 J2,3 ) 8.6 J2,3 ) 2.6 J2,3 )10.5
(R)Gc (S)Gc (R)Gt (S)Gt (S)Ac (R)Ac (S)At (R)At trans-tetraol cis-tetraol
6.66 6.64 6.55 6.5 6.6 6.6 6.49 6.58 6.54 6.66
J1,2 ) 8.7 J1,2 ) 8.9 J1,2 ) 8.5 J1,2 ) 7.3 J1,2 ) 7.2 J1,2 ) 7.2 J1,2 ) 8.5 J1,2 ) 9.2 J1,2 ) 8.3 J1,2 ) 7.9
5.65 5.68 5.65 5.64 5.73 5.73 5.8 5.77 5.6 5.65
anti J2,3 ) 2.0 J2,3 ) 1.9 J2,3 ) 2.3 J2,3 ) 2.1 J2,3 ) 2.1 J2,3 ) 2.0 J2,3 ) < 5 J2,3 ) 2.5 J2,3 ) 2.7 J2,3 ) 3.4
coupling constants observed and those previously reported for 5-methylchrysene-deoxyribonucleoside adducts (14, 15). For the deoxyadenosine and deoxyguanosine adducts from the syn dihydrodiol epoxide, comparison with the tetraol data and with the literature clarify whether the cis or trans orientation resulted from epoxide ring opening (22). The pattern of chemical shifts for the four protons in the partially-saturated ring and the pattern of the coupling constants J1,2, J2,3, and J3,4 were very distinct for the cis and trans isomers because of the diaxial arrangement of the protons on C2 and C3 in the cis compounds (J2,3 ∼ 8-11 Hz) and the diequatorial arrangement of these protons in the trans compounds (J2,3 ∼ 2.5-3.5 Hz) (21). Also, C4-H in the cis compounds is further downfield (by about 0.5-1.0 ppm) than in the trans compounds, as has been found empirically for all cis/trans pairs of tetraols and adducts studied to date (22). This downfield shift can be attributed to the C4-H proton being forced into the plane of the aromatic ring in the cis configuration. Thus, the cis or trans configuration of the adducts derived from the syn dihydrodiol epoxide was clearly established solely on the basis of the NMR data. The situation for adducts derived from the anti dihydrodiol epoxides was more complex. The NMR data established the primary structure, but the cis or trans assignment was not really obvious from the NMR data alone. Coupling constants are similar for both the cis and trans adducts and tetraols (see Table 2). Although the chemical shift difference is very small (∼0.1 ppm), the compounds assigned the cis configuration have the proton on C4 further downfield than in the compounds assigned a trans configuration, in concert with the empirical rule mentioned above. Additional evidence that supports the cis and trans assignments made for the anti adducts comes from the relative adduct yields and their chromatographic properties. Thus, it is known that hydrolysis of anti dihydrodiol epoxides yields almost exclusively a trans tetraol, and the cis tetraol had to be prepared from osmium tetroxide oxidation of the dihydrodiol. The configurations of the tetraols were known therefore. It could be seen then, as
methine protons C3-H
C4-H
5.89 5.9 5.36 5.42 5.77 5.77 4.91 4.92 5.42 5.34
J3,4 ) 4.0 J3,4 ) 3.6 J3,4 ) 3.4 J3,4 ) 3.5 J3,4 ) 2.5 J3,4 ) 4.0 J3,4 ) 2.6 J3,4 ) 2-4 J3,4 ) 3.3 J3,4 ) 2.6
6.07 6.03 7.07 7.1 6.37 6.38 7.03 7.05 6.8 7.43
6.34 6.21 6.21 6.16 6.16 6.15 6.08 6.09 5.81 5.85
J3,4 ) 3.9 J3,4 ) 4.6 J3,4 ) 4.5 J3,4 ) 5.2 J3,4 ) 4.4 J3,4 ) 4.2 J3,4 ) < 5 J3,4 ) 4.1 J3,4 ) 4.8 J3,4 ) 3.9
6.65 6.85 6.43 6.33 7.12 7.14 6.95 7.0 7.02 7.18
J4-NH ) 5.1 J4-NH ) 6.6 J4-NH ) 4.6 J4-NH ) 4.4 J4-NH ) 9.4 J4-NH ) 9.1
N-H
7.11 6.22 6.21 7.17 7.19
5.56 7.6 6.51
J4-NH ) 7.4
7.56 7.58 7.15 7.44
in all other cases studied so far, that the chemical shift of C4-H in the cis tetraol was slightly further downfield than in the trans tetraol (0.16 ppm). A similar difference in chemical shift was also present for the adducts; a tentative assignment of cis and trans configurations for the adducts could be made on this basis. These assignments are supported by the fact that trans adducts are the major adducts produced by anti dihydrodiol epoxides in DNA (8, 14, 22, 24-27, 31) and, as seen in Figure 2b (top panel), (R)At, (S)At, and (S)Gt are the major adducts formed. If the assignment of (S)Gt is correct, then the (R)Gt assignment follows from this because of the mirror image relationship between their CD spectra. Additionally, the results from reactions of optically active dihydrodiol epoxides with nucleotides (Figure 4b) show that one major and one minor adduct are produced from each anti dihydrodiol epoxide enantiomer and each purine nucleotide. The major adduct is the trans adduct in each case, as would be expected. Another line of reasoning is that the sequence of elution of adducts from HPLC for other non-planar compounds, such as the benzo[c]phenanthrene dihydrodiol epoxides (23), follows the sequence of cis eluting before trans, as in the present case. Thus, while the assignments of cis and trans configurations in the anti dihydrodiol epoxide reactions are less clear-cut than in the syn dihydrodiol epoxide reactions, the assignments made are consistent with all expectations from previous studies. The analysis of the deoxyribonucleoside adducts was complicated by broadening of several resonances in many of the adduct spectra and by a strong temperature dependence of the appearance of the proton spectra of these resonances. For each series of compounds, NMR spectra were obtained at several temperatures in order to determine the optimal conditions for analysis. For example, the resonances of the (R)At adduct from the anti dihydrodiol epoxide were noticeably broadened at room temperature but at -20 °C were relatively sharp. However, the (S)Ac syn adduct revealed dramatically broader lines at -50 °C than at room temperature. Previous work also shows evidence of broadening of some peaks in the NMR spectra of deoxyadenosine adducts (27, 28).
5,6-Dimethylchrysene-DNA Adducts
Chem. Res. Toxicol., Vol. 10, No. 4, 1997 383 Table 3. Adduct Distributions (%) in Reactions of Racemic Dihydrodiol Epoxides with DNA and Nucleotides DNAa dGuo R,S,R,S S,R,S,R R,S,S,R S,R,R,S
nucleotides dAdo
dGuo
dAdo
cis
trans
cis
trans
cis
trans
cis
trans
10.5 10.9 7.2 1.5
6.6 4 31.7 7.6
26.7 16 1.7 4.7
4 21.3 29.5 16.1
43.9 41.4 11.9 11
8.1 6.6 37.4 39.7
43.3 41.9 7.2 7.6
7.6 7.2 40.5 44.6
a Approximately 32% of the racemic anti and 24% of the racemic syn dihydrodiol epoxides were trapped by DNA in these reactions. For each reaction of DNA or nucleotide with a racemic dihydrodiol epoxide, the total products with both enantiomers sum to 100%.
Figure 4. HPLC separations of nucleotide adducts from reaction of racemic syn (a) and anti (b) 5,6-dimethylchrysene dihydrodiol epoxides and their optically pure enantiomers with deoxyguanylic acid and deoxyadenylic acid. Note that the adducts described in this figure are nucleotides even though the same abbreviations used elsewhere in this paper to describe nucleoside adducts are used to describe these phosphorylated nucleosides.
The assignment of absolute configuration in hydrocarbon-deoxyribonucleoside adducts has sometimes been possible using an empirical relationship between CD spectra and the configuration of the carbon involved in binding to the nucleoside (26). However, the comparison of CD spectra with those of closely related compounds, whose configurations are known, has seemed more definitive when such related spectra were available. Both these approaches were tried with the CD spectra of these 5,6-dimethylchrysene-deoxyribonucleoside adducts but confident configurational assignments could not be made. For this reason, the optically active dihydrodiol epoxides were prepared, and these were reacted separately with purine deoxyribonucleotides to assign configurations. These reaction products were separated as nucleotides (Figure 4), and CD spectra were recorded for each nucleotide adduct to establish the common origins of the nucleotide adducts in Figure 4 and the nucleoside adducts in Figure 2. The relative sequence of elution of nucleoside and nucleotide adducts was the same except for the syn dihydrodiol epoxide-deoxyguanosine/deoxyguanylic acid adducts. The absolute configurations for each of the adducts were clear from the data in Figure 4, and these are reflected in the abbreviations assigned to each adduct throughout this manuscript. Based on the above assignments for nucleoside adducts obtained from the reactions with purine nucleotides, each enantiomer of the anti dihydrodiol epoxide (i.e., R,S,S,R and S,R,R,S) exhibited a strong preference for trans adduct formation whereas each syn enantiomer exhibited an equally strong preference for cis adduct formation (Table 3). Preferential cis adduct formation with nucleotides by syn dihydrodiol epoxides has been characteristic of most dihydrodiol epoxides studied to date (15, 22, 3133) with the sole exception of the syn dihydrodiol epoxide from benzo[g]chrysene (28), which shows a strong preference for trans adduct formation. The syn dihydrodiol epoxides of benzo[c]phenanthrene, 5,6-dimethylchrysene, and benzo[g]chrysene are all similar in that all have a crowded bay or a fjord region, thus the trans adduct formation by the latter compound may be attributed to effects of the extra benzene ring that is present on the reactivity of the epoxide through presumably enhanced delocalization of developing charge. Comparison of the DNA reaction data with those for other dihydrodiol epoxides investigated earlier indicates similarities with other compounds derived from nonplanar hydrocarbons in that extensive reaction with deoxyadenosine residues was found (dAdo/dGuo adduct ratios 68/32 and 52/48 for the racemic syn and anti dihydrodiol epoxides, respectively), but each dihydrodiol epoxide is nevertheless unique in its overall adduct formation with DNA, and
384 Chem. Res. Toxicol., Vol. 10, No. 4, 1997
as noted for the nucleotide reactions, the 5,6-dimethylchrysene derivatives are more similar to the benzo[c]phenanthrene than to the benzo[g]chrysene derivatives. The present studies have allowed the adducts formed with DNA by the four configurationally isomeric 5,6dimethylchrysene 1,2-dihydrodiol 3,4-epoxides to be characterized. Adduct formation followed expectations in that reaction occurred primarily with the amino groups of the purine bases in DNA and that reaction with both deoxyadenosine and deoxyguanosine was extensive for these nonplanar hydrocarbon-derived reactive derivatives. The source of steric crowding for the compounds studied herein is the presence of a methyl group in the bay region, and in this regard, these agents are more similar to those derived from 7,12-dimethylbenz[a]anthracene (32, 33) than to those derived from fjord region-containing structures, such as benzo[c]phenanthrene and benzo[g]chrysene (21-23, 27). Nevertheless, with regard to cis/trans adduct ratios, the 5,6-dimethylchrysene derivatives are similar to those of benzo[c]phenanthrene, whereas benzo[c]phenanthrene derivatives are distinctly different from benzo[g]chrysene derivatives. We have previously reported mutational spectra for the syn and anti dihydrodiol epoxides of 5,6-dimethylchrysene (16). In general, these findings were consistent with the chemistry reported herein. Thus, the syn dihydrodiol epoxide exhibited a greater propensity to mutate AT pairs (77% of total mutations) than did the anti diastereomer (45% of total mutations). The chemical findings (Table 3) are that 68% of the reaction with DNA is with deoxyadenosine for the syn diastereomer as compared to 52% for the anti diastereomer. Although the data base of hydrocarbon dihydrodiol epoxide reactions with DNA and its constituents is becoming quite substantial, the chemistry involved is sufficiently complex that it is not yet possible to clearly predict all aspects of the outcome of these reactions. It seems clear that deoxyguanosine adducts will be the principal product with DNA if the parent hydrocarbon is planar and that both deoxyadenosine and deoxyguanosine adducts will be formed in substantial quantities if the parent hydrocarbon is non-planar. Moreover, the principal products from all anti dihydrodiol epoxides are thought to be trans adducts irrespective of whether they are deoxyguanosine or deoxyadenosine adducts. For the syn diastereomers, cis adducts predominate in all cases so far except for benzo[g]chrysene derivatives. If the greater size of the conjugated aromatic system dictates trans adduct formation, then trans adducts may predominate in the adducts derived from syn dihydrodiol epoxides of other large polycyclics, such as dibenzo[a,l]pyrene for example. Even in the situations where the outcome is fairly predictable, the chemical basis for these experimental findings generally needs to be better understood.
Acknowledgment. Research sponsored in part by the National Cancer Institute, DHHS, under contract with ABL.
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