Synthesis and Structure Determination of the Adducts Formed by

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Chem. Res. Toxicol. 1996, 9, 1264-1277

Synthesis and Structure Determination of the Adducts Formed by Electrochemical Oxidation of 1,2,3,4-Tetrahydro-7,12-dimethylbenz[a]anthracene in the Presence of Deoxyribonucleosides or Adenine Patrick P. J. Mulder,† Liang Chen,† B. Chandra Sekhar,† Mathai George,‡ Michael L. Gross,‡,§ Eleanor G. Rogan,† and Ercole L. Cavalieri*,† Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, Nebraska 68198-6805, and Nebraska Center for Mass Spectrometry, Department of Chemistry, University of NebraskasLincoln, Lincoln, Nebraska 68588-0362 Received June 19, 1996X

Study of DNA adducts formed with aromatic hydrocarbons is part of the strategy to elucidate the mechanisms of tumor initiation by these compounds. 1,2,3,4-Tetrahydro-7,12-dimethylbenz[a]anthracene (THDMBA) is of special interest because it allows discrimination between the pathways of bioactivation by one-electron oxidation and monooxygenation. To study and identify adducts formed biologically, synthetic adducts are needed as reference standards. THDMBA was electrochemically oxidized in the presence of deoxyadenosine (dA), adenine (Ade), deoxyguanosine (dG), or deoxycytidine (dC). In the presence of dA, four adducts were isolated: 7-methyl-1,2,3,4-tetrahydrobenz[a]anthracene-12-CH2-N7Ade (7-MTHBA-12-CH2N7Ade, 3.6%), 12-MTHBA-7-CH2-N7Ade (4.2%), 7-MTHBA-12-CH2-N6dA (5.8%), and 12-exomethylene-7-MTHBA-7-N6dA (22.8%); a dehydrogenated product, 7,12-di-exo-methylene-THBA (44.2%), was also obtained. In the presence of Ade, nine adducts were synthesized: 7-MTHBA12-CH2-N7Ade (1.1%), 12-MTHBA-7-CH2-N7Ade (2.4%), 7-MTHBA-12-CH2-N1Ade (10.2%), 12MTHBA-7-CH2-N1Ade (13.2%), 7-MTHBA-12-CH2-N3Ade (1.7%), 12-MTHBA-7-CH2-N3Ade (1.7%), 7-exo-methylene-12-MTHBA-12-N3Ade (11.2%), 12-exo-methylene-7-MTHBA-7-N3Ade (27.9%), and 12-exo-methylene-7-MTHBA-7-N6Ade (12.1%), as well as the dehydrogenated product 7,12-di-exo-methylene-THBA (16.7%). In the presence of dG, three adducts were produced: 7-MTHBA-12-CH2-N7Gua (24.2%), 12-MTHBA-7-CH2-N7Gua (12.2%), and 7-MTHBA12-CH2-N2dG (3.7%), as well as the dehydrogenated product 7,12-di-exo-methylene-THBA (38.9%). Anodic oxidation in the presence of dC yielded a large amount of 7,12-di-exo-methyleneTHBA (80.4%), but no adducts. The structure of the adducts was elucidated by using UV, NMR, and MS. The N-7 positions in dG, dA, and Ade, the 2-NH2 in dG, and the N-1 position in Ade form exclusively methyl-linked adducts. In contrast, the 6-NH2 group of dA and Ade and the N-3 of Ade prefer to attack the meso-anthracenic positions rather than the methyl groups. The order of reactivity of dG and dA in the formation of methyl-linked THDMBA adducts agrees well with that previously found for 7,12-dimethylbenz[a]anthracene [RamaKrishna et al. (1992) J. Am. Chem. Soc. 114, 1863-1874].

Introduction Understanding the mechanisms of activation of polycyclic aromatic hydrocarbons (PAH)1 in relation to the process of tumor initiation has been a research topic of longstanding interest. It has been proposed that most PAH are activated by monooxygenation to produce bayregion diol epoxides, which react with DNA to form * To whom correspondence should be addressed. † Eppley Institute for Research in Cancer and Allied Diseases. ‡ Nebraska Center for Mass Spectrometry. § Present address: Department of Chemistry, Washington University, One Brookings Dr., St. Louis, MO 63130. X Abstract published in Advance ACS Abstracts, October 1, 1996. 1 Abbreviations: Ade, adenine; BP, benzo[a]pyrene; CA, collisional activation; CAD, collisionally activated decomposition(s); 6-CH3BP, 6-methylbenzo[a]pyrene; COSY, two-dimensional chemical shift correlation spectroscopy; dA, deoxyadenosine; DB[a,l]P, dibenzo[a,l]pyrene; dG, deoxyguanosine; DMBA, 7,12-dimethylbenz[a]anthracene; DMF, dimethylformamide; FAB MS/MS, fast atom bombardment tandem mass spectrometry; Gua, guanine; MBA, methylbenz[a]anthracene; Me2SO, dimethyl sulfoxide; MTHBA, methyl-1,2,3,4-tetrahydrobenz[a]anthracene; NOE, nuclear Overhauser effect; PAH, polycyclic aromatic hydrocarbon(s); PDA, photodiode array; THBA, 1,2,3,4tetrahydrobenz[a]anthracene; THDMBA, 1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene.

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adducts (1-3). Evidence is accumulating, however, that the major mechanism of activation for the most potent carcinogenic PAH is one-electron oxidation to form intermediate radical cations (4, 5). These radical cations may either react with DNA directly, in the case of unsubstituted PAH, or they may first be converted to radical and carbocation intermediates in the case of mesomethyl-substituted PAH. Typically, one-electron oxidation will lead to activation of positions in the molecule that are different from those activated by monooxygenation. Furthermore, no oxygen is incorporated in the PAH moiety of the adducts formed by one-electron oxidation. For these reasons, study of the PAH-DNA adducts formed in vitro and in vivo will establish the relative contribution of one mechanism with respect to the other (6-11). The very potent carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) activated by rat liver microsomes reacts with DNA to produce almost exclusively depurinating adducts (9). Moreover, when applied to mouse skin, a very similar picture is obtained (10). Activation of DMBA mainly leads to formation of DNA adducts in © 1996 American Chemical Society

One-Electron Oxidation of 1,2,3,4-Tetrahydro-DMBA

which a covalent bond is formed between the 12-CH3 group of DMBA and the N-7 position of adenine (Ade) or guanine (Gua). These adducts are lost from DNA by depurination after cleavage of the glycosidic bond. A small amount of stable adducts (approximately 1%) is obtained (9, 10). Three stable DNA adducts arising from reaction of the bay-region diol epoxides of DMBA with dA and dG were previously identified (11, 12). It is possible that DMBA diol epoxide also forms depurinating adducts because such adducts are formed by other PAH (6, 8, 13, 14). Because the 1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene (THDMBA) angular ring is hydrogenated, the bay-region diol epoxide cannot be formed and, thus, the diol epoxide cannot be involved in the biological activation of THDMBA. Still THDMBA is a potent carcinogen in mouse skin (15-17) and especially in rat mammary gland (16). Thus, study of the mechanism of activation of THDMBA is worthwhile, because its activation can serve as a model for the one-electron oxidation pathway. The anodic oxidation potential of THDMBA (1.01 V) is 0.19 V lower than that of DMBA (18), making it a readily oxidizable compound in chemical and biological systems. There are potentially three positions of THDMBA that can be biologically activated, as indicated in a recent metabolism study: the 7- and 12-methyl groups and C-1 in the angular ring (19). To determine the adducts formed in biological systems, it is necessary to obtain well-characterized synthetic adducts that can serve as reference standards. A convenient way to do this is to oxidize electrochemically the parent PAH in the presence of nucleosides and to isolate the adducts formed. Anodic oxidation of DMBA in the presence of deoxyadenosine (dA) yields two Ade adducts without deoxyribose: 7-methylbenz[a]anthracene (MBA)12-CH2-N7Ade and 12-MBA-7-CH2-N3Ade (20). Reaction with deoxyguanosine (dG) produces three deoxyribosefree adducts: 7-MBA-12-CH2-N7Gua, 12-MBA-7-CH2N7Gua, and 7-MBA-12-CH2-C8Gua (a secondary product), and one adduct with the sugar: 7-MBA-12-CH2C8dG. In this article, the synthesis and structure determination of adducts formed by electrochemical oxidation of THDMBA in the presence of deoxyribonucleosides are reported. Because it has been apparent from the work on DMBA (20) and other PAH (21-23) that the reaction with dA does not produce all the Ade depurinating adducts of biological interest, electrochemical oxidation of THDMBA in the presence of Ade itself was also explored. Structure determination of the adducts was accomplished by using UV, NMR, and fast atom bombardment tandem mass spectrometry (FAB MS/MS).

Experimental Section Caution: THDMBA is a hazardous chemical and should be handled in accordance with NIH guidelines (24). Materials. THDMBA was synthesized according to a published procedure (25). It was purified by recrystallization from CH3OH prior to use. dG, dA (TCI, Portland, OR), deoxycytidine (dC), Ade, and 3-methylAde (Aldrich, Milwaukee, WI) were desiccated over P2O5 under vacuum at 110 °C for 48 h prior to use. Anhydrous dimethylformamide (DMF, Aldrich) and HPLCgrade organic solvents (EM Science, Gibbstown, NJ) were used as obtained. 1H NMR. Proton and homonuclear two-dimensional chemical shift correlation spectroscopy (COSY) NMR spectra were recorded on a Varian Unity 500 at 499.835 MHz; the solvent was

Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1265 deuterated dimethyl sulfoxide (Me2SO-d6) at 30 °C. Chemical shifts (δ) are reported relative to Me2SO (2.49 ppm), and the coupling constants (J) are given in hertz (Hz). Nuclear Overhauser effect (NOE) spectra were recorded in Me2SO-d6 at 60 °C. FAB MS/MS. Collisionally activated decomposition (CAD) spectra were obtained by using a VG ZAB-T, a four-sector tandem mass spectrometer of BEBE design. The first stage (MS1) is a standard high-resolution ZAB SE of reverse geometry. MS2, also capable of high resolving power, is of a prototype Mattuch-Herzog type design, incorporating a standard magnet and an inhomogeneous planar electrostatic analyzer. This design allows the use of a photodiode array (PDA) for simultaneous detection of ions over a variable mass range and a singlepoint detector for scanning experiments. For experiments reported here, sample quantities were sufficiently large so that the single-point detector was adequate. Samples were dissolved in 25 µL of Me2SO, and a 1-µL aliquot was placed on the probe along with 1 µL of matrix, a 1:1 mixture of 3-nitrobenzyl alcohol and glycerol. A Cs+ gun operated at 30 keV was used to desorb the ions from the probe. The instrument accelerating voltage was 8 kV. CAD spectra were obtained after precursor ion activation in the third field-free region (between MS1 and MS2) by collisions with He, which had been added to the collision cell to attenuate the ion beam by 50%. MS1 was operated at a resolving power of 1000; MS2 resolving power was set at 1200 (full width at halfheight definition). Ten to fifteen 25-s scans were signalaveraged for each spectrum. Data acquisition and data workup were controlled by using a VAX 3100 workstation operating with OPUS software. HPLC. Analytical HPLC was conducted on a Waters 600E solvent delivery system equipped with a Waters 700 WISP autoinjector. Eluents were monitored for UV absorbance (254 nm) with a Waters 996 PDA detector, and the data were collected on a NEC Powermate 486/33i computer. Runs were conducted on a YMC (YMC, Overland Park, KS) ODS-AQ 5 µm column (6.0 × 250 mm). The column was eluted with either a CH3OH/H2O, a CH3CN/H2O, or an C2H5OH/H2O gradient [5 min 30% organic solvent in H2O, followed by a 70-min curvilinear gradient (CV5 for CH3OH, CV6 for CH3CN or C2H5OH) to 100% organic solvent] at 1 mL/min. Preparative HPLC was conducted on a Waters 600E solvent delivery system coupled with a Waters 990 PDA detector. Runs were conducted on a YMC ODS-AQ 5 µm column (20 × 250 mm) at a flow rate of 6 mL/min and varying gradients, depending on the nature of the adducts and the complexity of the reaction mixtures. Electrochemical Synthesis of Adducts. Electrochemical synthesis was conducted with a previously described apparatus (EG&G Princeton Applied Research, Princeton, NJ) (26). The electrolysis potential for THDMBA was 1.01 V (18), the anodic peak potential measured in DMF by cyclic voltammetry (Model CV27, Bioanalytical Systems, Lafayette, IN). The potential used for the synthesis of THDMBA adducts was 0.95 V. The individual deoxyribonucleosides and Ade have anodic peak potentials >1.20 V. Therefore, during adduct synthesis none of them was oxidized. Glassware, syringes, needles, electrochemical cell, and platinum working and reference electrodes were dried at 150 °C prior to use. The electrochemical cell and working electrodes were assembled while hot and then cooled under argon. Coupling between THDMBA and the nucleophiles was accomplished by selective anodic oxidation of the PAH in the presence of the nucleoside or free base. To determine the optimal amount of nucleoside or Ade, the reaction was carried out on a small scale (5-10 mg of THDMBA) with various amounts of nucleoside or Ade present. The reaction of THDMBA with dA was tried with 20, 25, 30, 60, and 100 equiv. Adding 25 equiv of dA to the reaction mixture led to a substantially higher yield of adducts than adding 20 equiv, but an increase to 30, 60, or 100 equiv did not further improve the yields. Similarly, yields were not much improved when more than 15 equiv of dG was added. For the reaction with Ade, 10

1266 Chem. Res. Toxicol., Vol. 9, No. 8, 1996 equiv produced the same amounts of adducts as 15 equiv, probably because in the latter case Ade did not fully dissolve. Finally, reacting THDMBA in the presence of 25 equiv of dC produced 7,12-di-exo-methylene-THBA (80%) but no significant amount of adducts. The reaction with dA, dG, or Ade was repeated on a larger scale (20 mg of THDMBA). In a typical preparation, a stirred solution of DMF (40 mL) containing 0.5 M KClO4 as the supporting electrolyte was preelectrolyzed at +1.45 V, while argon was bubbled in the cell, until no appreciable current could be detected (ca. 45 min). An aliquot of the DMF (5-10 mL) was taken out and used to dissolve THDMBA (20 mg, 0.077 mmol) and either dG (300 mg, 1.12 mmol), dA (500 mg, 2.00 mmol), or Ade (100 mg, 0.74 mmol) under sonication. The resulting mixture was added back to the cell, and stirring was continued until the solution was clear. The cell was switched on, and the electrode potential was gradually raised from 0 to 0.95 V and kept constant during the electrolysis. The reaction was stopped when the output current (I) had decreased to ca. 1% of the initial value (typically 1 h). In all cases, a charge of 1.9-2.1 times the theoretical charge had been passed, and essentially all starting material had reacted. After the reaction was complete, DMF was removed under vacuum, and the adducts were extracted three times from the solid (KClO4) by using a solvent mixture of ethanol/chloroform/ acetone (2:1:1). The resulting extract was evaporated under vacuum, and the residue was dissolved in 4 mL of Me2SO/C2H5OH (50:50), filtered through a 0.45-µm filter, and analyzed by analytical HPLC in each of the three solvent systems. Separation and purification of the adducts were conducted by preparative HPLC in two or three different solvent systems, depending on the solubility and separation characteristics of each adduct. The purity of the adducts after preparative HPLC was checked by analytical HPLC in two solvent systems and by 1H NMR. THDMBA and its derivatives were found to be prone to lightcatalyzed autoxidation of the central aromatic ring. Therefore, they were kept under argon and were protected from light as much as possible during handling. The yields of the various adducts were determined by flow radioactivity analysis. Custom-synthesized [3H]THDMBA (specific activity 800 mCi/mmol) was obtained from Chemsyn Science Laboratories (Lexena, KS). Anodic oxidation of low activity [3H]THDMBA (5 mg, 0.019 mmol, specific activity 10 mCi/mmol) in DMF (25 mL) containing 0.5 M KClO4 was carried out in the presence of dG (75 mg, 0.28 mmol), dA (125 mg, 0.50 mmol), and Ade (25 mg, 0.18 mmol), respectively. Detection and quantification of the products by radioactivity measurement were carried out on a Radiomatic Flo-one/Beta radiation monitor, A250 series (Radiomatic, Tampa, FL). THDMBA: 1H NMR, δ (ppm): 1.63 (m, 2H, 2-H2), 1.87 (m, 2H, 3-H2), 2.95 (t, 2H, J3,4 ) 6.8 Hz, 4-H2), 2.96 (s, 3H, 7-CH3), 3.07 (s, 3H, 12-CH3), 3.25 (t, 2H, J1,2 ) 5.8 Hz, 1-H2), 7.15 (d, 1H, J5,6 ) 9.0 Hz, 5-H), 7.50 (m, 2H, 9-H, 10-H), 8.03 (d, 1H, J5,6 ) 9.0 Hz, 6-H), 8.26 (m, 2H, 8-H, 11-H). UV, λmax (nm): 267.2, 363.8, 382.0, 402.8. 7-Methyl-1,2,3,4-tetrahydrobenz[a]anthracene (MTHBA)12-CH2-N7Ade: 1H NMR, δ (ppm): 1.44 (m, 2H, 2-H2), 1.79 (m, 2H, 3-H2), 2.95 (t, 2H, J3,4 ) 6.3 Hz, 4-H2), 3.02 (m, 2H, 1-H2), 3.10 (s, 3H, 7-CH3), 6.36 (bs, 2H, 12-CH2), 7.14 [s, 2H, 6-NH2 (Ade)], 7.25 (d, 1H, J5,6 ) 9.2 Hz, 5-H), 7.51 (m, 1H, 10-H), 7.52 [s, 1H, 8-H (Ade)], 7.55 (m, 1H, 9-H), 7.88 (d, 1H, J10,11 ) 8.8 Hz, 11-H), 8.21 (d, 1H, J5,6 ) 9.2 Hz, 6-H), 8.25 [s, 1H, 2-H (Ade)], 8.42 (d, 1H, J8,9 ) 8.5 Hz, 8-H). UV, λmax (nm): 266.0, 367.0, 384.8, 404.6. FAB MS, (M + H)+, C25H24N5: calcd m/z 394.2032; obsd m/z 394.2023. 12-MTHBA-7-CH2-N7Ade: 1H NMR, δ (ppm): 1.66 (m, 2H, 2-H2), 1.88 (m, 2H, 3-H2), 2.91 (t, 2H, J3,4 ) 6.4 Hz, 4-H2), 3.18 (s, 3H, 12-CH3), 3.29 (m, 2H, 1-H2), 6.45 (s, 2H, 7-CH2), 6.86 [s, 1H, 8-H (Ade)], 7.19 (d, 1H, J5,6 ) 9.1 Hz, 5-H), 7.25 [s, 2H, 6-NH2 (Ade)], 7.53 (m, 1-H, 9-H), 7.57 (m, 1-H, 10-H), 7.96 (d, 1H, J5,6 ) 9.1 Hz, 6-H), 8.20 (d, 1H, J8,9 ) 8.3 Hz, 8-H), 8.22 [s, 1H, 2-H (Ade)], 8.39 (d, 1H, J10,11 ) 8.5 Hz, 11-H). UV, λmax

Mulder et al. (nm): 266.8, 365.6, 383.6, 403.8. FAB MS, (M + H)+, C25H24N5: calcd m/z 394.2032; obsd m/z 394.2034. 7-MTHBA-12-CH2-N6dA: 1H NMR, δ (ppm): 1.56 (m, 2H, 2-H2), 1.83 (m, 2H, 3-H2), 2.29 (m, 1H, 2′-H), 2.76 (m, 1H, 2′-H), 2.94 (t, 2H, J3,4 ) 6.6 Hz, 4-H2), 2.95 (s, 3H, 7-CH3), 3.41 (m, 2H, 1-H2), 3.52 (m, 1H, 5′-H), 3.62 (m, 1H, 5′-H), 3.89 (m, 1-H, 4′-H), 4.42 (m, 1H, 3′-H), 5.19 (bs, 1H, 5′-OH), 5.30 (bs, 1H, 3′OH), 5.38 (bs, 2H, 12-CH2), 6.39 (t, 1H, J1′,2′ ) 7.0 Hz, 1′-H), 7.19 (d, 1H, J5,6 ) 9.0 Hz, 5-H), 7.48 (m, 2H, 9-H, 10-H), 8.11 (d, 1H, J5,6 ) 9.0 Hz, 6-H), 8.16 (d, 1H, J8,9 ) 9.3 Hz, 8-H), 8.34 [m, 2H, 11-H, 8-H (Ade)], 8.39 [bs, 1H, 2-H (Ade)], 8.48 [bs, 1H, 6-NH (Ade)]. UV, λmax (nm): 265.2, 363.2, 381.8, 402.2. FAB MS, (M + H)+, C30H32O3N5: calcd m/z 510.2505; obsd m/z 510.2500. 12-exo-Methylene-7-MTHBA-7-N6dA: 1H NMR, δ (ppm): 1.38 (m, 1H, 2-H), 1.56 (s, 3H, 7-CH3), 1.62 (m, 1H, 3-H), 1.87 (m, 1H, 3-H), 2.00 (m, 1H, 2-H), 2.24 (m, 1-H, 2′-H), 2.69 (m, 1H, 4-H), 2.75 (m, 1H, 4-H), 2.77 (m, 1H, 2′-H), 2.98 (m, 1H, 1-H), 3.04 (m, 1H, 1-H), 3.49 (m, 1H, 5′-H), 3.59 (m, 1H, 5′-H), 3.86 (m, 1H, 4′-H), 4.39 (m, 1H, 3′-H), 5.10 (bs, 1H, 5′-OH), 5.27 (bs, 1H, 3′-OH), 5.56 (s, 1H, 12-CHb2), 5.81 (s, 1H, 12-CHa2), 6.33 (t, 1H, J1′,2′ ) 7.0 Hz, 1′-H), 6.83 (d, 1H, J5,6 ) 8.1 Hz, 5-H), 7.08 (d, 1H, J5,6 ) 8.1 Hz, 6-H), 7.10 (m, 1H, 9-H), 7.17 (d, 1H, J8,9 ) 7.6 Hz, 8-H), 7.21 (m, 1H, 10-H), 7.61 (d, 1H, J10,11 ) 7.6 Hz, 11-H), 7.65 [s, 1H, 2-H (Ade)], 8.39 [s, 1H, 6-NH (Ade)], 8.45 [s, 1H, 8-H (Ade)]. UV, λmax (nm): 266.0. FAB MS, (M + H)+, C30H32O3N5: calcd m/z 510.2505; obsd m/z 510.2497. 7,12-Di-exo-methylene-1,2,3,4-tetrahydrobenz[a]anthracene (THBA): 1H NMR, δ (ppm): 1.67 (m, 2H, 2-H2), 1.76 (m, 2H, 3-H2), 2.80 (t, 2H, J3,4 ) 6.6 Hz, 4-H2), 2.95 (t, 2H, J1,2 ) 6.1 Hz, 1-H2), 5.52 (s, 1H, 12-CHb), 5.68 (s, 1H, 7-CHb), 5.74 (s, 1H, 7-CHa), 5.82 (s, 1H, 12-CHa), 7.08 (d, J5,6 ) 7.9 Hz, 5-H), 7.36 (m, 2H, 9-H, 10-H), 7.55 (d, J5,6 ) 7.9 Hz, 6-H), 7.65 (m, 1H, 11-H), 7.71 (m, 1H, 8-H). UV, λmax (nm): 236.0, 282.2. FAB MS, (M + H)+, C20H19: calcd m/z 259.1487; obsd m/z 259.1488. 7-MTHBA-12-CH2-N1Ade: 1H NMR, δ (ppm): 1.45 (m, 2H, 2-H2), 1.77 (m, 2H, 3-H2), 2.93 (t, 2H, J3,4 ) 6.8 Hz, 4-H2), 3.05 (m, 2H, 1-H2), 3.07 (s, 3H, 7-CH3), 6.31 (s, 2H, 12-CH2), 7.24 (d, 1H, J5,6 ) 9.1 Hz, 5-H), 7.44 (m, 1H, 10-H), 7.51 (m, 1H, 9-H), 7.51 [s, 1H, 2-H (Ade)], 7.79 [bs, 1H, 6-NH (Ade)], 7.83 (d, 1H, J10,11 ) 8.8 Hz, 11-H), 7.87 [s, 1H, 8-H (Ade)], 7.93 [bs, 1H, 6-NH (Ade)], 8.20 (d, 1H, J5,6 ) 9.1 Hz, 6-H), 8.40 (d, 1H, J8,9 ) 8.3 Hz, 8-H). UV, λmax (nm): 266.6, 368.4, 386.4, 405.6. FAB MS, (M + H)+, C25H24N5: calcd m/z 394.2032; obsd m/z 394.2036. 12-MTHBA-7-CH2-N1Ade: 1H NMR, δ (ppm): 1.63 (m, 2H, 2-H2), 1.86 (m, 2H, 3-H2), 2.90 (t, 2H, J3,4 ) 6.9 Hz, 4-H2), 3.15 (s, 3H, 12-CH3), 3.27 (t, 2H, J1,2 ) 6.1 Hz, 1-H2), 6.45 (s, 2H, 7-CH2), 7.17 (d, 1H, J5,6 ) 9.2 Hz, 5-H), 7.41 [s, 1H, 2-H (Ade)], 7.55 (m, 2H, 9-H, 10-H), 7.76 [bs, 1H, 6-NH (Ade)], 7.87 [bs, 1H, 6-NH (Ade)], 7.92 [s, 1H, 8-H (Ade)], 8.15 (d, 1H, J5,6 ) 9.2 Hz, 6-H), 8.37 (m, 1H, 8-H), 8.41 (m, 1H, 11-H). UV, λmax (nm): 266.6, 367.2, 384,8, 404.8. FAB MS, (M + H)+, C25H24N5: calcd m/z 394.2032; obsd m/z 394.2028. 12-exo-Methylene-7-MTHBA-7-N3Ade: 1H-NMR, δ (ppm): 1.41 (m, 1H, 2-H), 1.64 (m, 1H, 3-H), 1.87 (m, 1H, 3-H), 1.97 (s, 3H, 7-CH3), 1.99 (m, 1H, 2-H), 2.69 (m, 1H, 4-H), 2.76 (m, 1H, 4-H), 3.03 (m, 2H, 1-H2), 5.67 (s, 1H, 12-CHb), 5.93 (s, 1H, 12CHa), 6.16 (d, 1H, J5,6 ) 8.1 Hz, 6-H), 6.36 (d, 1H, J8,9 ) 7.8 Hz, 8-H), 6.83 (d, 1H, J5,6 ) 8.1 Hz, 5-H), 7.12 (m, 1H, 9-H), 7.22 [s, 2H, 6-NH2 (Ade)], 7.30 (m, 1H, 10-H), 7.64 [s, 1H, 8-H (Ade)], 7.68 (d, 1H, J10,11 ) 7.7 Hz, 11-H), 8.58 [s, 1H, 2-H (Ade)]. UV, λmax (nm): 259.0. FAB MS, (M + H)+, C25H24N5: calcd m/z 394.2032; obsd m/z 394.2033. 7-exo-Methylene-12-MTHBA-12-N3Ade: 1H NMR, δ (ppm): 0.87 (m, 1H, 1-H), 1.13 (m, 1H, 3-H), 1.24 (m, 1H, 2-H), 1.43 (m, 1H, 2-H), 1.63 (m, 1H, 3-H), 2.08 (s, 3H, 12-CH3), 2.58 (m, 1H, 1-H), 2.63 (m, 1H, 4-H), 2.67 (m, 1H, 4-H), 5.71 (s, 1H, 7-CHa), 5.78 (s, 1H, 7-CHb), 6.19 (d, 1H, J10,11 ) 7.6 Hz, 11-H), 7.07 (d, 1H, J5,6 ) 8.1 Hz, 5-H), 7.11 (m, 1H, 10-H), 7.19 [s, 2H, 6-NH2 (Ade)], 7.26 (m, 1H, 9-H), 7.62 (d, 1H, J5,6 ) 8.1 Hz, 6-H), 2 Letters a and b are assigned in clockwise manner: 12-CHa points toward 11-H, and 12-CHb toward 1-H2.

One-Electron Oxidation of 1,2,3,4-Tetrahydro-DMBA 7.69 [bs, 1H, 8-H (Ade)], 7.79 (d, 1H, J8,9 ) 7.8 Hz, 8-H), 8.58 [bs, 1H, 2-H (Ade)]. UV, λmax (nm): 261.4. FAB MS, (M + H)+, C25H24N5: calcd m/z 394.2032; obsd m/z 394.2027. 7-MTHBA-12-CH2-N3Ade: 1H NMR, δ (ppm): 1.46 (m, 2H, 2-H2), 1.79 (m, 2H, 3-H2), 2.95 (t, 2H, J3,4 ) 6.8 Hz, 4-H2), 3.08 (m, 2H, 1-H2), 3.08 (s, 3H, 7-CH3), 6.11 (s, 2H, 12-CH2), 7.23 [s, 2H, 6-NH2 (Ade)], 7.24 (d, 1H, J5,6 ) 9.0 Hz, 5-H), 7.38 [s, 1H, 2-H (Ade)], 7.46 (m, 1H, 10-H), 7.52 (m, 1H, 9-H), 7.92 (d, 1H, J10,11 ) 9.0 Hz, 11-H), 8.20 (d, 1H, J5,6 ) 9.0 Hz, 6-H), 8.27 [s, 1H, 8-H (Ade)], 8.40 (d, 1H, J8,9 ) 8.5 Hz, 8-H). UV, λmax (nm): 266.4, 365.4, 384.0, 404.0. FAB MS, (M + H)+, C25H24N5: calcd m/z 394.2032; obsd m/z 394.2027. 12-MTHBA-7-CH2-N3Ade: 1H NMR, δ (ppm): 1.63 (m, 2H, 2-H2), 1.87 (m, 2H, 3-H2), 2.91 (t, 2H, J3,4 ) 6.8 Hz, 4-H2), 3.14 (s, 3H, 12-CH3), 3.26 (m, 2H, 1-H2), 6.22 (s, 2H, 7-CH2), 7.19 (d, 1H, J5,6 ) 9.1 Hz, 5-H), 7.21 [s, 2H, 6-NH2 (Ade)], 7.37 [s, 1H, 2-H (Ade)], 7.56 (m, 2H, 9-H, 10-H), 8.24 (d, 1H, J5,6 ) 9.1 Hz, 6-H), 8.31 [s, 1H, 8-H (Ade)], 8.35 (m, 1H, 11-H), 8.49 (m, 1H, 8-H). UV, λmax (nm): 266.8, 365.0, 382.8, 403.0. FAB MS, (M + H)+, C25H24N5: calcd m/z 394.2032; obsd m/z 394.2031. 12-exo-Methylene-7-MTHBA-7-N6Ade: 1H NMR, δ (ppm): 1.39 (m, 1H, 2-H), 1.51 (s, 3H, 7-CH3), 1.63 (m, 1H, 3-H), 1.87 (m, 1H, 3-H), 2.01 (m, 1H, 2-H), 2.69 (m, 1H, 4-H), 2.75 (m, 1H, 4-H), 2.99 (m, 1H, 1-H), 3.05 (m, 1H, 1-H), 5.55 (s, 1H, 12-CHb), 5.81 (s, 1H, 12-CHa), 6.79 (d, 1H, J5,6 ) 8.1 Hz, 5-H), 7.00 [bs, 1H, 6-NH (Ade)], 7.08 (m, 1H, 9-H), 7.14 (d, 1H, J5,6 ) 8.1 Hz, 6-H), 7.20 (m, 1H, 10-H), 7.23 (d, 1H, J8,9 ) 7.8, 8-H), 7.35 [s, 1H, 2-H (Ade)], 7.60 (d, 1H, J10,11 ) 7.6, 11-H), 7.74 [s, 1H, 8-H (Ade)]. UV, λmax (nm): 266.6. FAB MS, (M + H)+, C25H24N5: calcd m/z 394.2032; obsd m/z 394.2027. 7-MTHBA-12-CH2-N7Gua: 1H NMR, δ (ppm): 1.50 (m, 2H, 2-H2), 1.81 (m, 2H, 3-H2), 2.95 (t, 2H, J3,4 ) 6.2 Hz, 4-H2), 3.06 (m, 2H, 1-H2), 3.08 (s, 3H, 7-CH3), 6.20 [s, 2H, 2-NH2 (Gua)], 6.23 (s, 2H, 12-CH2), 7.12 [s, 1H, 8-H (Gua)], 7.24 (d, 1H, J5,6 ) 9.1 Hz, 5-H), 7.53 (m, 2H, 9-H, 10-H), 7.96 (m, 1H, 11-H), 8.19 (d, 1H, J5,6 ) 9.1 Hz, 6-H), 8.40 (m, 1H, 8-H), 10.90 [bs, 1H, 1-NH (Gua)]. UV, λmax (nm): 265.6, 365.8, 384.0, 403.6. FAB MS, (M + H)+, C25H24ON5: calcd m/z 410.1981; obsd m/z 410.1979. 12-MTHBA-7-CH2-N7Gua: 1H NMR, δ (ppm): 1.63 (m, 2H, 2-H2), 1.85 (m, 2H, 3-H2), 2.91 (t, 2H, J3,4 ) 6.2 Hz, 4-H2), 3.13 (s, 3H, 12-CH3), 3.26 (m, 2H, 1-H2), 6.24 [s, 2H, 2-NH2 (Gua)], 6.40 (d, 2H, 7-CH2), 6.90 [s, 1H, 8-H (Gua)], 7.20 (d, 1H, J5,6 ) 9.1 Hz, 5-H), 7.55 (m, 2H, 9-H, 10-H), 8.18 (d, 1H, J5,6 ) 9.1 Hz, 6-H), 8.34 (m, 1H, 8-H), 8.41 (m, 1H, 11-H). UV, λmax (nm): 266.4, 364.4, 382.6, 403.2. FAB MS, (M + H)+, C25H24ON5: calcd m/z 410.1981; obsd m/z 410.1981. 7-MTHBA-12-CH2-N2dG: 1H NMR, δ (ppm): 1.61 (m, 2H, 2-H2), 1.86 (m, 2H, 3-H2), 2.25 (m, 1H, 2′-H), 2.74 (m, 1H, 2′-H), 2.96 (t, 2H, J3,4 ) 6.4 Hz, 4-H2), 3.03 (s, 3H, 7-CH3), 3.34 (m, 2H, 1-H2), 3.53 (m, 1H, 5′-H), 3.59 (m, 1H, 5′-H), 3.81 (m, 1H, 4′-H), 4.40 (m, 1H, 3′-H), 4.84 (d, 1H, J ) 3.9 Hz, 3′-OH), 5.22 (bs, 2H, 12-CH2), 5.25 (t, 1H, J ) 5.6 Hz, 5′-OH), 6.24 (t, 1H, J1′,2′ ) 7.0 Hz, 1′-H), 7.10 [bs, 1H, 2-NH (Gua)], 7.21 (d, 1H, J5,6 ) 9.0 Hz, 5-H), 7.54 (m, 1H, 9-H), 7.60 (m, 1H, 10-H), 7.95 [s, 1H, 8-H (Gua)], 8.12 (d, 1H, J5,6 ) 9.0 Hz, 6-H), 8.23 (d, 1H, J10,11 ) 8.8 Hz, 11-H), 8.37 (d, 1H, J8,9 ) 8.5 Hz, 8-H), 10.17 [bs, 1H, 1-NH (Gua)]. UV, λmax (nm): 265.8, 363.4, 382.8, 402.2. FAB MS, (M + H)+, C30H32O4N5: calcd m/z 526.2454; obsd m/z 526.2453. 7,12-Dioxo-THDMBA: 1H NMR, δ (ppm): 1.61 (m, 2H, 2-H, 3-H), 1.68 (m, 1H, 3-H), 1.84 (m, 1H, 2-H), 2.00 (s, 3H, 7-CH3), 2.19 (s, 3H, 12-CH3), 2.69 (m, 1H, 4-H), 2.76 (m, 1H, 4-H), 2.97 (m, 1H, 1-H), 3.05 (m, 1H, 1-H), 6.99 (d, 1H, J5,6 ) 8.0 Hz, 5-H), 7.19 (d, 1H, J5,6 ) 8.0 Hz, 6-H), 7.29 (m, 2H, 9-H, 10-H), 7.41 (m, 1H, 8-H), 7.48 (m, 1H, 11-H). UV, λmax (nm): 218.6, 264.6. FAB MS, (M + H)+, C20H21O2: calcd m/z 293.1541; obsd m/z 293.1551.

Results Synthesis and Purification of Adducts. The adducts produced in the electrochemical oxidation of

Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1267

Figure 1. HPLC chromatograms of the products obtained by anodic oxidation of THDMBA in the presence of (A) dA; (B) Ade; (C) dG. CH3OH/H2O gradient for (A) and (C), C2H5OH/H2O gradient for (B).

THDMBA in the presence of dA or dG were separated by preparative HPLC. Elution with a CH3OH/H2O gradient (Figure 1A for dA, Figure 1C for dG), collection of appropriate peaks, and their subsequent elution with a CH3CN/H2O gradient resulted in complete separation of the adducts. The mixture of products formed in the reaction with Ade, however, was first subjected to an C2H5OH/H2O gradient (Figure 1B). This was done because the N1Ade adducts eluted very slowly with a CH3OH/H2O gradient and not at all with a CH3CN/H2O gradient. The N1Ade adducts were further purified with an C2H5OH/H2O mixture under isocratic conditions,

1268 Chem. Res. Toxicol., Vol. 9, No. 8, 1996

Mulder et al.

Scheme 1. Electrochemical Oxidation of THDMBA in the Presence of dA

whereas the other products were subjected to a CH3CN/ H2O gradient, in some cases followed by a CH3OH/H2O gradient to obtain full separation. Subsequently, a more effective separation was developed for preparative purification of the adducts. First, the THDMBA-Ade adducts were eluted in 60% CH3CN in H2O for 5 min, followed by a 75-min linear gradient to 100% CH3CN. Then, the eluent was changed to 100% C2H5OH in 10 min, and the column was eluted with C2H5OH for 35 min. All of the THDMBA-Ade adducts were purified in the CH3CN/H2O gradient, except for 7-MTHBA-12-CH2-N1Ade and 12MTHBA-7-CH2-N1Ade, which eluted in 100% C2H5OH. These two isomeric N1Ade adducts were purified in a separate C2H5OH/H2O gradient, as described in the Experimental Section. Because a number of the adducts have an altered aromatic nucleus and, therefore, very different UV spectra, it was impossible to quantify the adducts by UV alone. Quantification by weight was equally impossible because the amounts isolated were small and the adducts did not readily form crystals. Because [3H]THDMBA was available, a small amount of this material was used for electrochemical oxidation. The product mixtures derived from the reaction with dG, dA, and Ade were separated by analytical HPLC in the same way as discussed above except that quantification was by detection with a radiation flow monitor. THDMBA and its derivatives are sensitive to autoxidation. 7,12-Dioxo-THDMBA is often present in small amounts in samples of the starting material. When not properly stored, older samples of adducts developed significant amounts of their oxygenated form. Keeping the samples under argon, protecting them from light, and storing them at -20 °C largely eliminated this problem. At an anodic potential of 0.95 V, THDMBA was readily oxidized in the presence of dA, dG, or Ade and converted into products within 1 h. Regardless of the nucleophile used, 1.9-2.1 equiv of charge was consumed. Anodic oxidation of THDMBA in the presence of 25 molar equiv of dA (Scheme 1) yielded five major products: 7-MTHBA12-CH2-N7Ade (3.6%), 12-MTHBA-7-CH2-N7Ade (4.2%), 7-MTHBA-12-CH2-N6dA (5.8%), 12-exo-methylene-7-

MTHBA-7-N6dA (22.8%), and 7,12-di-exo-methyleneTHBA (44.2%). Electrochemical oxidation of THDMBA in the presence of 10 equiv of Ade (Scheme 2) produced ten products: 7-MTHBA-12-CH2-N7Ade (1.1%), 12MTHBA-7-CH2-N7Ade (2.4%), 7-MTHBA-12-CH2-N1Ade (10.2%), 12-MTHBA-7-CH2-N1Ade (13.2%), 7-MTHBA12-CH2-N3Ade (1.7%), 12-MTHBA-7-CH2-N3Ade (1.7%), 12-exo-methylene-7-MTHBA-7-N3Ade (27.9%), 7-exo-methylene-12-MTHBA-12-N3Ade (11.2%), 12-exo-methylene-7-MTHBA-7-N6Ade (12.1%), and 7,12-di-exo-methylene-THBA (16.7%). Four products were isolated from anodic oxidation of THDMBA in the presence of 15 equiv of dG (Scheme 3): 7-MTHBA-12-CH2-N7Gua (24.2%), 12-MTHBA-7-CH2N7Gua (12.2%), 7-MTHBA-12-CH2-N2dG (3.7%), and 7,12-di-exo-methylene-THBA (38.9%). The reaction in the presence of 25 equiv of dC yielded only one product: 7,12-di-exo-methylene-THBA (80.4%). In all the above reactions, trace amounts of products due to overoxidation were found at the potential used. The anodic potential had to be raised to 1.05 V to observe the formation of substantial amounts of secondary products. This resulted in very complex reaction mixtures, which were not pursued further. Only trace amounts of 7-MTHBA-12CH2OH and 12-MTHBA-7-CH2OH were detected in these oxidations. Structure Elucidation of Adducts. 7-MTHBA-12CH2-N7Ade. This compound has typical THDMBA UV absorptions at 266, 367, 385, and 405 nm. A small bathochromic shift of 2-3 nm is observed for the longwavelength absorptions with respect to the parent compound. FAB mass spectrometry shows an [M + H]+ ion at m/z 394, as expected for a THDMBA-Ade adduct. Loss of Ade gives the prominent fragment at m/z 259. As has been demonstrated for DMBA adducts, CAD spectra obtained by tandem mass spectrometry of the parent ion can distinguish between 7-CH2- and 12-CH2-linked adducts (20, 27). For DMBA, 12-CH2 adducts give a m/z 240 fragment more prominent than the m/z 239, whereas for 7-CH2 adducts the order is reversed. For THDMBA adducts, the same phenomenon occurs, except the m/z values are shifted to 243 and 244 (Figure 2). Indeed, the



Scheme 2. Electrochemical Oxidation of THDMBA in the Presence of Ade

Scheme 3. Electrochemical Oxidation of THDMBA in the Presence of dG

CAD spectrum of the m/z 394 ion of 7-MTHBA-12-CH2N7Ade shows both fragments, with the m/z 244 ion more abundant (Figure 2A). The absence of the deoxyribose proton chemical shifts from the NMR spectrum (Figure 3A) also indicates that the sugar moiety is lost. Two sharp singlets at 7.52 and 8.25 ppm, together with a D2O-exchangeable singlet (2H) at 7.14 ppm, confirm the presence of Ade. One methyl group is found in the upfield region of the spectrum (3.10 ppm, not shown), whereas the rather broad singlet (2H) at 6.36 ppm can be attributed to a CH2 group. NOE irradiation of the latter enhances the doublet at 7.88 ppm (11-H) and the multiplet at 3.02 ppm (1-H2, not shown).

This is evidence that addition has occurred at the 12CH3 group of THDMBA. In the same experiment, an NOE is also seen for the NH2 group and for the resonance giving the singlet at 7.52 ppm (8-H of Ade). Thus, mass spectrometry and NMR show that there is a covalent bond between the N-7 of Ade and the 12-CH2 of THDMBA. The NMR spectrum shows good correlation with that of the DMBA adduct 7-MBA-12-CH2-N7Ade (20), thereby further substantiating the assignment. In a manner similar to the NMR of the DMBA adduct, 11-H has shifted upfield by 0.38 ppm with respect to that of the parent compound, and this is attributed to the electronic shielding effect of the Ade moiety.


Mulder et al.

Figure 2. Collisionally activated decomposition spectra of [M + H]+ of (A) 7-MTHBA-12-CH2-N7Ade and (B) 12-MTHBA-7-CH2N7Ade.

Figure 3. 1H NMR spectrum (aromatic region) of (A) 7-MTHBA12-CH2-N7Ade; (B) 12-MTHBA-7-CH2-N7Ade.

12-MTHBA-7-CH2-N7Ade. FAB mass spectrometry shows an [M + H]+ ion at m/z 394 for THDMBA-Ade and a fragment at m/z 259, resulting from loss of Ade. The CAD spectrum of the [M + H]+ ion shows that the m/z 243 fragment is more abundant than the m/z 244 fragment, indicating that substitution has taken place at the 7-CH3 group (Figure 2B). In the NMR spectrum (Figure 3B) of the second deoxyribose-free adduct isolated from the reaction with dA, the NH2 group (2H, exchangeable with D2O) reso-

nates at 7.25 ppm. Two sharp singlets, at 8.22 and 6.86 ppm, can be attributed to the 2-H and 8-H of Ade. NOE irradiation of the CH2 group at 6.45 ppm affects the doublets at 7.96 (6-H) and 8.20 (8-H) ppm, the NH2 group, and the singlet at 6.86 ppm (8-H of Ade). This confirms that Ade is covalently bound at the N-7 position with the 7-CH2 of THDMBA. Anodic oxidation of DMBA in the presence of dA also produced a second adduct without deoxyribose, which was identified as 12-MBA-7-CH2-N3Ade (20). The NMR spectrum of this compound is very similar to that of 12MTHBA-7-CH2-N7Ade. For the DMBA adduct, two singlets are observed at 8.24 and 6.98 ppm, whereas the NH2 group resonates at 7.27 ppm. Assignment of this compound was mainly based upon the absence of an NOE between the 7-CH2 group and the NH2 group (20). NOE enhancement of an NH2 group can sometimes be rather small owing to the quadrupole effect of the nitrogen, so it may have been overlooked. The NOE experiment was repeated for the DMBA adduct, and this time a small but definite correlation was observed between both groups. This result affirms that the DMBA compound is also an N7Ade adduct and not an N3Ade adduct as reported previously (20). A similar approach was taken in the case of BP-6-CH2-N3Ade, the only depurinating adduct formed in the reaction between 6-methylbenzo[a]pyrene (6-CH3BP) and dA (23). It was first assigned as an N3Ade adduct based both on a comparison of its Ade chemical shifts with those of the Ade adducts in the DMBA series (20) and on the absence of an NOE between the 6-NH2 and the 6-CH2 groups. However, this time a small NOE enhancement was observed, and it has been reassigned as BP-6-CH2-N7Ade. 7-MTHBA-12-CH2-N6dA. FAB mass spectrometry shows an [M + H]+ ion at m/z 510 and a major fragment at m/z 259, the result of loss of dA from the [M + H]+ ion. Collisional activation (CA) of the m/z 510 ion causes it to give a more abundant m/z 244 fragment than m/z 243 fragment, which indicates a 12-CH2 adduct. The NMR spectrum of 7-MTHBA-12-CH2-N6dA also shows the deoxyribose ring is still attached to the Ade


Figure 4.

1H


NMR spectrum of (A) 12-exo-methylene-7-MTHBA-7-N6dA; (B) 7-MTHBA-12-CH2-N2dG.

base. The protons 1′-H, 2′-H, 3′-H, 4′-H, 5′-H2, 3′-OH and 5′-OH, are assigned by COSY and by comparison with the spectrum of dA. Only one D2O-exchangeable proton is observed in the aromatic region (8.48 ppm, 6-NH). The CH2 group resonates approximately 1 ppm upfield (5.38 ppm) with respect to methylene in the adducts bound at N-7, as described above. These two observations suggest an N6dA adduct attached to one of the methyl groups. NOE irradiation of the CH2 group enhances the multiplets at 8.16 (11-H) and 3.41 (1-H2) ppm, indicating that substitution has occurred at the 12-CH2 group. Only a weak shielding effect of the 11-H is observed compared to that of the analogous proton in 7-MTHBA-12-CH2N7Ade (Figure 3A). The weak shielding apparently occurs because the distance between the Ade base and the THDMBA moiety is increased by one bond length. 12-exo-Methylene-7-MTHBA-7-N6dA. exo-Methylene compounds are known and are relatively stable. In fact, these compounds can be obtained by acid-catalyzed tautomerization of fluorinated THDMBAs (28, 29). In our laboratory, 7,12-di-exo-methylene-THBA is stable in solution. In the presence of air, however, the compound is prone to decompose by autoxidation. FAB mass spectrometry shows a [M + H]+ ion at m/z 510 and its major fragment at m/z 259 (loss of dA). CA of the m/z 510 ion causes it to give a fragmentation spectrum that is identical to that of 7-MTHBA-12-CH2N6dA (see above): the m/z 244 fragment is more abundant than the m/z 243 fragment. This can be explained

by the fact that both compounds produce the same carbocationic intermediate upon loss of the dA fragment. Only one broad absorption band with a λmax of 266 nm is observed in the UV spectrum, indicating that the anthracene-like aromaticity has been lost. This is further substantiated by the NMR spectrum (Figure 4A); the six aromatic protons resonate at higher field than for the parent compound, a strong indication that the central ring of the anthracene nucleus is no longer aromatic. The resonances of the deoxyribose ring protons are present in the spectrum and are assigned by COSY. Three oneproton resonances at 7.65 (2-H), 8.39 (exchangeable with D2O, 6-NH), and 8.45 ppm (8-H) are attributed to the Ade base, suggesting that an N6dA adduct is very likely. Two very sharp proton signals at 5.56 and 5.81 ppm are prominent in the spectrum. Di-exo-methylene protons are expected to resonate in this part of the spectrum, and usually their geminal coupling constant is very small. Witiak et al. have also found separated proton signals at 5.5 and 5.8 ppm which are not coupled with each other (28). Irradiation of the 5.81 ppm singlet (12-CHa)2 by NOE enhances the singlet at 5.56 ppm (12-CHb)2 and the doublet at 7.61 ppm (11-H), while irradiation of the resonance singlet at 5.56 ppm produces an effect on the singlet at 5.81 ppm and the multiplet at 3.02 ppm (1H2), all of which is in line with a 12-exocyclic double bond. The resonance for the 7-CH3 group is found at 1.56 ppm, suggesting addition at the meso-anthracenic C-7 position with loss of aromaticity in the central benzene


ring. NOE irradiation of the 7-CH3 group enhances 6-H (7.08 ppm), 8-H (7.17 ppm), and 6-NH (8.39 ppm), which confirms the assignment. As a result of the lowered symmetry in this compound, the aliphatic protons of the angular ring are no longer equivalent and resonate at different chemical shifts. 7,12-Di-exo-methylene-THBA. This compound elutes just before the parent compound, which makes it unlikely to be an adduct. The UV spectrum shows two broad absorption bands at 236 and 282 nm, indicating that the anthracenic aromaticity has been lost. The FAB mass spectrum shows an [M + H]+ ion at m/z 259, whereas collisional activation of the ion in an MS/MS experiment produces an abundant m/z 244 fragment. The production of this fragment points to preferential protonation of the molecular ion at the 7-exocyclic double bond, producing a species that is very similar to that generated by loss of the substituent from a 12-CH2 adduct. In the NMR spectrum, the resonances of six aromatic protons are observed, together with four sharp singlets, found between 5.52 and 5.82 ppm, and the signals of a total of eight aliphatic protons, whereas no methyl group signals or D2O-exchangeable protons can be detected. The spectrum strongly suggests the presence of two exocyclic double bonds, one attached to the C-7 and one to the C-12 position. The double bond protons were irradiated in a series of NOE experiments; those experiments together with COSY provide sufficient data to complete the structural assignment. 7-MTHBA-12-CH2-N1Ade. FAB mass spectrometry shows an m/z 394 [M + H]+ ion and its m/z 259 fragment due to loss of Ade. CAD indicates the structure as a 12-CH2 adduct, because the m/z 244 fragment is more abundant than the m/z 243 fragment. The NMR spectrum taken in Me2SO at 30 °C displays two D2O exchangeable broad singlets at 7.79 and 7.93 ppm (Figure 5A). NOE irradiation of the methylene group at 6.31 ppm shows enhancement of the doublet at 7.83 ppm (11-H), the multiplet at 3.05 ppm (1-H2), and the singlet at 7.51 ppm (2-H of Ade). These findings confirm that the compound is a 12-CH2 adduct. Proton 11-H is shielded by 0.43 ppm with respect to the parent compound, an effect that is very similar to that observed for 7-MTHBA-12-CH2-N7Ade. The bonding of the 12-CH2 to the N-1 of Ade is suggested by the splitting of resonances of the NH2 protons (7.79 and 7.93 ppm). In fact, all of the PAH-N1Ade adducts characterized thus far exhibit this common feature (Table 1). This is attributed to the close proximity of the large aromatic moiety to the 6-amino group that creates a different chemical environment for the two protons. Furthermore, the 2-H of Ade is shifted upfield for the N1Ade adducts bonded to the meso-CH2, whereas it is shifted downfield for the N1Ade adducts linked to the aromatic ring (Table 1). 12-MTHBA-7-CH2-N1Ade. The FAB mass spectrum shows the [M + H]+ ion at m/z 394 and the expected fragment at m/z 259, due to loss of Ade. The CAD spectrum of the m/z 394 ion shows a more abundant m/z 243 than m/z 244 fragment, in accord with a 7-CH2 adduct. The NMR spectrum of this compound (Figure 5B) shows the splitting of the two proton resonances of the NH2 group (7.76 and 7.87 ppm), similarly to that seen for the NH2 group of 7-MTHBA-12-CH2-N1Ade (Figure 5A). This suggests that the Ade moiety is bonded at N-1. NOE irradiation of the methylene group at 6.45 ppm

Mulder et al.

Figure 5. 1H NMR spectrum (aromatic region) of (A) 7-MTHBA12-CH2-N1Ade; (B) 12-MTHBA-7-CH2-N1Ade. Table 1. Proton Chemical Shifts of PAH-N1Ade Adducts compound

H-2

H-8

6-NH2

7-MTHBA-12-CH2-N1Adea 12-MTHBA-7-CH2-N1Adea 7-MBA-12-CH2-N1Adeb 12-MBA-7-CH2-N1Adeb BP-6-CH2-N1Adeb 6-CH3BP-(1,3)-N1Adeb BP-6-N1Adec dibenzo[a,l]pyrene-10-N1Adeb dibenzo[c,g]carbazole-5-N1Adeb 7-methyldibenzo[c,g]carbazole-5-N1Adeb

7.51 7.41 7.67 7.54 7.64 8.66 8.71 8.74 8.61 8.62

7.87 7.92 7.92 7.94 7.98 7.94 7.62 7.64 7.64 7.79

7.79, 7.93 7.76, 7.87 7.92, 8.07 7.81, 7.94 7.83, 7.91 8.26, 8.35 8.51, 8.60 8.35, 8.50 8.19, 8.23 8.21, 8.29

a

In this article. b Unpublished results.3

c

Reference 8.

enhances the doublet at 8.15 ppm (6-H), the multiplet at 8.37 ppm (8-H), and the singlet at 7.41 ppm (2-H of Ade), indicating that the PAH moiety is bonded at the 7-CH2 group to the N1Ade. 12-exo-Methylene-7-MTHBA-7-N3Ade. The UV spectrum shows there is one broad absorption band at 259 nm. On the basis of the NMR spectrum, the adduct may be the 12-exo-methylene-7-N3Ade counterpart of 7-exomethylene-12-MTHBA-12-N3Ade (see below). Protons 12-CHa and 12-CHb of the exocyclic double bond are observed at 5.93 and 5.67 ppm, respectively. Assignments of the aromatic and aliphatic protons were made by COSY. Both 6-H (6.16 ppm) and 8-H (6.36 ppm) are strongly shielded by 1.39 and 1.35 ppm, respectively, with respect to their position in 7,12-di-exo-methylene-THBA, indicating a strong electrostatic effect of the Ade at these positions. An interaction of the Ade moiety with the angular ring seems to be absent. In fact, the chemical shifts of the angular ring protons are relatively unchanged with respect to 7,12-di-exo-methylene-THBA and resonate at almost identical positions as in 12-exomethylene-THBA-7-N6dA (see below). Irradiation of the


7-methyl group signal produces an NOE enhancement at 8.58 ppm, which is tentatively assigned as 2-H of Ade. Irradiation of the NH2 signal affords the same NOE enhancement at 8.58 ppm, corroborating the assignment as the 2-H of Ade. In fact, the average distance between the NH2 group and 2-H is 5.18 Å, whereas the average distance between the NH2 group and 8-H is 5.77 Å. These results are in line with an N3Ade adduct. The mass spectrometry data substantiate the NMR data. FAB mass spectrometry gives an [M + H]+ ion at m/z 394 and a major fragment at m/z 259 (loss of Ade), whereas the CAD of the [M + H]+ ion yields an m/z 244 fragment whose abundance is in excess of that of an m/z 243 fragment. 7-exo-Methylene-12-MTHBA-12-N3Ade. A broad absorption band at 261 nm is observed in the UV spectrum, a good indication that the aromaticity of the meso-anthracenic ring is lost. In the FAB mass spectrum, the [M + H]+ peak is seen at m/z 394, together with the fragment of m/z 259 (loss of Ade). In the CAD spectrum of the m/z 394 ion, the m/z 243 fragment is more abundant than the m/z 244 fragment, as expected for a 7-exo-methylene-C-12 adduct. Two sharp singlets at 5.71 and 5.78 ppm in the NMR spectrum can be attributed to an exocyclic double bond. Because their chemical shifts agree more closely with those of the 7-exo-methylene protons of 7,12-di-exomethylene-THBA than with the corresponding 12-exomethylene protons, it is reasonable to assign it as a 7-exocyclic double bond. NOE irradiation of each singlet confirms this assignment and makes it possible to assign the doublets at 7.62 and 7.79 ppm as due to 6-H and 8-H, respectively. Consequently, the Ade base must be located at the meso position 12. Irradiation of the 12-CH3 group enhances the broad singlet at 8.58 ppm, which is assigned as 2-H of Ade. It can be deduced from the NMR spectrum that the Ade moiety exercises a strong electronic effect on a number of protons of the THDMBA nucleus. In fact, 11-H is found at 6.19 ppm, 1.46 ppm upfield with respect to its position in 7,12-di-exo-methylene-THBA. This is a much larger shift than can be accounted for by changing the C-12 center from sp2 into sp3. Furthermore, one of the protons at C-1 of THDMBA is shielded to 0.87 ppm (2.08 ppm upfield compared to 7,12-di-exo-methyleneTHBA). Similarly, one proton of each pair at C-2 and C-3 experiences upfield shifts of 0.43 and 0.63 ppm, respectively. Substantial shielding effects, ranging between 0.7 and 1.0 ppm, have been observed on neighboring peri protons in N7Gua and N7Ade adducts of BP and in dibenzo[a,l]pyrene (DB[a,l]P) (21, 22, 26). For 7-exomethylene-12-MTHBA-12-N3Ade, in addition to a direct electrostatic effect of the Ade moiety on nearby protons, there is apparently an extended interaction between the Ade moiety and the angular ring of the THDMBA moiety. 7-MTHBA-12-CH2-N3Ade. The FAB mass spectrum gives the [M + H]+ ion at m/z 394 and the main fragment at m/z 259, whereas the CAD of the m/z 394 ion confirms this compound as a 12-CH2 adduct. In the NMR spectrum (Figure 5A) the aromatic protons are assigned by COSY and by comparison with the other adducts. The 11-H proton is tentatively assigned to the doublet at 7.92 ppm, denoting an upfield shift of 0.34 ppm for this proton compared to that of THDMBA. This is fully in line with the shielding observed for 11-H in 7-MTHBA-12-CH2-N7Ade and 7-MTHBA-12-CH2-N1Ade. Unfortunately, the key NOE experiment could not be performed to indicate the position of adenine substitution.


This was due to the close proximity of the 6-NH2 and 2-H signals of Ade. This adduct is, therefore, assigned by exclusion as the 7-MTHBA-12-CH2-N3Ade. In contrast to the N7Ade adduct in which the singlet of 12-CH2 displays considerable broadness (Figure 3A), the 12-CH2 resonance for this adduct is quite sharp and similar to the 7-MTHBA-12-CH2-N1Ade (Figure 5A). This suggests a less crowded environment for the 12-CH2 group, which is expected for an N3Ade adduct. 12-MTHBA-7-CH2-N3Ade. The 7-CH2 group of 12MTHBA-7-CH2-N3Ade resonates somewhat upfield (6.22 ppm) with respect to that of 12-MTHBA-7-CH2-N7Ade (6.45 ppm). The chemical shifts of the Ade protons 8-H (8.31 ppm), 2-H (7.37 ppm), and 6-NH2 (7.21 ppm) are in good accordance with an N3Ade adduct. Further evidence derives from irradiation of the 7-CH2 group in an NOE experiment: the protons 2-H(Ade), 6-H, and 8-H were enhanced. The FAB mass spectrum shows an [M + H]+ ion at m/z 394, which fragments upon CA to give additional evidence for a 7-CH2 adduct. 12-exo-Methylene-7-MTHBA-7-N6Ade. A broad absorption band at 267 nm is seen in the UV spectrum of this compound, its shape and λmax being almost identical to those of 12-exo-methylene-7-MTHBA-7-N6dA (see above). The FAB mass spectrum shows the [M + H]+ ion at m/z 394 and the fragment due to the loss of Ade at m/z 259, as expected. The m/z 244 fragment is more abundant than the m/z 243 fragment in the CAD spectrum of the m/z 394 ion, which points to a C-7 adduct. In the NMR spectrum, one D2O-exchangeable proton shows its resonance at 7.00 ppm; it is assigned to 6-NH of Ade. It is unclear why there is a considerable difference in chemical shift with respect to the 6-NH (8.39 ppm) of the corresponding N6dA compound. 2-H (7.35 vs 7.65 ppm) and 8-H (7.73 vs 8.45 ppm) also resonate at higher field for the N6Ade than for the N6dA derivative. In contrast, the protons belonging to the THDMBA moiety resonate within a range of 0.06 ppm in both compounds. No strong shielding of 6-H and 8-H is observed. This is another indication that the covalent bond is formed at the amino group. 7-MTHBA-12-CH2-N7Gua. It is evident from the NMR spectrum that the deoxyribose ring is lost, because none of its protons are present. The singlets at 10.90 ppm (D2O-exchangeable, 1-NH), 7.12 ppm (8-H), and 6.20 ppm (2H, D2O-exchangeable, 2-NH2) are assigned to the Gua base. The presence of both the NH2 group and the 8-H of Gua strongly suggests an N7Gua adduct. In an NOE experiment, irradiation of the resonance of the 12CH2 group (6.23 ppm) affords an enhancement of both 11-H (7.96 ppm) and 8-H of Gua (7.12 ppm), indicating that the adduct bond occurs at the 12-CH3 group. The shielding of 11-H (0.30 ppm with respect to THDMBA) is comparable to the 12-CH2 adducts as discussed above. When the NMR spectrum is compared to that of 7-MBA12-CH2-N7Gua (20), two notable differences are observed: the chemical shifts of the 2-NH2 group (1.00 ppm downfield in the DMBA derivative) and the 8-H of Gua (1.14 ppm downfield in the DMBA derivative). The spectrum of 7-MBA-12-CH2-N7Gua was recorded at 500 MHz, which provides higher resolution and sensitivity than at 300 MHz (20). The somewhat broad resonance of the NH2 group is very close to that of the CH2 group, as in 7-MTHBA-12-CH2-N7Gua, and was, for this reason, probably overlooked in the 300 MHz spectrum (20). The 8-H of Gua is assigned to the singlet at 7.20 ppm instead


of to the singlet at 8.26 ppm, which the 500 MHz spectrum shows to be an artifact. 12-MTHBA-7-CH2-N7Gua. The absence of deoxyribose protons and the presence of singlets at 6.90 ppm (8-H of Gua) and 6.24 ppm (2H, exchangeable with D2O, 2-NH2) suggest an N7Gua adduct, with linkage to the 7-CH2 group. An NOE experiment on the 7-CH2 group at 6.40 ppm confirms this assignment. With the exception of the 7-CH2 group (6.40 ppm for 12-MTHBA-7-CH2N7Gua vs 5.36 ppm for 12-MBA-7-CH2-N7Gua), the spectrum of this N7Gua adduct correlates well with the 300 MHz spectrum of 12-MBA-7-CH2-N7Gua (20). Unfortunately, there was insufficient material available to obtain a 500 MHz spectrum of this compound, so at present this discrepancy cannot be resolved. 7-MTHBA-12-CH2-N2dG. The FAB mass spectrum displays an [M + H]+ ion at m/z 526, together with the expected fragment at m/z 410 (loss of the deoxyribose ring) and a fragment at m/z 259 (loss of dG). The CAD of the [M + H]+ at the m/z 410 fragment produces an m/z 244 fragment that is more abundant than the m/z 243 fragment, which indicates a 12-CH2 adduct. The NMR spectrum (Figure 4B) shows the presence of the deoxyribose protons, which can be assigned by comparison with the spectrum of dG and by COSY. The sharp singlet at 7.95 (8-H) and the broad D2O-exchangeable singlets at 10.17 (1-NH) and 7.10 (2-NH) are attributed to the Gua base. The presence of the latter two provides strong support for an N2dG adduct. NOE irradiation of the singlet at 5.22 ppm enhances the doublet at 8.23 ppm (11-H) and the multiplet at 3.34 ppm (1-H2). Hence, the singlet is assigned as 12-CH2. Similarly to that of 7-MTHBA-12-CH2-N6dA, the 11-H is not shielded in this adduct, because the Gua base is farther away from the THDMBA moiety. A deoxyribose-containing 12-CH2 adduct was also obtained from the reaction of DMBA with dG, but this was assigned as a C8dG adduct rather than an N2dG adduct (20). This assignment was mainly based upon the absence of an 8-H singlet of Gua in the 300 MHz NMR spectrum. However, when recorded at 500 MHz, the spectrum of this adduct reveals the presence of an 8-H proton at 7.98 ppm. This chemical shift fits well with that of 7-MTHBA-12-CH2-N2dG and that of dG (7.89 ppm). Even at 500 MHz, the 8-H resonance is not very well resolved from the resonance of 4-H (and because of its long relaxation time, it integrates for less than one proton). Therefore, it is quite understandable that this signal was previously missed in the 300 MHz spectrum (20). Furthermore, from the 300 MHz spectrum, it is deduced that the 2-NH2 group resonates somewhere between 7.5 and 7.8 ppm. Because that region is very crowded in the 300 MHz spectrum, it is actually not possible to discriminate between a 2-NH2 and a 2-NH group. The 500 MHz spectrum, which was obtained later, is more clear. The 2-NH (1-H, exchangeable with D2O) resonates at 7.50 ppm. Structural evidence for a C8dG adduct is supported in part by the fact that this adduct could be electrochemically converted to a product identified as a C8Gua adduct (20). Unfortunately, there was insufficient material left to obtain a 500 MHz spectrum of this product. However, the 300 MHz spectrum is in accord with an N2Gua adduct, if we assume that the loss of the deoxyribose ring does not cause a dramatic change in the chemical shift of the 8-H proton of Gua. In that case, the 8-H of Gua may be obscured by the 4-H

Mulder et al.

resonance (in fact, the 300- MHz spectrum obtained after D2O exchange shows a somewhat better resolved 8-H of Gua3). The 2-NH resonance can either be hidden in the bulk of proton resonances in the 7.6-7.8 ppm region or be assigned to the resonance at 6.73 ppm, previously assigned as 1-NH or 9-NH. In the reaction of 6-CH3BP with dG, a C8dG adduct has also been isolated (23). When reexamined by 500 MHz NMR, it was found to be an N2dG adduct as well. Its 500 MHz spectrum demonstrates the presence of the 8-H (7.99 ppm) and 2-NH (7.07 ppm) of Gua. Finally, 500 MHz NMR fully supports the previous assignment of BP-6-C8dG as one of the major adducts of BP. Reassignment of 7-MBA-12-CH2-C8dG and BP-6-CH2C8dG as 7-MBA-12-CH2-N2dG and BP-6-CH2-N2dG, respectively, is consistent with the results of a hydrolysis study performed on these adducts and on BP-6-C8dG (21). BP-6-C8dG was found to hydrolyze very fast under moderate acidic (pH 3-4) conditions, whereas the other two adducts hydrolyze only to a small extent when subjected to a prolonged reaction time and a higher reaction temperature (21). A CH2-NH bond should in fact be much more resistant to acid hydrolysis than a CH2-C8 bond.

Discussion Formation of Adducts. Reaction of THDMBA with dG, dA, or Ade produces an array of adducts, which can be used as reference standards in the study of the biological formation of DNA adducts from THDMBA. The use of Ade in addition to dA enlarges the number of methylene-linked Ade adducts from 2 to 6, including the biologically relevant N3Ade adducts (8, 13). As discussed in the structure determination section, the putative N3Ade adducts previously proposed to be obtained from the reaction of DMBA (20) and 6-CH3BP (23) with dA are actually N7Ade adducts. This means that dA is unable to produce N3Ade adducts at least as observed in all the electrochemical oxidations performed to date, including those involving BP (21) and DB[a,l]P (22). In addition, N3Ade adducts are not obtained when dA is reacted with the diol epoxides of BP (21) or DB[a,l]P (30). Apparently, the deoxyribose ring in dA prevents nucleophilic attack of the N-3 position on the electrophilic THDMBA species. THDMBA is unique with respect to any of the other meso-methylated PAH studied so far because it has the ability to produce adducts covalently bound to the mesoanthracenic positions where a methyl group is substituted. These adducts are apparently formed only from the 6-NH2 of dA or Ade and the N-3 of Ade, but not from the NH2 group of dG, from the N-1 of Ade, or from the N-7 position of dA, Ade, or dG (Table 2). Of the two mesoanthracenic positions of THDMBA, the C-7 position is clearly favored. The 6-NH2 of dA and Ade appear to form a meso adduct only at C-7, whereas the N-3 of Ade is capable of forming both, with the C-7 adduct (27.9%) formed in preference to the C-12 (11.2%). The higher reactivity at C-7 vs C-12 may be the result of preferential loss of a proton from the 12-CH3 group in the THDMBA radical cation, as discussed below. No C8dG or C8Gua adducts could be isolated from the reaction with dG. As was outlined above in the description of the NMR results, the C8dG adducts previously identified from the oxidation of DMBA (20) and 6-CH3BP (23) are actually N2dG adducts. No C8dG adducts



Scheme 4. Mechanism of Adduct Formation by Initial One-Electron Oxidation of THDMBA

Table 2. Formation of THDMBA Adducts with dA, Ade, or dG adduct yield at the two electrophilic sites (%) CH2-linked meso combined

purine nucleoside or base

nucleophilic position of linkage in the purine

dA

N-7 6-NH2

7.8 5.8

N-7 N-1 N-3 6-NH2

3.5 23.4 3.4

N-7 2-NH2

36.4 3.7

Ade

dG

22.8

7.8 28.6

39.1 12.1

3.5 23.4 42.5 12.1 36.4 3.7

were obtained from the reaction of benzylic bromides or acetates of DMBA with dG in DMF (20). Furthermore, none were found when BP diol epoxide (21) or DB[a,l]P diol epoxide (30) was reacted with dG in DMF. The identity of the C8dG adduct of BP was confirmed by 500 MHz NMR, and there is no reason to doubt the identity of BP-6-C8Gua (21, 26) or that of DB[a,l]P-10-C8dG and DB[a,l]P-10-C8Gua (22). These observations suggest an interesting conclusion: C8dG adducts can be formed at the reactive aromatic positions of unsubstituted PAH, but not so far at the reactive benzylic positions of methylsubstituted PAH. No adducts were obtained when THDMBA was electrochemically oxidized in the presence of dC. This corroborates our findings for DMBA (20), BP (21), DB[a,l]P (22), and 6-CH3BP (23) and confirms that dC is much less nucleophilic than dG, dA, and Ade (31).

Reactivity of Nucleophilic Groups. When all the adducts isolated (methyl and meso adducts combined) in the oxidation of THDMBA are considered (Table 2), it is apparent that for dA most of the adducts are formed at the 6-NH2 group (29%), and considerably fewer at the N-7 position (8%). None of the other positions in dA display significant reactivity toward the electrophile. For Ade, N-3 reacts to give the largest total amount of adducts (42%), followed by the N-1 position (23%), then the 6-NH2 group (12%) and finally the N-7 position (4%) (Table 2). For dG, the N-7 position is by far the most reactive (36%), followed by the 2-NH2 group (4%). However, when only the methylene-linked adducts are taken into account, the order of reactivity for dA is more-orless reversed: the N-7 position (8%) is slightly more reactive than the 6-NH2 group (6%). For Ade, the change is even more dramatic: the N-1 position (23%) is far more reactive toward the methyl groups than the N-7 (4%) and N-3 (3%) positions. The 6-NH2 group does not display detectable reactivity toward the methyl groups. The order for dG is unchanged from previous studies because only methylene-linked adducts were isolated. For DMBA, only adducts covalently bound to the CH3 groups have been isolated (20). When the order of reactivity for the nucleophilic groups in dA and dG is considered, a picture similar to that of THDMBA is obtained. For dA, only the N-7 position is reactive, whereas for dG, the N-7 position is clearly more reactive than the 2-NH2 group (20). Similarly, in the electrochemical oxidation of 6-CH3BP (23), the N-7 position is the most reactive for dA; no 6-NH2 or other adducts were


isolated. For dG, the N-7 position is more reactive than the 2-NH2 group. In the reaction with dG, considerable amounts of 6-CH3BP-1- and 3-N7Gua were isolated, but no BP-6-CH2-N7Gua was found (23). When the reaction was repeated recently, BP-6-CH2-N7Gua was isolated; previously it escaped isolation because of its low solubility.3 In conclusion, the relative order of reactivity of the nucleophilic groups in dA, dG, and Ade is the same for THDMBA, DMBA, and 6-CH3BP in DMF when the PAH are electrochemically oxidized. Mechanism of Adduct Formation. Oxidation of THDMBA in the presence of nucleophiles proceeds with the consumption of 2 equiv of charge. At the potential used, oxidation of the solvent or of any of the nucleophiles can be excluded, and oxidation of primary adducts is negligible. These results indicate that an overall twoelectron oxidation process takes place. Radical cations of dimethylanthracene derivatives (32) and other substituted aromatic hydrocarbons (33) are known to be very strong acids, which can deprotonate easily in polar aprotic solvents such as Me2SO to produce free radicals. Deprotonation is thermodynamically and kinetically favorable if there are no great stereoelectronic constraints to forming an sp2 center coplanar with the aromatic π-system (34-37). Although in those cases deprotonation is not the rate-limiting step, its site does determine the selectivity with respect to product distribution (36, 37). Proton loss is generally an irreversible process, and small differences in stereoelectronic environment can lead to differences in individual deprotonation rate constants for nonequivalent methyl groups (36, 37). For THDMBA, deprotonation can occur from the 7- or the 12-CH3 group, generating two different radicals (Scheme 4). Although the 12-CH3 group is in a somewhat more crowded environment than the 7-CH3 group, its rate of deprotonation is higher. This can be rationalized as follows: generation of a 7-CH2 sp2 center increases unfavorable peri interactions with both 6-H and 8-H, whereas a 12-CH2 sp2 center increases only the peri interaction with 11-H. In fact, the steric hindrance of the 1-H2 protons is quite similar for a 12-CH3 group and a 12-CH2 group. The overall more abundant formation of 12-CH2 and 7-meso adducts vs 7-CH2 and 12-meso adducts substantiates this hypothesis. Proton loss from the 12-CH3 group results in a species in which the spin density is located largely at the 12methylene and the 7-meso positions, whereas deprotonation from 7-CH3 yields a species with high spin density at the 7-methylene and 12-meso positions (Scheme 4). Subsequent anodic oxidation of the radical to a carbocation should be a very efficient process, because an unpaired electron is lost (38). For THDMBA, two different carbocations will be formed, in which the positive charge is distributed in a similar way as the spin density in the radical. Overall, one-electron oxidation of THDMBA leads to the generation of four reactive centers in the molecule, as is substantiated by the product distribution. Isolation of considerable amounts of 7,12-di-exo-methylene-THBA (in many cases the major product) also speaks in favor of a carbocation as the reacting intermediate. This compound is most likely produced by deprotonation of the carbocation, rather than by loss of a hydrogen radical from the radical intermediate. The 3

Cavalieri et al. (unpublished results).

Mulder et al.

latter would involve the breaking of a strong C-H bond, which is energetically unfavorable. Formation of this dehydrogenated product suggests that deprotonation of the carbocation competes with the nucleophilic attack by the purine base. Large amounts of meso adducts are formed in the reaction with dA and Ade. These adducts can arise from nucleophilic attack on the radical cation and/or on the carbocation. Nucleophilic attack on the radical cation implies that the relative ratio of meso to methyl-linked adducts should be related to the amount of nucleophile present. When the concentration of nucleophile is increased, it should become more efficient in reacting with the radical cation at its meso positions before it can deprotonate and eventually form methyl-linked adducts. However, the ratio of meso to methyl adducts in the reaction with dA does not change much going from 20 to 100 equiv. Thus, nucleophilic attack on the radical cation is likely to be at best a minor pathway. Moreover, if dA and Ade are capable of reacting with THDMBA radical cation at its meso positions, it would be logical to expect that dA and Ade should be able to do so with DMBA (20) and with 6-CH3BP (23). No such products, however, have been identified. On the basis of these observations, it is reasonable to conclude that the meso and methyl-linked adducts formed in the electrochemical oxidation of THDMBA arise from nucleophilic attack on carbocation intermediates.

Acknowledgment. This research was supported by U.S. Public Health Service Grants P01-CA49210, awarded to both research groups, and R01-CA44686. Core support at the Eppley Institute was from the National Cancer Institute (P30-CA36727).

References (1) Sims, P., and Grover, P. L. (1981) Involvement of dihydrodiols and diol epoxides in the metabolic activation of polycyclic hydrocarbons other than benzo[a]pyrene. In Polycyclic Hydrocarbons and Cancer (Gelboin, H. V., and Ts’o, P. O. P., Eds.) Vol. 3, pp 117-181, Academic Press, New York. (2) Conney, A. H. (1982) Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons. G. H. A. Clowes Memorial Lecture. Cancer Res. 42, 4875-4917. (3) Hall, M., and Grover, P. L. (1990) Polycyclic aromatic hydrocarbons: metabolism, activation and tumor initiation. In Chemical Carcinogenesis and Mutagenesis I (Cooper, C. S., and Grover, P. L., Eds.) pp 327-372, Springer-Verlag, Berlin. (4) Cavalieri, E. L., and Rogan, E. G. (1992) The approach to understanding aromatic hydrocarbon carcinogenesis. The central role of radical cations in metabolic activation. Pharmacol. Ther. 55, 183-199. (5) Cavalieri, E. L., and Rogan, E. G. (1995) Central role of radical cations in metabolic activation of polycyclic aromatic hydrocarbons. Xenobiotica 25, 677-688. (6) Devanesan, P. D., RamaKrishna, N. V. S., Todorovic, R., Rogan, E. G., Cavalieri, E. L., Jeong, H., Jankowiak, R., and Small, G. J. (1992) Identification and quantitation of benzo[a]pyrene-DNA adducts formed by rat liver microsomes in vitro. Chem. Res. Toxicol. 5, 302-309. (7) Rogan, E. G., Devanesan, P. D., RamaKrishna, N. V. S., Higginbotham, S., Padmavathi, N. S., Chapman, K., Cavalieri, E. L., Jeong, H., Jankowiak, R., and Small, G. J. (1993) Identification and quantitation of benzo[a]pyrene-DNA adducts formed in mouse skin. Chem. Res. Toxicol. 6, 356-363. (8) Chen, L., Devanesan, P. D., Higginbotham, S., Ariese, F., Jankowiak, R., Small, G. J., Rogan, E. G., and Cavalieri, E. L. (1996) Expanded analysis of benzo[a]pyrene-DNA adducts formed in vitro and in mouse skin: Their significance in tumor initiation. Chem. Res. Toxicol. 9, 897-903. (9) RamaKrishna, N. V. S., Devanesan, P. D., Rogan, E. G., Cavalieri, E. L., Jeong, H., Jankowiak, R., and Small, G. J. (1992) Mechanism of metabolic activation of the potent carcinogen 7,12dimethylbenz[a]anthracene. Chem. Res. Toxicol. 5, 220-226.

One-Electron Oxidation of 1,2,3,4-Tetrahydro-DMBA (10) Devanesan, P. D., RamaKrishna, N. V. S., Padmavathi, N. S., Higginbotham, S., Rogan, E. G., Cavalieri, E. L., Marsch, G. A., Jankowiak, R., and Small, G. J. (1993) Identification and quantitation of 7,12-dimethylbenz[a]anthracene-DNA adducts formed in mouse skin. Chem. Res. Toxicol. 6, 364-371. (11) Cheng, S. C., Prakash, A. S., Pigott, M. A., Hilton, B. D., Lee, H., Harvey, R. G., and Dipple, A. (1988) A metabolite of the carcinogen 7,12-dimethylbenz[a]anthracene that reacts predominantly with adenine residues in DNA. Carcinogenesis 9, 17211723. (12) Cheng, S. C., Prakash, 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[a]anthracene-adenine nucleoside adducts. Chem. Res. Toxicol. 1, 216-221. (13) Li, K.-M., Todorovic, R., Rogan, E. G., Cavalieri, E. L., Ariese, F., Suh, M., Jankowiak, R., and Small, G. J. (1995) Identification and quantitation of dibenzo[a,l]pyrene-DNA adducts formed by rat liver microsomes in vitro: Preponderance of depurinating adducts. Biochemistry 34, 8043-8049. (14) Cheh, A. M., Chadha, A., Sayer, J. M., Yeh, H. J. C., Yagi, H., Pannell, L. K., and Jerina, D. M. (1993) Structures of covalent nucleoside adducts formed from adenine, guanine, and cytosine bases of DNA and the optically active bay-region 3,4-diol 1,2epoxides of benz[a]anthracene. J. Org. Chem. 58, 4013-4022. (15) DiGiovanni, J., Diamond, L., Singer, J. M., Daniel, F. B., Witiak, D. T., and Slaga, T. J. (1982) Tumor initiating activity of 4-fluoro7,12-dimethylbenz[a]anthracene and 1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene in female SENCAR mice. Carcinogenesis 3, 651-655. (16) Cavalieri, E. L., Rogan, E. G., Higginbotham, S., Cremonesi, P., and Salmasi, S. (1990) Tumorigenicity of 7,12-dimethylbenz[a]anthracene, some of its fluorinated derivatives, and 1,2,3,4tetrahydro-7,12-dimethylbenz[a]anthracene in mouse skin and rat mammary gland. Polycyclic Aromat. Compd. 1, 59-70. (17) Nair, R. V., Walker, S. E., Sharma, P. K., Witiak, D. T., and DiGiovanni, J. (1992) Mouse skin tumor initiating activity of fluorination derivatives of 1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene. Chem. Res. Toxicol. 5, 153-156. (18) Cremonesi, P., Rogan, E., and Cavalieri, E. (1992) Correlation studies of anodic peak potentials and ionization potentials for polycyclic aromatic hydrocarbons. Chem. Res. Toxicol. 5, 346355. (19) Rinderle, S. J., Black, S. D., Sharma, P. K., and Witiak, D. T. (1992) Comparative metabolism in vitro of a novel carcinogenic polycyclic aromatic hydrocarbon, 1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracene, and its two regioisomeric B-ring fluoro analogues. Cancer Res. 52, 3035-3042. (20) RamaKrishna, N. V. S., Cavalieri, E. L., Rogan, E. G., Dolnikowski, G. G., Cerny, R. L., Gross, M. L., Jeong, H., Jankowiak, R., and Small, G. J. (1992) Synthesis and structure determination of the adducts of the potent carcinogen 7,12-dimethylbenz[a]anthracene and deoxyribonucleosides formed by electrochemical oxidation: Models for metabolic activation by one-electron oxidation. J. Am. Chem. Soc. 114, 1863-1874. (21) RamaKrishna, N. V. S., Gao, F., Padmavathi, N. S., Cavalieri, E. L., Rogan, E. G., Cerny, R. L., and Gross, M. L. (1992) Model adducts of benzo[a]pyrene and nucleosides formed from its radical cation and diol epoxide. Chem. Res. Toxicol. 5, 293-302. (22) RamaKrishna, N. V. S., Padmavathi, N. S., Cavalieri, E. L. Rogan, E. G., Cerny, R. L., and Gross, M. L. (1993) Synthesis and structure determination of the adducts formed by electrochemical oxidation of the potent carcinogen dibenzo[a,l]pyrene in the presence of nucleosides. Chem. Res. Toxicol. 6, 554-560. (23) RamaKrishna, N. V. S., Li, K.-M., Rogan, E. G., Cavalieri, E. L., George, M., Cerny, R. L., and Gross, M. L. (1993) Adducts of 6-methylbenzo[a]pyrene and 6-fluorobenzo[a]pyrene formed by electrochemical oxidation in the presence of deoxyribonucleosides. Chem. Res. Toxicol. 6, 837-845.

Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1277 (24) NIH Guidelines for the Laboratory Use of Chemical Carcinogens (1981) NIH Publication No. 81-2385, U.S. Government Printing Office, Washington, D.C. (25) Inbasekaran, M. N., Witiak, D. T., Barone, K., and Loper, J. C. (1980) Synthesis and mutagenicity of A-ring reduced analogues of 7,12-dimethylbenz[a]anthracene. J. Med. Chem. 23, 278-281. (26) Rogan, E., Cavalieri, E., Tibbels, S., Cremonesi, P., Warner, C., Nagel, D., Tomer, K., Cerny, R., and Gross, M. (1988) Synthesis and identification of benzo[a]pyrene-guanine nucleoside adducts formed by electrochemical oxidation and by HRP catalyzed reaction of benzo[a]pyrene with DNA. J. Am. Chem. Soc. 110, 4023-4029. (27) Dolnikowski, G. G., Gross, M. L., and Cavalieri, E. L. (1991) Isomer differentiation in 7,12-dimethylbenz[a]anthracene-pyridine adducts by fast atom bombardment tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 2, 256-257. (28) Witiak, D. T., Abood, N. A., Goswami, S., and Milo, G. E. (1986) The 5-, 6-, and 10-fluoro-1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracenes. Comparative acid-catalyzed isomerization to exomethylene tautomers: A 6-fluoro peri effect. J. Org. Chem. 51, 4499-4507. (29) Witiak, D. T., Goswami, S., and Milo, G. E. (1988) Peri fluoro steric effects: Synthesis and comparative acid-catalyzed isomerization of the 8-, 9-, and 11-fluoro-1,2,3,4-tetrahydro-7,12-dimethylbenz[a]anthracenes to exomethylene tautomers. J. Org. Chem. 53, 345-352. (30) Li, K.-M., RamaKrishna, N. V. S., Padmavathi, N. S., Rogan, E. G., and Cavalieri, E. L. (1994) Synthesis and structure determination of the adducts of dibenzo[a,l]pyrene diol epoxides and deoxyadenosine and deoxyguanosine. Polycyclic Aromat. Compd. 6, 207-213. (31) Royer, R. E., Lyle, T. A., Moy, G. G., Daub, G. H., and Vander Jagt, D. L. (1979) Reactivity-selectivity properties of reactions of carcinogenic electrophiles with biomolecules. Kinetics and products of the reaction of benzo[a]pyrenyl-6-methyl cation with nucleosides and deoxynucleosides. J. Org. Chem. 44, 3202-3207. (32) Zhang, X.-M., Bordwell, F. G., Bares, J. E., Cheng, J.-P., and Petri, B. C. (1993) Homolytic bond dissociation energies of the acidic C-H bonds in R-substituted and 10-substituted 9-methylanthracenes and their related radical anions. J. Org. Chem. 58, 3051-3059. (33) Bordwell, F. G., Cheng, J.-P., and Bausch, M. J. (1988) Acidities of radical cations derived from remotely substituted and phenylsubstituted fluorenes. J. Am. Chem. Soc. 110, 2867-2872. (34) Cavalieri, E. L., and Roth, R. (1976) Reaction of methylbenzanthracenes and pyridine by one-electron oxidation. A model for metabolic activation and binding of carcinogenic aromatic hydrocarbons. J. Org. Chem. 41, 2679-2684. (35) Tolbert, L. M., Khanna, R. K., Popp, A. E., Gelbaum, L., and Bottomley, L. A. (1990) Stereoelectronic effects in the deprotonation of arylalkyl radical cations: meso-ethylanthracenes. J. Am. Chem. Soc. 112, 2373-2378. (36) Baciocchi, E., Mattioli, M., and Romano, R. (1991) Anodic oxidation of R-substituted p-xylenes. Electronic and stereoelectronic effects of R-substituents in the deprotonation of alkylaromatic radical cations. J. Org. Chem. 56, 7154-7160. (37) Baciocchi, E., Dalla Cort, A., Eberson, L., Mandolini, L., and Rol, C. (1986) Substituent effects on intramolecular selectivity and free energy relationships in anodic and metal ion oxidations of 5-X1,2,3-trimethylbenzenes. J. Org. Chem. 51, 4544-4548. (38) Schlesener, C. J., Amatore, C., and Kochi, J. K. (1984) Kinetics and mechanism of aromatic oxidative substitutions via electron transfer. Application of Marcus theory to organic processes in the endergonic region. J. Am. Chem. Soc. 106, 3567-3577.

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