Synthesis of Adducts Formed by Iodine Oxidation of Aromatic

Chem. Res. Toxicol. 1998, 11, 1201-1208

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Synthesis of Adducts Formed by Iodine Oxidation of Aromatic Hydrocarbons in the Presence of Deoxyribonucleosides and Nucleobases Aaron A. Hanson, Eleanor G. Rogan, and Ercole L. Cavalieri* Eppley Institute for Research in Cancer and Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805 Received June 4, 1998

Polycyclic aromatic hydrocarbons (PAH) undergo two main pathways of metabolic activation related to the initiation of tumors: one-electron oxidation to give radical cations and monooxygenation to yield bay-region diol epoxides. Synthesis of standard adducts is essential for identifying biologically formed adducts. Until recently, radical cation adducts were synthesized by oxidation of the PAH in an electrochemical apparatus, not readily available in many organic chemistry laboratories. We have developed a convenient and efficient method for synthesizing PAH-nucleoside adducts by using I2 as the oxidant. Adducts of benzo[a]pyrene (BP), dibenzo[a,l]pyrene (DB[a,l]P), and 7,12-dimethylbenz[a]anthracene were synthesized with deoxyguanosine (dG), deoxyadenosine, guanine (Gua), or adenine in either Me2SO or dimethylformamide (DMF) with or without AgClO4. When, for example, the potent carcinogen BP was dissolved in DMF in the presence of 3 equiv of I2, 5 equiv of dG, and 1 equiv of AgClO4, 45% of the BP was converted to BP-6-N7Gua. When BP was placed under the same reaction conditions in the absence of AgClO4, the extent of formation of BP-6-N7Gua decreased to 30%. When the potent carcinogen DB[a,l]P was dissolved in DMF in the presence of 3 equiv of I2, 5 equiv of dG, and 1 equiv of AgClO4, 43% of the DB[a,l]P was converted to DB[a,l]P-10-N7Gua. In the more polar solvent Me2SO under the same reaction conditions, however, the yield of DB[a,l]P-10-N7Gua was only 20%. Synthesis of adducts with the oxidant I2 is more convenient and, in some cases, more efficient than synthesis by electrochemical oxidation. This method simplifies the synthesis of PAH-nucleoside and nucleobase adducts that are essential for studying biologically formed PAH-DNA adducts.

Introduction Polycyclic aromatic hydrocarbons (PAH)1 undergo two main pathways of metabolic activation related to tumor initiation: one-electron oxidation to give radical cations and monooxygenation with formation of bay-region diol epoxides (1, 2). To establish radical cations as key intermediates in metabolic activation of PAH, several approaches have been investigated. Anodic oxidation of PAH, such as benzo[a]pyrene (BP), dibenzo[a,l]pyrene (DB[a,l]P), and 7,12-dimethylbenz[a]anthracene (DMBA), in the presence of nucleosides has been used to generate PAH-nucleoside adducts (3-7). Some of these adducts have been obtained when the covalent binding of BP, DB[a,l]P, or DMBA to DNA is catalyzed by horseradish peroxidase or rat liver microsomal cytochrome P450 in vitro, and in mouse skin and rat mammary gland in vivo (8-12). Chemical synthesis of standard adducts is essential for identifying biologically formed adducts. Until recently, * To whom correspondence should be addressed. Telephone: (402) 559-7237. Fax: (402) 559-8068. E-mail: [email protected]. 1 Abbreviations: Ade, adenine; BP, benzo[a]pyrene; BP-6-N7Gua, 7-(benzo[a]pyren-6-yl)guanine; CAD, collisionally activated decomposition; 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; NOE, nuclear Overhauser effect; PAH, polycyclic aromatic hydrocarbon(s); TFA, trifluoroacetic acid.

radical cation-derived adducts were synthesized by anodic oxidation of the PAH in an electrochemical apparatus (3-7) not readily available in many organic chemistry laboratories. We have developed an alternative, convenient, and efficient method for synthesizing PAH radical cation-nucleoside adducts by using I2 as the oxidant and Me2SO or dimethylformamide (DMF) as the solvent. There is a large body of evidence that PAH are oxidized by I2 to form radical cations that readily undergo nucleophilic substitution. The first experiments in which PAH were oxidized in the presence of such nucleophiles as pyridine or nucleobases were conducted on the silica surface of a thin-layer plate (13, 14). The yields were very poor, and the products were not well identified when the nucleophiles were nucleic acid bases (14). Improvement in yield was obtained in homogeneous reactions in which the nucleophile pyridine was also the solvent (15) or was dissolved in various organic solvents (16). More recently, stable PAH radical cation salt complexes have been obtained in benzene by one-electron oxidation of the PAH with I2 in the presence of AgClO4 (17, 18). These salts are relatively stable and can be stored in an inert atmosphere or reacted with nucleophiles to form PAH-nucleophile adducts. In this article, we report the synthesis of PAHnucleoside adducts obtained by I2 oxidation of PAH in

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1202 Chem. Res. Toxicol., Vol. 11, No. 10, 1998

DMF or Me2SO. This alternative method is more convenient and, in some cases, more efficient than electrochemical synthesis.

Experimental Section Caution: The following chemicals are hazardous and should be handled carefully in accordance with NIH guidelines (19): BP, DB[a,l]P, and DMBA. Materials. BP was obtained from Aldrich (Milwaukee, WI) and DMBA from Eastman (Rochester, NY). BP and DMBA were purified by silica gel column chromatography with 5% CHCl3 in hexane. DB[a,l]P was synthesized in our laboratory according to a published procedure (20). The purity of the three PAH was >99% by HPLC analysis. Deoxyguanosine (dG) and deoxyadenosine (dA) (TCI, Portland, OR) were desiccated over P2O5 under vacuum at 110 °C for 48 h prior to use. Adenine (Ade) and guanine (Gua) (P-L Biochemicals, Milwaukee, WI), anhydrous DMF, Me2SO and I2 (Aldrich), HPLC-grade organic solvents (EM Science, Gibbstown, NJ), AgClO4 (GFS Chemicals, Columbus, OH), and Na2S2O3 (Fisher Scientific, Fair Lawn, NJ) were used as obtained. HPLC. Analytical, semipreparative, and preparative HPLC was conducted on a Waters 600E solvent delivery system. Elutants were monitored for UV absorbance (254 nm) with a Waters 990 photodiode array detector. Runs were conducted on a YMC (YMC, Overland Park, KS) ODS-AQ 5 µm 120 Å column (4.6 mm × 250 mm, analytical; 10 mm × 250 mm, semipreparative) at 1 mL/min for analytical runs and 4 mL/ min for semipreparative runs. Preparative HPLC was conducted on a YMC ODS-AQ 5 µm 120 Å column (20 mm × 250 mm) at a flow rate of 8 mL/min. NMR. Proton, homonuclear two-dimensional chemical shift correlation spectroscopy (COSY) and nuclear Overhauser effect (NOE) NMR spectra were recorded on a Varian Unity 500 spectrometer at 499.835 MHz in Me2SO-d6 (2.49 ppm) at 26 °C. Mass Spectrometry. Fast atom bombardment tandem mass spectrometry (FAB MS/MS) was performed at the Nebraska Center for Mass Spectrometry (University of Nebraskas Lincoln, Lincoln, NE) using a Micromass (Manchester, England) AutoSpec high-resolution magnetic sector mass spectrometer. The instrument was equipped with an orthogonal acceleration time-of-flight serving as the second mass spectrometer in the tandem experiment. Xenon was admitted to the collision cell at a level to attenuate the precursor ion signal by 75%. Data acquisition and processing were accomplished using OPUS software that was provided by the manufacturer (Microcasm). Samples were dissolved in 5-10 µL of CH3OH; 1 µL aliquots were placed on the probe tip along with 1 µL of a 1:1 glycerol/ thioglycerol mixture. Chemical Synthesis of Adducts. Glassware, syringes, and needles were dried at 150 °C prior to use. The reaction glassware was assembled while hot, cooled under vacuum, and pressurized under an inert atmosphere (argon or nitrogen). Syringes and needles were cooled under vacuum in a desiccator and pressurized under an inert atmosphere. Coupling between PAH and the nucleophiles was accomplished by combining 59 µmol of PAH with 5 equiv of dG, dA, Ade, or Gua dissolved in 3.5 mL of Me2SO or DMF in a 50 mL three-necked flask. Attached to the three-necked flask were a gas/vacuum line adapter, an addition funnel topped with a rubber septum, and a rubber septum. Three equivalents of I2 and 1 equiv of AgClO4 were weighed in separate 20 mL glass vials, covered with rubber septa, put under vacuum, pressurized under an inert atmosphere, and dissolved in Me2SO or DMF. To keep total solvent volumes the same in reactions with or without AgClO4, the amount of solvent used to dissolve I2 was varied. When a reaction mixture included AgClO4, 1 mL of solvent was used to dissolve I2 and 1.5 mL to dissolve AgClO4, whereas 2.5 mL of solvent was used to dissolve I2 when AgClO4 was absent. To the vigorously stirred PAH/deoxyribonucleoside or nucleobase mixture was slowly added I2 through the septum

Hanson et al. Table 1. Analytical, Semipreparative, and Preparative HPLC Gradients for the Separation of Adductsa BP or DB[a,l]P-dG analytical and preparative

DMBA-dG analytical

timeb

CH3OH

time

CH3CN

0-5 40

50%c

0-5 70

30% 100%

100%

DMBA-dG preparative

BP-Ade/dA analytical

time

CH3CN

time

C2H5OH/CH3CNd

0-5 70

20% 100%

0-15 40

50% 100%

BP-Ade/dA preparative time 0-10 40

C2H5OH/CH3

CNd

50% 100%

DB[a,l]P-Ade/dA preparative

DB[a,l]P-Ade/dA analytical time

C2H5OH/CH3CNd

0-8 40

60% 100%

DMBA-Ade/dA semipreparative

time

C2H5OH/CH3CNd

time

C2H5OH/CH3CNd

0-10 40

55% 100%

0-15 30-40 50

30% 70% 100%

a All gradients were linear, except for the DMBA-dG analytical gradient in which a convex (CV5) gradient was used. b Time is in minutes. c The organic phase starts as a mixture with water. d The C2H5OH/CH3CN organic phase is a 3:1 mixture.

with a syringe and 19 gauge needle. AgClO4 was added to the addition funnel through the rubber septum. The I2/PAH/ deoxyribonucleoside or I2/PAH/nucleobase mixture was allowed to react for 1.5 h before AgClO4 was added dropwise from the addition funnel. Once all the compounds were combined, the reaction was allowed to proceed for an additional 16.5 h and then quenched with 0.1 M Na2S2O3. The reaction mixture was then dried under vacuum at 50 °C, redissolved in 4 mL of Me2SO/CH3OH (1:1), and filtered through a 0.45 µm filter. The filter was then washed with an additional 500 µL of Me2SO. Determination of the yield of the synthesized adducts was accomplished by analytical or semipreparative HPLC, in either the CH3CN/H2O, CH3OH/H2O, or C2H5OH/CH3CN/H2O solvent system (Table 1), with monitoring at 254 nm for comparison of peak areas of the adduct(s), byproduct(s), and parent compound. The percentage of each adduct in Tables 2-4 was calculated by dividing the peak area of the adduct by the sum of the peak areas of the adduct(s), byproduct(s), and parent compound. Purification of the adducts was conducted by preparative or semipreparative HPLC, as described in Table 1. Reactions conducted with Ade were analyzed in a C2H5OH/ CH3CN/H2O solvent system due to the problems encountered in the isolation of PAH-N1Ade adducts in either the CH3CN/ H2O or CH3OH/H2O solvent system. A typical HPLC separation of BP-Ade adducts is shown in Figure 1. The CH3CN/H2O gradient in Figure 1A allowed separation of BP-6-N7Ade and BP-6-N3Ade, but gave only a broad peak for BP-6-N1Ade. Use of a C2H5OH/CH3CN/H2O gradient, however, enabled separation of all three adducts (Figure 1B). Known adducts were identified by comparing analytical HPLC retention time, UV, and NMR spectra to those previously obtained. Novel adducts were identified by UV spectra, NMR spectra (including one-dimensional NOE, D2O exchange, and COSY), and FAB MS/MS. 7-MBA-12-CH2-N1Ade. 1H NMR (ppm): δ 3.15 (s, 3H, 7-CH3), 6.44 (s, 2H, 12-CH2), 7.45 (m, 1H, 2-H), 7.59 (m, 1H, 10-H), 7.66 (m, 2H, 3-H, 9-H), 7.69 (s, 1H, 2-H[Ade]), 7.80 (d, J ) 9.0 Hz, 1H, 5-H), 7.92 (s, 1H, 8-H[Ade]), 7.93 (m, 3H, 1-H, 11-H, 6-NH[Ade]), 8.01 (d, J ) 6.5 Hz, 1H, 4-H), 8.07 (bs, 1H, 6-NH[Ade]), 8.24 (d, J ) 9.5 Hz, 1H, 6-H), 8.53 (d, J ) 8.0 Hz, 1H, 8-H). UV: λmax 266, 287, 297, 351, 367, 384 nm. FAB MS [M + H]+: calcd for C25H19N5 m/z 390.1719, found m/z 390.1716.

PAH-Nucleoside Adduct Synthesis by I2 Oxidation

Chem. Res. Toxicol., Vol. 11, No. 10, 1998 1203 7.93 (m, 1H, 3-H), 7.99 (s, 1H, 8-H[Gua]), 8.23 (d, J ) 9.0 Hz, 1H, 6-H), 8.44 (m, 1H, 11-H), 8.52 (m, 2H, 1-H, 8-H), 10.15 (bs, 1H, 1-NH[Gua]). UV: λmax 264, 286, 296, 349, 364, 380 nm. FAB MS [M + H]+: calcd for C30H27N5O4 m/z 522.2141, found m/z 522.2131.

Results and Discussion

Figure 1. HPLC separation of adducts formed by I2 oxidation of BP in the presence of Ade and AgClO4. (A) The HPLC column was eluted with 40% CH3CN in H2O for 15 min, followed by a 15 min linear gradient to 60% CH3CN in H2O, at which the mixture was held for 10 min, followed by a 10 min linear gradient to 100% CH3CN. (B) The HPLC column was eluted with 65% C2H5OH/CH3CN (3:1) in H2O for 15 min, followed by a 25 min linear gradient to 100% C2H5OH/CH3CN (3:1). 12-MBA-7-CH2-N1Ade. 1H NMR (ppm): δ 3.39 (s, 3H, 12CH3), 6.52 (s, 2H, 7-CH2), 7.54 (s, 1H, 2-H[Ade]), 7.65 (m, 3H, 3-H, 4-H, 9-H), 7.72 (m, 1H, 2-H), 7.75 (d, J ) 9.5 Hz, 1H, 5-H), 7.81 (bs, 1H, 6-NH[Ade]), 7.94 (m, 2H, 10-H, 6-NH[Ade]), 7.95 (s, 1H, 8-H[Ade]), 8.25 (d, J ) 9.5 Hz, 1H, 6-H), 8.48 (m, 2H, 1-H, 11-H), 8.54 (m, 1H, 8-H). UV: λmax 266, 288, 298, 352, 367, 384 nm. FAB MS [M + H]+: calcd for C25H19N5 m/z 390.1719, found m/z 390.1729. 7-MBA-12-CH2-N3Ade. 1H NMR (ppm): δ 3.14 (s, 3H, 7-CH3), 6.22 (s, 2H, 12-CH2), 7.32 (bs, 2H, 6-NH2[Ade]), 7.42 (m, 1H, 2-H), 7.56 (s, 1H, 2-H[Ade]), 7.59 (m, 1H, 10-H), 7.66 (m, 2H, 3-H, 9-H), 7.78 (d, J ) 10.0 Hz, 1H, 5-H), 7.97 (m, 3H, 1-H, 4-H, 11-H), 8.23 (d, J ) 9.0 Hz, 1H, 6-H), 8.29 (s, 1H, 8-H[Ade]), 8.51 (d, J ) 9.0 Hz, 1H, 8-H). UV: λmax 266, 286, 297, 347, 364, 379 nm. FAB MS [M + H]+: calcd for C25H19N5 m/z 390.1719, found m/z 390.1708. 12-MBA-7-CH2-N3Ade. 1H NMR (ppm): δ 3.39 (s, 3H, 12CH3), 6.28 (s, 2H, 7-CH2), 7.23 (bs, 1H, 6-NH[Ade]), 7.48 (s, 1H, 2-H[Ade]), 7.65 (m, 2H, 2-H, 3-H), 7.70 (m, 2H, 9-H, 10-H), 7.76 (d, J ) 9.0 Hz, 1H, 5-H), 7.94 (m, 1H, 4-H), 8.32 (s, 1H, 8-H[Ade]), 8.33 (d, J ) 8.5 Hz, 1H, 6-H), 8.46 (d, J ) 8.5 Hz, 1H, 11-H), 8.53 (m, 1H, 1-H), 8.57 (d, J ) 8.0 Hz, 1H, 8-H). UV: λmax 266, 285, 295, 348, 363, 379 nm. FAB MS [M + H]+: calcd for C25H19N5 m/z 390.1719, found m/z 390.1708. 7-MBA-12-CH2-N2dG. 1H NMR (ppm): δ 2.22 (m, 1H, 2′H), 2.70 (m, 1H, 2′-H), 3.10 (s, 3H, 7-CH3), 3.49 (m, 1H, 5′-H), 3.55 (m, 1H, 5′-H), 3.78 (m, 1H, 4′-H), 4.34 (m, 1H, 3′-H), 4.82 (m, 1H, 5′-OH), 5.20 (d, J ) 3.5 Hz, 1H, 3′-OH), 5.30 (m, 2H, 12-CH2), 6.19 (t, J ) 7.0 Hz, 1H, 1′-H), 7.48 (bs, 1H, 2-NH[Gua]), 7.58 (m, 1H, 9-H), 7.66 (m, 1H, 4-H), 7.73 (m, 3-H, 2-H, 5-H, 10-H), 7.99 (s, 1H, 8-H[Gua]), 8.16 (d, J ) 10.0 Hz, 1H, 11-H), 8.34 (d, J ) 9.0 Hz, 1H, 6-H), 8.48 (dd, J1,2 ) 9.0 Hz, J8,9 ) 10.0 Hz, 2H, 1-H, 8-H), 10.27 (bs, 1H, 1-NH[Gua]). UV: λmax 264, 287, 297, 349, 364, 380 nm. FAB MS [M + H]+: calcd for C30H27N5O4 m/z 522.2141, found m/z 522.2141. 12-MBA-7-CH2-N2dG. 1H NMR (ppm): δ 2.33 (m, 1H, 2′H), 2.69 (m, 1H, 2′-H), 3.33 (s, 3H, 12-CH3), 3.58 (m, 1H, 5′-H), 3.63 (m, 1H, 5′-H), 3.89 (s, 1H, 4′-H), 4.44 (s, 1H, 3′-H), 4.97 (bs, 1H, 5′-OH), 5.40 (s, 1H, 3′-OH), 5.43 (s, 2H, 7-CH2), 6.38 (t, J ) 6.75 Hz, 1H, 1′-H), 6.92 (bs, 1H, 2-NH[Gua]), 7.64 (m, 2H, 2-H, 4-H), 7.73 (m, 2H, 9-H, 10-H), 7.78 (d, J ) 9.5 Hz, 1H, 5-H),

Optimization of Reaction Conditions. Reactions were conducted to determine whether one-electron oxidation by I2 would produce adequate yields of PAHnucleoside adducts. To optimize the yield of PAHnucleoside adducts, 59 µmol (15 mg) of BP was added to varying equivalents (equiv) of dG, I2, and AgClO4 in 6 mL of DMF at room temperature for 18 h. The best ratio was found to be 1:5:3:1 PAH:dG:I2:AgClO4. Smaller amounts of I2 resulted in low yields ( N-3. Radical cations of the unsubstituted PAH BP and DB[a,l]P react with Ade to form adducts mostly at N-1, followed by N-3 and then by N-7 (Scheme 1 and Tables 2 and 3). This suggests that the reactivity of these nucleophilic groups with the unsubstituted BP and DB[a,l]P radical cations is affected by factors other than their nucleophilicity. Steric accessibility strongly influences the reactivity of methanediazonium ion, as theoretically determined by Kim et al. (24). Steric interference at N-7 in Ade arises through repulsion between the attacking methanediazonium ion and the exocyclic amino group. Thus, the steric interference at N-7, determined by the adjacent 6-amino group, renders the N-7 less reactive with the unsubstituted radical cations than is N-3. For the methyl-substituted DMBA, the reactivity is directly related to the nucleophilicity, with N-1 > N-7 > N-3 (Scheme 2 and Table 4). This suggests that the benzylic carbenium ion, where the electrophilicity is localized (7), is less repelled by the exocyclic amino group than the unsubstituted PAH radical cations, in which the

Acknowledgment. We gratefully thank Dr. Ronald Cerny at the Nebraska Center for Mass Spectrometry at the University of NebraskasLincoln for the valuable mass spectral data. A.A.H. was supported by predoctoral fellowships from the University of Nebraska Environmental Toxicology Graduate Program and the University of Nebraska Medical Center. This research was supported by U.S. Public Health Service grants from the National Cancer Institute (P01 CA49210 and R01 CA49917). Core support at the Eppley Institute was funded by NCI Laboratory Cancer Research Center Support (Core) Grant CA36727.

References (1) 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. (2) Cavalieri, E. L., and Rogan, E. G. (1998) Mechanisms of tumor initiation by polycyclic aromatic hydrocarbons in mammals. In The Handbook of Environmental Chemistry: PAHs and Related Compounds (Neilson, A. H., Ed.) Vol. 3J, pp 81-117, Springer, Heidelberg, Germany. (3) 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. (4) 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. (5) 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. (6) 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. (7) Mulder, P. P. J., Chen, L., Sekhar, B. C., George, M., Gross, M. L., Rogan, E. G., and Cavalieri, E. L. (1996) 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. Chem. Res. Toxicol. 9, 1264-1277. (8) 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

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(9)

(10)

(11)

(12)

(13) (14) (15)

(16)

and quantitation of benzo[a]pyrene-DNA adducts formed in mouse skin. Chem. Res. Toxicol. 6, 356-363. 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. 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. 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. Todorovic, R., Ariese, F., Devanesan, P., Jankowiak, R., Small, G. J., Rogan, E., and Cavalieri, E. (1997) Determination of benzo[a]pyrene- and 7,12-dimethylbenz[a]anthracene-DNA adducts formed in rat mammary glands. Chem. Res. Toxicol. 10, 941947. Rochlitz, J. (1967) Neue Reaktionen der carcinogenen Kohlenwasserstoffe. II. Tetrahedron 23, 3043-3048. Wilk, M., and Girke, W. (1972) Reactions between benzo[a]pyrene and nucleobases by one-electron oxidation. J. Natl. Cancer Inst. 49, 1585-1597. Cavalieri, E., 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. Johnson, M. D., and Calvin, M. (1973) Induced nucleophilic substitution in benzo[a]pyrene. Nature 241, 271-272.

Hanson et al. (17) Cremonesi, P., Stack, D. E., Rogan, E. G., and Cavalieri, E. L. (1994) Radical cations of benzo[a]pyrene and 6-substituted derivatives: Synthesis and reaction with nucleophiles. J. Org. Chem. 59, 7683-7687. (18) Stack, D. E., Cremonesi, P., Hanson, A., Rogan, E. G., and Cavalieri, E. L. (1995) Radical cations of benzo[a]pyrene and 6-substituted derivatives: Reaction with nucleophiles and DNA. Xenobiotica 25, 755-760. (19) National Institutes of Health (1981) NIH Guidelines for the Laboratory Use of Chemical Carcinogens, NIH Publication 812385, U.S. Government Printing Office, Washington, DC. (20) Masuda, Y., and Kagawa, R. (1972) A novel synthesis and carcinogenicity of dibenzo[a,l]pyrene. Chem. Pharm. Bull. 20, 2736-2737. (21) Chen, L., Devanesan, P. D., Byun, J., Gooden, J. K., Gross, M. L., Rogan, E. G., and Cavalieri, E. L. (1997) Synthesis of depurinating DNA adducts formed by one-electron oxidation of 7H-dibenzo[c,g]carbazole and identification of these adducts after activation with rat liver microsomes. Chem. Res. Toxicol. 10, 225233. (22) Dolnikowski, G. G., Cavalieri, E. L., and Gross, M. 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, 256-258. (23) Pullman, A., and Pullman, B. (1981) Molecular electrostatic potential of nucleic acids. Q. Rev. Biophys. 14, 289-380. (24) Kim, H. S., Yu, M., Jiang, Q., and LeBreton, P. (1993) UV photoelectron and ab initio quantum mechanical characterization of 2′-deoxyguanosine 5′-phosphate: Electronic influences on DNA alkylation patterns. J. Am. Chem. Soc. 115, 6169-6183.

TX980127Q