GuanineâAdenine DNA Cross-Linking by 1,2,3,4-Diepoxybutane

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Guanine-Adenine DNA Cross-Linking by 1,2,3,4-Diepoxybutane: Potential Basis for Biological Activity Soobong Park, Jacob Hodge, Christopher Anderson, and Natalia Tretyakova* Department of Medicinal Chemistry, University of Minnesota Cancer Center, Mayo Mail Code 806, Room 760 E CCRB, 420 Delaware Street SE, Minneapolis, Minnesota 55455 Received July 2, 2004

1,2,3,4-Diepoxybutane (DEB) is a prominent carcinogenic metabolite of 1,3-butadiene (1,3BD), an important industrial chemical and an environmental pollutant found in cigarette smoke and automobile exhaust. DEB is capable of inducing a variety of genotoxic effects, including point mutations, large deletions, and chromosomal aberrations. The mutagenicity and carcinogenicity of DEB are thought to result from its ability to form bifunctional DNA-DNA adducts by sequentially alkylating two nucleobases within the DNA double helix. We recently reported that DEB-induced DNA-DNA cross-linking leads to the formation of 1,4-bis-(guan7-yl)-2,3-butanediol (bis-N7G-BD) adducts [Park, S., and Tretyakova, N. (2004) Structural characterization of the major DNA-DNA cross-link of 1,2,3,4-diepoxybutane. Chem. Res. Toxicol. 17 (2), 129-136]. However, guanine-guanine cross-linking by DEB cannot explain the development of A:T base pair mutations following exposure to DEB and 1,3-BD. In the present work, four asymmetrical DNA-DNA cross-links involving both adenine and guanine nucleobases were identified in double-stranded DNA treated with racemic DEB. These novel lesions were assigned the structures of 1-(aden-1-yl)-4-(guan-7-yl)-2,3-butanediol (N1A-N7GBD), 1-(aden-3-yl)-4-(guan-7-yl)-2,3-butanediol (N3A-N7G-BD), 1-(aden-7-yl)-4-(guan-7-yl)-2,3butanediol (N7A-N7G-BD), and 1-(aden-N6-yl)-4-(guan-7-yl)-2,3-butanediol (N6A-N7G-BD), based on the comparison of their MS/MS spectra, HPLC retention times, and UV spectra with those of the corresponding authentic standards prepared independently. Although guanineadenine lesions are ∼10 times less abundant in DEB-treated double-stranded DNA than the corresponding bis-N7G cross-links, N1A-N7G-BD and N6A-N7G-BD are more hydrolytically stable and, if formed in vivo, may accumulate in target tissues. HPLC-ESI-MS/MS analysis of guanine-adenine DEB cross-links induced in synthetic DNA duplexes 5′-(GGT)5, 5′-(GT)7G, and 5′-(GAA)5 (+-strand) demonstrate that G-A cross-linking by DEB produces primarily 1,3interstrand N1A-N7G lesions. The formation of bifunctional guanine-adenine adducts is likely to contribute to AT base pair substitutions and deletion mutations following DEB exposure.

Introduction (BD)1

1,3-Butadiene is an important industrial chemical classified as a human carcinogen based on the increased cancer risk in occupationally exposed humans and its strong tumorigenic effects in laboratory animals (1, 2). Metabolic activation of BD to DNA reactive metabolites involves two epoxidation steps mediated by cytochrome P450 enzymes. The first oxidation catalyzed by CYP2E1 and CYP2A6 yields (R)- and (S)-3,4-epoxy1-butene (EB) (3, 4). EB can be hydrolyzed to 1-butene3,4-diol or undergo a second oxidation to yield D,L- and meso-1,2,3,4-diepoxybutane (DEB) (3, 5). Experimental evidence suggests that DEB is responsible for many of the adverse biological effects of BD. DEB is 2 orders of magnitude more genotoxic and mutagenic than its monoepoxide analogues, EB and 3,4-epoxy-1,2-butanediol (6, * To whom correspondence should be addressed. Tel: 612-626-3432. Fax: 612-626-5135. E-mail: [email protected]. 1 Abbreviations: BD, 1,3-butadiene; DEB, 1,2,3,4-diepoxybutane; EB, 3,4-epoxy-1-butene; HMBC, heteronuclear multiple bond correlation; gHMBC, gradient heteronuclear multiple bond correlation; N1AN7G-BD, 1-(aden-1-yl)-4-(guan-7-yl)-2,3-butanediol (1); N3A-N7G-BD, 1-(aden-3-yl)-4-(guan-7-yl)-2,3-butanediol (2); N7A-N7G-BD, 1-(aden7-yl)-4-(guan-7-yl)-2,3-butanediol (3); N6A-N7G-BD, 1-(aden-N6-yl)-4(guan-7-yl)-2,3-butanediol (4).

7). Efficient metabolic activation of BD to DEB in mice is considered the basis for the extreme sensitivity of this species to BD carcinogenesis (5). The potent genotoxic activity of DEB is likely a result of its bifunctional nature, giving rise to DNA-DNA and DNA-protein cross-links (Scheme 1). Indeed, the formation of bifunctional DNA lesions correlates with the cytotoxicity and genotoxicity of DEB (8). The only bifunctional DNA-DNA lesion of DEB characterized so far is the bis-N7-guanine adduct first discovered by Brookes and Lawley over 30 years ago (9). We have recently completed a detailed structural study of DEB-induced guanine-guanine cross-links, which were identified as 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) (10). However, guanine-guanine cross-linking cannot explain the observation of A f T transversion mutations in DEBtreated human cells (11). In the present study, we report the first synthesis and structural characterization of the four regioisomeric guanine-adenine DEB conjugates and demonstrate their formation in double-stranded DNA following incubation with racemic DEB. Furthermore, sequence preferences for the formation of regioisomeric guanine-adenine cross-links of DEB are investigated using synthetic DNA duplexes of defined sequence.

10.1021/tx0498206 CCC: $27.50 © 2004 American Chemical Society Published on Web 11/17/2004

DNA Cross-Linking by 1,2,3,4-Diepoxybutane

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Scheme 1. Formation of Bifunctional DNA Adducts of DEB [Adapted from Rajski and Williams (1998) Chem. Rev. 98, 2723]

Materials and Methods Chemicals and Equipment. D,L-DEB, 1,4-dichlorobutene, adenine, adenosine, osmium tetroxide, 6-chloropurine riboside, and calf thymus (CT) DNA were purchased from Sigma-Aldrich (Milwaukee, WI; St. Louis, MO). N2-Acetylguanine was obtained from TCI (Chicago, IL). DNA oligodeoxynucleotides were prepared by standard phosphoramidite chemistry using a DNA synthesizer at the University of Minnesota Microchemical Facility. N2-Acetyl-N7-(4-chloro-2-butenyl)guanine (5). Compound 5 was prepared according to the procedure of Hua et al. (12). White solid; yield, 35%; mp 34-35 °C (lit. 34-35 °C). 1H NMR (DMSO-d6): δ 2.17 (s, 3H, CH3CO), 4.19 (d, J ) 8.4, 2H, CH2), 4.95 (d, J ) 6.6, 2H, CH2), 5.73 (m, 1H, dCH), 6.09 (m, 1H, dCH), 8.16 (s, 1H, CH), 11.57 (s, 1H, NH), 12.11 (s, 1H, NH). ESI+-MS: m/z 282.5 [M + H]+. N2-Acetyl-N7-(4-azo-2-butenyl)guanine (6). To a solution 5 (71.3 mg, 0.253 mmol) in DMSO (4 mL) was added sodium azide (92.5 mg, 1.43 mmol), followed by stirring for 4 h at 50 °C. The solvent was removed in vacuo, and the residue was suspended in CHCl3 (20 mL) and filtered. The filtrate was evaporated, and the residue was purified by preparatory TLC eluting with CHCl3:MeOH ) 10:1 to give 73 mg (99% yield) of white solid. 1H NMR (DMSO-d6): δ 2.16 (s, 3H, CH3CO), 4.91 (d, J ) 6.0, 4H, 2 × CH2), 5.89-5.96 (m, 1H, dCH), 5.99-6.06 (m, 1H, dCH), 7.73 (s, 1H, CH), 7.86 (bs, 2H, NH2), 8.09 (s, 1H, CH), 8.29 (s, 1H, CH), 11.15 (ss, 2H, 2 × NH). ESI-MS obsd, m/z ) 289.2; calcd for C16H18N10O4, m/z ) 289.3 (M + H)+. ESI+MS/MS: m/z 289.2 f 194 [Ac-Gua + H] +, 219 [Ac-Gua CHdCH] +, 152 [Gua + H]+. N2-Acetyl-N7-(4-azo-2,3-dihydroxy)guanine (7). To a solution of 6 (70 mg, 0.243 mmol) in THF (4 mL) were added OsO4 (2.5 mL, 4% sol in H2O) and 4-methylmorpoline-N-oxide (287 mg, 2.45 mmol). The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was concentrated, and methanol (1 mL) was added. Following centrifugation, the supernatant was purified by preparatory TLC eluting with

CHCl3:MeOH ) 10:1. The compound was extracted from the excised silica (300 mL, 5:1 CHCl3:MeOH) to give 55.8 mg (71.5%) of compound 7. ESI+-MS obsd, m/z ) 323.2; calcd for C16H18N10O4, m/z ) 323.3 (M + H)+. ESI+-MS/MS: m/z 323 f 194 [Ac-Gua + H] +, 236 [Ac-Gua - CHdCHOH + H] +, 152 [Gua + H]+. N2-Acetyl-N7-(4-amino-2,3-dihydroxy)guanine (8). A solution of compound 7 (50 mg, 0.155 mmol) in MeOH (15 mL) was stirred under a hydrogen atmosphere (38 PSI) for 4 h in the presence of catalytic 10% palladium on charcoal (17.6 mg) until no starting compound was visible by TLC (10:1 CHCl3:MeOH). The reaction mixture was filtered through Celite, and the filtrate was concentrated in vacuo to give 45 mg (99%) of 8 as a colorless oil, which was used in the next step without further purification. The presence of 8 was confirmed by ESI+MS: obsd, m/z ) 297.2; calcd for C16H18N10O4, m/z ) 297.3 (M + H)+. ESI+-MS/MS: m/z 297.0 f 194 [Ac-Gua + H]+. 1-(N-9-Ribosyl-aden-N6-yl)-4-(N-2-acetyl-guan-7-yl)-2,3butanediol (9). 6-Choropurine riboside (149 mg, 0.519 mmol), 8 (40 mg, 0.135 mmol), and DIEPA (33 µL) were mixed in DMF (3 mL) and stirred at 60 °C for 30 h. The volatile material was removed in vacuo, and the residue was used in the next step without further purification. The presence of 9 in the solid residue was confirmed by MS. ESI-MS obsd, m/z ) 547.2; calcd for C16H18N10O4 m/z 547.5 (M + H)+. ESI+-MS/MS: m/z 547.2 f 415.1 [M - R + 2H]+. 1-(Aden-N6-yl)-4-(guan-7-yl)-2,3-butanediol (4). The crude 9 from the previous reaction was suspended in 1 N HCl (6 mL) and heated at 80-90 °C for 5 h. The solution was neutralized with NH4OH, and the precipitated brown solid was isolated by centrifugation and washed with water. The residue was suspended in formic acid solution (0.4% in H2O, 6 mL) and purified by HPLC on a Supelcosil LC C18 DB semipreparative column (10 mm × 250 mm, flow rate of 3 mL/min) with a gradient of (A) 150 mM ammonium acetate and (B) acetonitrile (from 0 to 10% B over the course of 6 min and from 10 to 20% B over the course of 20 min). Compound 4 eluted at 16.0 min. HRMS (Q-TOF) obsd, m/z ) 373.1465; calcd for C14H17N10O3, m/z ) 373.1480 (M + H)+. ESI+-MS/MS: m/z 373 f 356 [M + H -

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H2O]+, 222 [M + H - Gua]+, 152 [Gua + H]+. Estimated total yield, 4% (18% from compound 8). 1-(N-9-Ribosyl-aden-1-yl)-4-(N2-acetyl-guan-7-yl)-2-butene (10). To a stirred solution of 5 (109 mg, 0.387 mmol) in DMF (4 mL) was added adenosine (78.1 mg, 0.578 mmol), and it was heated at 125 °C for 18 h. After the solution was cooled to 5 °C in an ice bath, the reaction mixture was diluted with ethyl acetate (3 mL) and stirred for 30 min to allow the precipitation of the solid. The suspension was centrifugated, and the supernatant was removed. The yellowish precipitate was washed with ethyl acetate (5 mL), dried under N2, and used in the next step without further purification. The presence of 10 in the precipitate was determined by mass spectrometry. ESI+MS obsd, m/z ) 513.2; calcd for C21H24N10O6, m/z ) 513.5 (M+). ESI+-MS/MS: m/z 513.2 f 381 [M - R]+, 246 [M - R-Ade]+, 188 [M - R - Ac-Gua]+. 1-(N-9-Ribosyl-aden-1-yl)-4-(N2-acetyl-guan-7-yl)-2,3-butanediol (10a). To a solution of crude 10 (122 mg, 0.238 mmol) in THF (10 mL) were added OsO4 (2.5 mL, 4% sol in H2O) and 4-methylmorpoline-N-oxide (137 mg, 1.17 mmol). The resulting cloudy mixture was stirred at room temperature for 18 h. The volatile solvent was evaporated, and the residue was diluted with 20 mL of water and washed with CHCl3 (100 mL). The aqueous layer was evaporated and used in the next step without further purification. The presence of 10a in the precipitate was confirmed by MS. ESI+-MS obsd, m/z ) 547.2; calcd for C21H24N10O6, m/z ) 547.5 (M)+. ESI+-MS/MS: m/z 547.2 f 415 [M - R + H]+, 398 [M - R - H2O + 2H]+, 262 [Ac-Gua - CH2CHOH-CHdCH]+. 1-(Aden-1-yl)-4-(guan-7-yl)-2,3-butanediol (1). Compound 10a was suspended in 1 N HCl (6 mL) and heated at 80-90 °C for 5 h. The solution was then neutralized with NH4OH and evaporated in vacuo. The residue was suspended in formic acid (0.4% in H2O, 6 mL) and purified by HPLC on a Supelcosil LC C18 DB semipreparative column (10 mm × 250 mm; flow rate, 3 mL/min) with a gradient of (A) 20 mM ammonium acetate, pH 3.8, and (B) acetonitrile (from 0 to 6% B over the course of 6 min and from 6 to 10% B over the course of 20 min). The HPLC retention time of 1 was 15.1 min at these conditions. HRMS (Q-TOF) obsd, m/z ) 373.1478; calcd for C14H17N10O3, m/z ) 373.1480 (M + H)+. ESI+-MS/MS: m/z 373.2 f 356.1 [M + H - H2O]+, 265.0, 220 [M + H - Ade - H2O]+, 151.9 [Gua + H]+. Estimated yield, 2% (4% from compound 10). 1-(Aden-3-yl)-4-(N2-acetyl-guan-7-yl)-2-butene (11). To a stirred solution of 5 (109 mg, 0.387 mmol) in DMF (4 mL) was added adenine (78.1 mg, 0.578 mmol), and the solution was heated at 125 °C for 18 h. After it was cooled to 5 °C in an ice bath, the reaction mixture was diluted with ethyl acetate (3 mL) and stirred for 30 min to give yellowish solid precipitation. The suspension was centrifuged, and the supernatant was removed. The precipitate was washed with ethyl acetate (5 mL) and dried under N2. The residue was used in the next step without further purification. The presence of the desired product 11 in the precipitate was confirmed by ESI+-MS: obsd, m/z 381.2; calcd for C16H16N10O2, m/z ) 381.4 (M + H)+. ESI+-MS/MS: m/z 381 f 246 [M + H - Ade]+, 188 [M + H - Ac - Gua]+. 1-(Aden-3-yl)-4-(N2-acetyl-guan-7-yl)-2,3-butanediol (11a). To a solution of crude 11 (122 mg, 0.321 mmol) in THF (10 mL) were added OsO4 (2.5 mL, 4% sol in H2O) and 4-methylmorpoline-N-oxide (137 mg, 1.17 mmol). The resulting cloudy mixture was stirred at room temperature for 18 h. A 1 N HCl solution (1 mL) was added to the reaction mixture and then stirred at room temperature for an additional 5 h. The reaction mixture was neutralized with 1 N NaOH. The solid was filtered, washed with H2O (75 mL), and dried under N2. The resulting brown solid was used in the next step without further purification. The presence of the desired product 11a in the precipitate was confirmed by MS. ESI+-MS obsd, m/z ) 415.2; calcd for C16H18N10O4, m/z ) 415.4 (M + H)+. ESI+-MS/MS: m/z 415.2 f 397 [M + H - H2O]+, 373 [M + 2H - Ac]+, 222 [M + H - Ac - Gua]+.

Park et al. 1-(Aden-3-yl)-4-(guan-7-yl)-2,3-butanediol (2). Compound 11a was suspended in 1 N HCl (6 mL) and heated at 80-90 °C for 5 h. The solution was then neutralized with NH4OH and evaporated in vacuo. The residue was suspended in formic acid (0.4% in H2O, 6 mL) and purified by HPLC on a Supelcosil LC C18 DB semiprep column (10 mm × 250 mm, flow rate of 3 mL/ min) with a gradient of (A) 20 mM ammonium acetate (pH adjusted to 2 with formic acid) and (B) acetonitrile (from 0 to 6% B over the course of 6 min and from 6 to 10% B over the course of 20 min) by injection of the supernatant. The HPLC retention time of 2 under these conditions was 17.6 min. HRMS (Q-TOF) obsd, m/z ) 373.1466; calcd for C14H17N10O3, m/z ) 373.1480 (M + H)+. ESI+-MS/MS: m/z 373.2 f 356.1 [M + H - H2O]+, 238 [M + H - Ade]+, 222 [M + H - Gua]+, 152 [Gua + H]+. Estimated yield, 10% (12.5% from 11). N3-Benzyladenine. To a stirred solution of adenine (3.0 g) in 15 mL of DMF was added benzyl bromide (1.26 g, 0.00736 mol) dissolved in DMF (0.879 mL). The mixture was heated to 60 °C for 20 h. After the mixture was cooled, a precipitate formed with the addition of a copious amount of ethyl acetate. The solid was then filtered and washed with ethyl acetate (2 × 40 mL). The mass of the dry solid was 560 mg with a 28.6% yield. ESI+MS obsd, m/z 226.1 (M + H)+; calcd for C13H12N4, 225.11. 1-(3-Benzyl-aden-7-yl)-4-(N 2 -acetyl-guan-7-yl)-2-butene (12). Compound 5 (110 mg) was combined with N3benzyladenine (118 mg) in 10.5 mL of dimethylacetamide and stirred for 20 h at 60 °C. The reaction was continued for 24 additional hours at 80 °C. Dissolved solids were then precipitated by the addition of 50 mL of ethyl acetate. The precipitate was filtered to give 12 (108 mg, 58.8%). ESI+-MS obsd, m/z 471.3; calcd for C23H23N10O2+, 471.20. MS/MS m/z 471.3 f 278.1 [M + H - Ac - Gua]+, 246 [M + 2H - Bz - Ade] +. 1-(3-Benzyl-aden-7-yl)-4-(N2-acetyl-guan-7-yl)-2,3-butenediol (13). Compound 12 (84 mg) was dissolved in 2 mL of H2O and 2.5 mL of THF. To this was added 220 mg of 4-methylmorphomine-N-oxide. After this was stirred for 10 min at room temperature, 1 mL of osmium tetroxide (4% w/v in H2O) was added, forming a brown precipitate. The reaction was continued for 16 h at room temperature. The precipitate containing 13 was then removed by filtration. ESI+-MS obsd, m/z 505.1; calcd for C23H25N10O4+, m/z 505.21. MS/MS m/z 505 f 463 [M + 2H Ac] +, 312 [M + H - Ac - Gua]+. 1-(Aden-7-yl)-4-(N2-acetyl-guan-7-yl)-2,3-butenediol (14). Compound 13 was mixed with 1 mL of toluene and 70 µL of concentrated H2SO4, forming a two phase mixture. The mixture was stirred vigorously for 72 h. The toluene was then removed under nitrogen to yield 14. ESI+-MS obsd, m/z 415.0 [M + H]+; calcd for C16H9N10O4, 415.16 (M + H)+. ESI+-MS/MS: m/z 415 [M + H]+f 373.0 [M + H - Ac]+, 262 [M + H - Ade, H2O]+, 222.1 [M + H - Ac - Gua]+. 1-(Aden-7-yl)-4-(guan-7-yl)-2,3-butenediol (3). The solution of compound 14 in sulfuric acid was heated to 70 °C for 1 h. The reaction mixture was separated by HPLC with a Supelcosil LC-18-DB semipreparative column (10 mm × 250 mm; flow rate, 3 mL/min) using buffers (A) 20 mM ammonium acetate, pH 4.9, and (B) acetonitrile with a gradient of 0-10% B from 0 to 20 min, 10-20% B from 20 to 25 min, and 20-70% B from 25 to 30 min. Compound 3 eluted at 21.3 min. HRMS (Q-TOF) obsd, m/z 373.1488 [M + H]+; calcd for C14H17N10O3, m/z 373.1480 (M + H)+, m/z 373.2 f 222 [M + H - Gua]+, 220 [M + H - Ade - H2O]+, 356.1 [M + H - H2O]+, 204, 152 [Gua + H]+. The estimated total yield was 4% (9.4% from 12). CT DNA Treatment with DEB and Hydrolysis to Release Nucleobase Lesions. CT DNA (1 mg/mL solution in 10 mM Tris-HCl buffer, pH 7.2) was treated with racemic DEB (50.0 mM) at 37 °C for 24 h. The mixture was extracted with diethyl ether (2 × 400 µL) to remove any unreacted DEB and subjected to either neutral thermal or acid hydrolysis. Neutral thermal hydrolysis was performed at 80 °C for 1 h. The DNA backbone was removed by filtration through Microcon YM-30 membrane filters (Millipore, Bedford, MA). The filtrates were directly analyzed by HPLC-ESI-MS/MS as described below. Acid

DNA Cross-Linking by 1,2,3,4-Diepoxybutane hydrolysis was performed by heating the DNA in 0.1 N HCl for 30 min at 70 °C. The solution was neutralized with NH4OH. The DNA backbone was removed by ultrafiltration as described above and analyzed by capillary HPLC-ESI+-MS/MS as described below. Dimroth Rearrangement. Acid hydrolysates of DEBtreated CT DNA were neutralized and filtered through Microcon YM-30 membrane filters (Millipore). The filtrate was dried under reduced pressure. The residue was dissolved in 10 mM Tris (pH 7.3) and heated to 70 °C overnight. The solution was directly analyzed by capillary HPLC-ESI-MS/MS as described below. Dimroth rearrangement of the synthetic compound 1 was performed by heating at pH 12 (sodium hydroxide) overnight. DEB Treatment of Synthetic Oligonucleotides of Defined Sequence. DNA 15-mers, 5′-(GGT)5, 5′-(GT)7G, 5′-(GAA)5, and the complementary strands, were synthesized by standard phosphoramidite chemistry. DNA duplexes (10 nmol) were treated with 5-50 mM DEB in 20 µL of 0.3 mM NaOAc buffer (pH 5) at 37 °C for 3 h. The mixtures were extracted with diethyl ether and subjected to acid hydrolysis as described above. The solutions were neutralized with NH4OH and analyzed by capillary HPLC-ESI-MS/MS as described below. Capillary HPLC-ESI-MS/MS. An Agilent 1100 MSD capillary LC ion trap MS system (Wilmington, DE) was used for these analyses. Chromatographic separation was achieved using an Agilent Zorbax SB-C18 column (150 mm × 0.5 mm, 5 µm) eluted at a flow rate of 10 µL/min, with a mobile phase of 15 mM ammonium acetate, pH 5.5 (A), and 100% acetonitrile (B). A linear gradient was run from 0 to 30% B over a period of 40 min. HPLC effluent from the first 5 min of each run was diverted to waste. The mass spectrometer was operated in the positive ion mode with nitrogen as a nebulizing (15 psi) and drying gas (5 L/min). Electrospray ionization was achieved at a spray voltage of 3-3.5 kV, and the drying gas temperature was set to 200 °C. The [M + H]+ ions of the G-A butanediol conjugates (m/z ) 373.2) were isolated and subjected to collisioninduced dissociation (CID; fragmentation amplitude ) 0.89). The product ions were detected within the scan range of m/z ) 100500. The target ion abundance value was 50 000, and the maximum accumulation time was 250 ms.

Results Capillary HPLC-ESI+-MS/MS analysis of the acid hydrolysates of DEB-treated double-stranded DNA (CT DNA) revealed the presence of four isomeric adenineguanine butanediol cross-links [m/z 373.2 (M + H); Figure 1A]. These lesions were marked in the order of their elution from the HPLC column: AGBD1 (22.5 min, tR), AGBD2 (24.5 min, tR), AGBD3 (25.7 min, tR), and AGBD4 (29.9 min, tR). AGBD2 and AGBD3 are also observed in thermal hydrolysates of DEB-exposed DNA (Figure 1B), suggesting that they represent hydrolytically labile nucleobase lesions. For example, N3- and N7alkylation of adenine and N7-substitution of guanine are known to destabilize the glycosidic bond, leading to spontaneous depurination of the modified bases, and the concomitant production of abasic sites (13). When the protonated molecules of isomeric A-G DEB-DNA lesions (Figure 1) are subjected to CID (MS/ MS), characteristic fragment ions indicative of their structure are obtained (Figure 2). The MS/MS spectrum of AGBD1 is characterized by the major mass fragment at m/z 220 (Figure 2A). In contrast, MS/MS spectra of AGBD2 and AGBD 4 are dominated by the signal at m/z 222 (Figure 2B,D). The tandem mass spectrum of AGBD3 contains both product ions (m/z 220 and 222) with similar intensities (Figure 2C). When the ions at m/z 220 are isolated and subjected to further fragmentation (MS3), they produce secondary fragments at m/z 152 [Gua + H]+,


while MS3 experiments of the m/z 373.2 f 222 ions produce secondary fragments at m/z 136 [Ade + H]+ (Supporting Information S-1). These results indicate that the mass fragments at m/z 222 retain the adenine moiety in their structure and thus most likely correspond to the neutral loss of guanine from the [M + H]+ ions of the adduct ([M + H - Gua]+). In contrast, m/z 220 ions still contain the guanine base, consistent with a structure resulting from the removal of adenine and water from protonated molecules of the cross-link ([M + H - Ade H2O]+). In addition, MS/MS spectra of A-G DEB lesions contain less abundant signals at m/z 356.1 (M + H H2O)+, 238 (M + H - Ade)+, m/z 152 (Gua + H) +, and 136 (Ade + H)+ (Figure 2). The observed differences between the MS/MS fragmentation patterns of isomeric guanine-adenine butanediol conjugates (Figure 2) strongly suggest that they represent structural isomers, rather than diastereomers. Theoretically, these isomers may have different substitution sites at the guanine or adenine heterocycles or within the butanediol linker. Previous studies indicate that DEB reactions with double-stranded DNA are regiospecific for the N-7 position of guanine (10, 14). In contrast, adenine alkylation with DEB can occur at multiple sites, including N-1, N-3, N-7, and N6 (13, 15). Within DEB, nucleophilic substitution is expected to take place at the least sterically hindered terminal carbons, giving rise to 2,3butanediol derivatives (14-16). We thus hypothesized that the four isomeric G-A cross-links isolated from DEB-treated DNA correspond to 2,3-butanediol conjugates involving N7-substitued guanine and N1-, N3-, N7-, or N6-substituted adenine (Scheme 2). Initial evidence for the formation of N6- and N1adenine-substituted G-A DEB cross-links (e.g., 1 and 4 in Scheme 2) is provided by the HPLC-ESI-MS/MS analyses of hydrolysates of DEB-treated DNA following heating at basic pH (Figure 1C). These conditions induce the rearrangement of N1-substituted adenines to the corresponding N6-adenine derivatives (Dimroth rearrangement). As shown in Figure 1C, the size of the HPLC-ESI-MS/MS peak marked as AGBD1 decreases following Dimroth rearrangement, with a concominant increase of AGBD4 peak size (Figure 1C). These results suggest that AGBD1 and AGBD4 represent N1- and N6adenyl-substituted G-A cross-links, respectively. Peaks labeled as AGBD2 and AGBD3 were tentatively assigned the structures of N7- and N3-alkyladenines based on their facile depurination at neutral pH (Figure 1B) (13). Synthesis of the Putative Guanine-Adenine Conjugates. Because the amounts of G-A cross-links derived from DEB-treated DNA are insufficient to allow their detailed structural characterization by NMR, we chose to synthesize the putative G-A nucleobase conjugates by an independent route. Authentic standards of adenineguanine DEB conjugates 1-4 (Scheme 2) were prepared as shown in Schemes 3-5. In all cases, 5 (12) was a key intermediate. Following the coupling of 5 with the adenine moiety, the butene double bond was converted to the desired diol functionality by cis-oxidation with osmium tetroxide. When trans-dichlorobutene is employed as a starting material, this gives rise to a racemic mixture of bis-G-A adducts (Scheme 6, top). The same G-A cross-link stereochemistry is expected in CT DNA treated with racemic DEB (Scheme 6, bottom).

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Figure 1. Capillary HPLC-ESI+-MS/MS analysis of adenine-guanine cross-links in CT DNA treated with DEB (80 µM): acid hydrolysate (A), neutral thermal hydrolysate (B), and acid hydrolysate and following Dimroth rearrangement at pH 8 (C). An Agilent 1100 capillary LC ion trap MS system was operated in the ESI+ mode. A Zorbax SB-C18 column (150 mm × 0.5 mm, 5 µm) was eluted at a 15 µL/min with a gradient of acetonitrile in 15 mM NH4 OAc, pH 5.5. Product ions of m/z 373.2 (protonated molecules of A-G butanediol cross-links) were detected. Extracted ion chromatograms were constructed to monitor the transitions m/z 373.2 [M + H]+ f 220.0 [M + H - Ade - H2O]+, 222 [M + H - Gua]+.

N6A-N7G-BD (4 in Scheme 2) was obtained by coupling 8 with 6-chloropurine riboside (Scheme 3) (17). Intermediate 8 was synthesized by treating 5 (12) with sodium azide, followed by OsO4-mediated oxidation of the butene double bond to a diol (Scheme 3). Catalytic hydrogenation of 7 with 10% palladium on charcoal converted the azide to the corresponding amine 8. Following coupling of 8 with 6-chloropurine riboside (17), the N9-ribozyl and the N2-acetyl protective group were removed (1 N HCl), yielding the desired product 4 (Scheme 3). This compound was purified by semipreparative HPLC. Our synthetic scheme for N1- and N3-adenine derivatives 1 and 2 (Scheme 2) takes advantage of the selectivity for N-3 alkylation of the adenine free base and the preferential N-1 alkylation of adenosine nucleoside (Scheme 4) (18). Compound 1 was prepared by nucleophilic displacement of the allylic Cl in 5 by adenosine at the N1 position to yield 10 (Scheme 4, top). The olefin of 10 then was oxidized with OsO4 in the presence of 4-methylmorpholine-N-oxide producing the corresponding vicinal diol (10a). Finally, 10a was deprotected (1 N HCl) to produce racemic 1. Compound 2 was prepared by the same method as compound 1, except that adenine was used in place of adenosine (Scheme 4, bottom). A modified experimental strategy was required for the synthesis of 3 because of the inherently low reactivity of the N7-position of adenine (Scheme 5). The N3-position

of adenine was blocked with a benzyl group to allow for selective alkylation at the N7 (19). Following coupling of N3-benzyladenine with 5 and cis-oxidation of the double bond with OsO4 to give 13, the benzyl group was removed. Initial attempts to remove the benzyl group by Pd/C-catalyzed hydrogenation (19) resulted in the recovery of unreacted starting material. However, selective debenzylation was achieved by treating it with a mixture of concentrated H2SO4 and dry toluene (19). The latter reaction is thought to proceed through the formation of benzyl carbenium ions, which are trapped by transbenzylation with toluene (19). The N2-acetyl protective group was removed by acid hydrolysis, yielding 3 (Scheme 5). Structural Characterization of Synthetic Adenine-Guanine Conjugates. The structures of the synthesized compounds 1-4 (Scheme 2) were confirmed by a combination of UV spectroscopy, tandem mass spectrometry, and NMR experiments. High-resolution mass spectral analyses of synthetic 1-4 (Q-TOF MS) showed protonated molecules of the adducts at m/z 373.1466, 373.1465, 373.1488, and 373.1478, respectively. These results indicate that the molecular formula corresponding to protonated molecules of compounds 1-4 is C14H17N10O3, which is consistent with a structure containing guanine and adenine bases connected by a butanediol bridge (calcd m/z 373.1480, M + H+).



Figure 2. MS/MS spectra of the isomeric adenine-guanine DEB cross-links. The protonated molecule ions of each A-G conjugate (m/z ) 373.2, [M + H]+) were isolated and fragmented in the Agilent 1100 capillary HPLC ion trap MS system. The fragmentation amplitude was 1.0-1.5.

Scheme 2. Proposed Structures of Isomeric Adenine-Guanine Adducts of DEB

Each of the individual isomers exhibited characteristic UV spectra that were reminiscent of the corresponding substituted adenines (Table 1). This is consistent with the stronger UV absorption by the substituted adenine heterocycle as compared with the guanine chromophore. MS/MS fragmentation of adducts 1-4 (Table 1) reveals important differences between regioisomers. While MS/ MS spectra of compounds 2 and 4 show predominant fragments at m/z 222.0 [M + H - Gua]+, MS/MS fragmentation of compound 1 is dominated by the loss of adenine and water (m/z 220.0 [M + H - Ade - H2O]+). Compound 3 exhibits both fragmentation pathways with nearly equal frequency (m/z 373 f 222, 220). As discussed above, similar variations were observed for the fragmentation patterns of isomeric G-A adducts isolated

from DEB-treated DNA (Figure 2). The observed tendency of the protonated molecules of compound 1 to undergo the neutral loss of adenine and water (m/z 373 [M + H] f 220.0 [M + H - Ade - H2O]+) may be a result of their preferential protonation at the guanine heterocycle (pKa ) 3.49 for N7-alkylguanine and 2.97 for N1alkyladenine) (Supporting Information S-1). In contrast, compounds 2 and 4 retain a proton at the adenine portion of the molecule (pKa ) 3.94 for N6-alkyladenine, pKa ) 5.0 for N3-alkyladenine), leading to the neutral loss of guanine and the formation of product ions at m/z 222.0 [M + H - Gua]+. Guanine modification site in compounds 1-4 should be at N7 because they were prepared from 5 (12) (Schemes 3-5). In support of this assignment, similar

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Scheme 3. Synthesis of N6A-N7G-BD

Scheme 4. Synthesis of N1A-N7G-BD and N3A-N7G-BD

guanine signals are observed in proton NMR spectra of the four G-A conjugates (Table 2). The guanine heterocycle is characterized by the one proton singlet at 7.77.9 ppm (H-8) and a two proton singlet at 5.9-6.2 ppm (NH2) (Table 2). The diastereotopic methylene protons adjacent to the guanine base (CH2D) appear as two doublets of doublets at 4.15-4.22 and 4.29-4.37 ppm with a geminal coupling constant of 13.8 Hz (Table 2 and

Figure 3). The CHCOH methine proton appears as a broad doublet at 3.82-3.93 ppm. Gradient heteronuclear multiple bond correlation (gHMBC) experiments (Figure 3) show a cross-peak between the R-methylene protons of the linker (CH2D) and the C-8 and C-5 of guanine (δ 144.0-144.7 and 109-111, respectively), confirming the N-7 guanine substitution (Table 2). Interestingly, the long-range H-C coupling is more pronounced for one of



Scheme 5. Synthesis of N7A-N7G-BD

Scheme 6. Stereochemistry of Synthetic and DNA-Derived A-G DEB Cross-Links

the two methylene protons, even for NMR experiments performed at 60 °C (Figure 3). Long-range interactions are also observed between CH2D protons and CHBOH carbon (δ 70.5-72.5; see Figure 3 and Table 2). Adenine substitution in compound 4 is specific for the N6-position because it was obtained by the coupling reaction between 4 and commercial 6-choropurine 2′deoxyriboside (17) (Scheme 3). This structural assignment is confirmed further by the 1H NMR spectrum of 4 in DMSO-d6 (Table 2; Supporting Information S-2).

Adenine heterocycle protons appear as one proton singlets at 8.16 ppm (H-2 of Ade) and 8.07 ppm (H-8 of Ade), as well as a broad one proton signal corresponding to the exocyclic amino group (N6H, 7.36 ppm). These signals are analogous to those observed for commercially obtained N6-methyladenine (8.2, 8.05, and 7.4 ppm, respectively). Although the H-9 proton is difficult to see, close examination of the spectrum reveals an exteremely broad, low intensity peak at ∼11 ppm. The signal for the linker CHBOH methine group closest to adenine appears as a broad doublet at 3.64 ppm. The methylene group adjacent to adenine is obscured by a broad water signal in the spectrum obtained in DMSO but appears as two broad doublets at 3.3 and 3.5 ppm when the sample is dissolved in D2O (not shown). The points of butanediol linker attachment to the adenine heterocycle in compounds 1-3 were established by 1H NMR and gHMBC spectroscopy experiments. In addition to guanine signals discussed above, the 1H NMR spectrum of 1 displays signals at 8.21 ppm (broad two proton singlet, NH2-Ade), 7.98 ppm (s, 1H, H-8-Ade), and 7.75 ppm (s, 1H, H-2-Ade) (Table 2; Supporting Information S-3). The corresponding signals of commercial N1methyladenine appear at 8.4 (NH2-Ade) and 8.15 ppm (H-2 and H-8 of Ade). The diastereotopic protons of the CH2A methylene group in 1 are observed as a doublet of doublets at δ ) 4.08 and 4.24 ppm (Jgem ) 15 Hz; Jvic ) 10.2 and 4.8 Hz, respectively) (Table 2). The CHBOH methine proton appears as a broad triplet at 3.9 ppm. The CH2A methylene protons adjacent to adenine show couplings with the adenine carbons C6 (144.5 ppm) and C2 (150.2 ppm) in the gHMBC spectrum (Table 2), confirming N-1 substitution within the adenine heterocycle. Long-range interactions are also evident between CH2A and CHcOH carbons of the linker (68.1 ppm) (Table 2). When compound 1 is heated at pH 12 overnight, it is quantitatively converted to compound 4 via Dimroth rearrangement, providing additional evidence for the N1-

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Table 1. UV Absorption Maxima and ESI+-MS/MS Fragmentation Patterns of Synthetic Guanine-Adenine Cross-Links and Reference Compoundsa UVmax

a

compound

pH 1

pH 7

pH 10

MS/MS fragments m/z 373 f (%)

1 2 3 4 N1-Me-Ade N3-Me-Ade N7-Me-Ade N6-Me-Ade

258 276 272, 250 (sh) 274, 254 (sh) 259 274 273 267

266 256 (sh) 278 276, 250 (sh) 273 266 274 270 267

273 277 271 275 270 273 270 273

220 (100), 265.0 (10), 356.1 (10), 151.9 (3) 222 (100), 238 (5), 356.1 (2), 204 (2), 152 (2) 222 (100), 220 (90), 356 (40) 204 (20), 152 (10) 222 (100), 152 (3), 356 (2)

See Figure 1 for MS/MS details.

adenine substitution in compound 1 (Supporting Information S-4). In addition to guanine signals common for all four A-G regioisomers (see above), the 1H NMR spectrum of compound 2 shows adenine signals at 8.31 (s, 1H, H-2) and 8.21 (s, 1H; H-8) ppm and a broad two proton signal at 7.8-8.0 ppm characteristic for N3-alkyladenines (NH2) (Table 2; Supporting Information S-5). This peak broadening is a result of the presence of two tautomeric forms of N3-alkyladenines (15). Adenine chemical shifts in 2 are comparable with those in authentic N3-methyladenine: 8.24 (H-2-Ade), 7.71 (H-8-Ade), and 7.7-7.9 (NH2Ade) ppm. The signals attributable to the linker methylene protons closest to Ade (CH2A) appear as two doublets of doublets at 4.26 and 4.45 ppm. The broad one proton signal at 3.86 ppm corresponds to CHBOH methine protons. Our 1H NMR assignments are confirmed by the gHMBC spectrum of 2 (Figure 3), where CH2A protons show coupling with the C-2 and the C-4 carbons of the adenine heterocycle (150.5 and 144.5 ppm, respectively). The 1H NMR spectrum of compound 3 contains adenine signals at 8.16 (two proton singlet, H2 and H8 of Ade) and 6.72 ppm (s, 2H, NH2-Ade) (Table 2; Supporting Information S-6). The adenine-CH2A methylene group is observed as two doublets of doublets at 4.49 and 4.30 ppm (Jgem ) 15.0 Hz, Jvic ) 3.0 and 10.2 Hz). The CHBOH methine proton appears as a broad singlet at 3.69 ppm. The CH2A protons adjacent to adenine are coupled to the adenine carbons C8 (147.3 ppm) and C5 (113.4 ppm) in the gHMBC spectrum, confirming the N-7 substitution at the adenine heterocycle (Table 2; Supporting Information S-7). It should be noted that gradient HMBC spectra of all four A-G stereoisomers show a much stronger coupling between one of the two diastereotopic protons of the CH2A methylene group and adenine ring carbons (Figure 3; Supporting Information S-7). Similarly, a much more prominent cross-peak is observed for one of the CH2D protons with C-5 and C-8 of the guanine than the other (Figure 3; Supporting Information S-7). This may be explained by the conformational restraints in the A-G butanediol conjugate, leading to low coupling constants between one of the methylene protons and carbons within the purine heterocycle. A similar phenomenon was previously observed for HMBC spectra of isoprene monoepoxide N7-guanine adducts (20). The optical configuration of the two stereogenic centers within 2,3-butanediol linker in 1-4 is defined by the stereochemistry of butene double bond oxidation (Schemes 3-5). Because trans-1,4-dichlorobutene was used as a precursor of butanediol linker in all four G-A conjugates,

syn-oxidation with osmium tetroxide should lead to R,R and S,S products (Scheme 6). As shown in Scheme 6, guanine-adenine conjugates prepared by this pathway should have the same chirality as DNA-derived guanineadenine cross-links induced by racemic DEB. Formation of Guanine-Adenine Cross-Links 1-4 in Double-Stranded DNA. The results of capillary HPLC-ESI-MS/MS analyses of synthetic guanine-adenine DEB cross-links (compounds 1-4) in parallel with the acid hydrolysates of DNA treated with racemic DEB are shown in Figure 4. DNA-derived DEB cross-links AGBD1 (26.1 min, tR), AGBD2 (27.5 min, tR), AGBD3 (28.9 min, tR), and AGBD4 (33.1 min, tR) coelute with the synthetic standards of N1A-N7G-BD (compound 1 in Scheme 1), N3A-N7G-BD (2), N7A-N7G-BD (3), and N6AN7G-BD (4), respectively (Figure 4). Furthermore, MS/ MS fragmentation patterns and UV spectra of synthetic adenine-guanine DEB conjugates are identical to those of the corresponding cross-links observed in DEB-treated DNA (Table 1 and Figures 2 and 5). On the basis of this evidence, AGBD1, AGBD2, AGBD3, and AGBD4 (Figure 1) were identified as 1, 2, 3, and 4, respectively (Scheme 2). The estimated molar ratios of N1A-N7G-BD, N3AN7G-BD, N7A-N7G-BD, and N6A-N7G-BD produced in double-stranded DNA treated with racemic DEB (10 mM) were ∼1:0.1:0.6:0.1 based on the HPLC-ESI-MS/MS peak areas (Figure 1). Surprisingly, the contribution of N3AN7G-BD appears greater in thermal DNA hydrolysates than in acid DNA hydrolysates (Figure 1A,B, respectively). This discrepancy cannot be explained by decomposition of N3A-N7G-BD at acidic conditions, as heating in 0.1 N HCl did not have any effect on adduct amounts (not shown). More likely, this difference is caused by HPLC-ESI-MS/MS signal suppression, the presence of a large access of guanine, and adenine nucleobases released during acid hydrolysates. The hydrolytic stability of DNA-derived G-A DEB lesions is consistent with their structures (14, 15). The formation of N3-Ade, N7-Ade, and N7-Gua substitution destabilizes the glycosidic bonds due to the production of quaternary amines, leading to spontaneous depurination and abasic site formation (13). Therefore, 2 and 3 are expected to be hydrolytically labile. Indeed, as shown in Figure 1B, heating DEB-treated DNA at neutral pH quantitatively releases both cross-links from the DNA backbone. At physiological conditions, the half-lives of 2 and 3 in double-stranded DNA are 31 and 17 h, respectively (Supporting Information S-8). In contrast, adducts 1 and 4 are not released from DNA at neutral pH (Figure 1) and thus, if not repaired, are expected to accumulate in target tissues, contributing to the cytotoxic and genotoxic effects of DEB. While 1 can be converted to 4 via



Table 2. NMR Characterization of Compounds 1-4a

a

NMR characterization of synthetic adenine-guanine butanediol conjugates 1-4.

Dimroth rearrangement at pH 12 (Figure 1C; Supporting Information S-3), this process is slow under neutral

conditions and is unlikely to explain the formation of 4 in our experiments.

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Figure 3. Gradient HMBC spectrum of 2 dissolved in DMSO-d6 solvent. A 600 MHz Varian Inova NMR spectrometer was used at a temperature of 60 °C. HMQC data were obtained with 32 transients, 256 increments, a relaxation delay of 1.5 s, and a one-bond coupling constant of 140 Hz. HMBC experiments were performed with 32 transients, 512 increments, a relaxation delay of 2 s, and a one-bond coupling constant of 140 Hz.

Figure 4. Capillary HPLC-ESI+-MS/MS analysis of synthetic G-A cross-links in parallel with the acid hydrolysate of DEBtreated DNA following Dimroth rearrangement. A Zorbax SBC18 column (250 mm × 0.5 mm, 5 µm) was eluted at a 10 µL/ min flow rate with a gradient of acetonitrile in 15 mM NH4 OAc, pH 5.5. See Figure 1 for further MS/MS details.

Formation of 1-4 in Oligodeoxynucleotides of Defined Sequence. Double-stranded DNA 15-mers of repeating sequence 5′-GGT-3′, 5′-GT-3′, and 5′-GAA-3′ (+-strand) were employed to investigate sequence preferences for the formation of regioisomeric G-A DEB lesions. Each duplex was treated with racemic DEB,

followed by thermal or mild acid hydrolysis and HPLCESI-MS/MS analysis of the resulting G-A cross-links (Figure 6). Among the three sequences investigated, maximum numbers of G-A DEB adducts were produced in the 5′(GGT)5 duplex (Figure 6A). HPLC-ESI-MS/MS analyses indicated that these cross-links were primarily N1A-N7G lesions (Figure 6A). N1A-N7G cross-link yields were ∼20 times lower in the 5′-(GT)7G duplex (Figure 6B). While the 5′-(GT)7G oligomer contains more potential G-A cross-linking sites, it can accommodate only 1,2-interstrand adducts (Figure 6B, insert). These results suggest that interstrand DNA cross-linking by DEB produces primarily 1,3-N1A-N7G BD lesions. Interestingly, DEB treatment of the 5′-(GT)7G duplex gives rise to an additional small G-A cross-link peak, which does not coelute with any of our G-A cross-link standards (31.5 min, tR) (Figure 6B). This product was observed only in acid DNA hydrolysates and exhibited major MS/MS fragments at m/z 222 [M + H - Gua]+ and 356 [M + H - H2O] + (results not shown). On the basis of its hydrolytic stability and characteristic MS/MS fragmentation pattern, this compound was tentatively identified as 1-(aden-6-yl)-3-(guan-7-yl)-2,4-butanediol. Potential intrastrand cross-linking by DEB was investigated in the 5′-(GAA)5 duplex. The 5′-GAA sequence can theoretically accommodate either 1,2- or 1,3-intrastrand linkages. While moderate amounts of all four isomeric G-A DEB adducts were observed (Figure 6C), their amounts were much lower than in the 5′-(GGT)5 oligomer. Taken together, these results indicate that while


Figure 5. UV spectra of DNA-derived (dotted line) and synthetic (solid line) 1 (A), 2 (B), 4 (C), and 3 (D).

racemic DEB is capable of inducing both intrastrand and interstrand G-A lesions, the major asymmetrical adduct produced is 1,3-N1A-N7G-BD.

Discussion DEB is a typical SN2 type alkylating agent targeting nucleophilic sites in DNA, including the N-7-position of guanine, N-3-position of adenine, and N-1-position of adenine (14, 15). Initial reactions of DNA with DEB produce primarily N7-(2′-hydroxy-3′,4′-epoxybut-1′-yl)guanine intermediate (Scheme 1). Spontaneous hydrolysis of the second epoxide ring yields N7-(2′,3′,4′-trihydroxybut-1′-yl)guanine monoadducts. Alternatively, the second oxirane group can interact with another nucleophilic site within the DNA duplex, giving rise to bifunctional DNA lesions (Scheme 1). Although DNA-DNA cross-linking by DEB is a relatively rare event as compared with much more frequent monoadduct formation, bifunctional alkylation appears to be essential for its biological activity. For example, DEB is much more genotoxic and mutagenic than its monoepoxide analogues, EB and 3,4-epoxy-1,2-butanediol. It is also 50 times more effective in inducing sister chromatid exchanges in human lymphocytes than EB (6) and is 2 orders of magnitude more mutagenic than EB in TK6 lymphoblasts (7). All three metabolites appear to produce the same types of DNA monoadducts, but only DEB is capable of inducing DNA-DNA and DNAprotein cross-links. Previous research aimed at the elucidation of DNADNA cross-linking mechanisms by DEB has been ham-


pered by the relatively lower yields of such lesions relative to DNA monoadducts and by the complex structural possibilities inherent to the formation of bifunctional DNA lesions. Over 40 years ago, Lawley and Brookes isolated the guanine-guanine lesion of DEB, bisN7G-BD, from DEB-treated salmon sperm DNA (9). Recent structural studies conducted in our laboratory confirm this structural assignment (10). However, mutagenesis studies indicate that DEB induces mostly partial deletions and base substitutions at A:T base pairs, suggesting that biologically relevant cross-linked lesions are produced at adenine or thymine nucleobases (11, 21, 22). Furthermore, gel electrophoresis-based studies provide indirect evidence for the formation of small quantities of hydrolytically stable DNA-DNA cross-links of DEB, presumably at adenine nucleobases (23). We now report that in addition to the previously observed bis-N7G-BD DEB cross-link, DEB produces cross-linked lesions involving adenine nucleobases: N1AN7G-BD, N3A-N7G-BD, N7A-N7G-BD, and N6A-N7GBD. Although the amounts of these asymmetrical lesions in DEB-treated double-stranded DNA are ∼10 times lower than of the corresponding bis-N7G cross-links, N1A-N7G-BD and N6A-N7G-BD are hydrolytically stable and, if formed in vivo, may accumulate in target tissues. In contrast, N7A-N7G-BD and N3A-N7G-BD depurinate at a higher rate than bis-N7G-BD (t1/2 ) 31 and 17 h, respectively, as compared with 81 h for bis-N7G-BD). This is consistent with an increased glycosidic bond lability of the corresponding monoadducts [the t1/2 values for N7-Me-Gua, N3-Me-Ade, and N7-Me-Ade in doublestranded DNA at pH 7 are reported as 155, 26, and 3 h, respectively (13)]. Both N1A-N7G-BD and N6A-N7G-BD can undergo depurination of the N7-substituted guanine, giving rise to a bulky adduct containing a butanediol cross-link to a free guanine base and a neighboring abasic site. Such lesions are likely to play a role in mutagenesis by error-prone repair or polymerase bypass mechanisms. Our experiments with DNA duplexes of defined sequence indicate that asymmetrical N7G-N1A DEB crosslinks are preferentially formed at complementary 5′GNT-3′/3′-CNA-5′ trinucleotides (N ) any nucleotide) (Figure 6). This is analogous to guanine-guanine crosslinking by DEB, which is selective for the distal N7-dG atoms in 5′-GNC sequences (23). While this sequence specificity may be partially explained by the 3′-orientation of the side chain of the N7-(2′-hydroxy-3′,4′-epoxybut1′-yl)guanine intermediate as a result of unfavorable steric interactions between the side chain and the 5′residue (24, 25), the butanediol tether length (∼5 Å) is too short to bridge the spacing between the N7-G and the N1-A atoms in 5′-GNT sequences of B-DNA (8.6 Å). In theory, the formation of the N7-(2′-hydroxy-3′,4′epoxybut-1′-yl) intermediate at the 5′-guanine may induce a local distortion of the DNA duplex, bringing the N1-A atom closer to the epoxy group of the N7-guanyl intermediate. Published thermodynamic and structural studies of the N7-methyl-guanine incorporated into a selfcomplementary Dickerson dodecamer argue against structural distortion of the DNA duplex by the positively charged N7-alkylguanine intermediate (26). However, (2′hydroxy-3′,4′-epoxybut-1′-yl) group, at the N7-G may have a greater effect on DNA structure because of the possible hydrogen bonding between the 2′-hydroxy group and the phosphodiester backbone or neighboring nucleobases.

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Figure 6. Capillary HPLC-ESI+-MS/MS analysis of isomeric G-A cross-links in synthetic duplexes of defined sequence following incubation with racemic DEB. See Figure 1 for further HPLC-MS/MS details.

While the formation of cross-linked lesions of DEB involving adenine nucleobases is consistent with the induction of A:T base pair mutations by DEB and 1,3BD, their potential role in mutagenesis remains to be established. The biological fate of the regioisomeric G-A cross-links of DEB is strongly dependent on their type (intrastrand vs interstrand). In general, interstrand cross-links are considered highly cytotoxic because of their ability to block DNA replication and transcription (27-30), while intrastrand cross-links can be either cytotoxic [e.g., cis-diamminedichloro platinum(II)] (29) or mutagenic (e.g., UV-induced thymine dimers) (31, 32). Polymerase bypass of synthetic DNA templates containing N6-trihydroxybutyl adenine monoadducts of DEB induces low levels of A f G and A f C base substitutions (

GuanineâAdenine DNA Cross-Linking by 1,2,3,4-Diepoxybutane

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