Identification of Adducts Produced by the Reaction of 4

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Identification of Adducts Produced by the Reaction of 4-(Acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol with Deoxyguanosine and DNA Pramod Upadhyaya, Shana J. Sturla, Natalia Tretyakova, Rebecca Ziegel, Peter W. Villalta, Mingyao Wang, and Stephen S. Hecht* University of Minnesota Cancer Center, Minneapolis, Minnesota 55455 Received October 3, 2002

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a metabolite of the tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). NNAL is present in the blood and urine of people exposed to tobacco products and has carcinogenic activity in rodents similar to that of NNK. DNA adducts specific to NNAL have not been previously identified. Metabolic activation of NNAL by R-methyl hydroxylation, a pathway known to occur in rodent and human microsomes, would produce pyridylhydroxybutylating agents that could react with DNA. We investigated this possibility in the present study by allowing 4-(acetoxymethylnitrosamino)1-(3-pyridyl)-1-butanone (NNALCH2OAc) to react with dGuo and DNA. Products were identified by HPLC with UV detection, liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) and LC/ESI-tandem mass spectrometry (LC/ESI-MS/MS). In the dGuo reactions, selected ion monitoring for m/z 417, corresponding to pyridylhydroxybutylated dGuo, showed several peaks. One adduct was identified as 7-[1-hydroxy-1-(3-pyridyl)but-4-yl]dGuo (21) by neutral thermal hydrolysis, which converted it to 7-[1-hydroxy-1-(3-pyridyl)but-4-yl]Gua (22) and 4-hydroxy-1-(3-pyridyl)-1-butanol (16). Adduct 22 was identified by comparison of its LC/ ESI-MS and LC/ESI-MS/MS properties to those of standard 22. Two other adducts, O6-[1hydroxy-1-(3-pyridyl)but-4-yl]dGuo (17) and N2-[1-hydroxy-1-(3-pyridyl)but-4-yl]dGuo (19), were identified by comparison of their LC/ESI-MS and LC/ESI-MS/MS properties to those of standard 17 and 19. Further evidence for the identity of 17 and 19 was obtained by mild acid hydrolysis, which converted them to the corresponding Gua bases 18 and 20, identified by comparison to synthetic standards. Neutral thermal hydrolysis of DNA that had been reacted with NNALCH2OAc produced 22, identified by comparison to a standard. Adducts 17 and 19 were identified in enzyme hydrolysates of this DNA by comparison to standards. Thus, DNA that had been allowed to react with NNALCH2OAc contained adducts 17, 19, and 21. The results of this study provide markers for investigating the role of specific NNAL-DNA adducts in carcinogenesis by NNAL and NNK.

Introduction NNK (1),1 a tobacco specific nitrosamine, is a potent carcinogen in rodents (1). NNK induces lung tumors in rats, mice, and hamsters independent of the route of administration. Its carcinogenicity to the lung is particularly strong in rats, in which tumors have been induced at total doses as low as 6 mg/kg (2). Even lower doses have induced lung tumors in rats when considered as part of a dose-response trend (2). NNK also causes * To whom correspondence should be addressed. Tel: 612-624-7604. Fax: 612-626-5135. E-mail: [email protected]. 1 Abbreviations: COSY, 1H-1H correlation spectroscopy; HPB, 4-hydroxy-l-(3-pyridyl)-1-butanone; HMBC, heteronuclear multiple bond correlation spectroscopy; LC/ESI-MS, liquid chromatography/ electrospray ionization-mass spectrometry; LC/ESI-MS/MS, liquid chromatography/electrospray ionization/tandem mass spectrometry; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; NNALCH2OAc, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; O6-pyridylhydroxybutyl-dGuo, O6-[1-hydroxy-1-(3-pyridyl)but-4-yl]dGuo; O6-pyridylhydroxybutyl-Gua, O6-[1-hydroxy-l-(3-pyridyl)but-4-yl]Gua; N2-pyridylhydroxybutyl-dGuo, N2-[1-hydroxy-1-(3-pyridyl)but-4-yl]dGuo; N2-pyridylhydroxybutyl-Gua, N2-[1-hydroxy-1-(3-pyridyl)but-4-yl]Gua; 7-pyridylhydroxybutyl-Gua, 7-[1-hydroxy-l-(3-pyridyl)but-4-yl]Gua.

tumors of the nasal mucosa, liver, and pancreas in rats (1). When NNK was administered by swabbing to the rat oral cavity, in admixture with N′-nitrosonornicotine, oral tumors were induced (3). These carcinogenicity data, together with extensive studies clearly documenting the presence of substantial amounts of NNK in tobacco products, lead to the conclusion that NNK plays a significant role as a cause of cancer in people who use these products (4-6). NNK requires metabolic activation to produce DNA adducts that are associated with its mutagenicity and carcinogenicity (1). Cytochrome P450-mediated hydroxylation of NNK at the methyl carbon adjacent to the N-nitroso group produces intermediate 4, which spontaneously loses formaldehyde giving pyridyloxobutyldiazohydroxide 8 (Scheme 1). This intermediate ultimately reacts with DNA yielding pyridyloxobutyl adducts. The structure of one of these, O6-[1-oxo-1-(3-pyridyl)but-4-yl]dGuo (11), has been elucidated previously, and this adduct is highly mutagenic in bacterial and mammalian systems (7, 8). Evidence has also been presented for pyridyloxobutylation of DNA phosphate (9). The struc-

10.1021/tx0256376 CCC: $25.00 © 2003 American Chemical Society Published on Web 01/28/2003

Adducts of Reactions with Deoxyguanosine and DNA

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Scheme 1. DNA Adduct Formation from NNK and NNAL

tures of other pyridyloxobutyl DNA adducts of NNK have not been reported, but it is known that they release HPB (12) upon acid or neutral thermal hydrolysis (1). HPBreleasing DNA adducts have been detected in various tissues of NNK-treated animals and in the lungs of smokers (1, 10, 11). Hydroxylation of NNK at the methylene carbon adjacent to the N-nitroso group yields methanediazohydroxide (9). This metabolite ultimately methylates DNA producing O6-methylGua (13), 7-methylGua (14), and O4-methylThy (15), all of which have been detected in tissue DNA of NNK-treated animals (1). Compounds 13 and 14 have also been detected in human lung DNA (12). In rodents and humans, NNK is converted by carbonyl reductase enzymes such as 11βhydroxysteroid dehydrogenase to NNAL (2) (1, 13). The carcinogenic activity of NNAL in rats and mice is similar to that of NNK (1). Like NNK, NNAL undergoes hydroxylation at its methylene and methyl carbons adjacent to the N-nitroso group. Compounds 13 and 14 have been detected in the DNA of rats treated with NNAL (14). HPB-releasing adducts have also been detected in the DNA of NNAL-treated rats, presumably resulting from reconversion of NNAL to NNK, followed by R-methyl hydroxylation (14). However, adducts resulting from R-methyl hydroxylation of NNAL have never been characterized or detected. This route of metabolism is known to occur in rodents and humans, producing 4-hydroxy1-(3-pyridyl)-1-butanol (16) (15-17). Therefore, DNA adducts could be formed via intermediate 10 and may contribute to the carcinogenicity of NNAL and NNK. To investigate this pathway of DNA adduct formation, we studied the reactions of NNALCH2OAc (3) with dGuo and DNA. Compound 3 is a stable precursor to intermediate 7, which would be formed by R-methyl hydroxylation of NNAL.

Experimental Section Apparatus and Assay Conditions. HPLC analyses were carried out with a Waters Associates system equipped with a model 440 UV detector. The following solvent elution programs were used. System 1 used a 300 mm × 3.9 mm, 10 µm C18 Bondclone (Phenomenex, Torrance, CA) column eluted at 0.5 mL/min with 5% CH3OH in 40 mM ammonium phosphate buffer, pH 6.5, for 10 min, then with a gradient from 5 to 35% CH3OH over the course of 60 min, then from 35 to 50% CH3OH in 10 min, and held for 15 min. Detection was by UV (254 nm). System 2 used a 250 mm × 21.2 mm, 5 µm Luna C18 column (Phenomenex) with elution by a gradient from 0 to 30% CH3OH in H2O over the course of 30 min at a flow rate of 5 mL/min and UV detection (295 nm). System 3 used a 250 mm × 4.6 mm, 5 µm Luna C18 column with elution by a solvent gradient from 0 to 20% CH3OH in H2O over the course of 20 min at a flow rate of 0.5 mL/min with UV detection (295 nm). Capillary HPLC was carried out with an Agilent 1100 capillary HPLC (Agilent Technologies, Palo Alto, CA), and system 4, which used a 150 mm × 0.5 mm, 5 µm Zorbax SB-C18 column (Agilent) eluted at a flow rate of 15 µL/min. The solvent elution system was 15 mM ammonium acetate (A), pH 5.5, and acetonitrile (B) with a 3 min wash period (100% A) and a gradient of 0-20% B in 17 min, followed by 20-40% B in 15 min. LC/ESI-MS and LC/ESI-MS/MS analyses were carried out on a Thermo Finnigan LCQ Deca instrument (Thermo Finnigan LC/MS Division, San Jose, CA) interfaced with a Waters Alliance 2690 HPLC multisolvent delivery system and equipped with an SPD-IOA UV detector (Shimadzu Scientific Instruments, Columbia, MD). We used HPLC system 1, described above, without an initial 10 min hold and a convex-5 gradient. The ESI source was set as follows: voltage, 2.0 kV; capillary temperature, 275 °C. MS/MS data were acquired with the following parameters: parent mass, m/z 417, 301, and 168 amu; isolation width, 1.5 amu; activation amplitude, 30%; activation Q, 0.25; activation time, 30 ms. For quantitation of O6-pyridylhydroxybutyl-dGuo (17) and N2pyridylhydroxybutyl-dGuo (19) in DNA hydrolysates, an Agilent

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1100 series capillary HPLC ion trap MS system (Agilent) was used in conjunction with HPLC system 4. The mass spectrometer was operated in the positive ion mode with N2 as nebulizing (15 psi) and drying gas (5 mL/min). The dry gas temperature was set to 200 °C. The instrument was tuned to maximum sensitivity by direct infusion of 17. MS/MS data were acquired using the following parameters: target ion abundance, 50 000; maximum accumulation time, 300 ms; scan averages, 10; scan width, 2.0 amu; and fragmentation amplitude, 0.53. NMR spectra were recorded in DMSO-d6 using a Varian Unity (Varian, Inc., Palo Alto, CA) spectrometer operating at 600 or 800 MHz at 25 °C. An inverse detection triple-resonance 3 mm (800 MHz) or 5 mm (600 MHz) probe was used. Resonance assignments for adduct 26 were made based on 1H-1H COSY and 1H-13C HMBC experiments acquired at 600 MHz (18). The COSY was acquired with a spectral width of 9000 Hz, 16 scans for each fid, and 512 increments. The HMBC spectrum was acquired using sweep widths of 7000 and 25 000 Hz in the t1 and t2 dimensions, respectively, 64 scans for each fid, and 512 increments. The first delay was set to match a 140 Hz coupling constant, and the second delay was set to match a long-range coupling constant of 8 Hz. Chemicals and Enzymes. Compounds 3, 11, 4-oxo-4-(3pyridyl)-but-1-yl p-toluenesulfonate (23), and N2-[1-oxo-1-(3pyridyl)but-4-yl]dGuo were synthesized according to literature procedures (7, 19, 20). Phosphodiesterase II and micrococcal nuclease were acquired from Worthington Biochemcial Corporation (Lakewood, NJ). 2′,3′,5′-Triacetyl-Guo (24) was obtained from Acros Organics (Fisher Scientific, Pittsburgh, PA). All other chemicals and enzymes were obtained from Sigma Aldrich Chemical Co. or Fluka (Milwaukee, WI). O6-Pyridylhydroxybutyl-dGuo (17). Compound 11 (0.415 mg, 0.001 mmol) was dissolved in 1.0 mL of 40 mM ammonium acetate buffer, pH 7.0, and 1.0 mL of CH3OH. NaBH4 (200 mg) was added, and the reaction mixture was stirred for 3 h at room temperature. HPLC analysis (system 1) indicated complete disappearance of starting material. The pH of the reaction was adjusted to 7.0 by addition of 1 N HCl, and 17 was purified by HPLC using system 1 (312 mg, 75%). 1H NMR (D2O): δ 8.41 (bs, 1H, pyr-2H), 8.29 (d, J ) 4.8 Hz, 1H, pyr-6H), 7.9 (m, 1H, pyr-4H), 7.9 (s, 1H, dGuo-C8-H), 7.39 (dd, J ) 5.4, 2.4 Hz, 1H, pyr-5H), 6.2 (dd, J ) 8.4, 7.2 Hz, 1H, 1′-H), 4.82-4.77 (m, 1H, but-1H), 4.48-4.50 (m, 2H, 3′-H), 4.32 (m, 1H, but-4Hb), 4.29 (m, 1H, but-4Ha), 4.01-3.99 (m, 1H, 4′-H), 3.68 (dd, J ) 7.8, 5.6 Hz, 1H, 5′-Ha), 3.63 (m, 1H, 5′-Hb), 2.69 (m, 1H, 2′- Hb), 2.34 (m, 1H, 2′-Ha), 1.92-1.87 (m, 2H, but-2-H), 1.82-1.76 (m, 2H, but-3Ha), 1.72-1.65 (m, 1H, but-3Hb). UV (50% CH3OH: H2O): λmax 214.8, 249.5, 282. MS (positive ion ESI): m/z (relative intensity) 833 (2M + H, 25), 475 (M + 59, 11), 418 (M + 2, 23), 417 (MH+, 100), 301 (3). ESI-MS/MS of m/z 417; m/z 301 (100), 150 (8),152 (3). HRMS calcd for C19H25O5N6 (M + H): 417.1881; found, 417.1906. N2-Pyridylhydroxybutyl-dGuo (19). N2- [1-Oxo-1-(3-pyridyl)but-4-yl]dGuo (0.415 mg, 0.001 mmol) was reduced with NaBH4 using the above procedure yielding 312 mg (75%) of 19. 1H NMR (DMSO-d ): δ 8.53 (dd, J ) 2.4, 2.4 Hz, 1H, pyr-2H), 6 8.42 (dd, J ) 4.8, 1.0 Hz, 1H, pyr-6H), 7.73 (d, J ) 8.0 Hz, 1H, pyr-4H), 7.33 (dd, J ) 4.8, 3.2 Hz, 1H, pyr-5H), 7.86 (s, 1H, dGuo-C8-H), 6.96 (bs, 1H, N2-H), 6.13 (dd, J ) 7.2, 6.4 Hz, 1H, 1′-H), 5.49 (bs, 1H, 3′-OH), 4.87 (bs, 1H, 5′-OH), 4.62 (dd, 1H, J ) 6.4, 4.8 Hz, but-1H), 4.35 (m, 2H, 3′-H), 3.8 (m, 1H, 4′-H), 3.56 (dd, J ) 6.4, 5.6 Hz, 1H, 5′-Ha), 3.49 (dd, J ) 6.4, 5.6 Hz, 1H, 5′-Hb), 3.27 (m, 2H, but-4H), 2.62 (m, 1H, 2′Ha), 2.17 (m, 1H, 2′-Hb), 1.70-1.66 (m, 2H, but-2H), 1.62-1.58 (m, 1H, but3Ha), 1.52-1.48 (m, 1H, but-3Hb). UV (50% CH3OH:H2O): λmax 215, 257. MS (positive ion ESI): m/z (relative intensity) 833 (2M + H, 33), 475 (M + 59, 35), 417 (MH+, 100), 301 (8). ESIMS/MS of m/z 417, m/z 301 (100). HRMS calcd for C19H25O5N6 (M + H): 417.1881; found, 417.1888. 7-[1-Oxo-1-(3-pyridyl)-but-4-yl]Gua (26). 2′,3′,5′-Triacetylguanosine (24, 330 mg, 0.81 mmo1) and 23 (110 mg, 0.36 mmol) were combined in a dry round bottom flask under N2. Anhydrous

Upadhyaya et al. dimethylformamide (DMF, 2 mL) was added to the flask, and the reaction mixture was heated at 115 °C for 2 h. The reaction mixture was allowed to cool to room temperature, and DMF was removed under high vacuum over a period of 3 h. The resulting residue was dissolved in CHCl3 and filtered through a glass fiber filter. The filtrate was concentrated to provide a brown oil. The oil was dissolved in CH3OH and purified by HPLC system 2 (retention time, 58-61 min) followed by system 3 (retention time, 72 min) to give 100 µg (1%) of 26. 1H NMR (DMSO-d6): δ 10.68 (s, 1H, N1H), 9.05 (d, J ) 2.4 Hz, 1H, pyr-2H), 8.77 (dd, J ) 4.9, 2.4 Hz, 1H, pyr-6H), 8.21 (d, J ) 7.9 Hz, 1H, pyr-4H), 7.90 (s, 1H, Gua-C8-H), 7.53 (dd, J ) 7.9, 4.9 Hz, 1H, pyr-5H), 6.04 (s, 2H, NH2), 4.23 (t, J ) 6.7 Hz, 2H, but-4H), 3.05 (t, J ) 7.0 Hz, 2H, but-2H), 2.11-2.16 (m, 2H, but-3H). 13C NMR: δ 201.4 (but-C1), 156.3 (pyr-C6), 152.1 (pyr-C2), 146.2 (Gua-C8), 138.3 (pyr-C4), 126.9 (pyr-C5), 111.0 (Gua-C5), 48.4 (but-C4), 37.9 (but-C2), 27.8 (but-C3). The carbon resonances of C2, C4, and C6 of Gua were not detected. UV (CH3OH): λmax 216, 277 nm. MS (positive ion ESI): m/z (relative intensity) 357 (8), 299 (MH+, 100), 152 (2), 148 (10), 106 (2). ESI-MS/MS of m/z 299, m/z 152 (51), 148 (100), 106 (2). 7-Pyridylhydroxybutyl-Gua (22). Compound 26 was dissolved in CH3OH (1 mL), and NaBH4 (approximately 5 mg) was added. After 4 h, the reaction mixture was diluted with H2O, concentrated to 1 mL, and purified by HPLC system 3 (45 µg; retention time, 52 min). 1H NMR (DMSO-d6): δ 10.68 (br, 1H, N1H), 8.47 (s, 1H, pyr-2H), 8.41 (d, J ) 6.4 Hz, 1H, pyr-6H), 7.88 (s, 1H, Gua-C8-H), 7.65 (d, J ) 8.8 Hz, 1H, pyr-4H), 7.31 (dd, J ) 8.8, 6.4 Hz, 1H, pyr-5H), 6.05 (bs, 2H, NH2), 5.35 (s, 1H, but-1-OH), 4.55 (m, 1H, but-1H), 3.41 (t, 2H, J ) 6.0 Hz, but- 4H), 1.82-1.86 (m, 1H, but-2Hb), 1.70-1.75 (m, 1H, but2Ha), 1.51-1.55 (m, 1H, but-3Hb), 1.45-1.50 (m, 1H, but-3Ha). 13C NMR: δ 162.9 (Gua-C4), 151.1 (pyr-C6), 150.6 (pyr-C2), 149.9 (pyr-C3), 137.3 (C8, in D2O), 136.5 (pyr-C4), 126.5 (pyrC5), 110.0 (Gua-C5), 75.5 (but-C4), 72.7 (but-C1), 38.8 (but-C3), 30.1 (but-C2). The carbon resonances from C2 and C6 of Gua were not detected. UV (20% CH3OH:H2O): λmax 214, 250, and 285 (nm). MS (positive ion ESI): m/z (relative intensity) 623 (2M + Na, 23), 601 (2M + H, 43), 301 (MH+, 100); ESI-MS/MS of m/z 301; m/z 283 (100), 152 (25), 150 (30), 132 (12). HRMS calcd for C14H17O2N6 (M + H): 301.1408; found, 301.1401. O6-Pyridylhydroxybutyl-Gua (18). Compound 17 (2 mg, 4.8 mmol) was added to 0.1 N HCl (5 mL) and heated at 85 °C for 30 min. After it was cooled, the mixture was neutralized with 0.1 N NaOH and applied to a C18 Sep-pak cartridge (Waters Corp., Milford, MA). The product was eluted with H2O: CH3OH (1:1) to give 1.0 mg of 18 (75%). 1H NMR (DMSO-d6): δ 12.4 (s, 1H, N9H), 8.55 (d, 1H, J ) 2.4 Hz, pyr- 2H), 8.44 (dd, J ) 1.6, 1.6 Hz, 1H, pyr-6H), 7.78 (s, 1H, Gua-C8-H), 7.74 (dd, J ) 2.4, 2.4 Hz, 1H, pyr- 4H), 7.35 (dd, J ) 8.0, 4.0 Hz, 1H, pyr-5H), 6.19 (s, 2H, NH2), 5.41 (d, J ) 4.0, 1H, but-1-OH), 4.67 (m, 1H, but-1H), 4.4 (m, 2H, but-4H), 1.83 (m, 1H, but-3H), 1.75 (m, 1H, but-2H), 1.72 (m, 1H, but-3H). UV (50% CH3OH:H2O): λmax 244.6, 282 nm. MS (positive ion ESI): m/z (relative intensity) 623 (2M + Na, 18), 601 (2M + H, 24), 359 (M + 59, 17), 301 (MH+, 100), 152 (GuaH+, 9). ESI-MS/MS of m/z 301; m/z 283 (4), 152 (GuaH+, 42), 150 (100). HRMS calcd for C14H17O2N6 (M + H): 301.1408; found, 301.1424. N2-Pyridylhydroxybutyl-Gua (20). Compound 19 (2 mg, 4.8 mmol) was added to 0.1 N HCl (5 mL) and heated at 85 °C for 30 min. After it was cooled, the mixture was neutralized with 0.1 N NaOH and applied to a C18 Sep-pak cartridge (Waters Corp.) eluted with H2O:CH3OH (1:1) to give 1.0 mg of 20 (75%). 1H NMR (DMSO-d6): 10.3 (s, 1H, N1H), 8.52 (d, 1H, J ) 1.65, pyr-2H), 8.43 (dd, J ) 1.6, 1.6, 1H, pyr-6H), 7.72 (d, J ) 7.4, 1H, pyr-4H), 7.6 (s, 1H, Gua-C8-H), 7.34 (dd, J ) 7.4, 4.6, 1H, pyr-5H), 6.27 (bs, 1H, NH2), 5.37 (d, J ) 4.4, 1H, but1-OH), 4.62 (bs, 1H, but-1H), 3.3 (m, 2H, but-4H), 1.56-1.7 (m, 3H, but-2H + but-3Ha), 1.45-1.51 (m, 1H, but-3Hb). UV (50% CH3OH:H2O): λmax 205, 250 nm. MS (positive ion ESI): m/z (relative intensity) 601 (2M + H, 38), 359 (M + 59, 16), 301



Scheme 2. Synthesis of 22

(MH+, 100). ESI-MS/MS of m/z 301; m/z 283 (100), 132 (10). HRMS calcd for C14H17O2N6 (M + H): 301.1408; found, 301.1392. Reactions. Compound 3 and dGuo. Compound 3 (0.25 mmol) was allowed to react with dGuo (0.05 mmol) in 5 mL of 0.1 M phosphate buffer, pH 7.0, in the presence of esterase (90 units) at 37 °C for 1 h. The reaction mixture was extracted with an equal volume of CHCl3. The aqueous phase was analyzed using HPLC system 1, with detection by UV or ESI-MS. Initial results indicated that there were several peaks of interest in the region from 60 to 95 min. Hence, the fraction eluting from 60 to 95 min was collected and analyzed in detail by LC/ESIMS. Compound 3 and DNA. Compound 3 (50, 100, 200 µM or 50 mM) was allowed to react with calf thymus DNA (20 mg) in 10 mL of 0.1 M phosphate buffer, pH 7.0, in the presence of esterase (500 units) at 37 °C for 90 min. The reaction mixture was extracted with an equal volume of CHCl3. The modified DNA was precipitated by addition of ethanol and saturated aqueous NaCl and then washed sequentially with 70 and 100% ethanol. For neutral thermal hydrolysis, the modified DNA (5.0 mg) was dissolved in 1 mL of 40 mM ammonium acetate buffer, pH 6.5. The solution was heated at 100 °C for 1 h. For acid hydrolysis, the modified DNA (5.0 mg) was heated with 5 mL of 0.1 N HCl at 85 °C for 1 h as described (21). Enzyme hydrolysis of DNA was carried out by two methods. In method 1, modified DNA (2.5 mg) was dissolved in 1 mL of 10 mM TrisHCl/5mM MgCl2 buffer, pH 7.0. The mixture was incubated at 37 °C for 16 h with DNase I, phosphodiesterase I, and alkaline phosphatase as described (21). The hydrolysate was further purified by solid phase extraction on a C18 Sep-pak cartridge (Waters Corp.). The products were eluted with H2O:CH3OH (1: 1) and analyzed by LC/ESI-MS using HPLC system 1 or 4. In method 2, the modified DNA (2.5 mg) was dissolved in 1 mL of 10 mM Tris-HCl/15 mM MgCl2 buffer, pH 7.0. The mixture was incubated at 37 °C for 5 h with micrococcal nuclease (55 units) and phosphodiesterase II (1 unit) in 10 mM sodium succinate, 5 mM CaCl2, pH 6, followed by addition of alkaline phosphatase (375 units) and incubation for an additional 17 h. Products were analyzed as above (22).

Results On the basis of literature precedent, we expected that pyridylhydroxybutylation of dGuo would occur at the N2-, O6-, and 7-positions (23). Therefore, we synthesized appropriate standards. Compounds 17 and 19 were prepared by NaBH4 reduction of the known adducts O6pyridyloxobutyl-dGuo (11) and N2-pyridyloxobutyl-dGuo, respectively. Adducts 17 and 19 were converted to the

corresponding Gua derivatives 18 and 20 by mild acid hydrolysis. Compound 22 was prepared from tosylate 23 as shown in Scheme 2. No attempt was made to synthesize 21, because of its predicted instability.

The structures of adducts 17-20 and 22 were confirmed by their UV, MS, and NMR spectra. The UV spectra are presented in Figure 1. 1H NMR data are summarized in Table 1. Assignments were confirmed by COSY. The protons attached to carbon 4 of the pyridylhydroxybutyl group resonated at 4.3-4.4 ppm in the O6pyridylhydroxybutyl derivatives 17 and 18 and at 3.33.4 ppm in the N-substituted adducts 19, 20, and 22. In the case of 7-pyridyloxobutyl-Gua (26), obtained as shown in Scheme 2, pyridyloxobutylation of 24 could have occurred at the 3-position or after it was depurinated, at the 9-position of the resulting Gua. These possibilities were excluded by HMBC (24) of 26, which showed connectivity between the protons on the 4-position of the pyridyloxobutyl group and the C-5 and C-8 of Gua, as illustrated below. These spectral data, when considered together with the methods of synthesis, confirm the assigned structures of adducts 17-20 and 22. The reaction of 3 and dGuo was initially investigated by HPLC analysis with UV detection, producing the chromatogram shown in Figure 2. Further information about the identity of each peak was sought using LC/ESI-MS. Selected ion monitoring of m/z

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Figure 1. UV spectra of adducts 17-20 and 22.

Figure 2. Chromatogram obtained upon HPLC analysis of products formed in the reaction of 3 with dGuo. UV detector sensitivity is 32 times greater in the region from 70 to 95 min.

417 (M+ of 7-pyridylhydroxybutyl-dGuo (21) and M + 1 of adducts 17 and 19) was used to identify peaks corresponding to pyridylhydroxybutyl-dGuo. Peak numbers 9, 12, 14a,b, 16, 17, and 18a,b had m/z 417. Peaks 14a,b and 18a,b were separated only with new HPLC columns. After use, 14a,b was seen only as one peak, 14, and 18a,b was seen only as one peak, 18. Peak 1 was unreacted dGuo. Peak 4 was identified as diol 16 (Scheme 1), the major solvolysis product of 3. None of the other

peaks had MS data consistent with pyridylhydroxybutyldGuo. The compound giving rise to peak 9 of Figure 2 was collected, and its identity was investigated by LC/ESIMS and LC/ESI-MS/MS, before and after neutral thermal hydrolysis (100 °C, pH 6.5). The LC/ESI-MS results are presented in Figure 3. Panels A, C, and E show LC/ESIMS traces obtained by selected ion monitoring of m/z 417, 301, and 168, respectively, before neutral thermal hydrolysis while panels B, D, and F show traces from selected ion monitoring of the same ions after neutral thermal hydrolysis. Panels A and B demonstrate that the peak with m/z 417 (corresponding to peak 9 of Figure 2) disappears upon neutral thermal hydrolysis. Panel C shows that this adduct has an MS fragment at m/z 301 (the unshaded peak), and that another peak (shaded) with m/z 301 is also present. Panel D shows that the latter increases upon neutral thermal hydrolysis, while the former disappears. Panel E demonstrates that diol 16, with an M + 1 of m/z 168, is present in this mixture, presumably due to partial decomposition of 21. This peak increases upon neutral thermal hydrolysis (Panel F). dGuo was also observed as a product of neutral thermal hydrolysis (data not shown). Similar results were obtained upon acid (80 °C, 0.1 N HCl) hydrolysis of the collected material in peak 9. The peak eluting at 35 min in Panel D of Figure 3 was identified as 22 by comparison of its retention time, MS, and MS/MS to that of the standard, as shown in Figure 4. Selected ion monitoring of m/z 301 of standard 22 is shown in Panel A1, and the MS of the standard is illustrated in Panel A3. The MS has a base peak corresponding to M + 1, m/z 301, and prominent peaks corresponding to (2M + H)+ and (2M + Na)+. The MS of the isolated adduct (Figure 4B1,3) is essentially identical to that of the standard. Selected reaction monitoring of m/z 301 f 283 of standard 22 is shown in Figure 4A2, and the daughter ions of m/z 301 (M + H)+ are in Figure 4A4. The daughter ions are rationalized in Scheme 3. MS/



Table 1. 1H NMR Data for Adducts 17-20 and 22a

Pyridyl Protons adduct

2

4

O6-pyridylhydroxybutyl-dGuo

(17)

8.41 (bs)

7.9 (m)

N2-pyridylhydroxybutyl-dGuo

(19)

8.53 (dd) J ) 2.4, 2.4 8.55 (d) J ) 2.4 8.52 (d) J ) 1.65 8.47 (s)

7.73 (d) J ) 8.0 7.74 (dd) J ) 2.4, 2.4 7.72 (d) J ) 7.4 7.65 (d) J ) 8.8

O6-pyridylhydroxybutyl-Gua (18) N2-pyridylhydroxybutyl-Gua (20) 7-pyridylhydroxybutyl-Gua (22)

5

6

7.39 (dd) J ) 5.4, 2.4 7.33 (dd) J ) 4.8, 3.2 7.35 (dd) J ) 8.0, 4.0 7.34 (dd) J ) 7.4, 4.6 7.31 (dd) J ) 8.8, 6.4

8.29 (d) J ) 4.8 8.42 (dd) J ) 4.8, 1.0 8.44 (dd) J ) 1.6, 1.6 8.43 (dd) J ) 1.6, 1.6 8.41 (d) J ) 6.4

Hydroxybutyl Protons adduct

1

2

3

4

OH

O6-pyridylhydroxybutyl-dGuo (17)

4.82-4.77 (m)

1.92-1.87 (m)

N2-pyridylhydroxybutyl-dGuo (19)

1.70-1.66 (m)

4.32 (m) 4.29 (m) 3.27 (m)

5.4 (bs)

O6-pyridylhydroxybutyl-Gua (18)

4.62 (dd) J ) 6.4, 4.8 4.67 (m)

1.75 (m)

N2-pyridylhydroxybutyl-Gua (20)

4.62 (bs)

1.56-1.7 (m)

7-pyridylhydroxybutyl-Gua (22)

4.55 (m)

1.70-1.75 (m) 1.82-1.86 (m)

1.82-1.76 (m) 1.72-1.65 (m) 1.62-1.58 (m) 1.52-1.48 (m) 1.83 (m) 1.72 (m) 1.45-1.51 (m) 1.56-1.7 (m) 1.45-1.50 (m) 1.51-1.55 (m)

4.4 (m) 3.3 (m) 3.41 (t) J)6

5.41 (d) J ) 4.0 5.37 (d) J ) 4.4 5.35 (s)

dGuo or Gua Protons adduct O6-pyridylhydroxybutyl-dGuo (17) N2-pyridylhydroxybutyl-dGuo (19) O6-pyridylhydroxybutyl-Gua (18) N2-pyridylhydroxybutyl-Gua (20) 7-pyridylhydroxybutyl-Gua (22)

1

2-NH2 or 2-NH

8

10.3 (s) 10.7 (bs)

NOb 6.96 (bs) 6.19 (s) 6.27 (bs) 6.05 (s)

7.9 (s) 7.86 (s) 7.78 (s) 7.6 (s) 7.88 (s)

NO

9

12.4 (s) NO

2′-Deoxyribose Protons adduct

1′

2′

3′

4′

5′

O6-pyridylhydroxybutyl-dGuo (17)

6.2 (dd) J ) 8.4, 7.2

2.69 (m) 2.34 (m)

4.48-4.50 (m)

3.99-4.01 (m)

N2-pyridylhydroxybutyl-dGuo (19)

6.13 (dd) J ) 7.2, 6.4

2.62 (m) 2.17 (m)

4.35 (m)

3.8 (m)

3.68 (dd) J ) 7.8, 5.6 3.63 (m) 3.56 (dd) J ) 6.4, 5.6 3.49 (dd) J ) 6.4, 5.6

a Spectra determined at 800 MHz. Values are chemical shifts (ppm) and coupling constants (MHz). All spectra measured in DMSO-d 6 except 17 (D2O). b NO, not observed due to exchange or broadening.

MS of m/z 301 of the isolated adduct (Figure 4B2,4) was essentially identical to that of the standard. These results demonstrate that the material with m/z 417, eluting at 70 min in Figure 2 (peak 9) and 30.6 min in Figure 3A, is 21 while the shaded peaks in Figure 3C,D are 22. The results demonstrate that adduct 21 is thermally unstable and is converted to dGuo, 16, and 22 (Scheme 4). Some of this occurs during manipulations of 21, prior to neutral thermal hydrolysis. Neutral thermal and acid hydrolysis converted 21 to 16 and 22, but the relative proportions of each have not been determined. The retention time of peak 14 of Figure 2 was identical to that of 19. Figures 5A1,3 show that the LC/ESI-MS of standard adduct 19 was essentially the same as that of peak 14 (Figure 5B1,3). MS/MS of the M + H ion, m/z 417 gave predominantly m/z 301 (loss of 2′-deoxyribose and protonation), as shown in Figure 5A2,4. Essentially

identical results were obtained upon MS/MS analysis of collected peak 14 (Figure 5B2,4). Treatment of the material represented by peak 14 with 0.1 N HCl, 85 °C, converted it to the corresponding Gua derivative, which had an HPLC retention time identical to that of synthetic adduct 20. These results demonstrate that the compound giving rise to peak 14 of Figure 2 is 19. Peak 18 was similarly identified as 17 by comparison of its HPLC retention time, ESI-MS, and ESI-MS/MS to those of standard adduct 17 and by hydrolysis with acid and comparison of the retention time of the resulting product to that of adduct 18. LC/ESI-MS and LC/ESI-MS/MS were used to analyze enzyme, acid, and neutral thermal hydrolysates of DNA that had been allowed to react with 3. The results were similar to those shown for the reactions with dGuo. In enzyme hydrolysates, adducts 17 and 19 were identified

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Figure 3. LC/ESI-MS analysis of 21 and 22 formed in the reaction of 3 with dGuo before (panels A, C, E) and after (panels B, D, F) neutral thermal hydrolysis. Selected ion monitoring for m/z 417, the molecular ion of 21 (panels A and B); m/z 301, M + H of 22 (panels C and D); and m/z 168, M + 1 of diol 16 (panels E and F). Shaded peaks correspond to 21, 22, and 16. “Increased” means that the relative abundance of the shaded peak increased after neutral thermal hydrolysis.

Scheme 3. MS/MS Fragmentation of m/z 301 of 22

demonstrate the presence of adducts 17, 19, and 21 in DNA reacted with 3. When DNA that had been allowed to react with 50 mM 3 was enzymatically hydrolyzed using method 1, 19 (750 pmol/mg DNA) and 17 (178 pmol/mg DNA) were detected. Compound 21 was not observed. When the hydrolysate was heated at 100 °C for 1 h, 22 (38 pmol/mg DNA) was detected. When the DNA was directly heated at 100 °C for 1 h, the amount of 22 released was 69 pmol/mg DNA. When DNA was hydrolyzed enzymatically by method 2, 17 was quantified as follows (concentration of 3, adduct level in pmol/mg): 50 µM, 0.16; 100 µM, 0.35; 200 µM, 0.83; 50 mM, 229. Adducts 19 and 21 were not observed in these hydrolysates.

Discussion

by comparison of their retention times (by coinjection), ESI-MS, and ESI-MS/MS to those of standards. Acid hydrolysis of this DNA produced compounds that had retention times, ESI-MS, and ESI-MS/MS matching those of standard adducts 18 and 20 (see Supporting Information). Neutral thermal or acid hydrolysis of this DNA produced 22, identified by comparison of its LC/ ESI-MS, ESI-MS/MS, and retention time to those of the standard (Figure 4C). While adduct 21 could not be directly identified in enzyme hydrolysates, these results

The results of this study clearly demonstrate that NNALCH2OAc pyridylhydroxybutylates dGuo at its O6-, N2-, and 7-positions. Compounds 17 and 19 were identified by comparison of their HPLC retention times, LC/ ESI-MS, and LC/ESI-MS/MS properties to those of synthetic standards. Compound 21 was identified on the basis of its conversion to the Gua base 22 upon neutral thermal hydrolysis. The LC/ESI-MS and LC/ESI-MS/MS of this material matched that of synthetic 22. Similarly, 17, 19, and 21 were characterized as products of the reaction of 3 with DNA. These results will be useful in understanding mechanisms of NNAL carcinogenesis. NNAL has been tested for carcinogenicity in rats, mice, and hamsters (1). NNAL and NNK have similar carcinogenic activities in the rat lung (25). NNAL is signifi-



Figure 4. LC/ESI-MS analysis of standard 22 (panel A), material isolated from neutral thermal hydrolysates of the reaction of 3 with dGuo (panel B), and material isolated from neutral thermal hydrolysates of the reaction of 3 with DNA (panel C). In each panel, part 1 is selected ion monitoring of m/z 301, M + H of 22; part 2 is selected reaction monitoring of m/z 301 f 283; part 3 is the MS; and part 4 is the daughter ion spectrum of m/z 301.

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Figure 5. LC/ESI-MS analysis of standard 19 (panel A) and material isolated from the reaction of 3 with dGuo (panel B). In each panel, part 1 is selected ion monitoring of m/z 417, M + H of 19; part 2 is selected reaction monitoring of m/z 417 f 301; part 3 is the MS; and part 4 is the daughter ion spectrum of m/z 417.

Scheme 4. Hydrolysis of 21

cantly more active than NNK as a pancreatic carcinogen in rats, while it is a less effective carcinogen than NNK in rat liver and nasal mucosa (25). Racemic NNAL has approximately 30-70% as much activity as NNK for lung tumor induction in A/J mice (1). (S)-NNAL is equally as tumorigenic as NNK in A/J mouse lung while (R)-NNAL is less tumorigenic than NNK (26). Administration of

NNAL to hamsters did not result in tumors, although it did enhance pancreatic adenocarcinoma induced by N-nitrosobis-(2-oxopropyl)amine (27). NNK is extensively converted to NNAL in several types of organ culture systems and in vivo (1), but the role of NNAL in the carcinogenicity of NNK is not clear. NNAL is metabolically activated by R-hydroxylation in rodent


and human tissues, but it is also reconverted to NNK, which can then undergo metabolic activation. The relative importance of direct metabolic activation of NNAL vs conversion to NNK in NNAL/NNK carcinogenesis has been discussed (28, 29). Investigation of NNAL-DNA adducts in vivo would be one approach to determining mechanisms of NNAL carcinogenesis. There has only been one study of NNAL-DNA binding reported (14). In that study, levels of NNAL and NNK DNA adducts were compared in rat tissues after a single s.c. dose of each nitrosamine. There were comparable levels of 13 and 14 in liver DNA while the amounts of 13 and 14 in nasal mucosa and lung DNA were somewhat less after administration of NNAL than of NNK. NNAL and NNK both produced HPB-releasing DNA adducts in rat liver, indicating that DNA adduction by NNAL resulted, at least in part, from conversion to NNK. The adducts identified in the present study would be specifically formed by metabolic R-methyl hydroxylation of NNAL. Thus, investigations of their formation in vivo would allow differentiation of adducts resulting from NNAL and NNK and therefore could resolve some questions about mechanisms of carcinogenesis by NNAL and NNK. A considerable body of evidence demonstrates the presence of NNAL in the urine and blood of humans exposed to tobacco products (30). NNAL has been detected in smokers, snuff dippers, and tobacco chewers, and nonsmokers involuntarily exposed to tobacco products. Studies with human liver microsomes demonstrate that NNAL undergoes R-hydroxylation and reconversion to NNK, but there are no data available on the formation of specific NNAL-DNA adducts in humans (16, 17). Highly sensitive analytical methods will be necessary to investigate the presence of these adducts in humans. The adducts identified here all have a free hydroxyl group that can be derivatized as a key step in the development of such methods. We are currently investigating the use of accelerator mass spectrometry for detection of adduct 17 in human tissues. LC/ESI-MS was critical for detecting the adducts described here. Many studies of DNA adduct formation by alkylating agents have used HPLC with UV detection, which is particularly useful when the solvolysis products of the alkylating agents do not absorb in the UV. However, in the case of 3, the solvolysis products all contain a pyridine ring, which has UV properties similar to those of dGuo and other DNA nucleobases. Yields of adducts in these reactions are generally quite low. Therefore, even minor solvolysis products of 3 interfere with detection of adducts by HPLC-UV. The resulting chromatograms are complex, as illustrated in Figure 2. In contrast, the selectivity of LC/ESI-MS and LC/ESIMS/MS leads to clean chromatograms, as illustrated in Figures 3-5. Furthermore, these MS techniques provide significant amounts of structural information, particularly when standards are available. Peaks 14a,b and 18a,b of Figure 2 were observed as doublets only when new HPLC columns were used. Peaks 14a,b merged to one peak, 14, and 18a,b merged to one peak, 18, in subsequent analyses. Because NNAL has a chiral center at position 1, we speculate that 14a,b are two diastereomers of 19 while 18a,b are two diastereomers of 17. Further studies are required to investigate the stereochemistry of pyridylhydroxybutylation of dGuo and DNA. These studies will be carried out using enantiomers of 3.


LC/ESI-MS analysis demonstrated the presence of three other peakss12, 16, and 17 of Figure 2swith m/z 417 as products of the reaction of dGuo with 3. These are likely to be pyridylhydroxybutyl-dGuo adducts, but their identities are unknown at present. In addition, we have observed MS peaks corresponding to pyridylhydroxybutylation of other nucleobases in the enzymatic hydrolysates of the DNA reacted with NNALCH2OActreated DNA. The identity of these adducts is currently being investigated in our laboratory. The amount of 19 in enzymatic hydrolysates of DNA that had been reacted with 3 was strongly affected by the hydrolysis conditions. This adduct was not detected by method 2, which uses phosphodiesterase II (5′-exonuclease from bovine spleen) but was the major adduct detected by method 1, which uses phospodiesterase I (3′exonuclease from snake venom). Compound 21 was not detected in enzyme hydrolysates of this DNA, although neutral thermal hydrolysis produced 22. Our previous studies have uncovered the ability of 11 to completely block DNA digestion by phosphodiesterase I, while the activity of phosphodiesterase II was unaffected (31). Recent molecular modeling studies suggest that the blocking effect of 11 is a result of subtle structural changes in the DNA strand induced by the pyridyloxobutyl substituent, including C3′-endo sugar puckering and the loss of stacking interactions between the pyridyloxobutyl-substituted Gua and its flanking bases (32). It is likely that structurally homologous pyridylhydroxybutyl lesions have similar effects on nucleases, which could explain the varied results obtained with different hydrolysis conditions. Our future studies will investigate the use of a combination of several nucleases to completely release NNAL adducts from DNA for quantitative analyses (33). In summary, new dGuo and DNA adducts that could result from metabolic activation of NNAL have been identified in this study. Knowledge of these structures should be useful in further elucidating mechanisms of carcinogenesis by NNAL and NNK and in determining the role of these nitrosamines in human cancer.

Acknowledgment. This study was supported by Grant CA-81301 from the National Cancer Institute. S.S.H. is an American Cancer Society Research Professor, supported by ACS Grant RP-00-138. S.J.S. is supported by a Bowman Cancer Research Fellowship from the American Cancer Society. We thank Edward J. McIntee for his contributions to this study. Supporting Information Available: MS data supporting the identification of adducts 17 and 19 in DNA reacted with 3. This material is available free of charge via the Internet at http://pubs.acs.org.

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(32)

(33)

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TX0256376

Identification of Adducts Produced by the Reaction of 4

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