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Institute of EnVironmental and Occupational Toxicology, Casella Postale 108, CH-6780 Airolo, Switzerland,. Walther-Straub-Institut für Pharmakologie ...
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Chem. Res. Toxicol. 2006, 19, 1611-1618

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Biomarkers for Isocyanate Exposure: Synthesis of Isocyanate DNA Adducts Armin Beyerbach,†,‡,§ Peter B. Farmer,§ and Gabriele Sabbioni*,†,‡ Institute of EnVironmental and Occupational Toxicology, Casella Postale 108, CH-6780 Airolo, Switzerland, Walther-Straub-Institut fu¨r Pharmakologie und Toxikologie, Ludwig-Maximilians-UniVersita¨t Mu¨nchen, Nussbaumstrasse 26, D-80336 Mu¨nchen, Germany, and Cancer Biomarkers and PreVention Group, Biocentre, UniVersity of Leicester, UniVersity Road, Leicester LE1 7RH, United Kingdom ReceiVed April 23, 2006

Isocyanates are important intermediates in industrial manufacturing. DNA adducts and protein adducts are important tools to biomonitor people exposed to xenobiotics. In the present work, the formation of DNA adducts deriving from 4-chlorophenyl isocyanate (4CPI) and 4-methylphenyl isocyanate (4MPI) were explored. The adducts of 4CPI and/or 4MPI with 2′-deoxyadenosine, 2′-deoxyguanosine, and 2′-deoxycytidine were synthesized and characterized by NMR and MS. For low level detection, an LC-MS/MS method was developed. The formation of DNA adducts was confirmed in in Vitro reactions with DNA. Introduction Monoarylisocyanates are important intermediates in the manufacturing of pharmaceuticals and pesticides (1). Isocyanates cause severe irritation to the mucous membranes of the eyes and respiratory tract on inhalation exposure. The main target organ is the lung, where the damage to the bronchi, bronchioles, and alveoli occur, depending on the concentration and duration of exposure (2). The main damage after low levels of isocyanate exposure is among others lung sensitization in the form of asthma. The lung sensitization properties of 4-chlorophenyl isocyanate (4CPI1) have not been investigated (2). The corresponding aromatic amine of 4CPI, 4-chloroaniline, is carcinogenic in animal experiments (3). Arylisocyanates and arylamines can bind with proteins and/or DNA and lead to cytotoxic and genotoxic effects. It has been shown that blood protein adducts are excellent markers of exposure and potential dosimeters for the adducts in the target organ (4). Protein adducts of isocyanates might be involved in the etiology of sensitization reactions (5, 6). To improve the risk assessment for isocyanate exposed * Corresponding author. E-mail: [email protected]. † Institute of Environmental and Occupational Toxicology. ‡ Ludwig-Maximilians-Universita ¨ t Mu¨nchen. § University of Leicester. 1 Abbreviations: 4CPI, chlorophenyl isocyanate; 4MPI, 4-methylphenyl isocyanate, p-tolyl isocyanate; 4Cl-AcdG, N2-(N-p-chlorophenyl-carbamoyl)3′,5′-di-O-acetyl-2′-deoxyguanosine; 4Cl-dG, N2-(N-p-chlorophenyl-carbamoyl)-2′-deoxyguanosine; 4Cl-AcdA, N6-(N-p-chlorophenyl-carbamoyl)3′,5′-di-O-acetyl-2′-deoxyadenosine; 4Cl-dA, N6-(N-p-chlorophenyl-carbamoyl)-2′-deoxyadenosine; 4Cl-AcdC, N4-(N-p-chlorophenyl-carbamoyl)3′,5′-di-O-acetyl-2′-deoxycytidine; 4Cl-dC, N4-(N-p-chlorophenyl-carbamoyl)-2′-deoxycytidine; 4M-AcdG, N2-(N-p-methylphenyl-carbamoyl)-3′,5′di-O-acetyl-2′-deoxyguanosine; 4M-dG, N2-(N-p-methylphenyl-carbamoyl)2′-deoxyguanosine; 4M-AcdA, N6-(N-p-methylphenyl-carbamoyl)-3′,5′-diO-acetyl-2′-deoxyadenosine; 4M-dA, N6-(N-p-methylphenyl-carbamoyl)2′-deoxyadenosine; 4M-AcdC, N4-(N-p-methylphenyl-carbamoyl)-3′,5′-diO-acetyl-2′-deoxycytidine; 4M-dC, N4-(N-p-methylphenyl-carbamoyl)- 2′deoxycytidine; AcdA, 3′,5′-di-O-acetyl-2′-deoxyadenosine; AcdC, 3′,5′-diO-acetyl-2′-deoxycytidine; AcdG, 3′,5′-di-O-acetyl-2′-deoxyguanosine; dG, 2′-deoxyguanosine; dA, 2′-deoxyadenosine; dC, 2′-deoxycytidine; DNA, deoxyribonucleic acid; tR, retention time; NMR, nuclear magnetic resonance; UV, ultraviolet; HPLC, high-performance liquid chromatography; TLC, thin-layer chromatography; FAB/MS, fast atom bombardment mass spectrometry.

workers, it is important to develop dosimeters and to establish whether the toxic reactive intermediate is the isocyanate 4CPI or a metabolite of the corresponding arylamine, 4-chloroaniline, which may be formed from 4CPI. Aromatic amines are metabolized to highly reactive N-hydroxy arylamines (7) by mixed mono-oxygenases. N-hydroxy arylamines can be further metabolized to N-sulfonyloxy arylamines, N-acetoxy arylamines, or N-hydroxy arylamine N-glucuronides. These highly reactive intermediates, which bind covalently to biomolecules, are responsible for the genotoxic and cytotoxic effects of this class of compounds. In contrast to aromatic amines, isocyanates do not need any further activation to react with biomolecules (8). Arylamine-specific hemoglobin adducts are of the sulfinic acid amide type (9, 10). Isocyanate-specific adducts with hemoglobin have been found in the form of the reaction product with the N-terminal amino acid (11, 12, 13). The isocyanate-specific DNA adducts of 4-methylphenyl isocyanate (4MPI) and 4CPI are presently unknown. The arylamine-specific DNA adducts of 4MPI and 4CPI have been synthesized in Vitro (14, 15, 16) but have not been found in ViVo yet (17). DNA adducts of other isocyanates have been synthesized (18). The chemical structure of potential isocyanate adducts present in ViVo is not known. DNA adducts of isocyanates have been synthesized previously with methyl (19, 22), phenyl (20, 22), and 2-naphthyl isocyanate (21). Carbamoylation occurs exclusively at exocyclic amino groups. The level of acylation was 2 orders of magnitude higher at N4 of dC than that at N6 of adenine and 4 orders of magnitude higher than that at N2 of guanine (23). No adduct was detected with dT. Vock et al. (24) developed a 32P-postlabeling method for the detection of DNA adducts formed by 4,4′-methylene diphenyl diisocyanate (MDI). The adducts were analyzed using a two directional thin layer chromatography system (TLC). After incubation of the single 3′-monophosphate-deoxyriboside with MDI, five, seven, eight, and two reaction products were obtained for cytosine (C), adenine (A), guanine (G), and thymine (T), respectively. Incubation of calf thymus DNA with MDI yielded five, two, and one adducts attributable to C, A, and G, respectively. No attempt was made to isolate and characterize the adducts by NMR and MS. An analysis of DNA isolated

10.1021/tx0600853 CCC: $33.50 © 2006 American Chemical Society Published on Web 12/01/2006

1612 Chem. Res. Toxicol., Vol. 19, No. 12, 2006

from the epidermis of rats treated dermally with MDI showed an adduct pattern similar to the one seen in the in Vitro experiment with DNA. A total adduct level of 7 per 108 nucleotides was found. Isocyanates are not only present in the work environment, but they can be metabolically released from other products and lead to carbamoylation of biomolecules. For example, from the antitumor agent sulofenur, 4CPI was metabolically released (25). Carbamoylation reactions by N-methyl-N′-aryl-N-nitrosoureas and their corresponding phenyl isocyanates to the amino group of 5′-amino-5′-deoxythymidine have been kinetically studied in PBS (26). An important vehicle for isocyanates are reaction products with glutathione (27-29). The glutathione adducts release the isocyanate moiety to react with other nucleophiles, for example, proteins. Therefore, it appears that glutathione adducts are responsible for the transport of isocyanate to reactive sites away from the site of isocyanate uptake. Thus, in the present article, the DNA adducts of 4CPI and 4MPI were investigated. The developed methods can be applied to the analysis of other isocyanates that are commercially more important, such as MDI and toluene diisocyanate.

Materials and Methods Caution: 4CPI and 4MPI are skin and mucus membrane irritants and should be handled in a chemical Vapor hood with appropriate protectiVe equipment. Their hydrolysis products are aromatic amines, potential human carcinogens. Chemicals. 4-Chlorophenyl isocyanate (26000), 2′-deoxyguanosine monohydrate (31070), and 4-methylphenyl isocyanate (ptolyl isocyanate) (90125) were obtained from Fluka (Taufkirchen, Germany). Anhydrous pyridine (27, 097-0) was from Aldrich (Taufkirchen, Germany). 2′-Deoxyadenosine (D-7400), calf thymus DNA (D-1501), 2′-deoxycytidine (3897), acid phosphatase (P-3627), alkaline phosphatase (P-5931), and nuclease P1 (N-8630) were purchased from Sigma (Taufkirchen, Germany). Methanol for chromatography (106018) was obtained from Merck (Darmstadt, Germany). Dowex 50-X8 (20-50 U.S. mesh), acetic acid anhydride, dimethylformamide, dichloromethane, and trichloromethane were of highest lab quality. TLC was performed using Merck aluminum sheet silica gel 60 F254. The chromatograms were viewed under UV irradiation at 254 and 366 nm. HPLC instrumentation: Gilson and HP1100 with photodiodearray detector. Column. The samples were analyzed by HPLC on a reversed phase column (Lichrospher 125 × 4 mm, 5 µm, with precolumn, (Merck)) with a linear gradient of 20 min from 10% to 90% methanol in 50 mM ammonium formate buffer (pH 5.5). Mass Spectrometry and NMR. Fast atom bombardment (FAB) mass spectrometry was carried out using a VG 70-SEQ instrument with argon atoms at 8 keV acceleration. For positive FAB, the samples were introduced in a 3-nitrobenzylalcohol (NBA) or glycerol matrix and for negative FAB in an aminopropane diol matrix. Electrospray mass spectra were obtained on a Micromass Quattro BioQ tandem quadrupole mass spectrometer using either a Harvard Apparatus model 22 syringe pump or a Varian 9012 LC pump. For standard characterization, solutions were introduced via loop injection in water/acetonitrile. The dissociation of molecular ions was achieved using cone-induced fragmentation. LC-MS was carried out on the same instrument. In a typical experiment, the column was a Hypersil BDS (150 × 2.1 mm, 3 µm) with a flow rate of 200 µL/minute to the electrospray probe. The solvent system was solvent A, 0.15% acetic acid; solvent B, methanol; and a gradient 55-90% solvent B in 10 min. Selected reaction monitoring was carried out using a collision energy of 30 eV. Nuclear magnetic resonance spectra were recorded on a Bruker 250 MHz spectrometer. The degree of substitution of the C atoms was determined using the distorsionless enhancement by the polarization transfer (DEPT) method.

Beyerbach et al. Synthesis of 3′,5′-Di-O-acetyl-2′-deoxyguanosine (AcdG). 2′Deoxyguanosine monohydrate dG‚H2O (1.58 g, 5.54 mmol) was dissolved in acetic acid anhydride (10 mL), pyridine (10 mL), and dimethylformamide (5 mL). After 12 h of stirring at room temperature no dG was present. The solution was evaporated to dryness and coevaporated with toluene and acetone. Recrystallization of the white solid from ethanol/water ) 9:1 yielded AcdG (1.02 g, 2.9 mmol, 52%) as white crystals. TLC (silica gel, CHCl3/ MeOH ) 9:1): Rf ) 0.18 (dG), 0.83 (AcdG). FAB-MS (glycerol): m/z (%) ) 352 (36, [M + H]+), 336 (29), 152 (100, [MH - (diacetyl + dR)]+), 136 (60). 1H-NMR (250 MHz, [d ]DMSO): δ [ppm] )2.2 (s, 3 H, CH ), 6 3 2.3 (s, 3 H, CH3), 2.6-2.8 (m, 1 H, 2′-H), 3.1-3.25 (m, 1 H, 2′-H), 4.4-4.6 (m, 3 H, 4′-H, 2 × 5′-H), 5.6 (d, 1 H, 3′-H), 6.3 (‘t’, J ) 6. Hz, 1 H, 1′-H), 6.7 (br. s, 2 H, NH2), 8.2 (s, 1 H, 8-H), 10.9 (br. s, 1 H, NH). 13C-NMR (63 MHz, [d ]DMSO): δ [ppm] ) 20.9 (CH ), 21.1 6 3 (CH3), 35.9 (CH2-2′), 64.0 (CH2-5′), 74.8 (CH-3′), 81.9 (CH-1′), 83.0 (CH-4′), 117.2 (C-5), 135.6 (CH-8), 151.5 (C-4), 154.1 (C-2), 157.1 (C-6), 170.4 (CO), 170.5 (CO). Synthesis of 3′,5′-Di-O-acetyl-2′-deoxyadenosine (AcdA). 2′Deoxyadenosine monohydrate dA‚H2O (1 g, 3.73 mmol) was suspended in acetic acid anhydride (10 mL) and pyridine (10 mL). The dA dissolved within seconds. The reaction was monitored by TLC (silica gel, CH2Cl2/MeOH ) 95:5). After 20-30 min, the dA had reacted. The reaction was immediately quenched by the addition of water (50 mL) while vigorously stirring. The aqueous layer was extracted with CH2Cl2 (2 × 50 mL), and the combined extracts were evaporated to dryness, yielding a clear wax. Purification by flash chromatography on silica gel with CH2Cl2/MeOH ) 95:5 yielded AcdA (950 mg, 2.83 mmol, 75%) as a white foam. TLC (silica gel, CH2Cl2/MeOH ) 95:5): Rf ) 0 (dA), 0.31 (AcdA), 0.41 (di-O-Ac-NAc-dA), 0.53 (di-O-Ac-di-N-Ac-dA). 1H-NMR (250 MHz, [d ]DMSO): δ [ppm] )2.02 (s, 3 H, CH ), 6 3 2.10 (s, 3 H, CH3), 2.59 (m, 1 H, 2′-H), 3.15 (m, 1 H, 2′-H), 4.204.35 (m, 3 H, 4′-H, 2 × 5′-H), 5.41 (m, 1 H, 3′-H), 6.38 (‘t’, J ) 6.30 Hz, 1 H, 1′-H), 7.33 (s, 2 H, NH2), 8.18 (s, 1 H, 8-H), 8.35 (s, 1 H, 2-H). 13C-NMR (63 MHz, [d ]DMSO): δ [ppm] ) 20.9 (CH ), 21.1 6 3 (CH3), 35.6 (CH2-2′), 64.0 (CH2-5′), 74.7 (CH-3′), 82.0 (CH-1′), 83.9 (CH-4′), 119.6 (C-5), 139.9 (CH-8), 149.6 (C-4), 153.0 (CH-2), 156.5 (C-6), 170.4 (CO), 170.5 (CO). FAB-MS (NBA): m/z (%) ) 336 (48, [M + H]+), 136 (100, [MH - (diacetyl + dR)]+). Synthesis of 3′,5′-Di-O-acetyl-2′-deoxycytidine (AcdC). dC (2 g, 8.8 mmol) was suspended in acetic acid anhydride (5 mL) and pyridine (5 mL). After stirring for 40 min at room temperature, the reaction was complete. The clear solution was evaporated to dryness and coevaporated with toluene. The resulting N,3′,5′-triacetyl-2′-deoxycytidine was dissolved in CHCl3/MeOH ) 65:35, and 5 N hydrochloric acid (2.5 mL) was added. When only a faint spot of triacetylcytidine and a major spot of AcdC was detectable by TLC, the reaction mixture was evaporated to dryness in Vacuo. Repeated coevaporation with ethanol-toluene was carried out. The resulting white solid was purified by flash chromatography on silica gel with CHCl3/MeOH ) 85:15 yielding pure AcdC (700 mg, 2.25 mmol, 25%). TLC (silica gel, CHCl3/MeOH ) 8:2): Rf ) 0.05 (dC), 0.51 (AcdC), 0.74 (NAc-AcdC). FAB-MS (NBA): m/z (%) ) 623 (17, [2M + H]+), 312 (34, [M + H]+), 113 (23), 112 (100, [MH - (diacetyl + dR)]+). 13C-NMR (63 MHz, [d ]DMSO): δ [ppm] ) 20.9 (CH ), 21.1 6 3 (CH3), 36.9 (CH2-2′), 64.1 (CH2-5′), 74.6 (CH-3′), 81.5 (CH-1′), 85.6 (CH-4′), 94.9 (CH-5), 141.1 (CH-4), 155.3 (C-6), 166.0 (C-2), 170.4 (CO), 170.5 (CO). Reactions with Deoxynucleosides and DNA. General Procedure. All reactions with isocyanates were performed under argon. Isocyanate (0.75 mmol) and acetylated 2′-deoxynucleoside (0.37 mmol) were dissolved in anhydrous pyridine (3 mL). After 2-3 h of heating at 100 °C, all of the nucleoside had reacted and, the solution was evaporated to dryness. After coevaporation with

DNA Adducts of Isocyanates

Chem. Res. Toxicol., Vol. 19, No. 12, 2006 1613 Table 1. ESI-MS Spectra Obtained in the Positive and Ionization Mode for 4Cl-dC

isotope 35Cl/37Cl isotope 35Cl/37Cl isotope 35Cl/37Cl a

[M + Na]+ 403/405 [M + H]+ 381/383 [M - H]379/381

[(M - R) + Na]+ 287/289 [(M - dR) + H]+ 265/267 [(M -d R) - H]262/264

[dC + Na]+ 250 [dC + H]+ 228 nda [dC - H]226

[4CPI + Na]+ 176/178 [4CPI + H]+ 154nd/156nd

[dR + Na]+ 139 dR + H 117 nd

[cytosine + Na]+ 134 [cytosine + H]+ 112

nd ) not detected.

toluene, a gray solid was obtained. The solid was recrystallized from MeOH. The precipitate was filtered off with a glass frit and dried. Further purification by flash chromatography with CHCl3/ MeOH ) 9:1 yielded the O-acetylated isocyanate-nucleoside adduct (yield 32-89%). Deprotection. The solid was taken up in 4 M methanolic ammonia (20 mL), and the mixture was stirred for 14 h at room temperature. The solvents were removed, and the resulting white solid was thoroughly coevaporated with methanol, yielding the isocyanate nucleoside adduct (32-89%) in the form of white crystals. Synthesis of N2-(N-p-Chlorophenyl-carbamoyl)-2′-deoxyguanosine (4Cl-dG). 4CPI (115 mg, 0.75 mmol) and AcdG (131 mg, 0.373 mmol) in anhydrous pyridine (3 mL) were heated for 2-3 h at 90-100 °C, then evaporated, and coevaporated with toluene. The solid was recrystallized from MeOH filtration with a glass frit and purified by flash chromatography with CHCl3/MeOH ) 9:1, yielding 4Cl-AcdG (65 mg, 0.129 mmol, 35%). Deprotection. The solid was taken up in 4 M methanolic ammonia (20 mL), and the mixture was stirred for 14 h at room temperature. The solvents were removed, and the resulting white solid was thoroughly coevaporated with methanol, yielding 4Cl-dG (50 mg, 0.119 mmol, 32%) in the form of white crystals. 4Cl-AcdG. TLC (silica gel, CHCl3/MeOH ) 9:1): Rf ) 0.25. 4Cl-dG. TLC (silica gel, CHCl3/MeOH ) 8:2): rf) 0.10. 4Cl-AcdG. FAB-MS (NBA): m/z (%) ) 507, 37Cl, (23, [M + H]+); 505, 35Cl, (67, [M + H]+); 307, 37Cl, [43, MH - (diacetyl + dR)]+; 305, 35Cl, [100, MH - (diacetyl + dR)]+). Because of matrix peaks, relative intensities are reported only for masses greater than m/z 250. 4Cl-dG. FAB-MS (glycerol): m/z (%) ) 423, 37Cl, (14, [M + + H] ); 421, 35Cl, (35, [M + H]+); 307, 37Cl (37, [MH - dR]+; 305, 35Cl, (100, [MH - dR]+). Because of matrix peaks, relative intensities are reported only for masses greater than m/z 250. 4Cl-dG. ESI-MS: 445, 37Cl, [M + Na]+; 443, 35Cl, [M + Na]+; 423, 37Cl, [M + H]+; 421, 35Cl, [M + H]+; 329, 37Cl, [(M - dR) + Na]+; 327, 35Cl, [(M - dR) + Na]+; 307, 37Cl, [(M - dR) + H]+; 305, 35Cl, [(M - dR) + H]+; 200, [(guanine + CO) + Na]+;178, [(guanine + CO) + H]+; 174, [guanine + Na]+; 152, [guanine + H]+. 4Cl-dG. 1H-NMR (250 MHz, [d6]DMSO): δ [ppm] ) 2.272.34 (m, 1 H, 2′-H), 2.54-2.65 (m, 1 H, 2′-H), 3.53-3.62 (m, 2 H, 2 × 5′-H), 3.89 (m, 1 H, 4′-H), 4.46 (m, 1 H, 3′-H), 5.03 (br. s, 1 H, OH), 5.44 (br. s, 1 H, OH), 6.24 (‘t’, J ) 6.7 Hz, 1 H, 1′-H), 6.71 (br. s, 1 H, NH), 7.38 (d, J ) 8.7 Hz, 2 H, arom. 2-H, 6-H), 7.58 (d, J ) 8.7 Hz, 2 H, arom. 3-H, 5-H), 8.13 (s, 1 H, 8-H), 10.58 (br. s, 1 H, NH), 11.25 (br. s, 1 H, NH). 4Cl-dG. 13C-NMR (63 MHz, [d6]DMSO): δ [ppm] ) 40.2 (CH2-2′), 62.0 (CH2-5′), 71.1 (CH-3′), 83.3 (CH-1′), 88.1 (CH-4′), 119.8 (C-5), 120.7 (arom. CH-2, CH-6), 127.0 (arom. C-4), 129.2 (arom. CH-3, CH-5), 136.9 (CH-8), 137.8 (arom. C-1), 149.3, 150.2, 153.3 (C-4, C-2, CO, C-6, one signal is missing). 4Cl-dG. UV (HPLC-run: MeOH/ammonium formate pH 5.5): λ nm (E): 259 max (0.610, 100%), 277 shoulder (0.485, 80%) Synthesis of N6-(N-p-Chlorophenyl-carbamoyl)-2′-deoxyadenosine (4Cl-dA). The solid was recrystallized from MeOH filtration with a glass frit and purified by flash chromatography on silica gel with CHCl3/MeOH ) 9:1. The resulting 4Cl-AcdA (150 mg) was stirred in methanol with potassium carbonate for 12 h and then evaporated to dryness. Purification with flash chromatography on silica gel with CHCl3/MeOH ) 85:15 yielded 4Cl-dA (160 mg, 0.333 mmol, 89%).

TLC (silica gel, CHCl3/MeOH ) 9:1): Rf ) 0.51 (4Cl-AcdA), rf ) 0.13 (4Cl-dA). 4Cl-AcdA. FAB-MS (NBA): m/z (%) ) 491, 37Cl, (25, [M + H]+); 489, 35Cl, (62, [M + H]+); 291, 37Cl, (31, [MH - (diacetyl + dR)]+); 289, 35Cl, (100, [MH - (diacetyl + dR)]+). 4Cl-dA. FAB-MS (glycerol): m/z (%) ) 407, 37Cl (3, [M + H]+); 405, 35Cl (7, [M + H]+); 291, 37Cl (20, [MH - dR]+); 289, 35Cl (55, [MH - dR]+); 162 (42); 136 (100, [adenine + H]+); 115 (40, [dR]+). 4Cl-dA. ESI-MS: 429, 37Cl, [M + Na]+; 427, 35Cl, [M + Na]+; 313, 37Cl, [(M - dR) + Na]+; 311, 35Cl, [(M - dR) + Na]+; 184, [(adenine+CO)+Na]+; 158, [adenine+Na]+. 4Cl-dA. 1H-NMR (250 MHz, [d6]DMSO): δ [ppm] ) 2.342.41 (m, 1 H, 2′-H), 2.68-2.79 (m, 1′ H, 2′-H), signal for 5′-H hidden by HOD signal (3.3-3.8), 3.93 (m, 1 H, 4′-H), 4.47 (m, 1 H, 3′-H), 5.18 (br. s, 1 H, OH), 5.55 (br. s, 1 H, OH), 6.45 (‘t’, J ) 6.7 Hz, 1 H, 1′-H), 7.39 (d, J ) 7.5 Hz, 2 H, arom 2-H, 6-H), 7.50 (d, J ) 7.5 Hz, 2 H, arom, 3-H, 5-H), 8.67 (s, 1 H, 2-H or 8-H) 8.69 (s, 1 H, 2-H or 8-H), 10.28 (br. s, 1 H, NH), 11.83 (br. s, 1 H, NH). 4Cl-dA. 13C-NMR (63 MHz, [d6]DMSO): δ [ppm] ) 39.8 (CH2-2′), 62.4 (CH2-5′), 71.6 (CH-3′), 84.7 (CH-1′), 88.9 (CH-4′), 121.6 (arom CH-2, CH-6), (signal for C-5 at ca. 121.0 is missing, probably covered by the former peak or broadened), 127.4 (arom C-4), 129.5 (arom C-3, C-5), 138.5 (arom C-1), 143.3 (CH-8), 151.2 (CH-2), 150.3, 150.8, 151.9 (C-4, C-6 and CO). 4Cl-dA. UV (HPLC-run: MeOH/ammonium formate pH 5.5): λ nm (E): 239max (0.077, 25%), 250min (0.154, 49%), 281max (0.314, 100%). Synthesis of N4-(N-p-Chlorophenyl-carbamoyl)-2′-deoxycytidine (4Cl-dC). The solid was taken up in CHCl3/MeOH ) 1:1 (10 mL) and 5 M sodium hydroxide (250 µL, 10 drops from a Pasteur pipet) was added, and after 5 min, the solution was neutralized with Dowex 50-X8 (20-50 U.S. mesh) [H+ - form]. The Dowex was filtered off, and the solution was evaporated in Vacuo yielding a white solid (160 mg), which was purified by flash chromatography on silica gel with CHCl3/MeOH ) 85:15, yielding 4Cl-dC (68 mg, 0.179 mmol, 48%) in the form of white crystals. TLC (silica gel, CHCl3/MeOH ) 9:1): Rf ) 0.69 (4Cl-AcdC), rf ) 0.11 (4Cl-dC). 4Cl-dC. FAB-MS (NBA): m/z (%) ) 383, 37Cl, (8, [M + H]+); 381, 35Cl (23, [M + H]+); 267, 37Cl, (13, [MH - dR]+); 265, 35Cl, (38, [MH - dR]+); 118 (100, [dR]+). 4Cl-dC. ESI-MS: see Table 1. 4Cl-dC. 1H-NMR (250 MHz, [d6]DMSO): δ [ppm] )2.012.11 (m, 1 H, 2′-H), 2.27-2.36 (m, 1 H, 2′-H), 3.50-3.72 (m, 2 H, 2 × 5′-H), 3.88 (m, 1 H, 4′-H), 4.26 (m, 1 H, 3′-H), 5.12 (dd, J ) 4.7 Hz, J ) 4.8 Hz, 1 H, OH), 5.33 (d, J ) 3.9, 1 H, OH), 6.16 (‘t’, J ) 6.3 Hz, 1 H, 1′-H), 6.58 (d, J ) 6.9 Hz, 1 H, 5-H), 7.38 (d, J ) 8.8 Hz, 2 H, arom. 2-H, 6-H), 7.54 (d, J ) 8.8 Hz, 2 H, arom. 3-H, 5-H), 8.30 (d, J ) 7.4 Hz, 1 H, 6-H), 10.32 (br. s, 1 H, NH), 11.47 (br. s, 1 H, NH). 4Cl-dC. 13C-NMR (63 MHz, [d6]DMSO): δ [ppm] ) 41.1 (CH2-2′), 61.3 (CH2-5′), 70.3 (CH-3′), 86.5 (CH-1′), 88.3 (CH-4′), 95.3 (CH-5), 121.0 (arom. CH-2, CH-6), 127.2 (arom. C-4), 129.2 (arom. CH-3, CH-5), 137.6 (arom. C-1), 144.5 (CH-6), 151.6 (CO), 153.9 (C-2), 162.6 (C-4). 4Cl-dC. UV (HPLC-run: MeOH/ammonium formate pH 5.5): λ nm (E): 232max (0.503, 100%), 262min (0.292, 58%), 293max (0.379, 75%). Synthesis of N2-(N-p-Methylphenyl-carbamoyl)-2′-deoxyguanosine (4M-dG). 4MPI (99 mg, 0.746 mmol) + AcdG (131 mg,

1614 Chem. Res. Toxicol., Vol. 19, No. 12, 2006 0.37 mmol) were kept in anhydrous pyridine (3 mL). After 6 h at 100 °C, they were evaporated to dryness and coevaporated with toluene, and 235 mg of a white solid was obtained. Purification was by flash chromatography on silica gel with CHCl3/MeOH ) 9:1, yielding 4M-AcdG (90 mg, 0.204 mmol, 55%) as a white solid. This was suspended in CHCl3/MeOH ) 1:1 (10 mL), 5 M sodium hydroxide (250 µL, 10 drops from a Pasteur pipet) was added, and after 5 min, the solution was neutralized with Dowex 50-X8 (20-50 U.S. mesh) [H+ - form]. The Dowex was filtered off, washed with 10% formic acid (30 mL), and the solution was evaporated in Vacuo, yielding a white solid, which was pure 4M-dG (72 mg, 0.18 mmol, 49%) in the form of white crystals. 4M-AcdG. TLC (silica gel, CHCl3/MeOH ) 9:1): Rf ) 0.33. 4M-dG. TLC (silica gel, CHCl3/MeOH ) 8:2): Rf ) 0. 4M-AcdG. FAB-MS (NBA): m/z (%) ) 529 (43, [MH + 2Na]+), 507 (84, [MH + Na]+), 485 (42, [M + H]+), 307 (98, [MH + Na - (diacetyl + dR)]+), 285 (100, [MH - (diacetyl + dR)]+). 4M-dG. FAB-MS (glycerol): m/z (%) ) 401 (39, [M + H]+), 285 (100, [M - dR]+), 115 (43, [dR]+). 4M-dG. ESI-MS: m/z) 423 [M + Na]+, 401 [M + H]+, 316 [(dG + CO) + Na]+, 307 [(M - dR) + Na]+, 294 [(dG + CO) + H]+, 285 [(M - dR)] + H]+, 200 [(guanine + CO) + Na]+, 178 [(guanine + CO) + H]+, 174 [guanine + Na]+, 152 [guanine + H]+, 139 [dR + Na]+, 117 [dR + H]+. 4M-dG. 1H-NMR (250 MHz, [d6]DMSO): δ [ppm] ) 2.28 (s, 3 H, CH3), 2.28-2.33 (m, 1 H, 2′-H), 2.56-2.66 (m, 1 H, 2′-H), 3.52-3.63 (m, 2 H, 2 × 5′-H), 3.87 (m, 1 H, 4′-H), 4.39 (m, 1 H, 3′-H), 4.97 (br. s, 1 H, OH), 5.33 (br. s, 1 H, OH), 6.22 (‘t’, J ) 6.6 Hz, 1 H, 1′-H), 7.16 (d, J ) 8.2 Hz, 2 H, arom 2-H, 6-H), 7.39 (d, J ) 8.31 Hz, 2 H, arom. 3-H, 5-H), 8.17 (s, 1 H, 8-H), 9.69 (br. s, 1 H, NH), 10.25 (br. s, 1 H, NH), two signals for NH are missing. This was also reported for a similar adduct by Tamura et al. (22) . 4M-dG. 13C-NMR (63 MHz, [d6]DMSO): δ [ppm] ) 20.1 (CH3), 40.2 (CH2-2′), 61.9 (CH2-5′), 70.9 (CH-3′), 83.5 (CH-1′), 88.2 (CH-4′), 119.7 (arom. CH-2, CH-6), (117-120: C-5 signal not seen), 129.8 (arom. CH-3, CH-5), 132.9 (arom. C-4), 135.5 (arom. C-1), 137.4 (CH-8), 148.9, 152.9 (C-4, C-2, C-6, CO, between ca. 148-160, two signals are missing because of low signal intensities). 4M-dG. UV (HPLC-run: MeOH/ammonium formate pH 5.5): λ nm (E): 259 max (0.534, 100%), 277 shoulder (0.424, 79%) Synthesis of N6-(N-p-Methylphenyl-carbamoyl)-2′-deoxyadenosine (4M-dA). 4MPI (99 mg, 0.746 mmol) + AcdA (125 mg, 0.37 mmol) were in anhydrous pyridine (3 mL). After 2.5 h at 100 °C, they were evaporated to dryness, coevaporated with toluene, and 218 mg of a white solid was obtained. This was dissolved in CHCl3, and MeOH was added to precipitate the 4M-AcdA. The mixture was centrifuged, and the supernatant was decanted. Again, the white solid was taken up in CHCl3 and precipitated with MeOH. The precipitate was dried thoroughly, yielding 4M-AcdA. The solid was taken up in CHCl3/MeOH ) 1:1 (10 mL), 5 M sodium hydroxide (250 µL, 10 drops from a Pasteur pipet) was added, and after 10 min, the solution was neutralized with Dowex 50-X8 (20-50 U.S. mesh) [H+ - form]. The Dowex was filtered off, and the solution was evaporated in Vacuo yielding a white solid, which was pure 4M-dA (84 mg, 0.218 mmol, 59%) in the form of white crystals. 4M-AcdA. TLC (silica gel, CHCl3/MeOH ) 9:1): Rf ) 0.72. 4M-dA. TLC (silica gel, CHCl3/MeOH ) 8:2): Rf ) 0.37. 4M-AcdA. FAB-MS (NBA): m/z (%) ) 469 (64, [M + H]+), 269 (100, [MH - (diacetyl + dR)]+). 4M-dA. FAB-MS (NBA): m/z (%) ) 385 (55, [M + H]+), 269 (100, [MH - dR]+). 4M-dA. ESI-MS: m/z ) 407 [M + Na]+, 385 [M + H]+, 300 [dA + CO + Na]+, 291 [(M - dR) + Na]+, 269 [(M - dR) + H]+, 184 [(adenine + CO) + Na]+, 162 [(adenine + CO) + H]+, 158 [adenine + Na]+, 139 [dR + Na]+, 136 [adenine + H]+, 117 [dR + H]+.

Beyerbach et al. 4M-dA. 1H-NMR (250 MHz, [d6]DMSO): δ [ppm] ) 2.28 (s, 3 H, CH3), 2.36-2.43 (m, 1 H, 2′-H), 2.75-2.81 (m, 1 H, 2′-H), 3.57-3.66 (m, 2 H, 2 × 5′-H), 3.93 (m, 1 H, 4′-H), 4.47 (m, 1 H, 3′-H), 5.04 (m, J ) 5.5 Hz, 1 H, OH), 5.38 (d, J ) 4.1, 1 H, OH), 6.47 (‘t’, J ) 6.7 Hz, 1 H, 1′-H), 7.16 (d, J ) 8.3 Hz, 2 H, arom 2-H, 6-H), 7.52 (d, J ) 8.3 Hz, 2 H, arom H-3, H-5), 8.69 (s, 1 H, 2-H or 8-H), 8.70 (s, 1 H, 8-H or 2-H), 10.06 (br. s, 1 H, NH), 11.73 (br. s, 1 H, NH). 4M-dA. 13C-NMR (63 MHz, [d6]DMSO): δ [ppm] ) 20.8 (CH3), 40.2 (CH2-2′), 61.9 (CH2-5′), 71.0 (CH-3′), 84.2 (CH-1′), 88.4 (CH-4′), 119.8 (arom. CH-2, CH-6), 120.8 (C-5), 129.7 (arom. CH-3, CH-5), 132.5 (arom. C-4), 136.2 (arom. C-1), 142.7 (CH-8), 151.1 (CH-2), 150.3 150.6 151.3 (C-4, C-6, CO). 4M-dA. UV (HPLC-run: MeOH/ammonium formate pH 5.5): λ nm (E): 236max (0.198), 250min (0.174, 53%), 281max (0.330, 100%). Synthesis of N4-(N-p-Methylphenyl-carbamoyl)-2′-deoxycytidine (4M-dC). 4MPI (98 mg, 0.74 mmol) + AcdC (115 mg, 0.37 mmol) were in anhydrous pyridine (3 mL). After 3 h at 100 °C, they were evaporated to dryness, coevaporated with toluene, and 225 mg of a white solid was obtained. (Using flash chromatography on silica gel with CHCl3/MeOH ) 97:3 no good separation was achieved.) The solid was taken up in CHCl3/MeOH ) 1:1 (10 mL), 5 M sodium hydroxide (250 µL, 10 drops from a Pasteur pipet) was added, and after 5 min, the solution was neutralized with Dowex 50-X8 (20-50 U.S. mesh) [H+ - form]. The Dowex was filtered off, and the solution was evaporated in Vacuo, yielding a white solid (110 mg), which was purified by flash chromatography on silica gel with CHCl3/MeOH ) 85:15, yielding 4M-dC (88 mg, 66%) in the form of white crystals. TLC (silica gel, CHCl3/MeOH ) 9:1): Rf ) 0.70 (4M-AcdC), 0.15 (4M-dC). 4M-dC. FAB-MS (NBA): m/z (%) ) 361 (28, [M + H]+), 245 (100, [MH - dR]+). 4M-dC. ESI-MS: m/z ) 383 [M + Na]+, 361 [M + H]+, 267 [(M - dR) + Na]+, 250 [dC + Na]+, 245 [(M - dR) + H]+, 160 [(cytosine + CO) + Na]+, 134 [cytosine + Na]+, 112 [cytosine + H]+. 4M-dC. 1H-NMR (250 MHz, [d6]DMSO): δ [ppm] )2.012.12 (m, 1 H, 2′-H), 2.26 (s, 3 H, CH3), 2.26-2.36 (m, 1 H, 2′-H), 3.55-3.68 (m, 2 H, 2 × 5′-H), 3.88 (m, 1 H, 4′-H), 4.26 (m, 1 H, 3′-H), 5.09 (br. s, 1 H, OH), 5.30 (br. s, 1 H, OH), 6.16 (‘t’, J ) 6.3 Hz, 1 H, 1′-H), 6.50 (d, J ) 6.9 Hz, 1 H, 5-H), 7.14 (d, J ) 8.2 Hz, 2 H, arom. 2-H, 6-H), 7.39 (d, J ) 8.2 Hz, 2 H, arom. 3-H, 5-H), 8.28 (d, J ) 7.5 Hz, 1 H, 6-H), 10.22 (br. s, 1 H, NH), 11.28 (br. s, 1 H, NH). 4M-dC. 13C-NMR (63 MHz, [d6]DMSO): δ [ppm] ) 22.8 (CH3), 41.1 (CH2-2′), 61.3 (CH2-5′), 70.3 (CH-3′), 86.5 (CH-1′), 88.2 (CH-4′), 95.3 (CH-5), 119.6 (arom. CH-2, CH-6), 129.7 (arom. CH-3, CH-5), 132.7 (arom. C-4), 136.0 (arom. C-1), 144.3 (CH-6), 151.6 (CO), 153.9 (C-2), 162.6 (C-4). 4M-dC. UV (HPLC-run: MeOH/ammonium formate pH 5.5): λ nm (E): 232max (0.528, 100%), 262min (0.261, 49%), 295max (0.403, 76%). Reaction of Isocyanates with DNA. The isocyanate was added to DNA (5 mg) in water (2 mL) and THF (2 mL, HPLC quality). After an incubation time of 2 h at 37 °C, the reaction mixture was extracted with diethyl ether and ethyl acetate (3-4 × 6-8 mL). After centrifugation (3000g), the aqueous solution was transferred with a Gilson pipet into another tube. The solution was buffered with 50 mM Bis-tris and 1 mM MgCl2 at pH 6.5. Per 1 mg of DNA and 1 mL of solution, the following three enzymes were added: 24 units of nuclease P1 (310 units/mg solid in 260 µL of 1 mM ZnCl2 ) 1.2 units/µL), 2.4 units of alkaline phosphatase (50 units in 100 µL of water ) 0.5 units/µL), 0.3 units of acid phosphatase (10 mg of solid (8 units) in 80 µL of water ) 0.1 units/µL). The mixture was incubated for 8 h at 50 °C. After denaturation of the enzymes at 100 °C for 5 min, the enzymes were removed by centrifugation. 4CPI Reaction with DNA. CHCl3 (4 mL) was added, and the mixture was extracted. Centrifugation at 3000g for 5 min yielded

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Figure 1. Reactions of 4CPI and 4MPI with dA, dG, and dC.

Figure 2. MS/MS experiment with 4M-dG, ESI [M + H]+, 70 and 120 eV collision energy.

a clear aqueous and CHCl3 layer. The precipitate sedimented between the two layers. Both layers were evaporated and analyzed by HPLC-UV. The HPLC chromatogram from the CHCl3 extract had no adduct peaks. The HPLC chromatogram from the water layer had 2 peaks with tR ) 11.1 min and tR ) 14.0 min, which belong to 4Cl-dG and 4Cl-dA. The UV spectra confirmed the statement. 4MPI Reaction with DNA. The workup was the same as that for the 4CPI reaction. The HPLC chromatogram from the water layer had 1 adduct peak, which belonged to 4M-dC (tR ) 9.23 min, UV the same as standard). The CHCl3 extract had no adduct peak.

Results and Discussion Adducts of isocyanates, which might be formed in ViVo, were synthesized in Vitro. The single deoxynucleosides were reacted with the isocyanates 4CPI and 4MPI (Figure 1). Isocyanate and the O-acetyl protected nucleoside were heated for 2-3 h in pyridine. The reactions were monitored by TLC. After evaporation of the solvent, the reaction products were recrystallized from methanol. Further purification by flash chromatography yielded the O-acetylated isocyanate-nucleoside adducts. The adducts were obtained in 32-89% yield. Acetyl groups of the

1616 Chem. Res. Toxicol., Vol. 19, No. 12, 2006

Figure 3. MS-fragmentation of the 4M-dA adduct.

nucleosides were cleaved with ammonia, potassium carbonate, or sodium hydroxide. The products of 4CPI and 4MPI with dG, dA, and dC were isolated and characterized. The 4CPI and 4MPI adducts were characterized by NMR and MS. All of the spectroscopic data are listed in the experimental section. The 1H-NMR signals were assigned in most cases according to similar compounds (14, 19, 22, 30, 31) and using increment rules for the prediction of chemical shifts (32, 33). The chemical shift for the protons (1-H′-5′-H) in the sugar moiety were almost identical for all deacetylated adducts. The protons were present as multiplets, except for 1′-H, which appeared as a triplet at 6.2-6.4 ppm. The protons 2-H′ were found between 2.2 and 2.7 ppm, the protons 5′-H were present at 3.5-3.7 ppm, the proton 4′-H was present at 3.8-3.9 ppm, and the proton 3-H′ at 4.4-4.6 ppm. This corresponds to the results for other dG adducts found in the literature (14, 19, 22). The signals of the protons from the nucleic bases did not shift significantly compared to those of the natural deoxynucleosides. For the dG adducts, the 8-H of guanine was found at ca. 8.15 in both adducts. For the dA adducts, the protons 2-H and 8-H were very close, 8.67-8.70, and could not be assigned. These chemical shifts are similar to the results found for the methyl-

Beyerbach et al.

isocyanate and/or phenylisocyanate adducts with AcdA and AcdG (22). For the dC adducts, the protons 5-H and 6-H were at 6.5-6.6 and 8.28-8.3 ppm. Similar chemical shifts were reported in ref 19 for the adduct of methylisocyanate with dC. The protons in the aromatic ring of 4-chloroaniline (4CA) and 4-methylaniline (4MA) were very similar through all adducts. The signals of the aromatic protons of the arylamine moiety were estimated with the increment rules (30, 31) and assigned accordingly. The chemical shift of 2/6-H was at a higher field than that of the 3/5-H protons. The coupling of these aromatic protons should correspond to an AA′XX′-spectrum. Mostly, only 4 lines were visible instead of 20. Only JAX (JA′X′) could be assigned. For 4CA, the chemical shift of 2/6-H was at 7.387.39 and for 3/5-H at 7.50-7.58. The signals in the 4MA were found at 7.14-7.16 for 2/6-H and at 7.39-7.50 for 3/5-H. For the 13C-NMR spectra, the signals of the carbons in the sugar unit were similar for all adducts except for C-1′ in dC adducts, which were shifted to a lower field by 3 ppm in comparison to dG adducts. The signals of the carbons for C-2′, C-5′, C-3′, C-1′, and C-4′ were found at 40.2-41.1, 61.3-62.4, 70.3-71.6, 83.3-86.5, and 88.1-88.9 ppm, respectively. This corresponds to the chemical shifts in unmodified deoxynucleosides (34-36). The chemical shifts of the guanine-carbons in 4Cl-dG and 4M-dG could only be assigned for C-5 and C-8 at 120 and 137, respectively. In comparison to unmodified dG, the signal of C-5 shifted ca. 3 ppm upfield as expected from the transformation of the attached amino group to a urea moiety (increment rules for single substituted aromatic compounds, 34, 35). The chemical shift for C-8 remained the same as that in unmodified dG. The chemical shift of the remaining quaternary carbons were different compared to unmodified dG. The largest shift upfield would be expected for C-2. We assigned C-2 tentatively at 150, which would correspond to a 4 ppm upfield shift in comparison to dG. The carbonyl C-6 shifted upfield by ca. 4 ppm. In the dA adducts, 4Cl-dA and 4M-dA, the signals for adenine could be assigned only for C-5, C8, and C-2. The other signals were too close to each other. The signal for C-8 was shifted ca. 3 ppm downfield and C-6, the site of the amino group that reacts with the isocyanate, ca. 5 ppm upfield in comparison to the unmodified dA. In the dC adducts, 4Cl-dC and 4M-dC, the chemical shift of C-5 was the same as that in dC. The signal of C-6 shifted 3 ppm downfield and C-4, the

Figure 4. LC-MS/MS chromatogramm of 20 fmol of 4M-dA (m/z 385 f 269), 4M-dG, (m/z 401 f 285) 4M-dC (m/z 361 f 245) obtainded by multiple reaction monitoring. The HPLC conditions were Hypersil BDS C18 (3 µm, 150 × 2.1 mm I.D.), methanol in 0.1% acetic acid, at t ) 0-10-11 min, and 55-90-90% MeOH. The lowest trace is the total ion chromatogram (TIC) of the mixture.

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4Cl-dG and the 4Cl-dA were obtained. The reaction of 4MPI with DNA yielded 4M-dC. This is in contrast to other studies. The reaction with 2-naphthyl isocyanate yielded mainly adducts with dC and a very small amount of dA adducts (21). However, no adduction was observed for dG residues of DNA. Methylisocyanate and phenylisocyanate reacted predominantly with dC and to a much lesser degree with dA and dG (22). The application of MDI to rat skin yielded DNA adducts with dA, dG, and dC, which corresponded to the 2D-TLC pattern produced by adducts generated in Vitro (24).

Conclusions New 2′-deoxynucleoside-adducts of 4CPI and 4MPI could be synthesized. The adducts can be detected at low levels by LC-MS/MS. Adducts were isolated from CPI and MPI reacted 2′-deoxynucleosides. Because isocyanates are direct-acting acylating agents, which do not require metabolic activation, the same adducts would be expected to form in ViVo as shown by Vock et al. (24) for MDI applied to rat skin. Figure 5. LC-MS/MS analysis of 4MPI adducts. The average and range of two determinations were plotted. The correlation coefficient was >0.99 for all compounds. The following ions were monitored: 4M-dG, (m/z 401 f 285) 4M-dC (m/z 361 f 245), 4M-dA (m/z 385 f 269).

site of the amino group that reacts with the isocyanate, shifted 3 ppm upfield in comparison to the unmodified dC. C-2 and the CO of the urea could not be unambiguously assigned. The carbon signals of 4CA were assigned according to Mo¨ller et al. (30). The signals of C-2/6, C-4, C-3/5, and C-1 ranged from (0.8, (0.2, (0.15, and (0.45 ppm, respectively. The signals of 4MA were assigned according to Sabbioni et al. (31) and ranged from (0.1, (0.05, (0.2, and (0.35 ppm, respectively. The mass spectrometric properties were investigated with FAB-MS and ESI-MS. The data of the FAB-MS analyses are presented in the Materials and Methods section. The ions [M + H]+ and [(M - dR) + H]+ formed the characteristic MS-pattern of the compounds. The ESI-MS analyses were performed in the positive ionization mode and in a few cases also in the negative ionization mode. A typical fragmentation pattern is listed in Table 1 and in (Figures 2-4). Most fragments are clusters with Na+. In the negative ESI experiment, only the mass ion and two other fragments were detected. HPLC-MS Analysis of the Adducts. An LC-MS/MS spectrum of 4M-dG is presented in Figure 3. More fragmented MS spectra are seen at higher voltage. Typically for adducts with nucleosides, the main fragment results from the cleavage of the bond between the sugar and the nucleic base unit. For quantitation of adducts, a fast and simple HPLC method was developed and optimized for 2-deoxynucleoside adducts of 4MPI: 4M-dG, 4M-dC, and 4M-dA. Tandem mass spectrometry was used to improve the sensitivity and selectivity of the analysis, using multiple reaction monitoring of the loss of the sugar (dR) moiety. The limit of detection of the dC and dA adducts was 10 fmol and of the dG adduct was 20 fmol (Figure 4). Ten fmol of adduct in 100 µg DNA is equivalent to approximately 1 adduct per 3.1 × 107 total nucleotides. The calibration curves for all adducts were linear in the range 10-100 fmol (Figure 5). At higher concentrations, the calibration of the 4M-dA level was lower than the expected linear increase. Reaction with DNA in Vitro. In a further step, DNA was modified in Vitro with the isocyanates 4CPI and 4MPI. The DNA was hydrolyzed enzymatically and analyzed by HPLC with UV and ESI-MS detection. For the reaction of 4CPI, the

Acknowledgment. We acknowledge the financial support of the European Community (BMH4-CT95-09559). The analytical costs in Leicester were partially supported by the Medical Research Council. We thank J. H. Lamb for help with mass spectrometry.

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