Synthesis, Characterization, and 32P-Postlabeling of N

Torsten Haack, Gernot Boche, Christian Kliem, Manfred Wiessler, Dieter Albert, and Heinz H. Schmeiser*. Fachbereich Chemie, Philipps-Universität Marb...
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Chem. Res. Toxicol. 2004, 17, 776-784

Synthesis, Characterization, and 32P-Postlabeling of N-(Deoxyguanosin)-4-aminobiphenyl 3′-Phosphate Adducts Torsten Haack,† Gernot Boche,† Christian Kliem,‡ Manfred Wiessler,‡ Dieter Albert,§ and Heinz H. Schmeiser*,‡ Fachbereich Chemie, Philipps-Universita¨ t Marburg, Hans-Meerweinstrasse, 35032 Marburg, Germany, and Division of Molecular Toxicology and Central Spectroscopy Department, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany Received December 19, 2003

The 32P-postlabeling assay is an extremely sensitive technique for detecting carcinogenDNA adducts. However, for the assignment of DNA adduct structures and the accurate determination of DNA adduct levels by 32P-postlabeling, authentic adduct standards are needed. For most 32P-postlabeling applications, such verified synthetic standard compounds are required in the form of their deoxynucleoside 3′-phosphates because they represent substrates for the polynucleotide kinase for transfer of [32P]phosphate from [γ-32P]ATP. Three N-(deoxyguanosin)4-aminobiphenyl 3′-phosphate adducts were prepared and fully characterized by 1H NMR and mass spectroscopy to serve as standards for the 32P-postlabeling assay. Apart from the C8and the N2-deoxyguanosine 3′-phosphate adducts of the mutagenic human bladder carcinogen 4-aminobiphenyl (dG3′p-C8-4-ABP and dG3′p-N2-4-ABP), the C8-deoxyguanosine 3′-phosphate adduct of the nonmutagenic 4′-tert-butyl-4-aminobiphenyl (dG3′p-C8-4′tBu-4-ABP) was included in the study. Both C8-deoxyguanosine 3′-phosphate adducts were prepared by the in situ formation of deoxyguanosine 3′-phosphate and its subsequent reaction with the appropriate electrophilic amination agent (N-acetoxy compound). The N2-deoxyguanosine 3′-phosphate adduct was obtained by a modification of a previously described procedure for the synthesis of N2-deoxyguanosine adducts of aromatic amines. The three adduct standards were added at different concentrations to calf thymus DNA, and adduct recoveries were determined by the 32P-postlabeling assay under conditions routinely used in the standard methodology, enhancement by nuclease P1 digestion and enhancement by butanol extraction. The dG3′p-C8-4-ABP adduct was recovered irrespective of the concentration with approximately 30% in both the standard and the butanol extraction version of the assay. Both C8-deoxyguanosine 3′-phosphate adducts were sensitive to nuclease P1 digestion resulting in recoveries of only 1-3%. In contrast, the dG3′p-N2-4-ABP adduct was resistant to nuclease P1 digestion; however, recovery in all three versions was poor (1-2%) resulting in a detection limit of one adduct/106 nucleotides. These results demonstrate that the 32P-postlabeling assay underestimates the level of DNA adducts formed by 4-ABP and indicates that there is a need to determine the recovery for each adduct to be analyzed by the 32P-postlabeling technique.

Introduction 1

The human carcinogen 4-ABP (1) has been shown to be a major etiological factor in human bladder cancer (2, 3) and is also regarded as a significant factor of human * To whom correspondence should be addressed. Tel: ++49-6221423348. Fax: ++49-6221-423375. E-mail: [email protected]. † Fachbereich Chemie, Philipps-Universita ¨ t Marburg. ‡ Division of Molecular Toxicology, German Cancer Research Center. § Central Spectroscopy Department, German Cancer Research Center. 1 Abbreviations: 4-ABP, 4-aminobiphenyl; RAL, relative adduct labeling; DMSO, dimethyl sulfoxide; dG3′p, 2′-deoxyguanosine 3′phosphate; dG-C8-4-ABP, N-(2′-deoxyguanosin-8-yl)-4-aminobiphenyl; dG3′p-C8-4-ABP, N-(2′-deoxyguanosin-3′-phospho-8-yl)-4-aminobiphenyl; dG3′p-C8-4′tBu-4-ABP, N-(2′-deoxyguanosin-3′-phospho-8-yl)4′-tert-butyl-4-aminobiphenyl; dG-N2-4-ABP, N-(2′-deoxyguanosin-N2yl)-4-aminobiphenyl; dG3′p-N2-4-ABP, N-(2′-deoxyguanosin-3′-phosphoN2-yl)-4-aminobiphenyl; dG-N2dN-4-ABP, N-(2′-deoxyguanosin-N2-yl)4-azobiphenyl; N-OAc-4-ABP, N-acetoxy-4-aminobiphenyl; N-OH-4ABP, N-hydroxy-4-aminobiphenyl; COSY, correlation spectroscopy; ROESY, rotational Overhauser effect spectroscopy.

breast cancer (4). In experimental animals, 4-ABP is a potent urinary bladder carcinogen and it induces mammary tumors in rats (5). Like other aromatic amines, 4-ABP is an environmental and occupational contaminant. Environmental exposure to 4-ABP is mainly by cigarette smoke (1), hair dye usage (6), and exhaust of fossil fuels (7). To exert its carcinogenic effect, 4-ABP must undergo metabolic activation to reactive electrophilic intermediates that bind to DNA, forming covalent DNA adducts (8). The most persistent DNA adduct induced by 4-ABP in vivo in animals and identified in humans is dG-C84-ABP (Figure 1A) (8, 9). This C8-deoxyguanosine adduct is formed as the predominant DNA adduct by 4-ABP, but several minor adducts, N2-deoxyguanosine-4-ABP adducts and one C8-deoxyadenosine-4-ABP adduct (dA-C84-ABP; Figure 1C), have also been detected in treated cells by the 32P-postlabeling assay (10, 11). Recently,

10.1021/tx0342666 CCC: $27.50 © 2004 American Chemical Society Published on Web 04/23/2004

4-Aminobiphenyl Adduct Standards for

32P-Postlabeling

Figure 1. DNA adducts formed by 4-ABP. (A) dG-C8-4-ABP, (B) dG-N2-4-ABP, (C) dA-C8-4-ABP, (D) 3dG-N2-4-ABP, and (E) dG-N2dN-4-ABP.

Swaminathan and Hatcher (12, 13) reported the identification of three minor N2-deoxyguanosine adducts generated by reaction of N-OAc-4-ABP with dG3′p, namely, 3-(deoxyguanosin-N2-yl)-4-aminobiphenyl (3dG-N2-4-ABP; Figure 1D), dG-N2-4-ABP (Figure 1B), and dG-N2dN-4ABP (Figure 1E). Using the 32P-postlabeling procedure, dG-C8-4-ABP, dA-C8-4-ABP, dG-N2dN-4-ABP, and dGN2-4-ABP were found in human uroepithelial cells after treatment by N-OH-4-ABP, the proximate metabolite of 4-ABP (11-14). Its high sensitivity, versatility, and the fact that only microgram amounts of DNA are required have made the 32 P-postlabeling assay developed by Randerath, Reddy, and Gupta (15, 16) the most widely used method for the detection of DNA adducts. Thus, numerous studies on the detection of DNA adducts generated by 4-ABP in experimental animals and humans have used the 32Ppostlabeling assay (9, 17-21). The basic procedure comprises four steps: (i) enzymatic hydrolysis of carcinogenmodified DNA to nucleoside 3′-phosphates; (ii) an adduct enrichment step, usually nuclease P1 digestion or butanol extraction; (iii) labeling of all nucleoside 3′-phosphates, normal and modified ones, by polynucleotide kinasemediated transfer of 32P-orthophosphate from [γ-32P]ATP to form 5′-32P-labeled 3′,5′-bisphosphates; and (iv) chromatographic separation by TLC or HPLC followed by detection and quantitation of adduct spots by 32P-decay. Major weaknesses of the 32P-postlabeling assay are its inability to provide structural information of adducts and the inaccuracy of measurements of DNA adduct levels. Although some conclusions on the chemical structure can be drawn based on the chromatographic mobilities of adduct spots, assignment of adduct structures has to be

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accomplished indirectly by cochromatographic analyses with synthetic adduct standards. Quantitation of DNA adducts is typically achieved by RAL, in which the ratio of the adduct-associated radioactivity to the radioactivity found in normal nucleotides is calculated. However, some assumptions are inherent when quantifying DNA adduct levels by RAL, the foremost being that adducted and normal nucleoside 3′-phosphates are labeled with equal efficiency. Several studies have demonstrated that the labeling efficiency is adduct-dependent showing that certain adducts are better (22) but in most cases poorer substrates (23, 24) for the polynucleotide kinase than the normal nucleotides. Moreover, when using the nuclease P1 version, it is assumed that the adducts are completely resistant to the 3′-phosphatase activity of nuclease P1; likewise, the butanol extraction version assumes that adducts are extracted with 100% efficiency. Therefore, the 32P-postlabeling assay generally underestimates adduct levels due to incomplete DNA digestion, inefficiency of adduct labeling by polynucleotide kinase, and/or loss of adducts during enrichment and chromatography stages. As shown by an interlaboratory trial of 32P-postlabeling procedures, quantitation of 4-ABP-modified DNA using the RAL method strongly underestimated the adduct level (25). Likewise, when DNA containing dG-C8-4-ABP was used as a standard to compare adduct quantitation by different detection methods, adduct levels determined by 32Ppostlabeling were approximately 4% of those found with the 3H measurements (17). One approach to improve the significance of the 32Ppostlabeling assay is the use of synthetically prepared adduct standards from the carcinogen of interest. Because adduct recoveries will differ from adduct to adduct, well-characterized individual adduct standards are needed for determining adduct recoveries and defining correction factors. In the typical 32P-postlabeling procedure, adducts are excised from DNA as deoxyribonucleoside 3′-phosphates before they are labeled at the 5′-OH by polynucleotide kinase. Therefore, adduct standards may be prepared as deoxyribonucleoside 3′-phosphates. Of the known ABP-DNA adducts, only two minor adducts were unambiguously structurally characterized as deoxyribonucleoside 3′-phosphates, namely, 3dG-N2-4-ABP and dGN2dN-4-ABP (12, 13). Definitive structural identification was reported for dG-C8-4-ABP as nucleoside (26) and as 3′,5′-bisphosphate (27) and for dG-N2-4-ABP also as nucleoside by Swaminathan and Hatcher (12) and Scheer et al. (28). The most common strategy for the synthesis of 4-ABPDNA adducts involves the reaction of N-OAc-4-ABP with purine bases, purine nucleosides, and nucleotides or DNA. These procedures, which mimic the metabolic activation pathway for arylamines, produce C8 deoxyguanosine adducts as the major products, but the yields are typically very low (12, 13, 17, 29). Recently, synthetic strategies for the preparation of dG-C8 adducts with amino and nitro-arenes, involving palladium-catalyzed C-N bond formation (30, 31) and the generation of arylnitrenes (32), have been reported. In view of the current relevance of the 32P-labeling methodology as a human biomonitoring method for the detection and quantitation of 4-ABP-DNA adducts, we here describe the synthesis and the characterization of 3′-phosphate adducts to serve as standard compounds. We also report the recoveries of the major C8-deoxy-

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Table 1. 1H NMR Chemical Shifts and Coupling Constants of N-(Deoxyguanosin)-4-ABP 3′-Phosphate Adduct Standards dG3′p-C8-4-AB (5) δ (ppm) N-1-H amine-H N2-H A2,6-H A3,5-H B2,6-H B3,5-H B4-H 1′-H 2′-Ha 2′-Hb 3′-H 4′-H 5′-Ha 5′-Hb 8-H t-Bu NCHCH2 NCHCH2 CH2CH3 CH3

10.55 (bs, 1H) 8.83 (bs, 1H) 6.48 (bs, 2H) 7.88 (d, 2H) 7.56 (d, 2H) 7.61 (d, 2H) 7.41 (t, 2H) 7.28 (t, 1H) 6.35 (dd, 1H) 2.21 (dd, 1H) 2.56-2.51 (bm, 1H) 4.93 (bs, 1H) 4.22 (bs, 1H) 3.71 (bd, 1H) 3.89 (bd, 1H) 3.16-3.13 (m, 8H) 1.58-1.53 (m, 8H) 1.30 (sext., 8H) 0.92 (t, 12H)

dG3′p-C8-4′-tBu-4-ABP (6) J (Hz)

8.5 8.78 7.4 7.8 7.3 10.0, 5.4 12.7,5.0

7.3 7.3

δ (ppm) 10.80 (bs, 1H) 8.85 (bs, 1H) 6.64 (bs, 2H) 7.90 (d, 2H) 7.53 (d, 2H) 7.54 (d, 2H) 7.42 (d, 2H) 6.34 (dd, 1H) 2.15 (dd, 1H) 2.54-2.41 (m, 1H) 4.95-4.87 (m, 1H) 4.21-4.15 (m, 1H) 3.92-3.89 (m, 1H) 3.92-3.89 (m, 1H) 1.30 (s, 9H) 3.20-3.09 (m, 8H) 1.58-1.50 (m, 8H) 1.33-1.26 (m, 8H) 0.91 (t, 12 H)

guanosine 3′-phosphate adduct (dG3′p-C8-4-ABP) and a N2-deoxyguanosine 3′-phosphate adduct (dG3′p-N2-4ABP) from 4-ABP in the three routinely used versions of the 32P-postlabeling assay.

Materials and Methods Caution: N-Arylamines, N-hydroxyarylamines, and Nacetoxyarylamines are mutagenic and carcinogenic and should be handled with care. Exposure to 32P should be avoided, by working in a confined laboratory area, with protective clothing, plexiglass shielding, Geiger counters, and body dosimeters. Wastes must be discarded according to appropriate safety procedures. Instrumentation. 1H and 13C NMR spectra were recorded on a Bruker spectrometer AV-600, ARX-200, or AM-400 and are referenced to tetramethylsilane as an internal standard. Phosphorus spectra were referenced to phosphoric acid as an external standard. Electrospray mass spectra (ESI) were obtained on a Hewlett-Packard HP 5889 system. The melting points are uncorrected. Column chromatography was carried out on Merck silica gel 60. Chemicals. 2′-Deoxyguanosine and 4-nitrobiphenyl were acquired from Fluka Chemicals. Calf thymus DNA was obtained from Boehringer (Mannheim, Germany), and spleen phosphodiesterase was obtained from Calbiochem. Micrococcal nuclease, nuclease P1, and 4-ABP were purchased from Sigma-Aldrich. N-OH-4-ABP was prepared as previously described (33). 4′-tertButyl-4-aminobiphenyl was prepared by the reduction of 4′-tertbutyl-4-nitrobiphenyl using SnCl2/HCl (34). 5′-tert-Butyl-dimethylsilyl-2′-deoxyguanosine 3′-diallyl phosphate 4 was prepared as described (35). 4-Hydrazobiphenyl was prepared according to ref 36, and diethylammonium hydrogencarbonate was synthesized as described (37). All other enzymes and chemicals for the 32P-postlabeling assay were obtained commercially from sources described previously (38). dG3′p-C8-4-ABP (5). To a solution of 4 (0.6 g, 1.11 mmol) and formic acid (670 µL, 804 mg, 17.5 mmol) in THF (20 mL) was added n-butylamine (440 µL, 324 mg, 4.43 mmol), triphenylphosphine (204 mg, 0.78 mmol), and tetrakis(triphenylphosphine)palladium(0) (128 mg, 0.11 mmol). The mixture was stirred under an argon atmosphere for 3 h at 50 °C. The solvent was removed in vacuo, and the residue was dissolved in a 1 M solution of tetrabutylammoniumfluoride in THF and was then stirred at room temperature for 1 h. After evaporation of the solvent, the residue was suspended in water (50 mL). The aqueous phase was extracted with ethyl acetate (3 × 50 mL) and n-butanol (1 × 50 mL) and concentrated to

J (Hz)

8.6 8.6 8.3 8.3 10.1, 5.2 12.1, 4.8

7.3

dG3′p-N2-4-ABP (8) δ (ppm) 10.63 (bs, 1H) 8.05 (s, 1H) 9.27 (bs, 1H) 6.87 (d, 2H) 7.49 (d, 2H) 7.55 (d, 2H) 7.37 (t, 2H) 7.24 (t, 1H) 6.11 (t, 1H) 2.45-2.39 (bm, 1H) 2.57-2.51 (m, 1H) 4.79 (bs, 1H) 3.96 (bs, 1H) 3.60 (bs, 1H) 3.60 (bs, 1H) 7.92 (s, 1H) 3.16-3.14 (m, 8H) 1.56 (bqint., 8H) 1.29 (sept., 8H) 0.92 (t, 12H)

J (Hz)

8.5 8.5 7.3 7.7 7.3 6.6

7.82 7.3 7.4

approximately 30 mL. In a second flask, acetyl cyanide (0.39 mL, 0.38 g, 5.5 mmol) was added dropwise at -78 °C to a stirring solution of N-OH-4-ABP (1.0 g, 5.4 mmol) and triethylamine (0.75 mL, 0.55 g, 5.4 mmol) in THF (60 mL). The mixture was stirred at -78 °C for 20 min and then added to the first flask containing the aqueous solution at room temperature. Stirring was continued at room temperature for 3 h and then, the organic solvent was removed in vacuo. The aqueous residue was extracted with ethyl acetate (2 × 30 mL) and with n-butanol (5 × 30 mL). The combined butanol layers were dried over magnesium sulfate and evaporated to dryness. The residue was purified by HPLC (40-50% methanol in water) to afford 18 mg (2.1%) of 5 as its tetrabutylammonium monosalt; pale yellow solid; mp 160-161 °C. UV/vis (MeOH): λmax 206, 306 nm; λmin 230 nm, consistent with the UV spectrum of the bisphosphate adduct (11). The molar extinction coefficient was measured experimentally in methanol (306nm ) 24 800) and found to be comparable to the value (301nm ) 28 430) reported for N-(guanosin-8-yl)-4-aminobiphenyl-5′-monophosphate (39). 1H NMR (DMSO-d6, 600 MHz) in Table 1. 13C NMR (DMSO-d6, 100 MHz): δ 155.9, 153.3, 149.7, 143.3, 140.4, 140.3, 132.4, 129.0, 126.8, 126.7, 126.2, 118.0, 112.5, 86.4, 83.2, 75.6, 61.6, 57.7, 37.3, 23.2, 19.4, 13.6. 31P NMR (CDCl3, 80 MHz): δ 0.1. MS (ESI negative): m/z 513 (anion). dG3′p-C8-4′-tBu-4-ABP (6). Compound 6 was prepared according to the procedure described above for 5, using 4′-tertbutyl-4-hydroxyaminobiphenyl instead of N-OH-4-ABP; 20 mg (2.2) % pale yellow solid; mp 165-167 °C. UV/vis (MeOH): λmax 205 nm, 305 nm, λmin 235 nm. The molar extinction coefficient was measured experimentally in methanol (305nm ) 16 300). 1H NMR (DMSO-d6, 400 MHz) in Table 1. 13C NMR (DMSO-d6, 100 MHz): δ 156.0, 153.4, 149.7, 149.0, 143.3, 140.2, 137.5, 132.3, 126.6, 125.8, 125.7, 118.0, 112.2, 86.9, 83.2, 74.8, 61.4, 57.7, 37.7, 34.3, 31.3, 23.2, 19.4, 13.6. 31P NMR (CDCl3, 160 MHz): δ 0.2. MS (ESI negative): 569 (anion). O 6 -Allyl-5′-(tert-butyl-dimethylsilyl)-2′-deoxyguanosine 3′-Diallyl Phosphate. To a solution of 4 (2.09 g, 3.86 mmol), allyl alcohol (0.45 mL, 384 mg, 6.61 mmol), and triphenylphosphine (2.53 g, 9.65 mmol) in THF (35 mL) was slowly added azodicarboxylic acid diethylester (1.5 mL, 1.66 g, 9.53 mmol) at room temperature. After it was stirred for 1 h at room temperature, the solvent was removed in vacuo and the residue was purified by column chromatography (ethyl acetate) to give 1.40 g (63%) of the product as a colorless oil. 1H NMR (CDCl3, 200 MHz): δ 7.90 (s, 1H), 6.36 (t, 1H, J ) 7.1 Hz), 5.86-6.21 (m, 3H), 5.23-5.46 (m, 6H), 5.09-5.18 (m, 1H), 4.99 (d, 2H, J ) 5.8 Hz), 4.86 (bs, 2H), 4.54-4.61 (m, 4H), 4.29-4.34 (m, 1H), 0.89 (s, 9H), 0.08 (s, 6H). 13C NMR (CDCl3, 50 MHz): δ 160.9,

4-Aminobiphenyl Adduct Standards for

32P-Postlabeling

Chem. Res. Toxicol., Vol. 17, No. 6, 2004 779

Table 2. Recoveries of the Synthesized Adduct Standards under Typical 32P-postlabeling

assay

standard procedure nuclease P1 digestion butanol extraction

adduct level (adduct/ normal nucleotides) 1/103 1/104 1/105 1/105 1/106 1/107 1/105 1/106 1/107 1/108

dG3′p-C8-4-ABP 17 (12-25) 20 (15-21) 30 (25-45) 3 (1-5) 2 (1-3) ND 20 (16-30) 35 (30-60) 35 (10-50) 30 (5-80)

32P-Postlabeling

adduct recovery in % (range) dG3′p-C8-4'tBu-4-ABP

Conditionsa dG3′p-N2-4-ABP

3 (2-5) 4 (3-5) ND 1 (1-3) ND

2 (2-5) 2 (2-5) ND 1 (0.4-2) ND

10 (5-30) 12 (10-19) 15 (12-22) ND

1 (0.5-3) 2 (1-4) ND

a Adduct recoveries were calculated by RAL values from the means and ranges from at least three independent labelings analyzed in duplicate; ND, not detected.

159.2, 153.7, 137.2, 132.7, 132.2, 132.1, 118.7, 118.7, 118.2, 115.8, 85.9, 83.5, 78.7, 68.5, 68.4, 67.2, 63.2, 39.4, 25.9, 18.3, -5.3, -5.5. 31P NMR (CDCl3, 80 MHz): δ -1.1. O 6 -Allyl-5′-(tert-butyl-dimethylsilyl)-2′-deoxyxanthosine 3′-Diallyl Phosphate. tert-Butyl nitrite (15 mL) was added to O6-allyl-5′-(tert-butyl-dimethylsilyl)-2′-deoxyguanosine 3′-diallyl phosphate (1.4 g, 2.41 mmol) at 0 °C. After all of the solid had dissolved, the solution was concentrated in vacuo and the residue was purified by column chromatography (ethyl acetate); 350 mg (25%) colorless solid. 1H NMR (CDCl3, 200 MHz): δ 7.97 (s, 1H), 6.41 (t, 1H, J ) 6.6 Hz), 5.82-6.17 (m, 3H), 5.20-5.46 (m, 6H), 5.09 (d, 2H, J ) 6.0 Hz), 5.00-5.10 (m, 1H), 4.52-4.61 (m, 4H), 4.26-4.30 (m, 1H), 3.82-3.90 (m, 2H), 2.44-2.58 (m, 1H), 2.74-2.83 (m, 1H), 0.87 (s, 9H), 0.07 (s, 6H). 13C NMR (CDCl , 50 MHz): δ 161.8, 159.3, 149.3 (C4), 137.1, 3 132.1, 131.9, 131.8, 119.0, 118.6, 118.6, 114.7, 85.9, 84.2, 77.2, 68.8, 68.5, 68.4, 62.8, 40.3, 25.9, 18.3, -5.5, -5.6. 31P NMR (CDCl3, 80 MHz): δ -1.3. O6-Allyl-5′-(tert-Butyl-dimethylsilyl)-O2-trifluormethanesulfonyl-2′-desoxyguanosine 3′-Diallyl Phosphate (7). To an ice-cold solution of O6-allyl-5′-(tert-butyl-dimethylsilyl)-2′deoxyxanthosine 3′-diallyl phosphate (350 mg, 0.6 mmol), triethylamine (290 mg, 2.9 mmol), and a catalytic amount of 4-(dimethylamino)pyridine in CH2Cl2 (8 mL) was added triflic chloride (77 µL, 122 mg, 0.7 mmol). After the mixture was stirred at 0 °C for 30 min, the solvent was removed and the residue was purified by column chromatography (ethyl acetate) to afford 260 mg (61%) of 7 as a colorless solid. 1H NMR (CDCl3, 200 MHz): δ 8.32 (s, 1H), 6.42 (dd, 1H, J ) 8.0, 5.8 Hz), 5.856.20 (m, 3H), 5.25-5.52 (m, 6H), 5.07 (d, 2H, J ) 5.8 Hz), 5.065.14 (m, 1H), 4.52-4.62 (m, 4H), 4.36 (dd, 1H, J ) 4.5 Hz, 2.8 Hz), 3.86 (t, 2H, J ) 3.5 Hz), 2.58-2.84 (m, 2H), 0.86 (s, 9H), 0.07 (s, 6H). 13C NMR (CDCl3, 50 MHz): δ 161.5, 152.2, 151.9, 141.8, 132.1, 132.0, 131.1, 121.1, 119.0, 118.8, 118.8, 118.5 (q, J ) 320 Hz), 86.5, 84.7, 78.4, 69.2, 68.5, 68.4, 63.1, 40.0, 25.8, 18.2, -5.6. 31P NMR (CDCl3, 80 MHz): δ -1.1. N-(O6-Allyl-5′-(tert-butyl-dimethylsilyl)-2′-desoxyguanosin-N2-yl)-4-aminobiphenyl 3′-Diallyl Phosphate. A solution of 7 (260 g, 0.36 mmol) and 4-hydrazobiphenyl (270 mg, 1.46 mmol, 4 equiv) in 1 mL of DMF was stirred under argon atmosphere at room temperature for 3 days. Then, the solvent was removed in vacuo and the residue was purified by column chromatography on silica gel (ethyl acetate) to give 226 mg (84%) of the product as a pale yellow powder. 1H NMR (CDCl3, 200 MHz): δ 7.92 (s, 1H), 7.52 (d, 2H, J ) 7.3 Hz), 7.45 (d, 2H, J ) 8.5 Hz), 7.37 (t, 2H, H3), 7.33-7.41 (m, 1H, H4), 7.02 (bs, 1H), 7.00 (d, 2H, J ) 8.5 Hz), 6.33 (t, 1H, J ) 6.9 Hz), 6.27 (bs, 1H), 5.82-6.10 (m, 3H), 5.20-5.41 (m, 6H), 5.10-5.18 (m, 1H), 4.90 (d, 2H, J ) 6.0 Hz), 4.51-4.59 (m, 4H), 4.21-4.30 (m, 1H), 3.743.82 (m, 2H), 2.61-2.67 (m, 2H), 0.88 (s, 9H), 0.05 (s, 6H). 13C NMR (CDCl3, 50 MHz): δ 160.8, 159.5, 153.3, 148.8, 140.9, 137.7, 133.2, 132.3, 132.1, 131.9, 128.5, 127.6, 126.3, 126.2, 118.6, 118.6, 118.3, 113.7, 85.7, 83.6, 78.3, 68.4, 68.2, 67.3, 63.1, 39.1, 25.8, 18.2, -5.6, -5.7. 31P NMR (CDCl3, 80 MHz): δ -1.1. dG3′p-N2-4-ABP (8). To a solution of N-(O6-allyl-5′-(tertbutyl-dimethylsilyl)-2′-desoxyguanosin-N2-yl)-4-aminobi-

phenyl 3′-diallyl phosphate (221 mg, 0.3 mmol) and diethylammonium hydrogencarbonate (319 mg, 2.36 mmol) in CH2Cl2 was slowly added a solution of tetrakis(triphenylphosphine)palladium(0) (75 mg, 0.065 mmol) and triphenylphosphine (11 mg, 0.04 mmol) in CH2Cl2 (7 mL) at room temperature, and the resulting mixture was stirred for 30 min. The solvent was evaporated, and the residue was treated with a solution of tetrabutylammonium fluoride in THF (1M, 15 mL, 15 mmol) and stirred for another 1 h. The solvent was removed in vacuo, and the residue was suspended in water (80 mL) and extracted with ethyl acetate (3 × 50 mL) and then n-butanol (3 × 50 mL). The combined butanol layers were dried over magnesium sulfate and concentrated to dryness, and the residue was dissolved in methanol (20 mL). After the addition of a catalytic amount of palladium/charcoal, the mixture was stirred under a hydrogen atmosphere for 1 h, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (methanol/water 1:1), yielding 20 mg (9%) of 8 as its tetrabutylammonium monosalt; yellow solid. UV (MeOH): λmin 230, λmax 272 nm in agreement with the value reported (12). The molar extinction coefficient was measured experimentally in methanol (272nm ) 27 700). 1H NMR (DMSO-d6, 600 MHz) in Table 1. MS (ESI negative): m/z 513 (anion). 32P-Postlabeling Analysis. The concentration of adduct standards was determined spectrophotometrically. The standard procedure was essentially performed as described previously (16, 38) except that 37.5 mU instead of 7.5 mU of spleen phosphodiesterase was used for digestion. Adduct standards dissolved in 0.15 mmol/L sodium citrate and 1.5 mmol/L sodium chloride (0.01 xSSC) were added to 1 µg of calf thymus DNA, digested, and analyzed as reported (38). For the nuclease P1 enrichment version and the butanol extraction-mediated enrichment procedure, adduct standards dissolved in 0.15 mmol/L sodium citrate and 1.5 mmol/L sodium chloride (0.01 xSSC) were added to calf thymus DNA samples (12.5 mg) and analyzed as described (38) except that the amount of spleen phosphodiesterase was increased to 62.5 mU. For resolution of adduct spots, the chromatographic conditions were used as follows: D1, 1 M sodium phosphate, pH 6.5; D3, 3.5 M lithium formate, 8.5 M urea, pH 3.5; D4, 0.8 M LiCl, 0.5 M Tris-HCl, 8.5 M urea, pH 8.0; and D5, 1.7 M NaH2PO4, pH 6.0. Adducts and normal nucleotides were detected and quantitated by an Instant imager (Packard). Count rates of adducted fractions were determined from maps after subtraction of count rates from adjacent blank areas. Excess [γ-32P]ATP after the postlabeling reaction was confirmed. Adduct levels were calculated in units of RAL, which is the ratio of cpm of adducted nucleotides to cpm of total nucleotides in the assay. These RAL values were used for the quantitation of recovery of adduct standards shown in Table 2.

Results Preparation of N-(Deoxyguanosin)-4-ABP 3′-Phosphate Adduct Standards. 1. Synthesis of the C8Deoxyguanosine 3′-Phosphate Adducts (dG3′p-C8-

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Figure 2. Synthesis of the C8-deoxyguanosine 3′-phosphate adducts (dG3′p-C8-4-ABP 5 and dG3′p-C8-4′tBu-4-ABP 6). BuNH2, n-butylamine; Pd(PPh3)4, tetrakis(triphenylphosphine)palladium(0); TBAF, tetrabutylammoniumfluoride; and Ar-NHOAc, N-acetoxy compound.

Figure 3. Synthesis of the N2-deoxyguanosine 3′-phosphate adduct (dG3′p-N2-4-ABP) 8. DEAD, azodicarboxylic acid diethylester; tBuONO, tert-butyl nitrite; Tf O, triflicanhydride; DMAP, 4-(dimethylamino)pyridine; Pd(PPh ) , tetrakis(triphenylphosphine)2 3 4 palladium(0); (Et2NH2)2CO3, diethylammoniumcarbonate; and TBAF, tetrabutylammoniumfluoride.

4-ABP 5 and dG3′p-C8-4′tBu-4-ABP 6). The nucleotide adduct 5 (Figure 2) was prepared by the reaction of N-OAc-4-ABP with dG 3′-phosphate. Previous reports about the synthesis of 5 (12, 13, 17, 23, 24) did not provide complete spectroscopic data, presumably because amounts were insufficient. Yields in these amination reactions are generally low, and the starting material dG3′p is expensive. We prepared dG3′p following a literature procedure in gram quantities (35). Thus, 5′-silylated dG was treated with diallyl phosphorochloridate to afford 4, which served as the starting material for the subsequent amination reaction. Deprotection and in situ reaction with N-OAc4-ABP afforded the desired adduct 5 in milligram quantities. Adduct 6, which represents an adduct of a nonmutagenic amine, was synthesized by the same way replacing N-OH-4-ABP by 4′-tert-butyl-4-hydroxyaminobipheny. Both dG 3′-phosphate adducts were purified by reverse phase HPLC and obtained as their tetrabutylammonium salts. High-resolution 1H NMR spectroscopy at 600 MHz of 5 (Figure 4) and at 400 MHz of 6 were consistent with their identities as dG3′p-C8-4-ABP in agreement with data reported for the nucleoside adduct by Kadlubar et al. (26) and dG3′p-C8-4′tBu-4-ABP. 2. Synthesis of the N2-Deoxyguanosine 3′-Phosphate Adduct (dG3′p-N2-4-ABP) 8. The synthesis of the nucleoside adduct dG-N2-4-ABP utilizing a nucleophilic substitution in the C2-position was reported by

Steinbrecher et al. (40). To synthesize the corresponding nucleotide adduct, we have modified this approach by using the phosphonated compound 4 as the starting material (Figure 3). Allyl-protection of O6 in Mitsunobu fashion, hydrolysis of the C2-N bond with tert-butyl nitrite, and subsequent reaction of the resulting xanthosine with triflic chloride afforded the C2-activated compound 7. 4-Hydrazobiphenyl was then easily introduced in the C2-position resulting in the protected N2adduct, which after deprotection, reduction, and HPLC purification furnished the desired dG3′p-N2-4-ABP 8. The structural characterization of this product as dG3′p-N24-ABP was established by 1H NMR spectroscopy at 600 MHz with the aid of COSY and ROESY experiments (Figure 5) and is in good accordance with the data provided for the corresponding nucleoside adduct (28). The hydrazo linkage turned out to be sensitive to oxidation leading to the strongly colored azo derivative identified by Hatcher and Swaminathan (13) and observed as impurity in the NMR spectrum of 8 (Figure 5). Recoveries of N-(Deoxyguanosin)-4-ABP 3′-Phosphate Adduct Standards by 32P-Postlabeling. The three fully characterized deoxyguanosine 3′-phosphate adducts were added as their tetrabutylammonium salts at different concentrations to calf thymus DNA and tested for their recoveries in the 32P-postlabeling assay. Adduct recoveries were determined with the three com-

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Figure 4. Downfield region of the 1H 600 MHz NMR spectrum of dG3′p-C8-4-ABP 5. The lettering designates the specific protons of the structure given in the inset.

Figure 5. Downfield region of the 1H 600 MHz NMR spectrum of dG3′p-N2-4-ABP 8. The lettering designates the specific protons of the structure given in the inset. An asterisk indicates signals of dG3′p-N2dN-4-ABP.

monly used versions of the assay that is the standard procedure, the enrichment by nuclease P1 digestion or butanol extraction all performed under typical conditions. 1. 32P-Postlabeling Standard Procedure. The standard procedure of the 32P-postlabeling assay is suitable for the detection of DNA adduct levels as low as 1 adduct per 106 normal nucleotides. In a typical experiment, 1 µg of DNA (3.08 nmol dNps) is digested to deoxynucleoside 3′-phosphates and an aliquot of the

digest (0.5 nmol dNps) is labeled and analyzed. Therefore, adduct standards were added to calf thymus DNA to result in adduct levels of 1/103 (3.08 pmol standard), 1/104 (0.308 pmol standard), and 1/105 (30.8 fmol standard) adduct/normal nucleotides. Then, the mixture was digested, labeled, separated by TLC, and analyzed. As shown in Figure 6, when analyzed by the standard procedure, all 3′-phosphate adduct standards resulted in one major adduct spot. However, in digests with adduct

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adduct and to 10-15% of the dG3′p-C8-4′tBu-4-ABP adduct over a 100-fold range of adduct concentrations. It is clear that adduct standard dG3′p-N2-4-ABP was recovered poorly by either 32P-postlabeling version (ca. 2%). The detection limit for adduct dG3′p-C8-4-ABP by enhancement with nuclease P1 digestion was determined at one adduct in 106 nucleotides for adduct dG3′p-C84′tBu-4-ABP and dG3′p-N2-4-ABP at one adduct in 105 nucleotides. The limit of detection for all three adduct standards tested was improved by the butanol extraction version (one adduct in 108 nucleotides for adduct dG3′pC8-4-ABP and one adduct in 107 nucleotides for adduct dG3′p-C8-4′tBu-4-ABP and one adduct in 106 nucleotides for adduct dG3′p-N2-4-ABP).

Discussion

Figure 6. Autoradiographic profiles obtained from digests of calf thymus DNA and adduct standards dG3′p-C8-4-ABP (A), dG3′p-N2-4-ABP (B), dG3′p-C8-4′tBu-4-ABP (C), and solvent only (D). The standard procedure of the 32P-postlabeling assay was used for analysis. Origins are in the bottom left-hand corner. Instant Imager analysis was for 6 min. Chromatographic conditions: D1, 1 M sodium phosphate, pH 6.5; D3, 3.5 M lithium formate, 8.5 M urea, pH 3.5; D4, 0.8 M LiCl, 0.5 M TrisHCl, 8.5 M urea, pH 8.0; and D5, 1.7 M NaH2PO4, pH 6.0.

standard dG3′p-N2-4-ABP, a radioactive smear in the D3 direction was observed probably due to partial decomposition of the compound during the procedure. Whereas the two 4-ABP-derived adducts migrated to the same location, the more lipophilic adduct dG3′p-C8-4′tBu-4ABP stayed close to the origin of the TLC plate (Figure 6C). Control experiments with calf thymus DNA and solvent alone did not show adduct spots (Figure 6D). As shown in Table 2 for adduct standard dG3′p-C8-4-ABP recovery by the standard procedure was between 20 and 30% independent of the amount added to the unmodified DNA. In contrast, the other two adduct standards were only recovered with around 2-3%. Under conditions usually used in the standard procedure, the limit of detection for adduct dG3′p-C8-4-ABP was reached at approximately 1/105 and for the other adducts at 1/104 adduct/normal nucleotides. At these adduct levels, the amount of radioactivity of the adduct spot was only 30 cpm above background. 2. Enhancement Methods. The recovery of the three adduct standards was also determined by the commonly used enrichment procedures of the 32P-postlabeling assay, digestion by nuclease P1, and butanol extraction. Typically, 12.5 µg of DNA (38.5 nmol dNps) is used for both versions. To create adduct levels commonly observed in in vivo samples, adduct standards were added to calf thymus DNA so that ratios in a range between 1/105 (0.385 pmol standard) and 1/108 (0.385 fmol standard) adduct/normal nucleotides were achieved. The autoradiograms obtained were identical to those obtained by the standard procedure presented in Figure 6. All adduct standards produced one major adduct spot that did not occur in the solvent control sample. Both dG-C8 adducts were sensitive to digestion by nuclease P1 resulting in recoveries of 2% or less (Table 2). In contrast, enrichment by butanol extraction led to a recovery of approximately 30% for the dG3′p-C8-4-ABP

In this work, we describe the synthesis and the definitive identification of three deoxyguanosine 3′phosphate adducts dG3′p-C8-4-ABP 5, dG3′p-N2-4-ABP 8, and dG3′p-C8-4′tBu-4-ABP 6. Two of those adducts (5 and 8) are formed by in vivo reaction of the strong mutagen 4-ABP with DNA. The third adduct 6 differs from 5 by an additional tert-butyl group at the biphenyl moiety. In comparison to 4-ABP, 4′-tert-butyl-4-aminobiphenyl was previously shown to be essentially nonmutagenic in the Ames test (41, 42). Both C-8 deoxyguanosine adducts were prepared by amination reaction of dG3′p, and structural assignment was based on spectroscopic analysis, including MS, NMR (COSY and ROESY experiments), and UV spectroscopy. The 1H NMR spectrum of dG3′p-C8-4-ABP 5 presented in Figure 4 revealed the following features: (i) all nine protons associated with the biphenyl ring were detected and assigned by COSY and ROESY experiments in agreement with data reported for the bisphosphate derivative (27), (ii) a broad singlet with a characteristic chemical shift (δ 6.48) for the amino group of dGp was found, whereas (iii) a signal with a chemical shift corresponding with the singlet C-8 proton of purine was missing, indicating that covalent linkage is through the amino group of 4-ABP to the C8 position of dGp. The 1H NMR spectrum of dG3′p-N2-4-ABP 8 (Figure 5) shows singlet resonances at 10.63, 9.27, and 8.05 ppm for three different N-H groups, indicating the loss of one proton at the amino group of dGp. In contrast to 5, 8 exhibits a singlet at 7.92 ppm for the C8 proton and two singlets at 8.05 and 9.27 ppm, which were assigned to the hydrazo linkage. The signals between 6.87 and 7.55 ppm were unambiguously assigned to the nine protons of the biphenyl ring completing results reported by Swaminathan and Hatcher (12). We found strong crosspeaks in the ROESY spectrum between the C8 proton and the sugar protons 1′-H and 2′-Hb (syn conformation) and weak cross-peaks between the C8 proton and the 3′-H and 5′-H2 (anti conformation) implying an equilibrium of syn and anti conformational isomers. Moreover, in the range of the aromatic protons, several minor signals were detected and assigned to the azo adduct dG3′p-N2dN-4-ABP previously characterized by Hatcher and Swaminathan (13). This azo adduct is formed by oxidation of the hydrazo linkage of 8 and formation increased when NMR spectra were recorded at higher temperatures (310 K) (data not shown) indicating that 8 is quite unstable. When comparing the 1H NMR data published for 8 (13) with those of the present study, it

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becomes evident that the chemical shifts for the protons of the hydrazo linkage differ considerably (6.74 vs 6.90 ppm and 8.05 vs 9.27 ppm). A possible explanation for this discrepancy is a bent conformation of 8 due to an interaction between purine and biphenyl system as already suggested by Famulok et al. (29). Because in our case 8 is present as the tetrabutylammonium salt, those cations with unpolar ends could inhibit this bending. Moreover, recoveries of the three adduct standards were determined using the standard 32P-postlabeling method as well as the two commonly used enhancement methods. All adduct standards resulted in one major adduct spot accompanied by small amounts of other spots when analyzed by either version of the 32P-postlabeling assay (Figure 6), indicating the purity of the synthetically prepared standard compounds. In line with reports by others (11, 24, 43, 44), 5 and 6 were sensitive to digestion with nuclease P1 (recoveries of 2% or less) indicative for dG-C8 arylamine DNA adducts. Enrichment by butanol extraction led to a substantial improvement in recovery rates for both dG-C8 adducts. As already evident from results obtained with the standard procedure, the dG3′p-C8-4′tBu-4-ABP adduct exhibited a lower recovery rate also by butanol extraction as compared to the less lipophilic dG3′p-C8-4-ABP adduct. The observed recovery of 30% for the dG3′p-C8-4ABP adduct by enrichment with butanol extraction is approximately 3-fold higher than the value reported by Phillips and Castegnaro (25) from a 32P-postlabeling interlaboratory trial. However, in this study, the adduct level of the major 4-ABP adduct was determined in liver DNA from 4-ABP-treated mice. Thus, inefficient digestion of the adducted DNA might be one of the reasons for this discrepancy. In particular, the spleen phosphodiesterase in the postlabeling procedure has been shown to encounter difficulties in releasing adducts quantitatively (45). Also using synthetic dG3′p-C8-4-ABP, Hemminki et al. (24) reported a quantitative labeling efficiency and an adduct recovery of 30% (range 19-47) after nuclease P1 digestion for the same concentration range that we used. However, in this study, dG3′p-C8-4-ABP was used as the only substrate in the labeling reaction. Adduct standard dG3′p-N2-4-ABP is recovered poorly by either 32P-postlabeling version (ca. 2%). At this point, it is not clear whether this poor recovery is primarily due to an instability during DNA digestion, labeling reaction or chromatographic procedures, or to a low efficiency of labeling by T4 polynucleotide kinase or both in combination. Instability of this adduct was evident by a radioactive smear during the postlabeling assay, presumably due to the oxidation of the hydrazo linkage forming the corresponding azo adduct dG-N2dN-4-ABP (Figure 1E) consistent with our NMR data. In summary, DNA adducts formed by the human carcinogen 4-ABP were prepared and definitively identified as their deoxynucleoside 3′-phosphates to serve as adduct standards for the 32P-postlabeling assay. In all three commonly used versions of the 32P-postlabeling method, these adduct standards were poorly recovered and showed differences in recovery rates. Because 4-ABP is an ubiquitous environmental contaminant and its DNA adducts have been detected in human tissues (9, 46), the availability of 4-ABP adduct standards allows identification and accurate quantitation of human 4-ABP-DNA adducts in 32P-postlabeling analyses and is therefore of

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great utility in monitoring human exposure to 4-ABP and in estimating cancer risk.

Acknowledgment. We thank W. E. Hull for valuable comments. Supporting Information Available: COSY and ROESY spectra of 5 (dG3′p-C8-4-ABP) and 8 (dG3′p-N2-4-ABP). This material is available free of charge via the Internet at http:// pubs.acs.org.

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