Synthesis, Characterization, and Immunochemical Detection of O6

Jun 16, 1997 - DNA modification in carcinogen risk assessment in relation to diet: recent advances and some perspectives from a MAFF workshop. David E...
0 downloads 9 Views 281KB Size
Download PDF
652

Chem. Res. Toxicol. 1997, 10, 652-659

Synthesis, Characterization, and Immunochemical Detection of O6-(Carboxymethyl)-2′-deoxyguanosine: A DNA Adduct Formed by Nitrosated Glycine Derivatives Kathryn L. Harrison,† Neil Fairhurst,‡ Brian C. Challis,‡ and David E. G. Shuker*,† MRC Toxicology Unit, Hodgkin Building, University of Leicester, P.O. Box 138, Lancaster Road, Leicester LE1 9HN, U.K., and Department of Chemistry, The Open University, Walton Hall, Milton Keynes MK9 6AA, U.K. Received December 12, 1996X

O6-(Carboxymethyl)-2′-deoxyguanosine (O6-CMdG) is formed in DNA by nitrosated glycine derivatives and appears to be nonrepairable by O6-alkylguanine transferases. O6-CMdG has been synthesized by an unambiguous route involving the introduction of a methyl glycolate moiety at C6 of a 3′,5′-bis-O-(methoxyacetyl)dGuo derivative by displacement of a quinuclidinium ion. Methanolysis of the methoxyacetyl groups and calcium hydroxide-mediated hydrolysis of the methyl ester afforded the calcium salt of O6-CMdG in good yield. A similar route was used to synthesize O6-(carboxymethyl)guanosine (O6-CMGuo), which was used to prepare protein conjugates for immunization. Rabbit antisera were prepared, and a quantitative competitive ELISA was developed which showed 50% inhibition at 2 pmol of O6-CMdG/ well. O6-CMGuo was 30 times less cross-reactive (50% inhibition at 60 pmol/well), and normal nucleosides and carboxymethylated purines did not cross-react to any significant extent. The antiserum was also used to prepare reusable immunoaffinity columns which retained O6-CMdG. The binding of O6-CMdG was so strong that conditions used to elute the adduct (1 M trifluoroacetic acid) resulted in partial hydrolysis (becoming quantitative on heating) of the glycosidic bond to give O6-CMguanine which was detected by HPLC with fluorescence detection. DNA treated with azaserine (5 mmol), N-(N′-acetyl-L-prolyl)-N-nitrosoglycine (5 mmol), and potassium diazoacetate (5 mmol) afforded O6-CMdG at levels of 7.3, 393.9, and 496 µmol of O6-CMdG/mol of dG. The antiserum also recognized O6-CMdG in intact DNA.

Introduction Alkylation of DNA is considered to be a key step in the induction of cancer by many different chemicals (1). For many compounds including N-alkyl-N-nitroso compounds, alkylation at O6 of 2′-deoxyguanosine (dGuo) appears to be the major mutagenic lesion, although O4alkylthymidines may also be mutagenic (2). In both prokaryotic and eukaryotic cells, efficient specific repair mechanisms exist for the removal of O6-methyldGuo residues as well as some higher homologues, albeit more slowly (3). Among the many N-alkyl-N-nitroso compounds that are known to be carcinogenic, there are a number which share a common feature of being carboxymethylating agents. N-Nitrosoglycocholic acid (NOGC1 ) is a carcinogenic and mutagenic derivative of the naturally occurring bile acid conjugate glycocholic acid (4-6). Incubation of NOGC with calf thymus DNA in vitro gave rise * Author to whom correspondence should be addressed. † University of Leicester. ‡ The Open University. X Abstract published in Advance ACS Abstracts, May 1, 1997. 1 Abbreviations: NOGC, N-nitrosoglycocholic acid; 7-CMG, 7-(carboxymethyl)guanine; 3-CMA, 3-(carboxymethyl)adenine; O6-CMG, O6(carboxymethyl)guanine; O6-CMGuo, O6-(carboxymethyl)guanosine; APNG, N-(N′-acetyl-L-prolyl)-N-nitrosoglycine; AS, azaserine; O6CMdG, O6-(carboxymethyl)guanine-2′-deoxyguanosine; BSA, bovine serum albumin; OV, ovalbumin; PBS, phosphate-buffered saline; TFA, trifluoroacetic acid; TEA, triethylammonium acetate; DBU, 1,8diazabicyclo[5.4.0]undecane; ELISA, enzyme-linked immunosorbant assay; IgG, immunoglobulin G; TPase, thymidine phosphorylase; PNPase, purine nucleoside phosphorylase; KDA, potassium diazoacetate.

S0893-228x(96)00203-2 CCC: $14.00

to N-7-(carboxymethyl)guanine (7-CMG), N-3-(carboxymethyl)adenine (3-CMA), and O6-(carboxymethyl)guanine (O6-CMG) (7). Furthermore, administration of [14C]NOGC to rats gave rise to dose dependent excretion of 7-CMG in urine (7). N-Nitroso peptides which are C-terminal in glycine, such as N-(N′-acetyl-L-prolyl)-Nnitrosoglycine (APNG), are mutagenic (8) and carcinogenic (9) and would be expected to be carboxymethylating agents by analogy with NOGC. Similarly, N-nitroso-N(carboxymethyl)urea is a gastrointestinal carcinogen (10, 11). Azaserine (AS), a pancreatic carcinogen, is also known to carboxymethylate DNA in vivo. [14C]-7-CMG was detected in DNA extracted from acinar cells treated in vitro with [14C]azaserine (12). Recently, O6-(carboxymethyl)-2′-deoxyguanosine (O6CMdG) has attracted our interest because of its apparent lack of repair by bacterial and mammalian O6-alkylguanine alkyltransferases (13). In an analogous manner to many N-nitroso compounds, NOGC gives rise to alkylation mainly at purine nitrogen atoms, and the level of O6-CMdG is about 10% of that at N-7 of guanine (13). In order to study the occurrence of O6-CMdG in DNA, the synthesis and characterization of the previously unknown O6-CMdG were required for its use as an authentic compound. In addition, antibodies to O6-CMdG were required for detection and analysis of this adduct in human tissues. Previous experience (14, 15) has shown that antibodies are extremely useful not only in quantitation of adducts but also in the isolation, purification, and concentration of the adducts using immunoaffinity techniques. In the case of O6-alkyldG adducts, antigens © 1997 American Chemical Society

Immunochemical Detection of O6-CMdG

can be prepared from the corresponding guanosine analogue (16), and this approach is described along with the use of the antibodies to immunopurify O6-CMdG prior to detection by HPLC fluorescence.

Materials and Methods Warning: APNG and AS have been shown to be carcinogenic in experimental animals and should be treated with extreme caution. Unused solutions of APNG were decomposed by overnight treatment with 0.1 M NaOH in a fume cupboard. Unused solutions of AS and potassium diazoacetate were decomposed by overnight treatment with 10% aqueous acetic acid. General. Melting points were measured on a Gallenkamp hot-stage apparatus and are uncorrected. Infrared spectra were recorded on Perkin-Elmer 298 and 1420 grating spectrophotometers. 1H-NMR spectra were recorded on either a Jeol FX90Q spectrometer or a Jeol FX400Q in the solvents indicated. Mass spectra were recorded by using VG-7070 and VG-20-250 instruments. Microanalyses were provided by Medac Ltd., Brunel University, London. 2′-Deoxyguanosine and guanosine were supplied by Cruachem Ltd. (Aired, Glasgow, U.K.). Methyl glycolate (Kodak) was purified by distillation. Quinuclidine (Aldrich) was purified by sublimation. Azaserine, bovine serum albumin (BSA), ovalbumin (OV), horseradish peroxidase-linked goat anti-rabbit immunoglobulin G (IgG), thymidine, thymidine phosphorylase (Tpase, E.C. 2.4.2.4), purine nucleoside phosphorylase (PNPase, E.C. 2.4.2.1), calf thymus DNA, nuclease P1 (E.C. 3.1.30.1), alkaline phosphatase type III (Escherichia coli, E.C. 3.1.3.1), and acid phosphatase type I (E.C. 3.1.3.2) were purchased from Sigma Chemical Co. [3H]Thymidine was purchased from Amersham Life Sciences. Buffer salts and solvents were purchased from Fisons and were of an analytical grade. 3′,5′-Bis-O-(Methoxyacetyl)-2′-deoxyguanosine (1a). 2′Deoxyguanosine (1.0 g, 3.60 mmol) was suspended in anhydrous N,N-dimethylformamide (DMF) (16 mL) containing pyridine (4 mL), methoxyacetic anhydride (17) (2.45 g, 15 mmol) was added, and the mixture was heated at 40 °C for 2 h. The solution became homogeneous and was allowed to cool to room temperature. Methanol (0.5 mL) was then added, and the solution stirred for a further 30 min at room temperature. The mixture was concentrated under reduced pressure, and the residue was filtered and then washed with cold ethanol. Recrystallization from ethanol gave 1a as white crystals: yield 1.18 g (81%); mp 176-177 °C; νmax (Nujol) 3450 (N-H str), 3350 (N-H str), 1750 (CdO ester str), 1700 (CdO amide str), 1230 cm-1 (C-O str); NMR (DMSO-d6) δ 2.50 (1H, m, 2′-H), 2.93 (1H, m, 2′-H), 3.30 (6H, s, 2 × CH3), 3.92 (4H, 2s, 2 × CH2), 4.10 (2H, s, 5′-CH2), 4.30 (1H, m, 4′-H), 5.38 (1H, m, 3′-H), 6.17 (1H, t, 1′-H), 6.63 (2H, brs, NH2), 7.95 (1H, s, 8-H), 10.70 (1H, brs, NH); MS m/z (FAB positive ion) 412 (MH+) 20, 152 (MH+ - 260) 100. O6-[(2,4,6-Trimethylphenyl)sulfonyl]-3′,5′-bis-O-(methoxyacetyl)-2′-deoxyguanosine (1b). 3′,5′-Bis-O-(methoxyacetyl)-2′-deoxyguanosine (1.0 g, 2.4 mmol), 2-mesitylsulfonyl chloride (1.2 g, 5.5 mmol), and 4-(dimethylamino)pyridine (15 mg, 1.2 mmol) were suspended in CH3CN (10 mL). Triethylamine (1.15 g, 11.4 mmol) was added dropwise, and the suspension stirred at room temperature. After 2 h, this solution was concentrated under reduced pressure; the residue was redissolved in CHCl3/CH3OH (90:10, v/v, 10 mL) and then separated on silica gel (60 g) using CHCl3 (70 mL) and then CHCl3/CH3OH (97:3, v/v) as eluants, to give the product 1b as a brown glass. This glass was dissolved in the minimum volume of CH2Cl2 and precipitated with petroleum ether (40-60 °C) to give 1b as pale yellow crystals: yield 1.35 g (95%); mp 140152 °C; νmax (Nujol) 3450 (N-H str), 3350 (N-H str), 1750 (CdO ester str), 1350 (SO2-o str), 1230 (C-O str), 1160 cm-1 (SO2str); NMR (CDCl3) δ 2.31 (3H, s, p-CH3), 2.50 (1H, m, 2′-H), 2.75 (6H, s, 2 × o-CH3), 2.93 (1H, m, 2′-H), 3.35 (6H, 2s, 2 × CH3), 3.92 (4H, 2s, 2 × CH2), 4.10 (2H, s, 5′-CH2), 4.40 (1H, m, 4′-H), 4.95 (2H, brs, NH2), 5.50 (1H, m, 3′-H), 6.30 (1H, t, 1′-H), 6.95 (2H, brs, arom), 7.90 (1H, s, 8-H); MS m/z (EI) 593 (M+) 12, 333 (M+ - 260) 30.

Chem. Res. Toxicol., Vol. 10, No. 6, 1997 653 O6-(Carboxymethyl)-2′-deoxyguanosine, Methyl Ester (1e). 1b (0.3 g, 0.5 mmol) was dissolved in dry THF (4 mL), quinuclidine (0.32 g, 2.5 mmol) was added, and the solution stirred at room temperature under dry nitrogen. Progress of the reaction was monitored by loss of 1b and formation of a blue fluorescent spot at the baseline (believed to be the quinuclidinium ion intermediate 1c), using silica gel TLC developed with EtOAc/CH3OH (85:15, v/v). The formation of 1c was usually complete in ca. 3 h at room temperature. Dry methyl glycolate (0.44 g, 5 mmol) in THF (3 mL) and 1,8-diazabicyclo[5.4.0]undecane (DBU; 0.24 g, 1.5 mmol) in THF (3 mL) were added to the mixture, and the solution stirred at room temperature for 3-4 h. After deprotection of 1d using methanolic Et3N and purification by column chromatography on silica gel (30 g) using CH3OH/EtOAc (15:85, v/v) as eluant, 1e was further purified by semipreparative LC on a Phase Sep S10 ODS2 C-18 silica gel column, using CH3CN/H2O (13:87, v/v) isocratic eluant system. After removal of the eluant by lyophilization, 1e was obtained as a white crystalline solid: yield 0.11 g (68%); mp 83-84 °C; λmax (pH ) 7) 248, 281; νmax (KBr) 3300 (N-H, O-H str), 2800 (C-H str),1752 (CdO str), 1650 (CdN str), 1580 (CdC str), 1250 (C-O str), 1065 cm-1 (C-O str); NMR (DMSO-d6) δ 2.30 (2H, m, 2′-H), 3.55 (2H, m, 5′-CH2), 3.61 (3H, s, OCH3), 3.80 (1H, m, 4′-H), 4.35 (1H, m, 3′-H), 4.81 (2H, s, CH2CO2CH3), 5.0 (1H, t, 5′-OH), 5.30 (1H, d, 3′-OH), 6.22 (1H, t, 1′-H), 6.45 (2H, brs, NH2), 8.10 (1H, s, 8-H); MS m/z (FAB positive ion) 340 (MH+) 10, 224 (MH+ - 116) 20, 152 (MH+ - alkyl - 116) 32. O6-(Carboxymethyl)-2′-deoxyguanosine (1f). 1e (0.042 g, 0.12 mmol) was dissolved in aqueous 4 mM Ca(OH)2 (30 mL), and the solution stirred at room temperature. Loss of starting material was monitored by TLC on silica gel using EtOAc/CH3OH (85:15, v/v) as eluant. After 18 h, lyophilization of the reaction solution gave 1f as a white fluffy solid. The solid was dissolved in absolute ethanol, and the insoluble residue was removed. The solvent was removed under reduced pressure, to give 1f as a white solid: yield 0.040 g (98%); mp >250 °C; λmax (pH ) 7) 248, 282; νmax (KBr) 3300 (N-H, O-H br str), 2860 (C-H str), 1650 (CdN str), 1620 (CdO str), 1580 (CdC str), 1250 (C-O str), 1065 cm-1 (C-O str); NMR (DMSO-d6) δ 2.30 (2H, m, 2′-H), 3.55 (2H, m, 5′-H), 3.80 (1H, m, 4′-H), 4.35 (1H, m, 3′-H), 4.64 (2H, s, CH2), 5.0 (1H, t, 5′-OH), 5.30 (1H, d, 3′-OH), 6.22 (1H, t, 1′-H), 6.45 (2H, brs, NH2), 8.09 (1H, s, 8-H); MS m/z (FAB negative ion) 324 (M-) 8, 151 (M- - alkyl - 116) 20. Anal. Calcd for C24H28N10O12Ca‚2H2O: C, 39.78; H, 4.42; N, 19.34; O, 30.94; Ca, 5.52. Found: C, 39.60; H, 4.44; N, 19.30. O 6 -[(2,4,6-Trimethylphenyl)sulfonyl]-2′,3′,5′-tri-Oacetylguanosine (2b). 2a (18) (1.0 g, 2.4 mmol), 2-mesitylsulfonyl chloride (1.05 g, 4.8 mmol), and 4-(dimethylamino)pyridine (130 mg, 1.06 mmol) were suspended in CH3CN (12 mL). Triethylamine (1.10 g, 9.6 mmol) was added dropwise, and the suspension stirred at room temperature. After 2 h, the residue was evaporated to dryness under reduced pressure, and the residue was redissolved in CHCl3/CH3OH (90:10, v/v) (2 mL) and separated on silica gel (60 g) using CHCl3 (70 mL) and then CHCl3/CH3OH (97:3, v/v) as eluants, to give a brown solid which was dissolved in the minimum volume of CH2Cl2 and precipitated with petroleum ether (40-60 °C) to give 2b as yellow crystals: yield 1.31 g (91%); mp 140-141 °C [lit. (17) mp 141142 °C]; νmax (Nujol) 3450 (N-H str), 3350 (N-H str), 1730 (CdO ester str), 1350 (SO2-O str), 1230 (C-O str), 1160 cm-1 (SO2-O str); NMR (CDCl3) δ 2.03 (3H, s, CH3CO), 2.07 (3H, s, CH3CO), 2.08 (3H, s, CH3CO), 2.30 (3H, s, p-CH3), 2.70 (6H, s, o-CH3), 4.35 (2H, brs, 5′-CH2), 5.60 (2H, m, 2′-H, 3′-H), 5.85 (1H, m, 2′-H), 6.00 (1H, brd), 6.50 (2H, brs, NH2), 6.9 (2H, s, arom), 7.8 (1H, s, m, 8-H); MS m/z (FAB positive ion) 592 (MH+) 18, 334 (MH+ - 258) 50. O6-(Carboxymethyl)guanosine, Methyl Ester (2e). 2b (0.3 g, 0.5 mmol) was dissolved in dry THF (4 mL), quinuclidine (0.32 g, 2.5 mmol) was added, and the solution was stirred at room temperature under dry N2. Progress of the reaction was monitored by loss of 2b and formation of a blue fluorescent spot at the baseline (presumably the quinuclidium salt 2c), using

654 Chem. Res. Toxicol., Vol. 10, No. 6, 1997 silica gel TLC developed with EtOAc/CH3OH (85:15, v/v). The formation of the quinuclidine ion intermediate 2c was usually complete in ca. 3-4 h at room temperature. Dry methyl glycolate (0.44 g, 5 mmol) in THF (5 mL) and DBU (0.24 g, 1.5 mmol) in THF (3 mL) were added to the reaction mixture, and the solution stirred at room temperature until the blue fluorescent spot disappeared. After 4 h, the solution was cooled to room temperature, the solvent removed under reduced pressure, and the resultant residue dissolved in methanol (5 mL), treated with 0.4 M methanolic triethylamine (9 mL), and then stirred at room temperature for 12 min. Evaporation of the solvent under reduced pressure gave a residual oil, which was separated on silica gel (40 g) using EtOAc/CH3OH (85:15, v/v) as eluant. The appropriate product-containing fractions identified by TLC on silica gel were combined, dried using Na2SO4, filtered, and evaporated under reduced pressure to give 2e as a white solid. 2e was further purified by semipreparative HPLC on a Phase Sep C-18 silica gel column using CH3OH/H2O (12:88, v/v) as eluant at 20 mL/min at room temperature. After removal of the solvent by lyophilization, 2e was isolated as a white crystalline solid: yield 0.11 g (62%); mp 123-124 °C; λmax (pH ) 7) 248, 281; νmax (KBr) 3300 (N-H, O-H str), 2860 (C-H str), 1752 (CdO str), 1580 (CdC str), 1250 (C-O str), 1065 cm-1 (C-O str); NMR (DMSO-d6) δ 3.58 (2H, m, 5′-H), 3.61 (3H, s, OCH3), 3.88 (1H, m, 4′-H), 4.10 (1H, m, 3′-H), 4.46 (1H, m, 2′H), 4.87 (2H, s, CH2CO2CH3), 5.14 (2H, m, 5′-OH, 2′-OH), 5.40 (1H, d, 3′-OH), 5.80 (1H, d, 1′-H), 6.52 (2H, brs, NH2), 8.11 (1H, s, 8-H); MS m/z (FAB positive ion), 356 (MH+) 12, 224 (MH+ 132) 30. O6-(Carboxymethyl)guanosine (2f). 2e (0.042 g, 0.11 mmol) was dissolved in 4 mM aqueous Ca(OH)2 solution (30 mL), and the solution stirred at room temperature. Loss of starting material was monitored by TLC on silica gel using EtOAc/CH3OH (85:15, v/v) as eluant. After 18 h, lyophilization of the reaction solution gave 2f as a white fluffy solid. The solid was dissolved in ethanol, and the insoluble residue was removed. The solvent was removed under reduced pressure to give 2f as a white solid: yield 0.04 g (97%); mp >250 °C; λmax (pH ) 7) 248 (10 020), 282 nm, (9880); νmax (KBr) 3300 (O-H, N-H str), 2880 (C-H str),1650 (CdN str), 1620 (CdO str), 1580 (CdC str), 1250 (C-O str), 1065 cm-1 (C-O str); NMR (400 MHz, DMSO-d6) δ 3.58 (2H, m, 5′-CH2), 3.88 (1H, m, 4′-H), 4.10 (1H, m, 3′-H), 4.46 (1H, m, 2′-H), 4.62 (2H, s, CH2), 5.14 (2H, m, 5′-OH, 2-OH), 5.40 (1H, m, 3′-OH), 5.82 (1H, d, 1′-H), 6.52 (2H, brs, NH2), 8.09 (1H, s, 8-H); MS m/z (FAB negative ion) 340 (M-) 7, 151 (M- - alkyl - 116) 20. Anal. Calcd for C24H28N10O14Ca‚H2O: C, 39.02; H, 4.07; N, 18.97; O, 32.50; Ca, 5.42. Found: C, 38.90; H, 4.35; N, 18.90. Preparation of [3H]-O6-CMdG. Enzymatic Coupling: This reaction was carried out essentially as described by Chapeau and Marnett (19) with the following modifications. O6CMG (0.214 µmol; prepared by hydrolysis of O6-CMdG in 10% HFBA followed by isolation using HPLC) and thymidine (0.66 µmol) were dissolved in 300 µL of 20 mM potassium phosphate buffer, and the pH was adjusted to 7.3. Tpase (0.22 unit), PNPase (6.6 units), sodium azide (final concentration 0.05%), and 66 µL of a solution containing [3H]thymidine (2.44 MBq) were added. The mixture was incubated at 38 °C for 2 weeks, over which time the progress of the reaction was monitored by RP-HPLC (employing the system outlined below) using a duplicate reaction mixture which had [3H]thymidine replaced by the same volume of water. HPLC Conditions: Analytical and preparative HPLC were performed on a Waters HPLC system [600E system controller and a UV spectrophotometric detector (Shimadzu SPD6A) at a wavelength of 278 nm] equipped with a Techspere 5 mm ODS reverse-phase column (250 × 4.6 mm) at a flow rate of 1 mL/ min. A step gradient elution system was employed as follows: 0-10 min 10% MeOH in 0.1 M TEA (pH ) 7), 10-20 min 15% MeOH in 0.1 M TEA (pH ) 7), 20-30 min 20% MeOH in 0.1 M TEA (pH ) 7), and going back to initial conditions over a further 10 min. The order of elution was thymine, O6-CMG, thymidine, and O6-CMdG.

Harrison et al. Purification of [3H]-O6-CMdG: Aliquots of the reaction mixture (3 × 100 µL) were loaded onto the HPLC system (run conditions as above), and the peak corresponding to O6-CMdG was collected and concentrated by freeze-drying. These fractions were then combined, concentrated to dryness, redissolved in 100 µL of 0.1 M TEA (pH ) 7), and reinjected onto the HPLC system. Fractions were collected every 30 s for 30 min, and 5 µL of each (in 3 mL of liquid scintillation fluid) was then counted on a Wallac 1410 liquid scintillation counter. The fraction corresponding in retention time to O6-CMdG showed an elevated level of radioactivity. Rechromatography of this fraction showed that no other peaks were present except for O6-CMdG, and on quantitation against a calibration curve, an isolated yield of 7.44% of [3H]-O6-CMdG was achieved. Preparation of O6-CMGuo Conjugated with Ovalbumin (OV) or Bovine Serum Albumin (BSA). 2f (Ca2+ salt, 10 mg) in H2O (0.5 mL) was treated with NaIO4 (21.4 mg, 100 µL) and stirred at room temperature for 15 min. Ethylene glycol (5 µL) was added to stop the reaction, and this solution was added to OV or BSA (10 mg) in H2O (1 mL) adjusted to pH ) 9.5 with 0.2 M Na2CO3 and kept at room temperature for 4 min. The Schiff base was stabilized by addition of NaCNBH4 (0.5 mL of 30 mg/mL freshly made in H2O) and left at 4 °C for 3 h, with 1 drop of octanol to prevent foaming. The resultant mixture was dialyzed overnight against PBS and then eluted through a Sephadex G-50 column using 0.15 M NaCl. The first eluting UV-absorbing fractions were collected and concentrated in a Micro-ProDiCon apparatus (Spectrum Microgon, Laguna Hills, CA) overnight and the samples lyophilized. The hapten-carrier ratio was determined by measuring the contribution of the nucleoside to the absorbance at 280 nm. The ratios were 3 for O6-CMGuo-BSA and 1 for O6-CMGuo-OV. Immunization of Rabbits and Preparation of Rabbit Antisera. O6-CMGuo-BSA conjugate (2.5 mg) dissolved in PBS (1.25 mL) was emulsified with Freunds complete adjuvant (1.25 mL); 1 mL of the emulsion was injected into multiple sites on the shaved backs of two rabbits (New Zealand White, female). After 3 weeks, the rabbits were given another injection of O6CMGuo-BSA adduct (1 mg in PBS/Freunds incomplete adjuvant, 1 mL/rabbit) in 500 µL aliquots into the hind quarters. Two months later, the rabbits were given a further booster injection of O6-CMGuo-BSA adduct (0.5 mg in PBS/Freunds incomplete adjuvant, 1 mL/rabbit) in 500 µL aliquots into the hind quarter. Two months later, the rabbits were given a further booster injection of O6-CMGuo-BSA adduct [1 mg in PBS/Freunds incomplete adjuvant (1:1, v/v, 2 mL)], 1 mL/rabbit, in 500 µL aliquots into the hind quarter. Two weeks later the rabbits were bled from the lateral ear vein, and the blood was stored at 4 °C overnight. The sera were then stored at 37 °C for 1 h and centrifuged at 1000g for 10 min. The supernatants were decanted off and stored in 1 mL aliquots at -30 °C. ELISA Procedure. Optimal conditions for ELISA were determined using a checkerboard procedure in which coating antigen levels of 1 ng-10 µg/well and antiserum dilutions of 1 in 10 to 1 in 106 were tested. A reasonable absorbance was found using 5 ng of O6-CMGuo-OV/well and a dilution of 1 in 200 000. Both antisera displayed similar properties in a competitive ELISA for O6-CMdGuo. The ELISA protocol was as follows: Polystyrene microtiter plates (96-well; Dynatech M129B) were filled with a solution of coating antigen (40 µL of PBS containing 5 ng of O6-CMGuo-OV) and dried overnight at 37 °C. Plates were stored at room temperature, protected from dust and in the dark. Plates were washed with PBS/0.005% Tween (6×) and then dried by tapping onto absorbant paper towels. Standard solutions of O6-CMdG or other inhibitor were prepared in PBS so that the concentration varied between 0.1 and 107 fmol/25 µL. An aliquot of O6-CMdG in PBS (25 µL) was pipetted onto the plate in rows (eight wells) for each of the standard solutions. Usually two rows on the ELISA plate were used for controls, and to each of these rows was added PBS (25 µL/well). A reference row containing PBS (50 µL/well) was also used. Polyclonal rabbit antiserum (CMG2; 25 µL of a l:200 000

Immunochemical Detection of O6-CMdG dilution in PBS of the neat antiserum) was added to each well in the plate except those in the reference row. The plate was then incubated at room temperature for 90 min. The supernatant liquid was then decanted from the ELISA plate, and the plate was washed in PBS/Tween (6×). Horseradish peroxidaselinked goat anti-rabbit immunoglobulin G (Sigma; 50 µL of a l:1000 dilution in PBS) was added to every well on the plate. The plate was reincubated at room temperature for 90 min. After 90 min, the supernatant was discarded and the plate washed with PBS/Tween (6×) and once with distilled water. Aqueous citrate buffer, pH ) 5.3 (50 µL), from a solution (10 mL) containing 3′,3′,5′,5′-tetramethylbenzidine (prepared by addition of 1 mg in 100 µL of dimethyl sulfoxide) and H2O2 (2 µL of 30%, w/w, solution), was added to each well of the plate, and the plate was incubated for 15 min at room temperature to allow color development to occur. HCl (1 M 50 µL) was added to each well, and the optical density at 450 nm of each well of the plate was measured by an automatic plate reader. Generally, the control wells of the plate gave an optical density of ca. 0.7 after subtraction of the reference (blank) wells. Preparation and Characterization of Immunoaffinity Columns for O6-CMdG. Immunoaffinity columns were prepared by covalently linking the ammonium sulfate-precipitated IgG fraction of sera to protein A-Sepharose CL4B and using the resultant gel to prepare small (1 mL) columns as described by Friesen et al. (20). [3H]-O6-CMdG (17.2 ng, 950 dpm) in PBS/ 0.02% azide (2 mL) was applied to a column followed by a further 3 mL of PBS/0.02% azide. The column was then washed with water (10 mL). One milliliter fractions of the column eluate were collected, and the radioactivity was measured by liquid scintillation counting using various elution conditions: 1 M acetic acid (5 mL), 50% aqueous MeOH (5 mL), 50% aqueous DMSO (5 mL), 1 M formic acid (5 mL), 1 M trifluroacetic acid (TFA) (5 mL), and 0.1 M TFA (5 mL). Quantitative elution of the [3H]-O6-CMdG was obtained by washing the column with 1M TFA (5 mL) with most of the radioactivity eluting in the first 2 mL. Determination of the Column Capacity for O6-CMdG: PBS/0.02% azide (2 mL) containing [3H]-O6-CMdG (17.2 ng, 950 dpm) and O6-CMdG (0-1000 ng) was applied to immunoaffinity columns. The columns were washed with PBS/0.02% azide (3 mL) and water (10 mL), elution with TFA (1 M, 5 mL) was then carried out, and the eluate was collected directly into scintillation vials. Liquid scintillation fluid (3 mL) was added to each vial and the radioactivity determined by scintillation counting. The results are expressed as the percentage of [3H]-O6-CMdG retained on the column. Treatment of DNA with Various Nitrosated Glycine Derivatives. Calf thymus DNA was dissolved in PBS (pH ) 7) (5 mg/mL), and APNG (21), AS, or potassium diazoacetate (22) was added to give a 5 mM solution which was left gently stirring (37 °C) in the dark overnight. After treatment, DNA was precipitated from the reaction medium with sodium acetate (0.1 volume, 2.5 M) and cold ethanol (2 volumes) and centrifuged gently (3000g for 5 min) and the DNA washed with ethanol. The DNA pellet was recovered, evaporated to dryness, and resuspended in water to the original volume. Determination of O6-CMdG in DNA: Enzyme hydrolysis was performed following the method described by Beranek et al. (23). DNA samples were hydrolyzed in 50 mM bisTris/1 mM MgCl2 (pH ) 6.5) at 50 °C for 8 h, using nuclease P1 (24 units), bacterial alkaline phosphatase (4.8 units), and wheat germ acid phosphatase (0.3 unit) per 1 mg of DNA (final concentration 1 mg of DNA/mL). The reaction was stopped by heating at 100 °C for 5 min, and the mixture was then centrifuged to remove the denatured enzyme protein. DNA hydrolysate (2.5 mg) in 2 mL of PBS/0.02% azide was loaded onto an O6-CMdG immunoaffinity column and then washed with PBS (3 mL) and water (10 mL). Elution was achieved with 1 M TFA (5 mL) which was collected and then heated for 1 h at 50 °C to quantitatively convert O6-CMdG to the more fluorescent base O6-CMG. The immunoaffinity column was then washed with PBS/0.02% azide (15 mL) for further use. The TFA fraction was evaporated

Chem. Res. Toxicol., Vol. 10, No. 6, 1997 655 to dryness (freeze-dried) and the residue redissolved in water (1 mL), which was in turn evaporated to dryness (Speedvac) and redissolved in 0.1% HFBA (25 µL) for HPLC analysis. The above procedure was also applied to untreated DNA. Analytical HPLC was performed on a 100 × 2 mm Hypersil BDS C18 (3 µm) column for both O6-CMG and dG quantitation. O6-CMG was quantitated by fluorescence detection (excitation at 286 nm, emission at 378 nm) using isocratic conditions (0.1% HFBA/2.7 mM EDTA:methanol, 90:10) at 0.2 mL/min. dG was determined in aliquots of DNA hydrolysate (10 µL) prior to immunoaffinity treatment by detection at 260 nm using isocratic conditions (0.1 M TEA, pH ) 4.5:methanol, 96:4) at 0.2 mL/min.

Results Synthesis of O6-CMdG. The synthesis of O6-CMdG (1f) is summarized in Scheme 1. The synthesis route was based on the approach developed by Gaffney and Jones (24), in which an O6-arylsulfonate is displaced by a trialkylamine, with the resulting trialkylammonium intermediate reacting with an alcohol to give the corresponding O6-alkylnucleoside. For our purposes, it was not necessary to protect the exocyclic N2-amino group, especially since removal of the N2-acyl group by concentrated ammonia can result in substitution of the O6-alkyl by an amino group (24). It was decided to protect the 3′,5′-hydroxyl groups of dGuo with more readily hydrolyzable methoxyacetyl as opposed to acetyl. However, as described below, acetyl protection is advantageous for the more labile esters of 2′-hydroxyl groups in ribonucleosides. Conversion of 1 to 1b proceeded cleanly in ca. 80% overall yield. Attempts to introduce the methyl glycolate moiety via trimethylammonium or N-methylpyrrolidinium ion intermediates were unsuccessful and resulted in the formation of substantial amounts of 6-(dimethylamino)- and 6-pyrrolidinodG products, respectively (results not shown). In contrast, the use of quinuclidine as tertiary amine resulted in a 65% yield of 1d. The methoxyacetyl protecting groups were readily removed by treatment with triethylamine/methanol at room temperature to give 1e. However, at this stage, an HPLC cleanup was carried out since a persistent contamination with mesitylenesulfonic acid was detected in the mass spectrum of 1e despite recrystallization. Finally, 1f was prepared as the dihydrated calcium salt by calcium hydroxide-mediated hydrolysis of 1e. Synthesis of O6-CMGuo for Use in Antiserum Preparation. O6-CMGuo (2f) was required in order to prepare antibodies to 1f. Initially, the same procedure for protection of ribose hydroxyls using methoxyacetic anhydride was used, but deprotection of the 2′-hydroxyl occurred in subsequent steps resulting in a mixture of products (data not shown). It was, therefore, decided to use the more stable acetyl protection. Thus, guanosine was acetylated to give 2a (18), and this was cleanly converted to 2b in 90% yield. Introduction of the O6carboxymethyl moiety proceeded smoothly and was followed by deprotection of 2d without isolation and an HPLC purification step to give 2e in 62% yield. Calcium hydroxide hydrolysis of 2e afforded 2f in 97% yield as the monohydrated calcium salt. Preparation of an O6-CMdGuo Antiserum and Development of an ELISA Protocol. 1f was conjugated to BSA using the standard method of Mu¨ller and Rajewsky (16), which results in the formation of an antigenic molecule with a structure resembling O6CMdGuo. Despite the low level of modification, both

656 Chem. Res. Toxicol., Vol. 10, No. 6, 1997

Harrison et al.

Scheme 1. Synthetic Routes for the Preparation of O6-CMGuo (2f) and O6-CMdG (1f)a

a Conditions: (i) 1, methoxyacetic anhydride, DMF, 2, acetic anhydride, DMF; (ii) mesitylenesulfonyl chloride, triethylamine, 4-(dimethylamino)pyridine, rt; (iii) quinuclidine, THF, rt; (iv) methyl glycolate, DBU, THF, 65 °C; (v) triethylamine, MeOH, rt; (vi) Ca(OH) 2.

Figure 1. Inhibition curve for O6-CMdGuo in competitive ELISA using the conditions described in the Materials and Methods section.

rabbits that were immunized produced antisera of similar titer. Optimal conditions for competitive ELISA were found using a checkerboard procedure. At a level of coating antigen (O6-CMGuo-OV) of 5 ng/well and an antiserum dilution of 1 in 200 000, a final absorbance at 450 nm of ca. 0.7-0.9 was obtained. Under conditions of a competitive ELISA, O6-CMdGuo was tested over a wide range (0.1-107 fmol/well), and the 50% inhibition was found to be 2 and 3 pmol for antisera CMG2 and CMG1, respectively. Figure 1 shows the inhibition curve for CMG2. Under the same conditions, a number of purines and nucleosides were tested for cross-reactivity using CMG2, and the results are summarized in Table 1. A standard curve for O6-CMdGuo was constructed between 0.2 and 10 pmol/well and is linear over this range (Figure 2). Immunoaffinity Purification of O6-CMdG and Its Detection in DNA. Immunoaffinity columns were prepared by covalently linking a total IgG fraction from

Figure 2. Standard curve for O6-CMdGuo in a competitive ELISA. Table 1. Cross-Reactivity of Rabbit Antiserum CMG2 in a Competitive ELISA

substrate

substrate conctn for 50% inhibition (pmol/well)

O6-(carboxymethyl)-2′-deoxyguanosine O6-(carboxymethyl)guanosine O6-methyl-2′-deoxyguanosine O6-ethyl-2′-deoxyguanosine 2′-deoxyguanosine 7-(carboxymethyl)guanine 3-(carboxymethyl)adenine 2′-deoxyadenosine 2′-deoxycytidine thymidine

2 60 >104 >104 >104 >104 >104 >104 >104 >104

rabbit antiserum to Protein A-Sepharose (19). O6-CMdG was retained by the columns (Figure 3) with a capacity of ca. 1 nmol (Figure 4). The binding was so strong that fairly drastic conditions (1 M TFA) were required for elution. Nonetheless, the columns can be recycled many times using this elution solvent without apparent dete-

Immunochemical Detection of O6-CMdG

Figure 3. Elution of O6-CMdG from CMG2 immunoaffinity columns. [3H]-O6-CMdG (17.2 ng, 950 dpm) was in PBS/azide (2 mL), and the column was washed with PBS/azide (3 mL), water (10 mL), and 1 M TFA (5 mL). The column eluate was collected as 1 mL aliquots which were added to scintillation fluid and counted directly.

Chem. Res. Toxicol., Vol. 10, No. 6, 1997 657

Figure 5. Calibration line for the determination of added standards of O6-CMdG in DNA following enzymatic hydrolysis, immunoaffinity cleanup, and HPLC fluorescence. The details are given in the Materials and Methods section. Table 2. Yield of O6-CMdG in Calf Thymus DNA Treated with Various Nitrosated Glycine Derivatives compounda AS APNG KDA

O6-CMdG (µmol/mol of dG)b 7.3 ( 0.35 39.9 ( 2.5 496 ( 58

a At a concentration of 5 mmol. b Mean ( SD of three separate reactions.

Figure 4. Determination of the column capacity of immunoaffinity columns for O6-CMdG. [3H]-O6-CMdG (17.2 ng, 950 dpm) was eluted through the column in the presence of increasing amounts of unlabeled O6-CMdG (0-1000 ng).

rioration in column performance. Under these conditions O6-CMdG was extensively hydrolyzed to the corresponding base O6-CMG. However, since O6-alkylguanines are slightly more fluorescent then the corresponding deoxynucleosides (24), this phenomenon was turned into an advantage by heating the column eluate to drive the hydrolysis to completion prior to HPLC analysis. Standard amounts of O6-CMdG were added to samples of DNA (0-20 pmol/mg) which were then hydrolyzed and analyzed using immunoaffinity purification with HPLC fluorescence as described above. A linear response was observed (Figure 5). Calf thymus DNA (5 mg/mL) which had been incubated with APNG (5 mmol), AS (5 mmol), or KDA (5 mmol) was analyzed using the above method and found to contain varying amounts of O6-CMdG (Table 2). The HPLC fluorescence chromatograms were free of interfering peaks, and a representative trace for APNG-treated DNA is shown in Figure 6.

Figure 6. HPLC fluorescence traces of O6-CMG in APNGtreated (solid line) and control (broken line) DNA. In each case the sample injected corresponded to 0.25 mg of DNA. The conditions are described in the Materials and Methods section.

Discussion The synthesis of O6-CMdG (1f) has been achieved by unambiguous insertion of a glycolate moiety into the 6-position of a suitably protected dG molecule (Scheme 1). 1f was used as an authentic standard to confirm the

658 Chem. Res. Toxicol., Vol. 10, No. 6, 1997

Harrison et al.

Chart 1. Structures of N-(N′-Acetyl-L-prolyl)-N-nitrosoglycine (APNG), Azaserine (AS), and Potassium Diazoacetate (KDA)

presence of the nonrepairable carboxymethylated adduct in DNA treated with N-nitrosoglycocholic acid (13). An antiserum was raised against 1f by using the corresponding guanosine analogue bound to a carrier protein, and a sensitive competitive ELISA procedure was developed. Good recognition of O6-CMdG was obtained compared to normal nucleosides and carboxymethylated DNA bases. Interestingly, O6-CMGuo was 30 times less efficient as an inhibitor than O6-CMdGuo, illustrating the selectivity of the antiserum. O6-Alkylated deoxyguanosine and unmodified nucleosides were at least 5000 times less efficient as inhibitors, as were the carboxymethylated purine bases. It is apparent that the antiserum is very selective for the O6-CMdG molecule. Attempts to increase the sensitivity of the ELISA by conducting part of the assay at 4 °C only resulted in a modest (2.5-fold) improvement (data not shown), in contrast to the much larger effects noted for alkylpurine ELISAs (26). This may be due to the fact that the polar (charged) carboxymethyl group is very strongly bound in the antigen-binding site of the antibody molecule as a result of a combination of van der Waals and electrostatic interactions (see below). Immunoaffinity columns were prepared and found to efficiently bind O6-CMdG. The capacity of the columns (∼1 nmol) was found to be comparable with those prepared using antibodies against other DNA adducts (19, 27-29). It is interesting to note that in these studies we have immunoaffinity columns which have good selectivity and capacity using both monoclonal antibodies and polyclonal antisera but that monoclonal antibodies have the advantage of continuous availability. The apparent high affinity of the antibody was illustrated by the rather drastic conditions (1 M TFA) required to elute O6-CMdG giving rise to partial hydrolysis to O6-CMGuo. However, since the free base is slightly more fluorescent than the 2′-deoxynucleoside (25), this proved advantageous for the sensitivity of the assay: the hydrolysis was driven to completion by heating the eluate prior to evaporation and HPLC analysis. The utility of the combination of immunoaffinity purification of O6-CMdG with HPLC fluorescence is illustrated by analysis of O6CMdG in calf thymus DNA treated with APNG, a model nitroso peptide which is a potent mutagen and carcinogen (9), AS, a potent pancreatic carcinogen (12), and KDA (22) (Chart 1). KDA is particularly interesting because it is a stable nitrosated derivative of glycine, one of the most common dietary amino acids. APNG, AS, and KDA are members of a family of nitrosated glycine derivatives

Figure 7. HPLC chromatograms of O6-CMdG in calf thymus DNA treated with KDA (5 mmol). DNA was either acid hydrolyzed (10% HFBA, 100 °C, 30 min) and injected directly (solid line) or enzymatically hydrolyzed and immunoaffinity purified as described in the Materials and Methods section (broken line). In both cases the amount of hydrolysate injected onto the column was equivalent to 25 µg of DNA.

Figure 8. Immunoslotblot assay of calf thymus DNA containing increasing amounts of O6-CMdG. KDA-treated calf thymus DNA was mixed with unmodified calf thymus DNA, and a constant amount of DNA (1 µg) was added to each well in duplicate. The values indicated are the levels of O6-CMdG determined by immunoaffinity HPLC fluorescence.

which decompose or rearrange to give carboxymethylating agents resulting in the formation of O6-CMdG but which also give rise to DNA methylation (13). The advantage of using immunoaffinity purification prior to HPLC fluorescence, compared to no prepurification, is shown in Figure 7. Fluorescence due to the normal DNA bases interfered substantially with the quantitation of O6-CMdG. The absolute limit of detection of the HPLC fluorescence assay was 0.1 pmol/injection. If 1 mg of DNA hydrolysate was used per injection, the limit of detection of the combined immunoaffinity-HPLC fluorescence assay corresponded to 1 O6-CMdG adduct/ 107 normal bases. While this level of sensitivity is suitable for experimental studies where amounts of DNA for analysis are fairly large (>0.1 mg), it is not sufficient for human studies where only small biopsies or blood volumes, which yield ca. 10 µg of DNA, are available. The lack of cross-reactivity of antiserum CMG2 with normal DNA bases (Table 1) suggested that O6-CMdG could be

Immunochemical Detection of O6-CMdG

detected in intact DNA. Accordingly, an immunoslotblot assay was developed where an equivalent sensitivity is obtained on small amounts (typically 1 µg) of DNA (Figure 8). The immunoslotblot assay was carried out as described by Nehls et al. (30), and full details will be published elsewhere.

Acknowledgment. This work has been supported, in part, by the U.K. Ministry of Agriculture, Fisheries and Food (Contract No. 1A025) and the Cancer Research Campaign. Neil Fairhurst gratefully acknowledges the award of a European Science Foundation Toxicology Programme Fellowship which enabled him to work on the development of immunoassays at the IARC in Lyon, France. Some of the results in this paper are taken from the Ph.D. Thesis (University of London, 1990) of Neil Fairhurst. We thank Dr. J. A. Challis for obtaining the mass spectra.

References (1) Miller, E. C. (1978) Some current perspectives on chemical carcinogenesis in humans and experimental animals: address. Cancer Res. 38, 1479-1496. (2) Saffhill, R., Margison, G. P., and O’Connor, P. J. (1985) Mechanisms of carcinogenesis induced by alkylating agents. Biochim. Biophys. Acta 823, 111-145. (3) Karran, P., and Lindahl, T. (1985) Cellular defense mechanisms against alkylating agents. Cancer Surveys 4, 585-599. (4) Shuker, D. E. G., Tannenbaum, S. R., and Wishnok, J. S. (1981) N-Nitrosobile acid conjugates. I. Synthesis, chemical reactivity and mutagenic activity. J. Org. Chem. 46, 2092-2096. (5) Song P., Shuker, D. E. G., Bishop, W. W., Falchuk, K. R., Tannenbaum, S. R., and Thilly, W. G. (1982) Mutagenicity of N-nitrosobile acid conjugates, N-nitrosoglycocholic acid and Nnitrosotaurocholic acid. Cancer Res. 42, 1367-1371. (6) Busby, W. F., Jr., Shuker, D. E. G., Charnley, G., Newberne, P. M., Tannenbaum, S. R., kand Wogan, G. N. (1985) Carcinogenicity of the nitrosated bile acid conjugates, N-nitrosoglycocholic acid and N-nitrosotaurocholic acid. Cancer Res. 45, 1367-1371. (7) Shuker, D. E. G., Howell, J. R., and Street, B. W. (1987) Formation and fate of nucleic acid and protein adducts from N-nitroso bile acid conjugates. In Relevance of N-nitroso Compounds for Human Cancer: Exposures and Mechanisms, IARC Scientific Publications No. 84, (Bartsch, H., O’Neill, I. K., and Schu¨lte-Hermann, R., Eds.) pp 187-190, International Agency for Research on Cancer, Lyon, France. (8) Challis, B. C. (1989) Chemistry and biology of nitrosated peptides. Cancer Surveys 8, 363-384. (9) Anderson, D., and Blowers, S. D. (1994) Limited cancer bioassay to test a potential food chemical. Lancet 344, 343-344. (10) Bulay, O., Mirvish, S. S., Garcia, H., Pelfrene, A. F., and Gold, B. (1979) Carciogenicity test of six nitrosamides and a nitrosocyanamide administered orally to rats. J. Natl. Cancer Inst. 62, 1523-1528. (11) Maekaiva, A., Ogiu, T., Matsuoka, C., Onodera, H., Furuta, K., Tangawa, H., and Odashima, S. (1983) Induction of tumours in the small intestine and mammary gland of female Donryu rats by continuous oral administration of N-carboxymethyl-N-nitrosourea. J. Cancer Res. Clin. Oncol. 106, 12-16. (12) Zurlo, J., Curphey, T. J., Hiley, R., and Longnecker, D. S. (1982) Identification of 7-carboxymethylguanine in DNA from pancreatic acinar cells exposed to azaserine. Cancer Res. 42, 1286-1288.

Chem. Res. Toxicol., Vol. 10, No. 6, 1997 659 (13) Shuker, D. E. G., and Margison, G. P. (1997) Nitrosated glycine derivatives as a potential source of O6-methylguanine in DNA. Cancer Res. 57, 366-369. (14) Wild, C. P. (1990) Antibodies to DNA alkylation adducts as analytical tools in chemical carcinogenesis. Mutat. Res. 233, 219233. (15) Shuker, D. E. G., and Bartsch, H. (1994) Detection of human exposure to carcinogens by measurement of alkyl-DNA adducts using immunoaffinity clean-up in combination with gas chromatography-mass spectrometry and other methods of detection. Mutat. Res. 313, 263-268. (16) Mu¨ller, R., and Rajewsky, M. (1981) Immunological quantification by high affinity atibodies of O6-ethyldeoxyguanosine in DNA exposed to ethylnitrosourea. Cancer Res. 41, 887-896. (17) Reese, C. B., Stewart, J. C. M., van Boom, J. H., de Leeum, H. P. M., Nagel, J., and de Rooy, J. F. M. (1975) The synthesis of oligoribonucleotides. Part XI Preparation of ribonucleoside 2′acetyl-3′-esters by selective deacylation. J. Chem. Soc., Perkin Trans. 1 934-942. (18) Bridson, P. K., Markiewicz, W. T., and Reese, C. B. (1977) Acylation of 2′,3′,5′-tri-O-acetylguanosine. Chem. Commun. 791792. (19) Chapeau, M.-C., and Marnett, L. J. (1991) Enzymatic synthesis of purine deoxynucleoside adducts. Chem. Res. Toxicol. 4, 636638. (20) Friesen, M. D., Garren, L., Prevost, V., and Shuker, D. E. G. (1991) Isolation of urinary 3-methyladenine using immunoaffinity columns prior to determination by low-resolution gas chromatography-mass spectrometry. Chem. Res. Toxicol. 4, 102-106. (21) Challis, B. C., Milligan, J. R., and Mitchell, R. C. (1990) Synthesis of N-nitrosopeptides. J. Chem. Soc., Perkin Trans. I 3103-3107. (22) Mu¨ller, E. (1908) U ¨ ber pseudo diazoessigsa¨ure. (On pseudo diazoacetic acid.) Chem. Ber. 41, 3116-3139. (23) Beranek, D. T., Weis, C. C., and Swenson, D. H. (1980) A comprehensive quantitative analysis of methylated and ethylated DNA using high pressure liquid chromatography. Carcinogenesis 1, 595-606. (24) Gaffney, B. L., and Jones, R. A. (1982) Synthesis of O6-alkylated deoxyguanosine nucleosides. Tetrahedron Lett. 23, 2253-2256. (25) Pauly, G. T., Powers, M., Pei, G. K., and Moschel, R. C. (1988) Synthesis and properties of H-ras DNA sequences containing O6substituted 2′-deoxyguanosine residues at the first, second or both positions of codon 12. Chem. Res. Toxicol. 1, 391-397. (26) Hemminki, K. (1980) Identification of guanine-adducts of carcinogens by their fluorescence. Carcinogenesis 1, 311-316. (27) Prevost, V., Shuker, D. E. G., Bartsch, H., Pastorelli, R., Stillwell, W. G., Trudel, L. J., and Tannenbaum, S. R. (1990) The determination of urinary 3-methyladenine by immunoaffinity chromatography-monoclonal antibody based ELISA: use in human biomonitoring studies. Carcinogenesis 11, 1747-1751. (28) Prevost, V., Shuker, D. E. G., Friesen, M. D., Eberle, G., Rajewsky, M. F., and Bartsch, H. (1993) Immunoaffinity purification and gas chromatography-mass spectrometric quantification of 3-alkyladenines in urine: metabolism studies and basal excretion levels in man. Carcinogenesis 14, 199-204. (29) Durand, M.-J., and Shuker, D. E. G. (1994) Preparation of compound-specific and group-specific antibodies to 7-methylguanine and related 7-alkylguanines and their use in immunoaffinity purification. Carcinogenesis 15, 957-961. (30) Nehls, P., Adamkiewicz, J., and Rajewsky, M. F. (1984) Immunoslot-blot: a highly sensitive immunoassay for the quantification of carcinogen-modified nucleosides in DNA. J. Cancer Res. Clin. Oncol. 108, 23-29.

TX960203U