Synthesis of Oligonucleotides Containing the Ethylene Dibromide

The carcinogen ethylene dibromide (EDB) is activated by enzymatic conjugation with GSH ... DNA adduct derived from EDB was previously characterized as...
0 downloads 0 Views 430KB Size
Download PDF
Chem. Res. Toxicol. 1997, 10, 1133-1143

1133

Synthesis of Oligonucleotides Containing the Ethylene Dibromide-Derived DNA Adducts S-[2-(N7-Guanyl)ethyl]glutathione, S-[2-(N2-Guanyl)ethyl]glutathione, and S-[2-(O6-Guanyl)ethyl]glutathione at a Single Site† Mi-Sook Kim and F. Peter Guengerich* Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 Received June 23, 1997X

The carcinogen ethylene dibromide (EDB) is activated by enzymatic conjugation with GSH to form S-(2-bromoethyl)GSH, which reacts with DNA via an episulfonium ion. The major DNA adduct derived from EDB was previously characterized as S-[2-(N7-guanyl)ethyl]GSH, and S-[2-(N2-guanyl)ethyl]GSH and S-[2-(O6-guanyl)ethyl]GSH are minor adducts [Cmarik, J. L., Humphreys, W. G., Bruner, K. L., Lloyd, R. S., Tibbetts, C., and Guengerich, F. P. (1992) J. Biol. Chem. 267, 6672-6679]. S-[2-(N7-Guanyl)ethyl]GSH has been incorporated at the G* site in d(5′-TGCTG*CAAG-3′), a site previously found to show GC to AT transitions following treatment of M13 phage with S-(2-chloroethyl)GSH, and the desired product was separated by HPLC. This was ligated to d(5′-GGTACCGAG-3′) to yield d(5′-TGCTG*CAAGGGTACCGAG3′). S-[2-(N2-Guanyl)ethyl]GSH was incorporated into the G* site of the oligonucleotide in d(5′-TGCTG*CAAGGGTACCGAG-3′) by reacting S-(2-aminoethyl)GSH with an oligomer containing 2-fluoro-O6-[(trimethylsilyl)ethoxy]deoxyinosine at the target site. The 5′-(dimethoxytrityl)-N2-(phenoxyacetyl)-N-[(fluorenylmethyl)formyl] derivative of S-[2-(O6-deoxyguanosyl)ethyl]GSH dimethyl ester was synthesized by Mitsunobu alkylation of 5′-(dimethoxytrityl)N2-(phenoxyacetyl)deoxyguanosine with N-[(fluorenylmethyl)formyl]-S-(2-hydroxyethyl)GSH dimethyl ester, modified to form the phosphoramidite derivative, and incorporated at the G* site of d(5′-TGCTG*CAAGGGTACCGAG-3′). The protective groups were removed with 0.10 N NaOH to give the modified oligonucleotide containing S-[2-(O6-guanyl)ethyl]GSH. Although the overall yields were low, the synthesis of a single set of target site oligonucleotides containing these three known guanyl adducts allows for in vitro site-specific misincorporation studies.

Introduction 1

Ethylene dibromide (EDB) has been popular in the past as a pesticide and lead scavenger in gasoline. However, EDB causes cancer, gene mutation, and reproductive damage in a variety of animal species. The human toxicity of EDB from low-level, long term exposure is not yet clear, but the deaths of two workers acutely exposed to EDB have been reported (1). The major metabolic pathway in vivo is oxidation via mixed function oxidases; however, GSH conjugation via an episulfonium ion (2) is responsible for the formation of DNA adducts. The major DNA adduct derived from EDB was characterized as S-[2-(N7-guanyl)ethyl]GSH, and S-[2-(N2-guanyl)ethyl]GSH, S-[2-(O6-guanyl)ethyl]GSH, and S-[2-(N1-adenyl)ethyl]GSH are minor adducts (Scheme 1) (3-7). A forward mutation assay using bacteriophage M13mp18 with the reactive GSH conjugate S-(2-chloroethyl)GSH revealed that base-pair substitution muta† This research was supported in part by United States Public Health Service Grant R35 CA44353 and Grant P30 ES00267. M.-S. Kim was supported in part by a Merck predoctoral fellowship. * Address correspondence to this author. Tel: (615) 322-2261. Fax: (615) 322-3141. E-mail: [email protected]. X Abstract published in Advance ACS Abstracts, September 15, 1997. 1 Abbreviations: EDB, ethylene dibromide; FMOC, fluorenylmethoxycarbonyl; FLEC, fluorenylethyl chloroformate; CGE, capillary gel electrophoresis; FAB, fast atom bombardment; DMTrCl, 4,4′-dimethoxytrityl chloride. The standard abbreviations for nucleic acid bases and nucleotides are defined in the current Instructions to Authors (see the January issue of Chem. Res. Toxicol.).

S0893-228x(97)00108-2 CCC: $14.00

tions occurred in the lacZ region and GC to AT transitions dominated (7). The same mutational changes were dominant in widely different biological systems such as Drosophila melanogaster, Chinese hamster ovary cells, and Escherichia coli after treatment with EDB (8-10). In previous studies in our laboratory, oligonucleotides containing S-[2-(N7-guanyl)ethyl]GSH were prepared and their physicochemical properties were characterized using UV, CD, and NMR spectroscopy (11-13). Base pairing of S-[2-(N7-guanyl)ethyl]GSH to Cyt in the complementary sequences was shown to be quite disrupted. However, no evidence was obtained that the modified Gua preferred to bind to a base other than Cyt. No N7guanyl derivative has clearly been shown to be miscoding except the guanyl adduct derived from aflatoxin B1 exo8,9-epoxide (14). However, the pKa of the N1 guanyl proton has been shown to drop from ∼9 to ∼7.5 upon N7alkylation (13). Whether this alteration of the pKa of the N1 atom, which is involved in base pairing, could induce mispairing by polymerases is not yet known, although speculation has existed for many years (15, 16). O6AlkylGua derivatives have been shown to be critical in tumorigenesis by many chemical carcinogens (17), and some pair with Thy as well as with Cyt (18-22). High miscoding potentials for some N2-guanyl adducts have also been reported (23, 24). In light of this information, we are interested in comparing the miscoding potentials of S-[2-(N7-guanyl)ethyl]GSH, S-[2-(N2-guanyl)ethyl]© 1997 American Chemical Society

1134 Chem. Res. Toxicol., Vol. 10, No. 10, 1997

Kim and Guengerich

Scheme 1. Bioactivation of EDB and Formation of DNA Adducts

GSH, and S-[2-(O6-guanyl)ethyl]GSH. The oligonucleotides containing EDB/GSH adducts at the N2, N7, and O6 positions of a single guanine were synthesized, allowing in vitro site-specific misincorporation studies.

Experimental Procedures Caution: The following chemicals are hazardous and should be handled carefully: S-(2-chloroethyl)GSH, 1-bromo-2-chloroethane, Diazald, 9-fluorenylmethyl chloroformate, and CH2N2. Chemicals. GSH, GSSG, 9-fluorenylmethyl chloroformate, and fluorenylethyl chloroformate (FLEC) were purchased from Sigma Chemical Co. (St. Louis, MO). 1-Bromo-2-chloroethane, bromoethylamine‚HBr, 4,4′-dimethoxytrityl chloride (DMTrCl), anhydrous 1H-tetrazole, 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite, anhydrous CH3OH, dioxane, pyridine, triethylamine and diisopropylethylamine, C2H5SH, and Diazald were purchased from Aldrich Chemical Co. (Milwaukee, WI). 2-Fluoro-O6-[(trimethylsilyl)ethyl]-2′-deoxyinosine was synthesized by Dr. M. Mu¨ller in this laboratory (25). Silica gel (70230 mesh, 60 Å; Aldrich) was used for flash chromatography. Enzymes. T4 endonuclease V was provided by Prof. R. S. Lloyd (University of Texas, Galveston, TX), and Fnu4HI was purchased from New England Biolabs (Beverly, MA). Nuclease P1 and alkaline phosphatase were purchased from Sigma. Crotalus adamanteus venom phosphodiesterase I was purchased from Pharmacia (Piscataway, NJ). Spectroscopy. UV spectra were recorded using a modified Cary 14/OLIS spectrophotometer (On-line Instrument Systems, Bogart, GA). Fast atom bombardment (FAB) mass spectra were obtained using a Kratos II HH instrument (Kratos, Manchester, U.K.) in the Vanderbilt facility with various matrices. NMR spectra were recorded with Bruker AM-300 and AM-400 instruments (Bruker, Billerica, MA). Capillary Gel Electrophoresis (CGE). The purity of oligonucleotides was analyzed using a Beckman P/ACE 2000 CGE system (Beckman, San Ramon, CA) (see Supporting

Information). Samples were applied to a ssDNA 100 gel capillary in Tris-borate-urea buffer (obtained from the manufacturer) at -5 kV and run at -10 kV (30 °C), with UV detection at 254 nm. Characterization of Oligonucleotides. Each oligonucleotide (N2- and O6-Gua derivatives) (0.10 A260) was digested with nuclease P1 (0.08 unit; Sigma) in 20 µL of 10 mM Tris-HCl buffer (pH 7.0) containing 10 mM MgCl2 at 37 °C for 3 h and then with snake venom phosphodiesterase (0.08 unit; Pharmacia) and alkaline phosphatase (0.08 unit; Sigma) in 20 µL of 0.10 M TrisHCl buffer (pH 9.0) for 3 h at 37 °C. Each sample was diluted with H2O (100 µL) and analyzed by reversed-phase HPLC using a YMC-Pack octadecylsilane (C18)-AQ column (4.6 × 250 mm, 5 µm). The column was eluted with 50 mM NH4CH3CO2, pH 4.5 (solvent A), and CH3OH (solvent B) with the gradient (flow rate 1.0 mL min-1): 0 min (100% A, 0% B), 5 min (100% A, 0% B), 25 min (90% A, 10% B), 35 min (80% A, 20% B), 45 min (50% A, 50% B), 55 min (50% A, 50% B), 60 min (100% A, 0% B). Examination of the Possibility of Sulfur Oxidation during Oligonucleotide Synthesis. Met (10 mM) was reacted with oxidizing agent (0.10 M I2 in a mixture of H2O: pyridine:tetrahydrofuran, 1:10:40, v/v/v, DNA synthesis gradient; Applied Biosystems) for 1.5 h, and the reaction was quenched with 0.3 M aqueous Na2S2O3. Met and Met sulfoxide were analyzed by TLC using 70% aqueous CH3CH2OH (v/v), with visualization using ninhydrin solution (17 mM in n-butanol containing 3% CH3CO2H, v/v) (Rf 0.57, Met; 0.47, Met sulfoxide). Examination of Preservation of Chirality of GSH under Basic Conditions. GSSG (10 mM) was treated with 0.10 N NaOH for 3 days at room temperature, and samples were concentrated using a centrifugal drier. The sample was hydrolyzed in 6 N HCl at 110 °C for 18 h under vacuum. Hydrolyzed samples were concentrated using a centrifugal drier and derivatized with (+)-FLEC (26) in sodium borate buffer (1.0 M, pH 8.0) for 4 min. The reaction mixture was extracted with pentane (300 µL, ×3) and (C2H5)2O (300 µL, ×2) to remove unreacted FLEC. The diastereomeric amino acid derivatives

Ethylene Dibromide-GSH-DNA Adduct Synthesis

Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1135

Scheme 2. Synthesis of Oligonucleotide Containing S-[2-(N7-Guanyl)ethyl]GSH

were analyzed by reversed-phase HPLC using a Beckman C18 column (4.6 × 250, 5 µm). Samples were eluted with NaCH3CO2 buffer (30 mM, pH 4.4) and CH3CN using the gradient (flow rate 1.0 mL min-1): 0 min (70% A, 30% B), 5 min (70% A, 30% B), 50 min (40% A, 60% B), 60 min (10% A, 90% B), 65 min (10% A, 90% B), and 80 min (70% A, 30% B). Preparation of Oligonucleotides Containing S-[2-(N7Guanyl)ethyl]GSH (Scheme 2). Oligonucleotides. The oligodeoxynucleotides d(5′-TGCTGCAAG-3′) (a), d(5′-GGTACCGAG-3′) (b), and d(5′-AAAAAAAACTCGGTACCCTTGCAGCAGCAAAAAAAAA-3′) (c) were synthesized using solid-phase phosphoramidate chemistry on an Applied Biosystems 391 synthesizer (Applied Biosystems, Foster City, CA). After deprotection with NH4OH for 18 h at 55 °C, the oligomers were purified by reversed-phase HPLC using a YMC-Pack octadecylsilane (C18)-AQ column (10 × 250 mm, 5 µm; YMC, Wilmington, NC). The oligomer a was eluted with a gradient of 7-11% (v/v) CH3CN in 50 mM aqueous NH4CH3CO2 (pH 7.0) over 40 min with a flow rate of 2.5 mL min-1. The oligomer b was purified with a gradient formed from 50 mM NH4HCO2, pH 6.2 (solvent A), and CH3OH (solvent B): 0 min (100% A, 0% B), 40 min (70% A, 30% B), 50 min (50% A, 50% B), 60 min (100% A, 0% B). The oligomer c was purified with a gradient formed from 50 mM NH4HCO2, pH 6.2 (solvent A), and CH3OH (solvent B): 0 min (85% A, 15% B), 50 min (60% A, 40% B), 60 min (85% A, 15% B). Ammonium salts were removed by repeated lyophilization. Preparation and Identification of the Derivatized Deoxyribonucleotide d(5′-TGCTG*CAAG-3′), where G* ) S-[2(N7-Guanyl)ethyl]GSH. S-(2-Chloroethyl)GSH was synthesized as described by Humphreys et al. (27). The purified oligodeoxynucleotide d(5′-TGCTGCAAG-3′) (2 mg) was reacted with a large excess (100-fold by weight) of S-(2-chloroethyl)GSH in 50 mM potassium phosphate buffer (pH 7.0) for 15 min at 37 °C (11). The modified oligomer was separated using the same conditions as for the purification of oligomer a (vide supra). The presence of d(5′-TGCTG*CAAG-3′), where G* ) S-[2-(N7-guanyl)ethyl]GSH, in the designated peak was confirmed by cleavage with 1.0 M piperidine at 90 °C for 40 min. After the reaction, the samples were dried using a SpeedVac centrifugal drier (Savant Instruments, Farmingdale, NY) twice to remove piperidine completely. The samples were dissolved in 60 mM NaCH3CO2 buffer (pH 5.2) and digested with prostatic acid phophatase (100 mU) in the presence of 0.10 mM ZnCl2 at 37 °C for 1 h. After filtration through an Ultrafree-MC 10000 NMWL filter unit (Waters/Millipore, Milford, MA), the cleaved samples were analyzed by reversed-phase HPLC using the same conditions as for purification, with d(5′-TGCT-3′) and d(5′CAAG-3′) as standards. The oligomer d(5′-TGCTG*CAAG-3′)

was further purified by anion exchange HPLC with a Phenomenex W-Porex DEAE column (10 × 250 mm, 10 µm; Phenomenex, Torrance, CA) using a linear gradient of 0.5 M NH4CH3CO2 (pH 7.0) to 1.4 M NH4CH3CO2 in 30% CH3OH (v/v) over 45 min. The salts were removed by Sephadex G-10 column chromatography (Pharmacia, Piscataway, NJ) (0.68 A260 unit of modified oligonucleotide was obtained). Ligation of d(5′-TGCTG*CAAG-3′) [G* ) S-[2-(N7-Guanyl)ethyl]GSH] with d(5′-GGTACCGAG-3′). The oligomer d(5′-GGTACCGAG-3′) (0.54 A260 unit) was phosphorylated at the 5′ terminus and ligated to d(5′-TGCTG*CAAG-3′) (0.34 A260 unit) using d(5′-AAAAAAAACTCGGTACCCTTGCAGCAGCAAAAAAAAA) (2.0 A260 unit) as a scaffold. The ligated product was separated on a 20% polyacrylamide (w/v) denaturing gel. The gels were crushed and extracted with H2O (500 µL) at 4 °C overnight. The solution was filtered through a 0.2 µm filter (Costar, Cambridge, MA), and the gels were washed with H2O (200 µL, ×2). The oligomer was precipitated with cold C2H5OH in the presence of 3 M NaCH3CO2. After centrifigation at 4.5 × 104 rpm (Beckman TL-100 ultracentrifuge, TLA 100.3 rotor, Beckman, Palo Alto, CA) for 30 min (4 °C), the pellet was washed with 70% aqueous C2H5OH (100 µL, ×2) and dried using a centrifugal evaporator. The presence of S-[2-(N7-guanyl)ethyl]GSH in the ligated oligomer was confirmed by restriction digestion using Fnu4HI and T4 endonuclease treatment (37 °C for 2 h) (0.05 A260 unit of ligated oligonucleotide was obtained). Preparation of Oligonucleotides Containing S-[2-(N2Guanyl)ethyl]GSH. Preparation of S-[2-(N2-Deoxyguanosyl)ethyl]GSH. S-(2-Aminoethyl)GSH was synthesized by mixing GSH (0.5 g) with 3.3 equiv of Na0 (125 mg) in 25 mL of anhydrous CH3OH and adding this mixture to a stirred solution of 660 mg of 2-bromoethylamine‚HBr and 75 mg of Na0, dissolved in anhydrous CH3OH. After 1 h, 280 µL of glacial CH3CO2H was added to the reaction mixture. The precipitate was collected after centrifugation and dissolved in H2O. The compound was purified by reversed-phase HPLC using a Beckman Ultrasphere octadecylsilane (C18) column (10 × 250 mm, 10 µm; Beckman, San Ramon, CA). The column was eluted with an aqueous mixture containing 3% CH3CN (v/v) and 0.1% CF3CO2H (v/v). The identity of S-(2-aminoethyl)GSH was characterized by FAB MS (assignment and relative abundance in parentheses): m/z 351.1 (MH+, 50), 373.1 ([M + Na]+, 100). 1H-NMR (300 MHz, 2H O): δ 2.16 (dd, 2 H, Glu β), 2.56 (m, 2 2 H, Glu γ), 2.87-2.96 (m, 3 H, -SCH2CH2NH2 plus Cys βa), 3.10 (m, 1 H, Cys βb), 3.24 (t, 2 H, S-CH2CH2NH2), 3.78 (s, 2 H, Gly R plus t, 1 H, Glu R), 4.61 (m, 1 H, Cys R). S-[2-(N2-Deoxyguanosyl)ethyl]GSH was synthesized by reacting 2-fluoro-O6-[(trimethylsilyl)ethyl]deoxyinosine with a 25-fold molar excess of S-(2-aminoethyl)GSH in 0.5 mL of (CH3)2SO at

1136 Chem. Res. Toxicol., Vol. 10, No. 10, 1997

Kim and Guengerich

Scheme 3. Synthesis of Oligonucleotide Containing S-[2-(N2-Guanyl)ethyl]GSH

50 °C for 2 days. S-(2-Aminoethyl)GSH was dried in vacuo in an Abderhalden apparatus for 18 h at 64 °C before use in the reaction. The reaction mixture was diluted with H2O and analyzed by reversed-phase HPLC using a Beckman Ultrasphere octadecylsilane (C18) column (10 × 250 mm, 10 µm). The solvent system consisted of 50 mM NH4CH3CO2, pH 4.5 (solvent A), and CH3CN (solvent B) with a flow rate of 2.5 mL min-1. Two different nucleoside adducts were separated with the following gradient: 0 min (95% A, 5% B), 5 min (95% A, 5% B), 20 min (80% A, 20% B), 30 min (70% A, 30% B), 40 min (50% A, 50% B), 50 min (30% A, 70% B), 60 min (0% A, 100% B). 1HNMR (of major nucleoside) (400 MHz, 2H2O): δ 2.06 (dd, 2 H, Glu β), 2.48 (m, 2 H, Glu γ), 2.86-2.92 (m, 5 H, 5′, 5′′, Gly R, Glu R), 4.07 (m, 1 H, 4′), 4.56 (m, 1 H, 4′), 4.62 (dd, 2 H, -SCH2CH2N), 4.76 (m, 1 H, Cys R), 6.35 (t, 1 H, 1′), 7.94 (s, 1 H, H-8). MS: m/z 601.1 (MH+, 85), 623.1 ([M + Na]+, 28). UV: λmax 253 and 290 nm (pH 4.5). The adducts were collected and treated by the addition of CH3CO2H (10%, v/v) for 30 min (23 °C) to remove the O6-(trimethylsilyl)ethyl group. Deprotected nucleosides were further purified using reversed-phase HPLC. The Beckman Ultrasphere octadecylsilane (C18) column (10 × 250 mm, 10 µm) was eluted with 50 mM NH4CH3CO2, pH 4.5 (solvent A), and CH3OH (solvent B) with a flow rate of 2.5 mL min-1 using the gradient: 0 min (100% A, 0% B), 5 min (100% A, 0% B), 25 min (90% A, 10% B), 35 min (80% A, 20% B), 45 min (100% A, 0% B). The identity of S-[2-(N2-deoxyguanosyl)ethyl]GSH was confirmed by amino acid analysis. The adducts were hydrolyzed in 6 N HCl at 110 °C for 18 h under vacuum. The amino acid phenyl isothiocyanate derivatives were analyzed in the Vanderbilt Protein Chemistry Core Facility using a Waters HPLC system (Waters, Milford, MA). Equimolar amounts of Gly and Glu were detected from the hydrolysis of the major nucleoside adduct. Preparation of Deoxyribooligonucleotide Containing S-[2-(N2-Deoxyguanosyl)ethyl]GSH (Scheme 3). 5′-O-(4,4′Dimethoxytrityl)-2-fluoro-O6-[(trimethylsilyl)ethyl]-2′-deoxyinosine. 2-Fluoro-O6-[(trimethylsilyl)ethyl]deoxyinosine (0.34 mmol, 120 mg) was dried by repeated treatment with anhydrous pyridine (3 mL, ×4), with in vacuo removal of the solvent, and kept under vacuum for 1 h. It was reacted with 1.2 equiv of DMTrCl and 1.4 equiv of N,N-diisopropylethylamine in 3 mL of anhydrous pyridine for 15 h under a dry Ar atmosphere. The reaction was quenched with 25 µL of CH3OH and stirred for 10 min. The reaction mixture was concentrated in vacuo. The orange compound was dissolved in CH2Cl2 (30 mL) and extracted with 10% K2CO3 (30 mL, w/v). The organic layer was dried over anhydrous K2CO3 and purified by flash chromatography on silica gel using CH2Cl2/MeOH, 99:1, v/v (TLC Rf 0.2). 3′-O-[(N,N-Diisopropylamino)(2-cyanoethyl)phosphinyl]5′-O-(dimethoxytrityl)-2-fluoro-O6-[(trimethylsilyl)ethyl]-

2′-deoxyinosine. 5′-O-(4,4′-Dimethoxytrityl)-2-fluoro-O6-[(trimethylsilyl)ethyl]-2′-deoxyinosine (156 mg, 0.23 mmol) was dried by treatment with anhydrous pyridine (2 mL, ×3) as above. After storage under vacuum for 3 h, it was reacted with diisopropylethylamine (120 mg, 0.92 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidate (82 mg, 0.34 mmol) in 4 mL of anhydrous CH2Cl2 for 40 min. The reaction mixture was transferred to 10% aqueous K2CO3 (15 mL, w/v) and extracted with CH2Cl2 (15 mL, ×2) and ethyl acetate (15 mL). The combined organic layers were dried over Na2SO4 and purified with flash chromatography on silica gel (CH2Cl2:ethyl acetate, v/v, 70:30), TLC Rf 0.6 (hexane:ethyl acetate, v/v, 50: 50). Oligonucleotides. Oligonucleotides [d(5′-TGCTG*CAAGGGTACCGAG-3′), where G* ) 2-fluoro-O6-[(trimethylsilyl)ethoxy]deoxyinosine] were synthesized on a 1-µmol scale using 4-tertbutylphenoxyacetyl protecting groups (PerSeptive Biosystems, Framingham, MA) with an Expedite Nucleic Acid Synthesis System (Millipore Corp., Bedford, MA) according to the manufacturer’s standard protocol. To deprotect oligonucleotides, the beads from each 1-µmol cassette were treated with 0.10 N NaOH (1 mL) for 15 h, stirring slowly. The supernatants were removed, and the beads were washed with H2O (1 mL, ×3). The combined aqueous fractions were neutralized with 0.05 M CH3CO2H and dried by lyophilization. The residues were dissolved in H2O (1 mL), and the solutions were filtered through a 0.22 µm filter and lyophilized again. Oligonucleotides (0.2 µmol) and S-(2-aminoethyl)GSH (15 mg) were dried in vacuo in an Abderhalden apparatus for 18 h at 64 °C and reacted in 0.5 mL of anhydrous (CH3)2SO for 2 days at 50 °C. The reaction mixture was diluted with H2O (2 mL) and analyzed using reversed-phase HPLC. The YMC-Pack octadecylsilane (C18)-AQ column (10 × 250 mm, 5 µm) was eluted with 100 mM NH4HCO2, pH 6.2 (solvent A), and CH3OH (solvent B) with the following gradient (flow rate 2.5 mL min-1): 0 min (95% A, 5% B), 25 min (80% A, 20% B), 35 min (80% A, 20% B), 45 min (70% A, 30% B), 50 min (0% A, 100% B), 60 min (95% A, 5% B). Two major fractions (tR 47 and 52 min) were collected, lyophilized, and treated with 10% aqueous CH3CO2H (v/v) at room temperature for 30 min to remove the O6-(trimethylsilyl)ethyl group. After concentration using a centrifugal drier, O6-deprotected oligonucleotides were purified under the same conditions, with the modified oligonucleotide eluting at tR 26.5 min and the unreacted oligonucleotide at tR 47.5 min (1.6 A260 unit of modified oligonucleotide was obtained from the 1-µmol cassette). Preparation of Oligonucleotides Containing S-[2-(O6Guanyl)ethyl]GSH. Preparation of N-[(Fluorenylmethyl)formyl]-S-(2-hydroxyethyl)GSH Dimethyl Ester (Scheme 4). GSSG (3.0 g) was dissolved in 10% aqueous Na2CO3 (w/v, 20 mL) at 0 °C, and dioxane (10 mL) was added to this solution.

Ethylene Dibromide-GSH-DNA Adduct Synthesis Scheme 4. Preparation of N-[(Fluorenylmethyl)formyl]-S-(hydroxyethyl)GSH Dimethyl Ester

9-Fluorenylmethyl chloroformate (5.0 g in 10 mL of dioxane) was slowly added to this mixture, and the reaction proceeded for 1 h on ice. The reaction mixture was poured into 70 mL of chilled H2O and extracted with (C2H5)2O (50 mL, ×5) to remove unreacted 9-fluorenylmethyl chloroformate (28, 29). The aqueous fraction was acidified to pH 2 with 1 N HCl, and the precipitate was collected after centrifugation [30 min, (4 × 103)g] and dried by lyophilization. The product was characterized by FAB MS: m/z 1057.3 (MH+, 20), 1079.3 ([M + Na]+, 100). N-[(Fluorenylmethyl)formyl]GSSG (1.0 g) was dissolved in 3 mL of CH3OH and reacted with CH2N2 (1.0 g) for 1 h; the excess CH2N2 was removed under a N2 stream. The product was characterized by FAB MS: m/z 1134.8 ([M + Na]+, 100). Alcohol-free ethereal solutions of CH2N2 were prepared according to the standard protocol from Aldrich: 2-(2-ethoxyethoxy)ethanol (12 mL) and (C2H5)2O (7 mL) were added to a solution of KOH (2.0 g) in H2O (3.3 mL). This solution was placed in a 100 mL long-necked distilling flask fitted with a dropping funnel, efficient condenser, and oil bath at 70 °C. As the distillation of (C2H5)2O proceeded, a solution of 7.0 g of Diazald® in 70 mL of (C2H5)2O was added slowly through the dropping funnel. During the distillation, the flask was occasionally shaken vigorously and the ethereal CH2N2 was collected. The resulting N-[(fluorenylmethyl)formyl]GSSG tetramethyl ester (0.40 g) was reduced to N-[(fluorenylmethyl)formyl]GSH dimethyl ester by reaction with C2H5SH (8.0 mL) in a mixture of dioxane (30 mL), CH3OH (20 mL), and aqueous NH4HCO3 (20 mL, 0.5 M, pH 7.7) for 30 min. The reaction mixture was dried in vacuo and lyophilized several times to remove remaining salts. FAB MS: m/z 558.2 (MH+, 100), 580.2 ([M + Na]+, 85). N-[(Fluorenylmethyl)formyl]-S-(2-hydroxyethyl)GSH dimethyl ester was synthesized by mixing N-[(fluorenylmethyl)formyl]GSH dimethyl ester (2.0 g) with 1 equiv of Na0 (86 mg) in 25 mL of anhydrous CH3OH and adding this mixture to a stirred solution of 2.6 mL of 2-bromoethanol in 10 mL of anhydrous CH3OH. After 6 h, the product was purified from the reaction mixture by flash column chromatography on silica gel using a gradient of CH2Cl2:CH3OH (98:2 to 90:10, v/v), TLC Rf 0.53 (CH2Cl2:CH3OH, 90:10, v/v). FAB MS: m/z 602.2 (MH+, 10), 624.2 ([M + Na]+, 100). Preparation of Derivatized S-[2-(O6-Deoxyguanosyl)ethyl]GSH (Scheme 5). 5′-(Dimethoxytrityl)-N2-(phenoxyacetyl)dGuo (200 mg, 0.26 mmol) was dried in vacuo by repeated treatment with anhydrous pyridine (1 mL, ×3), kept under

Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1137 Scheme 5. Synthesis of Oligonucleotide Containing S-[2-(O6-Guanyl)ethyl]GSH

vacuum overnight, and reacted with (trimethylsilyl)imidazole (150 µL, 1.0 mmol) in 2 mL of anhydrous dioxane for 2 h. The reaction mixture was subjected to flash chromatography on silica gel using a gradient of CH2Cl2:CH3OH:(C2H5)3N (97.5:2: 0.5 to 94.5:5:0.5, v/v/v), TLC Rf 0.47 (CH2Cl2:CH3OH, 90:10, v/v), and dried in vacuo by treatment with anhydrous pyridine (1 mL, ×3). After drying under vacuum for 18 h, the product (134 mg, 0.17 mmol) was dissolved in 1 mL of anhydrous dioxane and transferred to a 3-necked flask equipped with a condenser. Triphenylphosphine (68 mg, 0.26 mmol) was added, and the solution was heated to 100 °C. Diethyl azodicarboxylate (41 µL, 0.26 mmol) and N-[(fluorenylmethyl)formyl]-S-(2-hydroxyethyl)GSH dimethyl ester (156 mg, 0.26 mmol) were added successively to the mixture and reacted for 15 min at 100 °C under an Ar atmosphere. N-[(Fluorenylmethyl)formyl]-S-(2-hydroxyethyl)GSH dimethyl ester was dried in vacuo in an Abderhalden apparatus for 18 h at 64 °C before use in the reaction. The reaction mixture was cooled slowly, and the reaction was continued overnight. (CH3CH2CH2CH2)4N+F- (150 µL, 0.17 mmol) was added to the mixture and reacted for 45 min to deprotect the 3′-hydroxyl of the deoxyribose moiety. The reaction mixture was poured into H2O (5 mL) and extracted with CH2Cl2 (15 mL, ×3). The organic phase was dried over Na2SO4, concentrated in vacuo, and purified by preparative TLC using CH2Cl2:CH3OH:(C2H5)3N (89.5:10:0.5, v/v/v), TLC Rf 0.44. The band was extracted using CH2Cl2:CH3OH (90:10, v/v, 50 mL, ×3) and (CH3)2CO (50 mL, ×2). The identity of the 5′-(dimethoxytrityl)-N2-(phenoxyacetyl)-N-[(fluorenylmethyl)formyl] derivative of S-[2-(O6-deoxyguanosyl)ethyl]GSH dimethyl ester was confirmed by FAB MS: m/z 1287.2 (MH+, 30), 1309.2 ([M + Na]+, 100). To confirm O6 substitution, a small portion of the 5′-(dimethoxytrityl)-N2-(phenoxyacetyl)-N-[(fluorenylmethyl)formyl] derivative of S-[2-(O6-deoxyguanosyl)ethyl]GSH dimethyl ester was treated with 80% aqueous CH3CO2H (v/v) for 30 min to remove the dimethoxytrityl group and concentrated using a centrifugal drier. Phenoxyacetyl, (fluorenylmethyl)formyl, and dimethyl ester groups were removed by treatment with 0.10 N NaOH at room temperature for 15 h. After neutralization with 0.05 N aqueous CH3CO2H, the sample was lyophilized and purified using reversed-phase HPLC. The Beckman Ultrasphere octadecylsilane (C18) column (10 × 250 mm, 10 µm) was eluted with 50 mM NH4CH3CO2, pH 4.5 (solvent A), and CH3OH (solvent B) with a flow rate of 2.5 mL

1138 Chem. Res. Toxicol., Vol. 10, No. 10, 1997 min-1 and the gradient: 0 min (95% A, 5% B), 5 min (95% A, 5% B), 20 min (80% A, 20% B), 30 min (70% A, 20% B), 40 min (50% A, 50% B), 50 min (30% A, 70% B), 60 min (0% A, 100% B). The major peak (tR 24 min) was collected and analyzed by UV spectroscopy and FAB MS. UV: λmax 288 nm (pH 2.0), λmax 281 and 248 nm (pH 7.0), λmax 280 and 248 nm (pH 12). MS: m/z 600.1 (MH+, 5), 623.1 ([M + Na]+, 3). 3′-O-[(N,N-Diisopropylamino)(2-cyanoethyl)phosphinyl] Derivative of 5′-(Dimethoxytrityl)-N2-(phenoxyacetyl)-N-[(fluorenylmethyl)formyl]-S-[2-(O6-deoxyguanosyl)ethyl]GSH Dimethyl Ester. The 5′-(dimethoxytrityl)-N2(phenoxyacetyl)-N-[(fluorenylmethyl)formyl] derivative of S-[2(O6-deoxyguanosyl)ethyl]GSH dimethyl ester (25 mg, 19 µmol) was dried in vacuo by repeated treatment with anhydrous pyridine (1 mL, ×3) and left under vacuum for 18 h. The product was reacted with 1H-tetrazole (1.6 mg, 23 µmol) and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (9.3 µL, 29 µmol) in 1 mL of anhydrous CH2Cl2 for 2 h. The reaction was monitored by TLC (CH2Cl2:CH3OH, 90:10, Rf 0.48). The reaction mixture was poured into 5 mL of H2O and extracted with CH2Cl2 (5 mL, ×3). The organic phase was dried over Na2SO4, concentrated, and purified by flash column chromatography on silica gel using a gradient of CH2Cl2:CH3OH:pyridine (99.5: 0:0.5 to 79.5:20:0.5, v/v/v). The product was dissolved in CH3CN (PerSeptive Biosystems) and carefully concentrated in vacuo. Oligonucleotides. Oligonucleotides [d(5′-TGCTG*CAAGGGTACCGAG-3′), where G* ) S-[2-(O6-deoxyguanosyl)ethyl]GSH] were synthesized on a 1-µmol scale with an Expedite Nucleic Acid Synthesis System according to the manufacturer’s standard protocol except for the coupling time, which was increased from 2 to 15 min at the G* site. In order to deprotect oligonucleotides, the beads from one 1-µmol cassette were treated with 0.10 N NaOH (1.0 mL) for 15 h at room temperature, stirring slowly. The supernatant was removed, and the beads were washed with H2O (1 mL, ×3). The combined aqueous fractions were neutralized with 0.05 M aqueous CH3CO2H, lyophilized, and dissolved in H2O (1 mL). Solutions were filtered through a 0.22 µm filter, lyophilized again, and analyzed by reversed-phase HPLC. A YMC-Pack octadecylsilane (C18)-AQ column (10 × 250 mm, 5 µm) was eluted with 50 mM NH4HCO2, pH 6.0 (solvent A), and CH3OH (solvent B) with the gradient as follows (flow rate 2.5 mL min-1): 0 min (85% A, 15% B), 50 min (70% A, 30% B), 55 min (85% A, 15% B). The fraction eluted at 30 min was repurified using the same HPLC system (vide supra) except that the gradient was changed [0 min (80% A, 20% B), 50 min (75% A, 25% B), 55 min (80% A, 20% B)] (0.3 A260 unit of modified oligonucleotide was obtained from the 1-µmol cassette).

Results and Discussion General. In order to evaluate the genetic effects of DNA modification, the synthesis of an oligodeoxynucleotide containing a single lesion is a prerequisite. Both chemical and enzymatic techniques have been widely applied to prepare site-specifically modified oligodeoxynucleotides, which are used as probes for the structural and biological effects of DNA-damaging agents (25, 3033). We have synthesized oligonucleotides containing ternary EDB/GSH adducts at the N2, N7, and O6 positions of a single Gua. All have been isolated in a high state of purity as judged by CGE, an independent and highly discriminating method (see Supporting Information). The identity of the synthesized oligomers has been confirmed by nuclease digestion and HPLC of the products. Preparation of Oligonucleotides Containing S-[2(N7-Guanyl)ethyl]GSH. S-[2-(N7-Guanyl)ethyl]GSH, the major DNA adduct derived from EDB, was incorporated at the G* site in d(5′-TGCTG*CAAG-3′), a site previously found to show a GC to AT transition after treatment of M13 phage with S-(2-chloroethyl)GSH (7). Reaction of d(5′-TGCTGCAAG-3′) with S-(2-chloroethyl)-

Kim and Guengerich

Figure 1. Identification of d(5′-TGCTG*CAAG-3′) using piperidine cleavage at the N7-guanyl adduct site: (A) separation of oligomers by reversed-phase HPLC after incubation of d(5′TGCTGCAAG-3′) with S-(2-chloroethyl)GSH and (B) analytical HPLC profile of peak 4 (from part A) after treatment with hot piperidine and phosphatase.

GSH yielded a mixture of oligonucleotides, modified at each of the three guanines, and multiply-modified oligonucleotides (13). We were able to separate these oligonucleotides by reversed-phase HPLC. The identity of the oligomer d(5′-TGCTG*CAAG-3′) was characterized after heating the fractions in the presence of piperidine and removing phosphates enzymatically with prostatic acid phosphatase (Figure 1). The chemical and thermal lability of N7-guanyl adducts has been a serious problem that has hindered the study of their biological properties. Recently, Bailey et al. (34) reported the construction of a viral genome containing an unstable aflatoxin B1-N7-Gua adduct situated at a unique site. The genome was constructed within 3 h at 16 °C, found to be the optimal conditions for aflatoxin B1-N7-Gua adduct stability. S-[2-(N7-Guanyl)ethyl]GSH has been shown to be unstable at 60 °C. However, it appears to be stable at both 16 and 37 °C over 32 h (unpublished data). The oligomer d(5′-TGCTG*CAAG3′) was ligated to the oligomer d(5′-GGTACCGAG-3′) in the presence of a 34-base-long scaffold at 16 °C. S-[2-(N7-Guanyl)ethyl]GSH incorporation into the ligated 18-mer was characterized using the restriction enzyme Fnu4HI and T4 endonuclease V. Fnu4HI recognizes the site GC/NGC and cuts the double-stranded sequence of oligonucleotides as shown below:

5′-TGC/TG*CAAG-3′ 3′-ACGA/CGTTC-5′ Restriction digestion was totally inhibited by the presence of S-[2-(N7-guanyl)ethyl]GSH. T4 endonuclease V recognizes and cleaves apurinic sites. No depurination at the G* site was observed unless the oligomer was first heated at 100 °C. Preparation of Oligonucleotides Containing S-[2(N2-Guanyl)ethyl]GSH. S-[2-(N2-Guanyl)ethyl]GSH, a minor DNA adduct derived from EDB, was incorporated into the G* site in d(5′-TGCTG*CAAGGGTACCGAG-3′) using a strategy developed by Harris and his associates (25, 32). In this strategy an amino derivative of the

Ethylene Dibromide-GSH-DNA Adduct Synthesis

Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1139

Scheme 6. Two Possible N2-Deoxyguanosyl Adducts from the Reaction of S-(2-Aminoethyl)GSH with 2-Fluoro-O6-[(trimethylsilyl)ethyl]deoxyinosine

Figure 2. Chromatographic separation of nucleosides obtained from the enzymatic digestion of oligonucleotide containing S[2-(N2-deoxyguanosyl)ethyl]GSH: (A) standard nucleosides and (B) enzymatic digest of d(5′-TGCTS*CAAGGGTACCGAG-3′), where S* ) S-[2-(N2-deoxyguanosyl)ethyl]GSH. The structure of S* was characterized by qualitative and quantitative amino acid analysis. Equal molar amounts of Glu and Gly were detected, as expected. See the text for discussion of 1H-NMR shifts of the ethylene protons.

mutagen displaces the halogen of a halo-substituted deoxyinosine. We synthesized S-(2-aminoethyl)GSH and reacted this with an oligomer containing 2-fluoro-O6[(trimethylsilyl)ethoxy]deoxyinosine. Considering the structure of S-(2-aminoethyl)GSH, there are two amino groups available for the displacement of the 2-fluoro group, the R-amino group of Glu and the S-(2-aminoethyl) moiety. In fact, we observed two N2-guanyl-adducted nucleosides in the reaction of S-(2-aminoethyl)GSH with 2-fluoro-O6-[(trimethylsilyl)ethoxy]deoxyinosine (Scheme 6). Attempts to protect the R-amino group using 9-fluorenylmethoxycarbonyl (FMOC) groups to block reaction with 2-fluoro-O6-[(trimethylsilyl)ethoxy]deoxyinosine were not successful, because the FMOC group was rapidly removed during the requisite displacement reaction at the 2 position under basic conditions. Thus, the problem was characterization of the structures of these two different N2-guanyl adducts, which were readily separable by reversed-phase HPLC. The 1H-NMR spectra are consistent with the view that coupling to the purine occurred through the amine on the ethyl group, as opposed to the Glu R-amino. Key chemical shift assignments in the spectrum of S-(2aminoethyl)GSH were Glu R at δ 3.78, the -SCH2CH2NH2 protons at δ 2.90, and the -SCH2CH2NH2 protons at δ 3.24. If coupling of the ethylamine nitrogen occurred, the chemical shift of the closest methylene protons might be expected to be modified. In the product, the following assignments were made: Glu R, δ 3.7; -SCH2CH2N-, δ ∼2.9; -SCH2CH2N-, δ 4.62. Although the assignment was not confirmed by decoupling and a substitution study with 15N was not successful in shifting the 1H-NMR spectrum (vide infra), the appearance of the doublet of doublets at δ 4.62 (near the Cys R proton group) is only consistent with this assignment. For reference, in S-[2-(N7-guanyl)ethyl]GSH the relevant

Figure 3. UV spectra of O6-alkyldeoxyguanosines: (A) standard O6-methyldGuo and (B) synthetic S-[2-(O6-deoxyguanosyl)ethyl]GSH.

chemical shifts are Glu R, δ 3.61; -SCH2CH2N-, δ 2.91/ 2.95; -SCH2CH2N-, δ 4.28, 4.37 (3). In the dimethyl ester of that compound the shifts are Glu R, δ 3.57; -SCH2CH2N, δ 3.07; -SCH2CH2N, δ 4.44 (27). In S-[2-(N1deoxyguanosinyl)methyl]GSH, the Glu R proton is at δ 3.73 and the -N2CH2-S proton is at δ 4.61 (35). In order to obtain more unambiguous evidence for the assignment of the structure, we attempted to use 13C/ 15N-NMR spectroscopy on these adducts. The two N2guanyl adducts were synthesized by reacting 2-fluoro-

1140 Chem. Res. Toxicol., Vol. 10, No. 10, 1997

Kim and Guengerich

Figure 4. Mass spectra of O6-alkyl derivatives: (A) mass spectrum (FAB) of the 5′-(dimethoxytrityl)-N2-(phenoxyacetyl)-N[(fluorenylmethyl)formyl] derivative of S-[2-(O6-deoxyguanosyl)ethyl]GSH dimethyl ester and (B) mass spectrum (FAB) of S-[2-(O6deoxyguanosyl)ethyl]GSH.

O6-[(trimethylsilyl)ethoxy]inosine with (2-[15N]aminoethyl)GSH. [[15N]Potassium phthalimide was reacted with excess EDB, and the resulting [15N](bromoethyl)phthalimide was hydrolyzed using a mixture of CH3CO2H and HBr to give 15NH2CH2CH2Br‚HBr (36-39). S-(2-[15N]Aminoethyl)GSH was prepared by reacting 15NH CH CH Br‚HBr with GSH sodium thiolate.] Un2 2 2 fortunately, the natural abundance 13C-NMR spectra did not provide a strong signal for the C-2 atom of Gua, which was expected to be split by 15N derived from S-(2-[15N]aminoethyl)GSH. Previous work with S-[1-(N2-guanosyl)methyl]GSH indicated that the C-2 atom has the weakest signal in Gua (35). We were able to further characterize the structure of S-[2-(N2-guanyl)ethyl]GSH by quantitative amino acid analysis of this compound. Equimolar amounts of Glu and Gly were detected, indicating that the S-(2-aminoethyl) group was linked to the Gua ring. At the oligonucleotide level, we clearly showed that only the S-[2(N2-guanyl)ethyl]GSH compound was found in the modified oligonucleotide after digestion with nuclease P1,

snake venom phosphodiesterase, and alkaline phosphatase (Figure 2). Preparation of Oligonucleotides Containing S-[2(O6-Guanyl)ethyl]GSH. O6-Alkylated Gua derivatives have been shown to be critical in tumorigenesis caused by some chemical carcinogens (17) and often pair with Thy as well as with Cyt (18, 19, 21, 22, 40). Our interest in the mispairing potential of S-[2-(O6-deoxyguanosyl)ethyl]GSH led us to synthesize the oligonucleotide containing this modified base. O6-Alkylguanines can be synthesized by direct alkylation under basic conditions, but the yield is too low to be practical (7). Ever since Loveless (18) suggested that O6-alkylguanines were important premutagenic and precarcinogenic lesions, many laboratories have developed strategies for the synthesis of oligonucleotides containing O6-alkylguanines (41-48). There have been many difficulties in the synthesis of oligonucleotides containing O6-alkylguanines, and potential side reactions occurring during the synthesis have been well described by Borowy-Borowski and Chambers (49). The major problem in the synthesis

Ethylene Dibromide-GSH-DNA Adduct Synthesis

Figure 5. Chromatographic separation of nucleosides obtained from the enzymatic digestion of an oligonucleotide containing S-[2-(O6-deoxyguanosyl)ethyl]GSH: (A) standard nucleosides and (B) enzymatic digest of the oligonucleotide d(5′-TGCTS*CAAGGGTACCGAG-3′), where S* ) S-[2-(O6-deoxyguanosyl)ethyl]GSH. Extra peaks eluted before dCyd were from the enzyme digestion buffer.

of oligonucleotides containing O6-alkylguanines arises from the protection of the N2 atom of Gua. In the synthesis of oligonucleotides the N2 atom of Gua is usually protected with an isobutyryl moiety, and the alkyl group on the O6 atom greatly stabilizes the protecting group on the N2 atom. Prolonged exposure to NH4OH at 65 °C, up to 72 h, is required to remove the isobutyryl group (49). 2,6-Diaminopurine, derived by nucleophilic attack on the O6-alkylGua by NH4OH, is the most problematic impurity because it mispairs (50). Gaffney et al. (44) used methoxide ions as an alternative deblocking agent for oligomers containing O6-methylGua. However, methoxide is not suitable for the deprotection of oligomers containing O6-alkylguanines other than O6methyl, because it replaces the O6-alkyl group with a methoxy group (49). We used phenoxyacetyl group protection for the N2 atom, which is labile in NH4OH with a t1/2 of 45 min (45, 47). The 5′-(dimethoxytrityl)N2-(phenoxyacetyl)-N-[(fluorenylmethyl)formyl] derivative of S-[2-(O6-deoxyguanosyl)ethyl]GSH dimethyl ester was synthesized by Mitsunobu alkylation (25, 48, 51) of 5′-(dimethoxytrityl)-N2-(phenoxyacetyl)dGuo with N[(fluorenylmethyl)formyl]-S-(2-hydroxyethyl)GSH dimethyl ester. The O6 alkylation site was identified by UV as well as FAB MS (Figures 3 and 4). The 5′-(dimethoxytrityl)-N2-(phenoxyacetyl)-N-[(fluorenylmethyl)formyl] derivative of S-[2-(O6-deoxyguanosyl)ethyl]GSH dimethyl ester was incorporated at the G* site of d(5′-TGCTG*CAAGGGTACCGAG-3′) by phosphoramidite solid-phase oligonucleotide synthesis. The coupling efficiency at the modified base dropped to ∼30% of that observed for the normal Gua base, even with prolonged coupling time (15 min instead of 2 min). The oligonucleotide was deprotected by treatment with 0.10 N NaOH for 14 h at room temperature. The FMOC group, which was used as a transient base protecting group (28, 29, 52, 53), and the dimethyl esters were also deprotected during this step to give the modified oligonucleotide containing S-[2-(O6guanyl)ethyl]GSH (Figure 5). Examination of the Possibility of Oxidation of Sulfur in GSH during the Oligonucleotide Synthesis. During the DNA synthesis by DNA synthesizer, the unstable trivalent phosphorus of the newly formed internucleotide linkage is oxidized to stable pentavalent

Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1141

phosphorus using an I2 solution. We addressed the question of whether this step could cause the oxidation of sulfur in GSH during the synthesis of the oligonucleotide containing S-[2-(O6-deoxyguanosyl)ethyl]GSH, using Met as a model compound for GSH. Met was treated with the I2 solution under the conditions used in normal oligonucleotide synthesis, and the possibility of oxidation of Met was examined with TLC using Met sulfoxide as a standard. No oxidation of Met was detected after the reaction with the iodine solution for 1.5 h, and we do not feel that sulfur oxidation is an issue in the preparation of our O6-guanyl derivative. Preservation of Chirality of GSH under Basic Conditions. The replication fate of certain DNA adducts has been shown to be dependent on the chirality of the lesions as well as the local sequence context (54, 55). Therefore, we examined the possibility of racemization of GSH under the basic conditions which we used for the deprotection of oligonucleotides after DNA synthesis. The model GSSG was treated with 0.10 N NaOH for 3 days at room temperature, hydrolyzed with 6 N HCl under vacuum, and analyzed with reversed-phase HPLC after chiral derivatization with (+)-FLEC. The L stereochemistry of GSSG was preserved after treatment with 0.10 N NaOH; i.e., only the L peak was observed, compared to the two peaks (tR 32 min, D-Glu; 34 min, L-Glu) obtained from standard DL-Glu. Other studies showed the preservation of L stereochemistry during the preparation of S-(2-hydroxyethyl)GSH using Na0. No racemization was observed for glutamic acid or S-(2-hydroxyethyl)cysteine, which was characterized by reversed-phase HPLC after hydrolysis of S-(2-hydroxyethyl)GSH and chiral derivatization with (+)-FLEC.

Conclusions Methods were developed for the synthesis of the oligonucleotide d(5′-TGCTG*CAAGGGTACCGAG-3′) containing EDB/GSH adducts at the N2, N7, and O6 positions of a single Gua (G*), to be used in in vitro site-specific misincorporation studies to characterize mutagenesis caused by EDB. The oligonucleotide structures have some novelty in that they can also be considered DNApeptide cross-links. The general approaches described here may be applicable to some other adducts as well.

Acknowledgment. We thank Dr. C. M. Harris for valuable technical suggestions, Dr. M. Persmark for the studies on the stereochemistry of S-(2-hydroxyethyl)GSH, and Dr. M. Mu¨ller for providing 2-fluoro-O6-[(trimethylsilyl)ethyl]deoxyinosine and for acquiring NMR spectra. We also thank P. Horton for technical assistance in DNA synthesis. Supporting Information Available: CGE traces of the oligonucleotides (1 page). Ordering information is given on any current masthead page.

References (1) Letz, G. A., Pond, S. M., Osterloh, J. D., Wade, R. L., and Becker, C. E. (1984) Two fatalities after acute occupational exposure to ethylene dibromide. J. Am. Med. Assoc. 252, 2428-2431. (2) Peterson, L. A., Harris, T. M., and Guengerich, F. P. (1988) Evidence for an episulfonium ion intermediate in the formation of S-[2-(N7-guanyl)ethyl]glutathione in DNA. J. Am. Chem. Soc. 110, 3284-3291. (3) Koga, N., Inskeep, P. B., Harris, T. M., and Guengerich, F. P. (1986) S-[2-(N7-Guanyl)ethyl]glutathione, the major DNA adduct formed from 1,2-dibromoethane. Biochemistry 25, 2192-2198.

1142 Chem. Res. Toxicol., Vol. 10, No. 10, 1997 (4) Inskeep, P. B., Koga, N., Cmarik, J. L., and Guengerich, F. P. (1986) Covalent binding of 1,2-dihaloalkanes to DNA and stability of the major DNA adduct, S-[2-(N7-guanyl)ethyl]glutathione. Cancer Res. 46, 2839-2844. (5) Kim, D. H., and Guengerich, F. P. (1989) Excretion of the mercapturic acid S-[2-(N7-guanyl)ethyl]-N-acetylcysteine in urine following administration of ethylene dibromide to rats. Cancer Res. 49, 5843-5851. (6) Kim, D.-H., and Guengerich, F. P. (1990) Formation of the DNA adduct S-[2-(N7-guanyl)ethyl]glutathione from ethylene dibromide: effects of modulation of glutathione and glutathione S-transferase levels and the lack of a role for sulfation. Carcinogenesis 11, 419-424. (7) Cmarik, J. L., Humphreys, W. G., Bruner, K. L., Lloyd, R. S., Tibbetts, C., and Guengerich, F. P. (1992) Mutation spectrum and sequence alkylation selectivity resulting from modification of bacteriophage M13mp18 with S-(2-chloroethyl)glutathione. Evidence for a role of S-[2-(N7-guanyl)ethyl]glutathione as a mutagenic lesion formed from ethylene dibromide. J. Biol. Chem. 267, 6672-6679. (8) Foster, P. L., Wilkinson, W. G., Miller, J. K., Sullivan, A. D., and Barnes, W. M. (1988) An analysis of the mutagenicity of 1,2dibromoethane to Escherichia coli: influence of DNA repair activities and metabolic pathways. Mutat. Res. 194, 171-181. (9) Ballering, L. A. P., Nivard, M. J. M., and Vogel, E. W. (1994) Mutation spectra of 1,2-dibromoethane, 1,2-dichloroethane and 1-bromo-2-chloroethane in excision repair proficient and repair deficient strains of Drosophila melanogaster. Carcinogenesis 15, 869-875. (10) Graves, R. J., Coutts, C., and Green, T. (1995) Methylene chlorideinduced DNA damage: an interspecies comparison. Carcinogenesis 16, 1919-1926. (11) Oida, T., Humphreys, W. G., and Guengerich, F. P. (1991) Preparation and characterization of oligonucleotides containing S-[2-(N7-guanyl)ethyl]glutathione. Biochemistry 30, 1051310522. (12) Kim, M.-S., and Guengerich, F. P. (1993) Interactions of N7-guanyl methyl- and thioether-substituted d(CATGCCT) derivatives with d(AGGNATC). Chem. Res. Toxicol. 6, 900-905. (13) Persmark, M., and Guengerich, F. P. (1994) Spectroscopic and thermodynamic characterization of the interaction of N7-guanyl thioether derivatives of d(TGCTG*CAAG) with potential complements. Biochemistry 33, 8662-8672. (14) Bailey, E. A., Iyer, R. S., Stone, M. P., Harris, T. M., and Essigmann, J. M. (1996) Mutational properties of the primary aflatoxin B1-DNA adduct. Proc. Natl. Acad. Sci. U.S.A. 93, 15351539. (15) Lawley, P. D., and Brookes, P. (1961) Acidic dissociation of 7:9dialkylguanines and its possible relation to mutagenic properties of alkylating agents. Nature 192, 1081-1082. (16) Lawley, P. D., and Brookes, P. (1962) Ionization of DNA bases or base analogues as a possible explanation of mutagenesis, with special reference to 5-bromodeoxyuridine. J. Mol. Biol. 4, 216219. (17) Peterson, L. A., and Hecht, S. S. (1991) O6-Methylguanine is a critical determinant of 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone tumorigenesis in A/J mouse lung. Cancer Res. 51, 55575564. (18) Loveless, A. (1969) Possible relevance of O6-alkylation of deoxyguanosine to the mutagenicity and carcinogenicity of nitrosamines and nitrosamides. Nature 223, 206-207. (19) Sibghat-Ullah, and Day, R. S., III. (1992) Incision at O6-methylguanine:thymine mispairs in DNA by extracts of human cells. Biochemistry 31, 7998-8008. (20) Swann, P. F. (1990) Why do O6-alkylguanine and O4-alkylthymine miscode? The relationship between the structure of DNA containing O6-alkylguanine and O4-alkylthymine and the mutagenic properties of these bases. Mutat. Res. 233, 81-94. (21) Singer, B., Chavez, F., Goodman, M. F., Essigmann, J. M., and Dosanjh, M. K. (1989) Effect of 3′ flanking neighbors on kinetics of pairing of dCTP and dTTP opposite O6-methylguanine in a defined primed oligonucleotide when Escherichia coli DNA polymerase I is used. Proc. Natl. Acad. Sci. U.S.A. 86, 8271-8274. (22) Dosanjh, M. K., Singer, B., and Essigmann, J. M. (1991) Comparative mutagenesis of O6-methylguanine and O4-methylthymine in Escherichia coli. Biochemistry 30, 7027-7033. (23) Mackay, W., Benasutti, M., Drouin, E., and Loechler, E. L. (1992) Mutagenesis by (+)-anti-B[a]P-N2-Gua, the major adduct of activated benzo[a]pyrene, when studied in an Escherichia coli plasmid using site-directed methods. Carcinogenesis 13, 14151425. (24) Shibutani, S., Margulis, L. A., Geacintov, N. E., and Grollman, A. P. (1993) Translesional synthesis on a DNA template containing a single stereoisomer of dG-(+)- or dG-(-)-anti-BPDE (7,8-

Kim and Guengerich

(25)

(26)

(27)

(28)

(29) (30)

(31) (32)

(33)

(34)

(35)

(36)

(37)

(38)

(39)

(40)

(41)

(42)

(43)

(44)

(45)

dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene). Biochemistry 32, 7531-7541. Decorte, B. L., Tsarouhtsis, D., Kuchimanchi, S., Cooper, M. D., Horton, P., Harris, C. M., and Harris, T. M. (1996) Improved strategies for postoligomerization synthesis of oligodeoxynucleotides bearing structurally defined adducts at the N2 position of deoxyguanosine. Chem. Res. Toxicol. 9, 630-637. Einarsson, S., and Josefesson, B. (1987) Preparation of amino acid enantiomers and chiral amines using precolumn derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate and reversed-phase liquid chromatography. Anal. Chem. 59, 1191-1195. Humphreys, W. G., Kim, D.-H., Cmarik, J. L., Shimada, T., and Guengerich, F. P. (1990) Comparison of the DNA alkylating properties and mutagenic responses caused by a series of S-(2haloethyl)-substituted cysteine and glutathione derivatives. Biochemistry 29, 10342-10350. Carpino, L. A., and Han, G. Y. (1970) The 9-fluorenylmethoxycarbonyl function, a new base-sensitive amino-protecting group. J. Am. Chem. Soc. 92, 5748-5749. Carpino, L. A., and Han, G. Y. (1972) The 9-fluorenylmethoxycarbonyl amino-protecting group. J. Org. Chem. 37, 3404-3409. Basu, A. K., and Essigmann, J. M. (1988) Site-specifically modified oligodeoxynucleotides as probes for the structural and biological effects of DNA-damaging agents. Chem. Res. Toxicol. 1, 1-18. Preston, B. D., and Loeb, L. A. (1988) Enzymatic synthesis of sitespecifically modified DNA. Mutat. Res. 200, 21-35. Harris, C. M., Zhou, L., Strand, E. A., and Harris, T. M. (1991) New strategy for the synthesis of oligodeoxynucleotides bearing adducts at exocyclic amino sites of purine nucleosides. J. Am. Chem. Soc. 113, 4328-4329. Langoue¨t, S., Mu¨ller, M., and Guengerich, F. P. (1997) Misincorporation of dNTPs opposite 1,N2-ethenoguanine and 5,6,7,9tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine in oligonucleotides by Escherichia coli polymerases I exo- and II exo-, T7 polymerase exo-, human immunodeficiency virus-1 reverse transcriptase, and rat polymerase β. Biochemistry 36, 6069-6079. Bailey, E. A., Iyer, R. S., Harris, T. M., and Essigmann, J. M. (1996) A viral genome containing an unstable aflatoxin B1-N7guanine DNA adduct situated at a unique site. Nucleic Acids Res. 24, 2821-2828. Thier, R., Pemble, S. E., Taylor, J. B., Humphreys, W. G., Persmark, M., Ketterer, B., and Guengerich, F. P. (1993) Expression of mammalian glutathione S-transferase 5-5 in Salmonella typhimurium TA1535 leads to base-pair mutations upon exposure to dihalomethanes. Proc. Natl. Acad. Sci. U.S.A. 90, 8576-8580. Fink, R. M., Enns, T., Kimball, C. P., Silberstein, H. E., Bale, W. F., Maddes, S. C., and Whipple, G. H. (1944) Plasma protein metabolism-normal and associated with shock; observations using protein labeled by heavy nitrogen in lysine. J. Expt. Med. 80, 455475. Guengerich, F. P., and Broquist, H. P. (1973) Biosynthesis of slaframine, (1S,6S,8aS)-1-acetoxy-6-aminooctahydroindolizine, a parasympathomimetic alklaoid of fungal origin. II. The origin of pipecolic acid. Biochemistry 12, 4270-4274. Landidi, D., and Rolla, F. (1976) A convenient synthesis of N-alkylphthalimides in a solid-liquid two-phase system in the presence of phase transfer catalysts. Synthesis 389-390. Soai, K., Ookawa, A., and Kato, K. (1982) A facile one-step synthesis of N-substituted phthalimides using a catalytic amount of crown ether. Bull. Chem. Soc. Jpn. 55, 1671-1672. Tan, H. B., Swann, P. F., and Chance, E. M. (1994) Kinetic analysis of the coding properties of O6-methylguanine in DNA: the crucial role of the conformation of the phosphodiester bond. Biochemistry 33, 5335-5346. Fowler, K. W., Bu¨chi, G., and Essigmann, J. M. (1982) Synthesis and characterization of an oligonucleotide containing a carcinogenmodified base: O6-methylguanine. J. Am. Chem. Soc. 104, 10501054. Gaffney, B. L., and Jones, R. A. (1982) A new strategy for the protection of deoxyguanosine during oligonucleotide synthesis. Tetrahedron Lett. 22, 2257-2260. Gaffney, B. L., and Jones, R. A. (1982) Synthesis of O6-alkylated deoxyguanosine nucleosides. A new strategy for the protection of deoxyguanosine during oligonucleotide synthesis. Tetrahedron Lett. 23, 2253-2256. Gaffney, B. L., Marky, L. A., and Jones, R. A. (1984) Synthesis and characterization of a set of four dodecadeoxyribonucleoside undecaphosphates containing O6-methylguanine opposite adenine, cytosine, guanine, and thymine. Biochemistry 23, 56865691. Li, B. F. L., and Swann, P. F. (1989) Synthesis and characterization of oligodeoxynucleotides containing O6-methyl-, O6-ethyl-, and O6-isopropylguanine. Biochemistry 28, 5779-5786.

Ethylene Dibromide-GSH-DNA Adduct Synthesis (46) Smith, C. A., Xu, Y. Z., and Swann, P. F. (1990) Solid-phase synthesis of oligodeoxynucleotides containing O6-alkylguanine. Carcinogenesis 11, 811-816. (47) Xu, Y. Z., and Swann, P. F. (1991) Solid phase synthesis of oligodeoxynucleotides containing O6-alkylguanine and O4-alkylthymine. Nucleosides Nucleotides 10, 315-318. (48) Pongracz, K., Dosanjh, M. K., Singer, B., and Bodell, W. J. (1994) Synthesis of a 25 base oligonucleotide containing a styrene oxide modification at the O6 position of 2′-deoxyguanosine at a defined site and incorporation studies of the similarly modified 2′deoxyguanosine-5′-triphosphate. Carcinogenesis 15, 1371-1375. (49) Borowy-Borowski, H., and Chambers, R. W. (1987) A study of side reactions occurring during synthesis of oligodeoxynucleotides containing O6-alkyldeoxyguanosine residues at preselected sites. Biochemistry 26, 2465-2471. (50) Krieg, D. R. (1963) Specificity of chemical mutagenesis. Prog. Nucleic. Acid Res. Mol. Biol. 2, 125-168. (51) Mitsunobu, O. (1981) The use of diethyl azodicarboxylate and triphenylphosphine in synthesis and transformation of natural products. Synthesis 1-28. (52) Koole, L. H., Moody, H. M., Broedeers, N. H. L., Quaedflieg, P. J. L. M., Kuijpers, W. H. A., van Genderen, M. H. P., Coenen, J. J.

Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1143 M., van der Wal, S., and Buck, H. M. (1989) Synthesis of phosphate-methylated DNA fragments using 9-fluorenylmethoxycarbonyl as transient base protecting group. J. Org. Chem. 54, 16571664. (53) Zhou, Y., and Romano, L. J. (1993) Solid-phase synthesis of oligonucleotides containing site-specific N-(2′-deoxyguanosin-8yl)-2-(acetylamino)fluorene adducts using 9-fluorenylmethoxycarbonyl as the base-protecting group. Biochemistry 32, 1404314052. (54) Latham, G. J., Zhou, L., Harris, C. M., Harris, T. M., and Lloyd, R. S. (1993) The replication fate of R- and S-styrene oxide adducts on adenine N6 is dependent on both the chirality of the lesion and the local sequence context. J. Biol. Chem. 268, 23427-23434. (55) Chary, P., Latham, G. J., Robberson, D. L., Kim, S. J., Han, S., Harris, C. M., Harris, T. M., and Lloyd, R. S. (1995) In vivo and in vitro replication consequences of stereoisomeric benzo[a]pyrene7,8-dihydrodiol 9,10-epoxide adducts on adenine N6 at the second position of N-ras codon 61. J. Biol. Chem. 270, 4990-5000.

TX9701081