Aralkylation of Guanosine with Para-Substituted Styrene Oxides

Jan 19, 1998 - Willem F. Veldhuyzen, Anthony J. Shallop, Roger A. Jones, and Steven E. Rokita. Journal of the American Chemical Society 2001 123 (45),...
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Chem. Res. Toxicol. 1998, 11, 44-53

Aralkylation of Guanosine with Para-Substituted Styrene Oxides Thomas Barlow* and Anthony Dipple Chemistry of Carcinogenesis Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, P.O. Box B, Frederick, Maryland 21702 Received July 21, 1997X

To probe mechanisms of nucleoside aralkylation, product distributions and product stereochemistries were determined in reactions of optically active p-methyl- and p-bromostyrene oxide with guanosine. The proportion of 7-, N2-, and O6-substituted guanosine products was ∼0.32:0.62:0.06 in neutral, aqueous reactions with the (R)-p-methylstyrene oxide and ∼0.85: 0.09:0.04 in reactions with the (R)-p-bromostyrene oxide. The exocyclic positions opened the epoxide at the R-carbon. Epoxide ring opening by the nitrogen at the 7-position showed little preference for the R- or β-carbons in reactions with p-methylstyrene oxide. However, the p-bromostyrene oxide favored reaction at the β-carbon almost 4-fold over reaction at the R-carbon. Almost total inversion of stereochemistry was found to occur in reactions at the 7-position. In contrast, the ratio of inversion to retention of configuration in N2- and O6-substituted products was ∼2:1 and ∼1:1 for reactions with the p-methylstyrene oxide and ∼6:1 and ∼3:1 for reactions with p-bromostyrene oxide, respectively. These experiments suggest that an SN2 mechanism is in effect with reactions at the 7-position, whereas substrates of an increasingly ionic nature are involved in reactions at the N2- and O6-positions, respectively.

Introduction Chemical carcinogens initiate the carcinogenic process by binding to constituents of DNA (1). Many of these carcinogens are not reactive themselves but require metabolic activation to become DNA-reactive agents (2). These latter ultimate carcinogens are often formed by epoxidation of unsaturated bonds, and it is the resultant oxidation products that react with DNA to initiate mutagenic and carcinogenic processes (2). Although many ultimate carcinogens share the same reactive epoxide moiety, the spectrum of DNA adducts formed by these compounds is varied. Simple alkyl epoxides such as ethylene oxide (3) and propylene oxide (4) predominantly target the 7-position of guanine residues (5), whereas alkylnitrosoureas react at the O6-position in addition to the 7-position (1), and the dihydrodiol epoxides of some polycyclic aromatic hydrocarbons such as benzo[a]pyrene (6-8) and 5-methylchrysene (9-11) react almost exclusively at the exocyclic amino group of guanine in DNA. This differing site selectivity has prompted several studies exploring the underlying mechanisms involved (12-19). The sites on guanosine that are modified most extensively by electrophilic carcinogens are the 7-, N2-, and O6-positions (15). Studies using benzylating agents that modify all three positions indicated that reactions at the ring nitrogen follow a bimolecular displacement mechanism whereas modification of the exocyclic groups requires some degree of substrate ionization for reaction to occur (14-17). These indications were supported by later studies which probed reactions at these sites with styrene oxide (18). Styrene oxide, the ultimate carcinogen formed from metabolic oxidation of styrene (20-22), * Corresponding author. X Abstract published in Advance ACS Abstracts, December 15, 1997.

also reacted in aqueous solution with all three guanosine centers (23, 24). The 7-position opened the oxirane ring at both the R- and β-carbons, and N2- and O6-substituted products were formed at the R-carbon (18, 23, 24). Optically active forms of the epoxide have been used to provide additional mechanistic information from the stereochemical dimension (18). In the present study, mechanistic investigations were extended by using optically active p-methyl- and pbromostyrene oxide to further explore the factors controlling aralkylation of guanosine.

Experimental Section Caution: The para-substituted styrene oxides synthesized may be potentially mutagenic and/or carcinogenic and should be handled with care. Chemicals. All chemicals were obtained from Aldrich Chemical Co. (Milwaukee, WI) with the exception of hydroquinidine 1,4-phthalazinediyl diether [(DHQD)2PHAL],1 hydroquinine 1,4-phthalazinediyl ether [(DHQ)2PHAL], and potassium osmate dihydrate which were obtained from Fluka (Ronkonkoma, NY) and generally labeled [14C]guanosine (specific radioactivity 509 mCi/mmol) which was obtained from Amersham Searle (Arlington Heights, IL). 2-Chloro-6-hydroxypurine was prepared by the method of Shapiro et al. (25). TLC was carried out on 60 F 254 silica gel on aluminum plates obtained from Aldrich Chemical Co. (Milwaukee, WI) using 2:8 ethyl acetate/ hexane as a solvent system. Wet flash chromatography was carried out using silica gel 60 obtained from Aldrich Chemical Co. Ecoscint A scintillant was obtained from National Diagnostics (Atlanta, GA). Instrumentation. HPLC was carried out on a HewlettPackard model 1090 high-pressure liquid chromatography instrument equipped with a diode array detector and a YMC J′ 1 Abbreviations: CE, capillary electrophoresis; (DHQD) PHAL, hy2 droquinidine 1,4-phthalazinediyl diether; (DHQ)2PHAL, hydroquinine 1,4-phthalazinediyl diether; ee, enantiomeric excess.

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Mechanisms of Guanosine Aralkylation sphere ODS-M80 250- × 4.6-mm column (Wilmington, NC). Chiral separations were achieved by capillary electrophoresis on a Beckman instrument equipped with an XAA 47- × 50-mm column using 10 mM phosphate adjusted to pH 2.3 by triethylamine and 5 mM chiral selector sulfyl butyl ether (IV)-βcyclodextrin [β-CD-SBE (IV)] as the mobile phase. Ultraviolet absorption spectra were recorded with a Milton Roy Spectronic 3000 diode array spectrophotometer. Circular dichroism spectra were measured on a Jasco model J500A spectropolarimeter equipped with a data-processing system for signal averaging. 1H NMR spectra were obtained using either a Varian XL-200 instrument or a Varian-500S instrument. COSY homonuclear 2D spectra were obtained using the Varian-500S instrument. Samples were dissolved in CDCl3 or DMSO-d6 with tetramethylsilane as an internal standard. Positive ion (+ve) electron impact (EI) mass spectra (MS) were obtained with a VG Analytical 72-50 instrument, and +ve fast atom bombardment (FAB) MS were obtained with a reverse-geometry VG Micromass ZAB-2F spectrometer interfaced to a VG 203S data system using glycerol as the FAB matrix. For bromo compounds, the ratio of abundance of 79Br/81Br isotopes was in the range of 1.070.91. Synthesis of (()-Para-Substituted Phenyl-1,2-ethanediols. Potassium ferricyanide (37.44 g, 114 mmol, 3 equiv) and potassium carbonate (15.72 g, 114 mmol, 3 equiv) were dissolved in 50% (v/v) tert-butyl alcohol (380 mL), and potassium osmate dihydrate (27.8 mg, 75 mmol, 0.002 equiv) was added. The biphasic mixture was cooled to 0 °C, para-substituted styrene (38.2 mmol, 1 equiv) was added, and the mixture was stirred for 8 h at 0 °C and for an additional 16 h at room temperature. Sodium sulfite (57.3 g) and ethyl acetate (200 mL) were added, and the mixture was stirred for 30 min. The product was extracted with ethyl acetate (3 × 100 mL), and the combined organic phase was dried over magnesium sulfate and concentrated in vacuo to yield a white solid. (()-(p-Methylphenyl)-1,2-ethanediol: 4.46 g, 77%; Rf 0.17; 1H NMR (CDCl ) δ 7.22 (d, 2, Ar, J ) 8.1 Hz), 7.12 (d, 2, Ar, J 3 ) 8.1 Hz), 4.71 (dd, 1, R-CH, JR,β1,β2 ) 3.5, 4.6 Hz), 3.68-3.55 (m, 2, β-CH1,2), 3.43 (br s, 2, OH), 2.32 (s, 3, CH3); +ve EI-MS m/z 152.082 46 ([M]+; calcd for C9H12O2, 152.083 73). (()-(p-Bromophenyl)-1,2-ethanediol: 6.47 g, 78%; Rf 0.21; 1H NMR (CDCl ) δ 7.47 (d, 2, Ar, J ) 8.5 Hz), 7.24 (d, 2, Ar, J 3 ) 8.2 Hz), 4.78 (dd, 1, R-CH, JR,β1,β2 ) 3.5, 4.4 Hz), 3.78-3.55 (m, 2, β-CH1,2), 2.74 (br s, 1, OH), 2.21 (br s, 1, OH); +ve EI-MS m/z 217.978 17 ([M]+; calcd for C8H9O281Br, 217.976 54), 215.980 35 ([M]+; calcd for C8H9O279Br, 215.978 59). Synthesis of (R)-Para-Substituted Phenyl-1,2-ethanediols. These were prepared by the methods used for (()-parasubstituted phenyl-1,2-ethanediols but with the addition of (DHQD)2PHAL (272 mg, 0.38 mmol, 0.01 equiv) along with the potassium osmate dihydrate (ee >95%). (R)-(p-Methylphenyl)-1,2-ethanediol: 3.87 g, 67%; Rf 0.17; 1H NMR (CDCl ) δ 7.18 (d, 2, Ar, J ) 8.3 Hz), 7.12 (d, 2, Ar, J 3 ) 8.3 Hz), 4.70 (dd, 1, R-CH, JR,β1,β2 ) 3.5, 4.7 Hz), 3.67-3.54 (m, 2, β-CH1,2), 3.31 (br s, 2, OH), 2.32 (s, 3, CH3); +ve EI-MS m/z 152.084 83 ([M]+; calcd for C9H12O2, 152.083 73). (R)-(p-Bromophenyl)-1,2-ethanediol: 6.05 g, 73%; Rf 0.21; 1H NMR (CDCl ) δ 7.49 (d, 2, Ar, J ) 8.4 Hz), 7.25 (d, 2, Ar, J 3 ) 8.4 Hz), 4.80 (dd, 1, R-CH, JR,β1,β2 ) 3.6, 4.4 Hz), 3.80-3.57 (m, 2, β-CH1,2), 2.51 (br s, 2, OH); +ve EI-MS m/z 217.977 59 ([M]+; calcd for C8H9O281Br, 217.976 54), 215.979 33 ([M]+; calcd for C8H9O279Br, 215.978 59). Synthesis of (S)-Para-Substituted Phenyl-1,2-ethanediols. These were prepared by the methods used for (()-parasubstituted phenyl-1,2-ethanediols but with the addition of (DHQ)2PHAL (272 mg, 0.38 mmol, 0.01 equiv) along with the potassium osmate hydrate (ee >95%). (S)-(p-Methylphenyl)-1,2-ethanediol: 4.27 g, 74%; Rf 0.17; 1H NMR (CDCl ) δ 7.20 (d, 2, Ar, J ) 8.1 Hz), 7.12 (d, 2, Ar, J 3 ) 8.1 Hz), 4.72 (dd, 1, R-CH, JR,β1,β2 ) 3.6, 4.6 Hz), 3.69-3.55 (m, 2, β-CH1,2), 3.41 (br s, 1, OH), 3.11 (br s, 1, OH), 2.32 (s, 3,

Chem. Res. Toxicol., Vol. 11, No. 1, 1998 45 CH3); +ve EI-MS m/z 152.083 50 ([M]+; calcd for C9H12O2, 152.083 73). (S)-(p-Bromophenyl)-1,2-ethanediol: 6.13 g, 74%; Rf 0.21; 1H NMR (CDCl ) δ 7.50 (d, 2, Ar, J ) 8.5 Hz), 7.26 (d, 2, Ar, J 3 ) 8.3 Hz), 4.80 (dd, 1, R-CH, JR,β1,β2 ) 3.6, 4.4 Hz), 3.80-3.57 (m, 2, β-CH1,2), 2.21 (br s, 2, OH); +ve EI-MS m/z 217.978 16 ([M]+; calcd for C8H9O281Br, 217.976 54), 215.979 67 ([M]+; calcd for C8H9O279Br 215.978 59). Synthesis of Para-Substituted Phenylethane 1-Hydroxy2-tosylates. Para-substituted phenyl-1,2-ethanediol (6.9 mmol, 1 equiv) was coevaporated with anhydrous pyridine (3 × 10 mL) and then redissolved in anhydrous pyridine (20 mL). The solution was cooled to 0 °C before the dropwise addition of tosyl chloride (1.45 g, 7.6 mmol, 1.1 equiv) solution in anhydrous pyridine (10 mL) with vigorous stirring. The mixture was stirred for 16 h, then coevaporated with toluene, and concentrated in vacuo. The resulting gum was dissolved in ethyl acetate (50 mL) and washed with water (50 mL). The product was extracted into the organic phase with ethyl acetate (3 × 50 mL), and the combined organic phase was dried over magnesium sulfate, filtered, and concentrated in vacuo before purification by wet flash column chromatography (silica, eluted with a gradient of 0-40% ethyl acetate in hexane) to yield the product as a white solid (yield 81-91%). (p-Methylphenyl)ethane 1-hydroxy-2-tosylate: Rf 0.51; 1H NMR (CDCl ) δ 7.76 (d, 2, Ar, J ) 8.3 Hz), 7.38-7.12 (m, 6, 3 Ar, Artosyl), 4.93 (dd, 1, R-CH, JR,β1,β2 ) 3.3, 5.1 Hz), 4.15-4.00 (m, 2, β-CH1,2), 2.54 (br s, 1, OH), 2.44 (s, 3, CH3), 2.32 (s, 3, CH3); +ve EI-MS m/z 306.093 71 ([M]+; calcd for C16H18O4S, 306.092 58). (p-Bromophenyl)ethane 1-hydroxy-2-tosylate: Rf 0.64; 1H NMR (CDCl ) δ 7.75 (d, 2, Ar, J ) 8.3 Hz), 7.48-7.17 (m, 6, 3 Ar, Artosyl), 4.95 (dd, 1, R-CH, JR,β1,β2 ) 3.2, 4.4 Hz), 4.16-3.97 (m, 2, β-CH1,2), 2.52 (br s, 1, OH), 2.46 (s, 3, CH3); +ve EI-MS m/z 371.981 39 ([M]+; calcd for C15H15O481BrS, 371.985 40), 369.988 40 ([M]+; calcd for C15H15O479BrS, 369.987 44). Synthesis of Para-Substituted Styrene Oxides. Parasubstituted phenylethane 1-hydroxy-2-tosylate (1.30 mmol, 1 equiv) and sodium hydroxide (105 mg, 2.60 mmol, 2 equiv) were dissolved in methanol (20 mL) and stirred for 2 h at room temperature. The reaction mixture was concentrated in vacuo and water (20 mL) added. The product was extracted with diethyl ether (3 × 30 mL), and the combined organic phases were dried over magnesium sulfate and concentrated in vacuo. The product was purified by wet flash column chromatography (silica, eluted with a gradient of 0-5% ethyl acetate in hexane) to yield the product as a pale yellow liquid (yield 72-89%). p-Methylstyrene oxide: Rf 0.95; 1H NMR (CDCl3) δ 7.16 (s, 4, Ar), 3.82 (dd, 1, R-CH, JR,β2 ) 2.6 Hz, JR,β1 ) 4.1 Hz), 3.11 (dd, 1, β-CH1, Jβ1,R ) 4.1 Hz, Jβ1,β2 ) 5.5 Hz), 2.78 (dd, 1, β-CH2, Jβ2,R ) 2.6 Hz, Jβ2,β1 ) 5.5 Hz), 2.34 (s, 3, CH3); +ve EI-MS m/z 134.073 32 ([M]+; calcd for C9H10O, 134.073 16). p-Bromostyrene oxide: Rf 0.98; 1H NMR (CDCl3) δ 7.47 (d, 2, Ar, J ) 8.5 Hz), 7.15 (d, 2, Ar, J ) 8.4 Hz), 3.82 (dd, 1, R-CH, JR,β2 ) 2.6 Hz, JR,β1 ) 4.0 Hz), 3.14 (dd, 1, β-CH1, Jβ1,R ) 4.1 Hz, Jβ1,β2 ) 5.5 Hz), 2.75 (dd, 1, β-CH2, Jβ2,R ) 2.5 Hz, Jβ2,β1 ) 5.5 Hz); +ve EI-MS m/z 199.965 67 ([M]+; calcd for C8H7O81Br, 199.965 98), 197.967 48 ([M]+; calcd for C8H7O79Br, 197.968 03). Synthesis of Para-Substituted Phenyl-1,2-ethane Cyclic Carbonates. Para-substituted phenyl-1,2-ethanediol (4.6 mmol, 1 equiv) and sodium hydroxide (18 mg, 0.5 mmol, 0.1 equiv) were suspended in dimethyl carbonate (10 mL, 10 g, 118 mmol, 25 equiv) and stirred under reflux for 24 h. The reaction mixture was concentrated in vacuo, and the product was purified by wet flash column chromatography (silica, eluted with a gradient of 0-30% ethyl acetate in hexane). The product was obtained as a pale yellow solid (yield 71-98%). (p-Methylphenyl)-1,2-ethane cyclic carbonate: Rf 0.40; 1H NMR (CDCl ) δ 7.26 (s, 4, Ar), 5.64 (t, 1, R-CH, J 3 R,β1,β2 ) 8.0 Hz), 4.77 (dd, 1, β-CH1, Jβ1,R ) 8.2 Hz, Jβ1,β2 ) 8.5 Hz), 4.34 (dd,

46 Chem. Res. Toxicol., Vol. 11, No. 1, 1998 1, β-CH2, Jβ2,R ) 8.0 Hz, Jβ2,β1 ) 8.6 Hz), 2.38 (s, 3, CH3); +ve EI-MS m/z 178.062 44 ([M]+; calcd for C10H10O3, 178.062 99). (p-Bromophenyl)-1,2-ethane cyclic carbonate: Rf 0.55; 1H NMR (CDCl ) δ 7.59 (d, 2, Ar, J ) 8.5 Hz), 7.24 (d, 2, Ar, J 3 ) 8.5 Hz), 5.64 (t, 1, R-CH, JR,β1,β2 ) 8.1 Hz), 4.80 (t, 1, β-CH1, Jβ1,R ) 8.6 Hz), 4.30 (dd, 1, β-CH2, Jβ2,R ) 7.8 Hz, Jβ2,β1 ) 8.6 Hz); +ve EI-MS m/z 243.958 18 ([M]+; calcd for C9H7O381Br, 243.955 81), 241.959 56 ([M]+; calcd for C9H7O379Br, 241.957 86). Synthesis of Para-Substituted Phenyl-1-azidoethan-2ols. Para-substituted phenyl-1,2-ethane cyclic carbonate (2.5 mmol, 1 equiv), sodium azide (322 mg, 5 mmol, 2 equiv), and water (44 mL, 2.5 mmol, 1 equiv) were dissolved in DMF (30 mL) and stirred at 70 °C for 48 h. The reaction mixture was concentrated in vacuo and water (50 mL) added. The product was extracted into the organic phase with ethyl acetate (3 × 50 mL), dried over magnesium sulfate, filtered, and concentrated in vacuo to yield a white solid (yield 81-86%). (p-Methylphenyl)-1-azidoethan-2-ol: Rf 0.47; νmax/cm-1 2102 (N3); 1H NMR (CDCl3) δ 7.21 (s, 4, Ar), 4.63 (t, 1, R-CH, JR,β1,β2 ) 6.5 Hz), 3.73 (d, 2, β-CH1,2, Jβ1,β2,R ) 6.5 Hz), 2.36 (s, 3, CH3); +ve EI-MS m/z 177.089 62 ([M]+; calcd for C9H11N3O, 177.090 21). (p-Bromophenyl)-1-azidoethan-2-ol: Rf 0.56; νmax/cm-1 2102 (N3); 1H NMR (CDCl3) δ 7.54 (d, 2, Ar, J ) 8.5 Hz), 7.23 (d, 2, Ar, J ) 8.5 Hz), 4.64 (dd, 1, R-CH, JR,β1 ) 5.4 Hz, JR,β2 ) 7.2 Hz), 3.73 (t, 2, β-CH1,2, Jβ1,β2,R ) 5.8 Hz); +ve EI-MS m/z 240.986 28 ([M]+; calcd for C8H8N3O79Br, 240.985 07). Synthesis of Para-Substituted Phenylglycinols. Parasubstituted phenyl-1-azidoethan-2-ol (2 mmol, 1 equiv) was dissolved in anhydrous toluene (25 mL), and Red-Al (1 mL, 3.2 mmol, 1.6 equiv) was added dropwise with stirring, under anhydrous conditions, at room temperature. The reaction mixture was heated under reflux for 2 h and concentrated in vacuo. Water (50 mL) was added slowly, and the product was extracted into the organic phase with dichloromethane (3 × 50 mL), dried over magnesium sulfate, filtered, and concentrated in vacuo to yield a white solid (yield 69-74%). (p-Methylphenyl)glycinol: 1H NMR (CDCl3) δ 7.23-7.15 (m, 4, Ar), 4.00 (br s, 1, R-CH), 3.56-3.53 (m, 2, β-CH1,2), 2.34 (s, 3, CH3); +ve EI-MS m/z 151.099 70 ([M]+; calcd for C9H13NO, 151.099 71). (p-Bromophenyl)glycinol: 1H NMR (CDCl3) δ 7.45-7.30 (m, 4, Ar), 4.74 (t, 1, R-CH, J ) 5.4 Hz), 3.89-3.82 (m, 1, β-CH1), 3.49-3.39 (m, 1, β-CH2), 3.31 (br s, 3, NH2, OH); +ve EI-MS m/z 214.995 09 ([M]+; calcd for C8H10NO79Br, 214.994 57), 216.992 98 ([M]+; calcd for C8H10NO81Br, 216.992 52). Synthesis of 7-[2-Hydroxy-2-(p-methylphenyl)ethyl]guanosines (RβN-7MeSOG and SβN-7MeSOG) and 7-[2-Hydroxy-2-(p-bromophenyl)ethyl]guanosines (RβN-7BrSOG and SβN-7BrSOG). Guanosine (450 mg, 1.60 mmol, 1 equiv) and ammonium acetate (100 mg) were dissolved in 50% (v/v) ethanol (100 mL), and optically active para-substituted styrene oxide (3.2 mmol, 2 equiv) was added. The mixture was stirred in the dark, at 37 °C for 96 h. The reaction mixture was concentrated to dryness, and the solid residue was washed with diethyl ether (50 mL) before resuspension in methanol (30 mL). The suspension was filtered, and the filtrate was applied to a Sephadex LH-20 column (2.8 × 80 cm) eluted at 1 mL/min with methanol. Absorption of the eluate was monitored continuously at 254 nm, and 8-mL fractions were collected. The product eluted in fractions 45-56 and was identified by comparison of the UV spectra under neutral, basic, and acidic conditions with those of literature spectra for 7-substituted guanosines (26), and those fractions were pooled. The product was further purified by reversed-phase HPLC eluted isocratically with 14% methanol for RβN-7MeSOG (46 min), 14% methanol for SβN-7MeSOG (32 min), 25% methanol for RβN-7BrSOG (30 min), or 22% methanol for SβN-7BrSOG (48 min) in 50 mM ammonium formate, pH 5.7. RβN-7MeSOG: UV λmax (methanol) 259, 282 (sh) nm; 1H NMR (DMSO-d6) δ 8.55 (s, 1, H-8), 7.34 (d, 2, Ar, J ) 8.0 Hz), 7.18 (d, 2, Ar, J ) 7.8 Hz), 5.89 (d, 1, H1′, J ) 4.6 Hz), 5.13 (dd,

Barlow and Dipple 1, R-CH, JR,β1 ) 3.8 Hz, JR,β2 ) 8.9 Hz), 4.78 (dd, 1, β-CH1, Jβ1,R ) 3.4 Hz, Jβ1,β2 ) 13.2 Hz), 4.56 (t, 1, H2′, J ) 4.8 Hz), 4.36 (dd, 1, β-CH2, Jβ2,R ) 8.9 Hz, Jβ2,β1 ) 13.2 Hz), 4.24 (t, 1, H3′, J ) 4.6 Hz), 4.17-4.16 (m, 1, H4′), 3.92 (dd, 1, H5′a, J ) 2.5, 12.3 Hz), 3.75 (dd, 1, H5′b, J ) 2.5, 12.3 Hz), 2.31 (s, 3, CH3); +ve FABMS m/z 418.1770 ([M + H]+; calcd for C19H24N5O6, 418.1726). SβN-7MeSOG: UV λmax (methanol) 259, 282 (sh) nm; 1H NMR (DMSO-d6) δ 8.55 (s, 1, H-8), 7.34 (d, 2, Ar, J ) 7.9 Hz), 7.18 (d, 2, Ar, J ) 7.7 Hz), 5.89 (d, 1, H1′, J ) 4.6 Hz), 5.13 (dd, 1, R-CH, JR,β1 ) 3.5 Hz, JR,β2 ) 8.7 Hz), 4.78 (dd, 1, β-CH1, Jβ1,R ) 3.8 Hz, Jβ1,β2 ) 13.5 Hz), 4.56 (t, 1, H2′, J ) 4.7 Hz), 4.36 (dd, 1, β-CH2, Jβ2,R ) 8.9 Hz, Jβ2,β1 ) 13.3 Hz), 4.24 (t, 1, H3′, J ) 4.7 Hz), 4.18-4.16 (m, 1, H4′), 3.92 (dd, 1, H5′a, J ) 2.5, 12.5 Hz), 3.75 (dd, 1, H5′b, J ) 2.6, 12.4 Hz), 2.32 (s, 3, CH3); +ve FABMS m/z 418.1777 ([M + H]+; calcd for C19H24N5O6, 418.1726). RβN-7BrSOG: UV λmax (methanol) 259, 282 (sh) nm; 1H NMR (DMSO-d6) δ 9.01 (s, 1, H-8), 7.48 (d, 2, Ar, J ) 8.3 Hz), 7.29 (d, 2, Ar, J ) 8.5 Hz), 6.43 (br s, 1, NH, exchanges with D2O), 5.78 (d, 1, H1′, J ) 4.9 Hz), 5.19 (d, 1, R-CH, J ) 7.9 Hz), 4.70 (dd, 1, β-CH1, Jβ1,R ) 9.0 Hz, Jβ1,β2 ) 13.1 Hz), 4.45 (t, 1, H2′, J ) 4.8 Hz), 4.16 (dd, 1, β-CH2, Jβ2,R ) 7.9 Hz, Jβ2,β1 ) 13.1 Hz), 4.11 (t, 1, H3′, J ) 4.3 Hz), 3.98 (dd, 1, H4′, J ) 3.6, 7.4 Hz), 3.71 (dd, 1, H5′a, J ) 2.7, 12.0 Hz), 3.58 (dd, 1, H5′b, J ) 2.7, 12.0 Hz); +ve FAB-MS m/z 482.0631 ([M + H]+; calcd for C18H21N5O679Br, 482.0674). SβN-7BrSOG: UV λmax (methanol) 259, 282 (sh) nm; 1H NMR (DMSO-d6) δ 9.01 (s, 1, H-8), 7.59 (d, 2, Ar, J ) 8.3 Hz), 7.43 (d, 2, Ar, J ) 8.3 Hz), 6.41 (br s, 1, NH, exchanges with D2O), 5.82 (d, 1, H1′, J ) 4.8 Hz), 5.09 (d, 1, R-CH, J ) 8.7 Hz), 4.73 (dd, 1, β-CH1, Jβ1,R ) 2.3 Hz, Jβ1,β2 ) 13.2 Hz), 4.45 (t, 1, H2′, J ) 4.8 Hz), 4.16 (dd, 1, β-CH2, Jβ2,R ) 9.2 Hz, Jβ2,β1 ) 13.2 Hz), 4.11 (t, 1, H3′, J ) 4.4 Hz), 3.99 (dd, 1, H4′, J ) 3.5, 7.4 Hz), 3.71 (dd, 1, H5′a, J ) 2.6, 12.0 Hz), 3.58 (dd, 1, H5′b, J ) 11.5 Hz); +ve FAB-MS m/z 482.0620 ([M + H]+; calcd for C18H21N5O679Br, 482.0674). Synthesis of 7-[2-Hydroxy-1-(p-methylphenyl)ethyl]guanosines (RrN-7MeSOG and SrN-7MeSOG) and 7-[2-Hydroxy-1-(p-bromophenyl)ethyl]guanosines (RrN-7BrSOG and SrN-7BrSOG). Guanosine (450 mg, 1.60 mmol, 1 equiv) and para-substituted styrene oxide (3.2 mmol, 2 equiv) were dissolved in glacial acetic acid (50 mL), and the solution was stirred, in the dark, at 37 °C for 16 h. The reaction mixture was slowly added to diethyl ether (450 mL) with stirring, and the resulting precipitate was collected by filtration. The solid was dissolved in methanol (30 mL), applied to a Sephadex LH20 column (2.8 × 80 cm), and eluted at 1 mL/min with methanol. Absorption of the eluate was monitored continuously at 254 nm, and 8-mL fractions were collected. The product eluted in fractions 34-45 and was identified by comparison of the UV spectra under neutral, basic and acidic conditions with those of literature spectra for 7-substituted guanosines (26), and those fractions were pooled. The product was further purified by reversed-phase HPLC eluting isocratically with 14% methanol for RRN-7MeSOG (36 min), 14% methanol for SRN-7MeSOG (57 min), 30% methanol for RRN-7BrSOG (21 min), or 35% methanol for SRN-7BrSOG (23 min) in 50 mM ammonium formate, pH 5.7. RrN-7MeSOG: UV λmax (methanol) 259, 282 (sh) nm; 1H NMR (DMSO-d6) δ 9.38 (s, 1, H-8), 7.30 (d, 2, Ar, J ) 8.1 Hz), 7.18 (d, 2, Ar, J ) 7.9 Hz), 6.50 (dd, 1, R-CH, J ) 4.7 Hz), 5.82 (d, 1, H1′, J ) 4.7 Hz), 4.53 (t, 1, H2′, J ) 4.8 Hz), 4.12 (t, 1, H3′, J ) 4.6 Hz), 4.01-3.98 (m, 1, H4′), 3.76-3.51 (m, 4, β-CH1,2, H5′a,b), 2.31 (s, 3, CH3); +ve FAB-MS m/z 440.1529 ([M + Na]+; calcd for C19H23N5O6Na, 440.1545). SrN-7MeSOG: UV λmax (methanol) 259, 282 (sh) nm; 1H NMR (DMSO-d6) δ 9.41 (s, 1, H-8), 7.36 (d, 2, Ar, J ) 7.9 Hz), 7.21 (d, 2, Ar, J ) 8.0 Hz), 6.47 (dd, 1, R-CH, J ) 4.4 Hz), 5.93 (d, 1, H1′, J ) 4.1 Hz), 4.60 (t, 1, H2′, J ) 4.6 Hz), 4.33-4.27 (m, 2, H3′, β-CH1), 4.18-4.14 (m, 2, H4′, β-CH2), 3.95 (dd, 1, H5′a, J ) 2.2, 12.4 Hz), 3.78 (dd, 1, H5′b, J ) 1.8, 12.4 Hz), 2.31 (s, 3, CH3); +ve FAB-MS m/z 440.1586 ([M + Na]+; calcd for C19H23N5O6Na, 440.1546).

Mechanisms of Guanosine Aralkylation RrN-7BrSOG: UV λmax (methanol) 259, 282 (sh) nm; 1H NMR (DMSO-d6) δ 9.35 (s, 1, H-8), 7.59 (d, 2, Ar, J ) 8.5 Hz), 7.39 (d, 2, Ar, J ) 8.5 Hz), 6.45 (dd, 1, R-CH, JR,β2 ) 4.8 Hz, JR,β1 ) 8.0 Hz), 5.82 (d, 1, H1′, J ) 4.6 Hz), 4.54 (t, 1, H2′, J ) 4.6 Hz), 4.27 (dd, 1, β-CH1, Jβ1,R ) 8.1 Hz, Jβ1,β2 ) 11.8 Hz), 4.18 (t, 1, H3′, J ) 4.5 Hz), 4.04 (dd, 1, β-CH2, JR2,R ) 4.8 Hz, Jβ2,β1 ) 12.0 Hz), 4.00 (dd, 1, H4′, J ) 3.0, 7.2 Hz) 3.73 (dd, 1, H5′a, J ) 2.9, 12.2 Hz), 3.78 (d, 1, H5′b, J ) 12.2 Hz); +ve FABMS m/z 482.0674 ([M + H]+; calcd for C18H20N5O679Br, 482.0596). SrN-7BrSOG: UV λmax (methanol) 259, 282 (sh) nm; 1H NMR (DMSO-d6) δ 9.34 (s, 1, H-8), 7.58 (d, 2, Ar, J ) 8.5 Hz), 7.39 (d, 2, Ar, J ) 8.5 Hz), 6.49-6.46 (m, 1, R-CH), 5.82 (d, 1, H1′, J ) 4.8 Hz), 4.34 (t, 1, H2′, J ) 4.9 Hz), 4.30-4.22 (m, 1, β-CH1), 4.09 (dd, 1, H3′, J ) 5.0, 10.1 Hz), 4.05-4.00 (m, 2, β-CH2, H4′), 3.61-3.55 (m, 1, H5′a), 3.47-3.41 (m, 1, H5′b); +ve FAB-MS m/z 482.0664 ([M + H]+; calcd for C18H20N5O679Br, 482.0596). Synthesis of N2-[2-Hydroxy-1-(p-methylphenyl)ethyl]guanosines (RrN2MeSOG and SrN2MeSOG) and N2-[2-Hydroxy-1-(p-bromophenyl)ethyl]guanosines (RrN2BrSOG and SrN2BrSOG). Guanosine (150 mg, 0.5 mmol, 1 equiv) and sodium carbonate (200 mg, 2 mmol, 4 equiv) were dissolved in water (10 mL), and the solution was stirred at 90 °C for 3 h. Para-substituted styrene oxide (1 mmol, 2 equiv) was added, and the mixture was stirred at 90 °C for an additional 72 h. The reaction mixture was allowed to cool before addition of methanol (12 mL). The resulting solution was applied to a Sephadex LH-20 column (2.8 × 80 cm) and eluted at 1 mL/min with 40% (v/v) methanol. Absorption of the eluate was monitored continuously at 254 nm, and 8-mL fractions were collected. The product eluted in fractions 87-103 and was identified by comparison of the UV spectra under neutral, basic, and acidic conditions with those of literature spectra for N2-substituted guanosines (18), and those fractions were pooled. The product was further purified by reversed-phase HPLC eluting isocratically with 25% methanol for RRN2MeSOG (22 min) and SRN2MeSOG (28 min) and 30% methanol for RRN2BrSOG (25 min) and SRN2BrSOG (47 min) in water. RrN2MeSOG: UV λmax (methanol) 256, 276 (sh) nm; 1H NMR (DMSO-d6) δ 10.79 (br s, 1, NH, exchanges with D2O), 7.88 (s, 1, H-8), 7.34 (br s, 1, NH, exchanges with D2O), 7.20 (d, 2, Ar, J ) 7.9 Hz), 7.10 (d, 2, Ar, J ) 8.1 Hz), 5.63 (d, 1, H1′, J ) 5.5 Hz), 4.99-4.97 (m, 1, R-CH), 4.49-4.46 (m, 1, H2′), 4.394.37 (m, 1, H3′), 4.13-4.10 (m, 1, H4′), 3.74-3.70 (m, 1, β-CH1), 3.66-3.63 (m, 2, β-CH2, H5′a), 3.57-3.53 (m, 1, H5′b), 2.29 (s, 3, CH3); +ve FAB-MS m/z 440.1592 ([M + Na]+; calcd for C19H23N5O6Na, 440.1546). SrN2MeSOG: UV λmax (methanol) 256, 276 (sh) nm; 1H NMR (DMSO-d6) δ 10.85 (br s, 1, NH, exchanges with D2O), 7.88 (s, 1, H-8), 7.31 (br s, 1, NH, exchanges with D2O), 7.27 (d, 2, Ar, J ) 7.9 Hz), 7.15 (d, 2, Ar, J ) 8.1 Hz), 5.63 (d, 1, H1′, J ) 5.8 Hz), 4.96 (dd, 1, R-CH, J ) 5.4, 12.4 Hz), 4.39-4.38 (m, 1, H2′), 4.13-4.11 (m, 1, H3′), 4.13-4.10 (m, 1, H4′), 3.72 (dd, 1, β-CH1, J ) 4.4, 10.6 Hz), 3.66-3.63 (m, 2, H5′a, β-CH2), 3.57-3.53 (m, 1, H5′b), 2.29 (s, 3, CH3); +ve FAB-MS m/z 462.1369 ([M + Na2]+; calcd for C19H23N5O6Na2, 462.1366). RrN2BrSOG: UV λmax (methanol) 247, 278 (sh) nm; 1H NMR (DMSO-d6) δ 10.55 (br s, 1, NH, exchanges with D2O), 7.90 (s, 1, H-8), 7.52 (d, 2, Ar, J ) 8.4 Hz), 7.34 (d, 2, Ar, J ) 8.4 Hz), 7.11 (br s, 1, NH, exchanges with D2O), 5.63 (d, 1, H1′, J ) 5.4 Hz), 5.00 (dd, 1, R-CH, J ) 5.0, 9.1 Hz), 4.42 (dd, 1, H2′, J ) 5.8, 11.0 Hz), 4.04 (dd, 1, H3′, J ) 4.1, 8.4 Hz), 3.82 (dd, 1, H4′, J ) 4.3, 8.7 Hz), 3.76-3.73 (m, 1, β-CH1), 3.68-3.64 (m, 1, β-CH2), 3.53-3.49 (m, 1, H5′a), 3.34-3.29 (m, 1, H5′b); +ve FAB-MS m/z 504.0450 ([M + Na]+; calcd for C18H19N5O679BrNa, 504.0415). SrN2BrSOG: UV λmax (methanol) 247, 278 (sh) nm; 1H NMR (DMSO-d6) δ 10.62 (br s, 1, NH, exchanges with D2O), 7.87 (s, 1, H-8), 7.51 (d, 2, Ar, J ) 8.4 Hz), 7.32 (d, 2, Ar, J ) 8.3 Hz), 7.23 (br s, 1, NH, exchanges with D2O), 5.58 (d, 1, H1′, J ) 5.8 Hz), 4.95-4.93 (m, 1, R-CH), 4.33 (dd, 1, H2′, J ) 5.5, 10.5 Hz), 4.07 (dd, 1, H3′, J ) 4.1, 10.0 Hz), 3.84 (dd, 1, H4′, J ) 4.1, 8.0 Hz), 3.75-3.71 (m, 1, β-CH1), 3.64-3.59 (m, 2, β-CH2, H5′a),

Chem. Res. Toxicol., Vol. 11, No. 1, 1998 47 3.54-3.50 (m, 1, H5′b); +ve FAB-MS m/z 504.0460 ([M + Na]+; calcd for C18H19N5O679BrNa, 504.0415). Samples of the N2-[2-hydroxy-1-(p-substituted phenyl)ethyl]guanosines were converted to N2-[2-hydroxy-1-(p-substituted phenyl)ethyl]guanines by acid hydrolysis with 1 M HCl at 100 °C for 1 h. The products were purified by reversed-phase HPLC eluting with 35% methanol for N2-[2-hydroxy-1-(p-methylphenyl)ethyl]guanines (34 min) or 45% methanol for N2-[2-hydroxy1-(p-bromophenyl)ethyl]guanines (22 min) in water. Synthesis of Individual Diastereomers of N2-[2-Hydroxy-1-(p-methylphenyl)ethyl]guanines (RrN2MeSOGua and SrN2MeSOGua) and N2-[2-Hydroxy-1-(p-bromophenyl)ethyl]guanines (RrN2BrSOGua and SrN2BrSOGua). 2-Chloro-6-hydroxypurine (100 mg, 0.6 mmol, 1 equiv) and optically active para-substituted phenylglycinol (1.2 mmol, 2 equiv) were dissolved in DMSO (4 mL) and stirred at 90 °C for 164 h. The reaction mixture was concentrated in vacuo, suspended in methanol (10 mL), and filtered. The product was purified from the filtrate by reversed-phase HPLC eluting with 35% methanol for RRN2MeSOGua (34 min) and SRN2MeSOGua (34 min) or 45% methanol for RRN2BrSOGua (22 min) and SRN2BrSOGua (22 min) in water. RrN2MeSOGua: UV λmax (methanol) 249, 279 nm; 1H NMR (DMSO-d6) δ 10.51 (br s, 1, NH, exchanges with D2O), 7.60 (s, 1, H-8), 7.21 (d, 2, Ar, J ) 7.9 Hz), 7.12 (d, 2, Ar, J ) 8.0 Hz), 4.90 (m, 1, R-CH), 3.72-3.69 (m, 1, β-CH1), 3.60-3.57 (m, 1, β-CH2), 2.26 (s, 3, CH3); +ve FAB-MS m/z 286.1322 ([M + H]+; calcd for C14H16N5O2, 286.1304). SrN2MeSOGua: UV λmax (methanol) 249, 279 nm; 1H NMR (DMSO-d6) δ 7.64 (s, 1, H-8), 7.21 (d, 2, Ar, J ) 8.0 Hz), 7.12 (d, 2, Ar, J ) 7.9 Hz), 4.91-4.90 (m, 1, R-CH), 3.72-3.69 (m, 1, β-CH1), 3.61-3.57 (m, 1, β-CH2), 2.26 (s, 3, CH3); +ve EI-MS m/z 285.121 92 ([M]+; calcd for C14H15N5O2, 285.122 58). RrN2BrSOGua: UV λmax (methanol) 251, 276 nm; 1H NMR (DMSO-d6) δ 7.61 (s, 1, H-8), 7.50 (d, 2, Ar, J ) 8.4 Hz), 7.29 (d, 2, Ar, J ) 8.4 Hz), 4.90-4.89 (m, 1, R-CH), 3.73-3.70 (m, 1, β-CH1), 3.62-3.59 (m, 1, β-CH2); +ve FAB-MS m/z 350.0146 ([M + H]+; calcd for C13H13N5O279Br, 350.0126). SrN2BrSOGua: UV λmax (methanol) 251, 276 nm; 1H NMR (DMSO-d6) δ 7.64 (s, 1, H-8), 7.51 (d, 2, Ar, J ) 8.4 Hz), 7.30 (d, 2, Ar, J ) 8.4 Hz), 4.92-4.91 (m, 1, R-CH), 3.72-3.71 (m, 1, β-CH1), 3.62-3.60 (m, 1, β-CH2); +ve FAB-MS m/z 350.0209 ([M + H]+; calcd for C12H13N5O279Br, 350.0126). The N2-[2-hydroxy-1-(p-methylphenyl)ethyl]guanines (RRN2MeSOGua and SRN2MeSOGua) and N2-[2-hydroxy-1-(p-bromophenyl)ethyl]guanines (RRN2BrSOGua and SRN2BrSOGua) were used to assign the regio- and stereochemistry of the N2[2-hydroxy-1-(p-methylphenyl)ethyl]guanosines (RRN2MeSOG and SRN2MeSOG) and N2-[2-hydroxy-1-(p-bromophenyl)ethyl]guanosines (RRN2BrSOG and SRN2BrSOG) after the guanosines had been converted to guanines by acid hydrolysis by comparison of the 1H NMR and CD spectra. Synthesis of O6-[2-Hydroxy-1-(p-methylphenyl)ethyl]guanosines (RrO6MeSOG and SrO6MeSOG), O6-[2-Hydroxy2-(p-methylphenyl)ethyl]guanosines (RβO6MeSOG and SβO6MeSOG), O6-[2-Hydroxy-1-(p-bromophenyl)ethyl]guanosines (RrO6BrSOG and SrO6BrSOG), and O6-[2-Hydroxy-2-(p-bromophenyl)ethyl]guanosines (RβO6BrSOG and SβO6BrSOG). Optically active para-substituted phenylethanediol (4 mmol, 10 equiv) was melted at >100 °C, and sodium (23 mg, 1 mmol, 2.5 equiv) was added. After the sodium had dissolved, 2-amino-6-chloropurine riboside (120 mg, 0.4 mmol, 1 equiv) was added, and the reaction mixture was stirred at >100 °C for 96 h. The reaction mixture was allowed to cool and methanol (10 mL) added. The suspension was filtered, the filtrate was added to water (10 mL), and the resulting solution was applied to a Sephadex LH-20 column (2.8 × 80 cm) which was eluted at 1 mL/min with 50% (v/v) methanol. Absorption of the eluate was monitored continuously at 254 nm, and 8-mL fractions were collected. The products eluted in fractions 130155 and were identified by comparison of the UV spectra under neutral conditions with those of literature spectra for O6-

48 Chem. Res. Toxicol., Vol. 11, No. 1, 1998 substituted guanosines (27), and those fractions were pooled. The product was further purified by reversed-phase HPLC eluting isocratically with 35% methanol for RRO6MeSOG (51 min) and RβO6MeSOG (54 min), 40% methanol for SRO6MeSOG (26 min) and SβO6MeSOG (43 min), 35% methanol for RRO6BrSOG (79 min) and RβO6BrSOG (85 min), and 40% methanol for SRO6BrSOG (30 min) and SβO6BrSOG (46 min) in water. RrO6MeSOG: UV λmax (methanol) 248, 282 nm; 1H NMR (DMSO-d6) δ 8.10 (s, 1, H-8), 7.35 (d, 2, Ar, J ) 8.0 Hz), 7.16 (d, 2, Ar, J ) 7.9 Hz), 6.34-6.31 (m, 3, NH2, exchanges with D2O and R-CH), 5.76 (d, 1, H1′, J ) 6.0 Hz), 4.50-4.46 (m, 1, H2′), 4.09 (dd, 1, H3′, J ) 5.2, 10.4 Hz), 3.90-3.83 (m, 2, H4′, β-CH1), 3.73-3.68 (m, 1, β-CH2), 3.64-3.61 (m, 1, H5′a), 3.55-3.52 (m, 1, H5′b), 2.30 (s, 3, CH3); +ve FAB-MS m/z 440.1500 ([M + Na]+; calcd for C19H23N5O6Na, 440.1546). SrO6MeSOG: UV λmax (methanol) 248, 282 nm; 1H NMR (DMSO-d6) δ 8.10 (s, 1, H-8), 7.32 (d, 2, Ar, J ) 8.0 Hz), 7.13 (d, 2, Ar, J ) 7.9 Hz), 6.33-6.31 (m, 3, NH2, exchanges with D2O and R-CH), 5.76 (d, 1, H1′, J ) 6.0 Hz), 4.50-4.45 (m, 1, H2′), 4.11-4.08 (m, 1, H3′), 3.89-3.82 (m, 2, H4′, β-CH1), 3.73-3.68 (m, 1, β-CH2), 3.64-3.61 (m, 1, H5′a), 3.55-3.52 (m, 1, H5′b), 2.27 (s, 3, CH3); +ve FAB-MS m/z 440.1529 ([M + Na]+; calcd for C19H23N5O6Na, 440.1546). RβO6MeSOG: UV λmax (methanol) 248, 283 nm; 1H NMR (DMSO-d6) δ 8.10 (s, 1, H-8), 7.35 (d, 2, Ar, J ) 7.9 Hz), 7.16 (d, 2, Ar, J ) 8.0 Hz), 6.42 (br s, 2, NH2, exchanges with D2O), 5.78 (d, 1, H1′, J ) 6.0 Hz), 4.97 (dd, 1, R-CH, J ) 4.0, 7.8 Hz), 4.45-4.35 (m, 3, H2′, β-CH1,2), 4.09 (dd, 1, H3′, J ) 5.1, 10.3 Hz), 3.89 (dd, 1, H4′, J ) 3.9, 7.5 Hz), 3.64-3.61 (m, 1, H5′a), 3.553.51 (m, 1, H5′b), 2.30 (s, 3, CH3); +ve FAB-MS m/z 440.1523 ([M + Na]+; calcd for C19H23N5O6Na, 440.1546). SβO6MeSOG: UV λmax (methanol) 248, 283 nm; 1H NMR (DMSO-d6) δ 8.10 (s, 1, H-8), 7.35 (d, 2, Ar, J ) 8.1 Hz), 7.16 (d, 2, Ar, J ) 7.8 Hz), 6.42 (br s, 2, NH2, exchanges with D2O), 5.78 (d, 1, H1′, J ) 6.0 Hz), 4.97 (dd, 1, R-CH, J ) 3.9, 7.8 Hz), 4.44-4.35 (m, 3, H2′, β-CH1,2), 4.09 (dd, 1, H3′, J ) 5.1, 10.3 Hz), 3.89 (dd, 1, H4′, J ) 3.8, 7.4 Hz), 3.64-3.61 (m, 1, H5′a), 3.553.52 (m, 1, H5′b), 2.29 (s, 3, CH3); +ve FAB-MS m/z 440.1519 ([M + Na]+; calcd for C19H23N5O6Na, 440.1546). RrO6BrSOG: UV λmax (methanol) 248, 284 nm; 1H NMR (DMSO-d6) δ 8.10 (s, 1, H-8), 7.55 (d, 2, Ar, J ) 8.3 Hz), 7.44 (d, 2, Ar, J ) 8.4 Hz), 6.44 (s, 2, NH2, exchanges with D2O), 6.29 (dd, 1, R-CH, J ) 4.4, 7.1 Hz), 5.78 (d, 1, H1′, J ) 6.0 Hz), 4.50 (t, 1, H2′), 4.10-4.09 (m, 1, H3′), 3.90-3.87 (m, 1, H4′), 3.863.84 (m, 1, β-CH1), 3.76-3.72 (m, 1, β-CH2), 3.64-3.61 (m, 1, H5′a), 3.55-3.50 (m, 1, H5′b); +ve FAB-MS m/z 482.0610 ([M + H]+; calcd for C18H21N5O679Br, 482.0596). SrO6BrSOG: UV λmax (methanol) 248, 284 nm; 1H NMR (DMSO-d6) δ 8.12 (s, 1, H-8), 7.54 (d, 2, Ar, J ) 8.5 Hz), 7.39 (d, 2, Ar, J ) 8.5 Hz), 6.36 (s, 2, NH2, exchanges with D2O), 6.29 (dd, 1, R-CH, J ) 4.6, 7.2 Hz), 5.76 (d, 1, H1′, J ) 6.0 Hz), 4.484.45 (m, 1, H2′), 4.10-4.08 (m, 1, H3′), 3.89-3.83 (m, 2, H4′, β-CH1), 3.76-3.72 (m, 1, β-CH2), 3.64-3.60 (m, 1, H5′a), 3.543.50 (m, 1, H5′b); +ve FAB-MS m/z 482.0635 ([M + H]+; calcd for C18H21N5O679Br, 482.0596). RβO6BrSOG: UV λmax (methanol) 248, 284 nm; 1H NMR (DMSO-d6) δ 8.12 (s, 1, H-8), 7.53 (d, 2, Ar, J ) 8.3 Hz), 7.39 (d, 2, Ar, J ) 8.4 Hz), 6.38 (s, 2, NH2, exchanges with D2O), 5.77 (d, 1, H1′, J ) 6.0 Hz), 5.02-5.01 (m, 1, R-CH), 4.46-4.43 (m, 3, H2′, β-CH1,2), 4.09 (dd, 1, H3′, J ) 5.0, 10.0 Hz), 3.88 (dd, H4′, J ) 3.8, 7.5 Hz), 3.55-3.51 (m, 1, H5′a), 3.50-3.46 (m, 1, H5′b); +ve FAB-MS m/z 482.0633 ([M + H]+; calcd for C18H21N5O679Br, 482.0596). SβO6BrSOG: UV λmax (methanol) 248, 284 nm; 1H NMR (DMSO-d6) δ 8.10 (s, 1, H-8), 7.55 (d, 2, Ar, J ) 8.4 Hz), 7.44 (d, 2, Ar, J ) 8.4 Hz), 6.43 (s, 2, NH2, exchanges with D2O), 5.78 (d, 1, H1′, J ) 6.0 Hz), 5.01 (dd, 1, R-CH, J ) 3.5, 7.1 Hz), 4.474.43 (m, 3, H2′, β-CH1,2), 4.09 (dd, 1, H3′, J ) 5.2, 10.4 Hz), 3.903.88 (dd, H4′), 3.65-3.61 (m, 1, H5′a), 3.56-3.51 (m, 1, H5′b); +ve FAB-MS m/z 482.0630 ([M + H]+; calcd for C18H21N5O679Br, 482.0596).

Barlow and Dipple Aralkylation of [14C]Guanosine in Aqueous Solution by Optically Active Para-Substituted Styrene Oxides. Optically active para-substituted styrene oxide (5 mL) was added to a solution of [14C]guanosine (1 mCi) in 50 mM Tris‚HCl buffer, pH 7.0 (1 mL). The reaction mixture was stirred in the dark at 37 °C for 24 h. Aliquots (250 mL) were used to dissolve a mixture containing guanosine, the four 7-substituted isomers, the two N2-substituted isomers, and the four O6-substituted isomers of the respective para-substituted styrene oxide adducts. The mixture was separated by reversed-phase HPLC eluting at 1 mL/min using the following solvents: A, 50 mM ammonium formate, pH 5.7; B, methanol. The p-methylstyrene oxideguanosine adducts were resolved using the following gradient of B in A: 0 min, 10%; 10 min, 10%; 60 min, 20%; 90 min, 20%; 130 min, 24%; 220 min, 33%. Typical retention times for the individual products were as follows: guanosine, 9 min; SβN7MeSOG, 46 min; RRN-7MeSOG, 57 min; RβN-7MeSOG, 74 min; RRN2MeSOG, 78 min; SRN-7MeSOG, 108 min; SRN2MeSOG, 117 min; SRO6MeSOG, 155 min; SβO6MeSOG, 180 min; RRO6MeSOG, 183 min; RβO6MeSOG, 186 min. The p-bromostyrene oxide-guanosine adducts were resolved using the following gradient of B in A: 0 min, 20%; 80 min, 24%; 100 min, 32%; 240 min, 44%. Typical retention times for the individual products were as follows: guanosine, 5 min; SβN7BrSOG, 92 min; RRN-7BrSOG, 108 min; RβN-7BrSOG, 122 min; RRN2BrSOG, 126 min; SRN-7BrSOG, 144 min; SRN2BrSOG, 153 min; SRO6BrSOG, 188 min; SβO6BrSOG, 212 min; RRO6BrSOG, 215 min; RβO6BrSOG, 218 min. Fractions (1 mL) were collected throughout the elution, and the radioactivity in each fraction was determined by liquid scintillation counting.

Results Investigation of the effects of para-substituents on the yields and configurations of various products formed in reactions between guanosine and p-substituted styrene oxides required the preparation of the anticipated products as markers, as well as the establishment of their stereochemical configurations. Previous studies (18) with unsubstituted styrene oxide indicated that markers for five diastereomeric pairs of guanosine products would be necessary (Chart 1). Oxirane ring opening at the R- or β-carbons of each styrene oxide enantiomer by the ring nitrogen at the guanosine 7-position can give rise to two diastereomeric pairs, i.e., R- or SβN-7XSOG and R- or SRN-7XSOG (Chart 1). Epoxide ring opening at the R-carbon by the exocyclic N2- and O6-positions of guanosine gives rise to two more pairs (R- and SRN2XSOG and R- and SRO6XSOG, respectively), and equilibration of the RO6-substituted products with βO6-substituted products (27) requires preparation of R- and SβO6XSOG. Thus the required p-bromo- and p-methyl-substituted markers were prepared using strategies that allowed the stereochemical outcome of the reactions to be determined. Racemic p-bromo- and p-methylstyrene oxides were prepared by established procedures (28). The synthesis of optically active p-substituted epoxides employed asymmetric dihydroxylation of p-substituted styrenes (29) to produce optically active diols with an enantiomeric excess of >95%. The epoxides were prepared from these diols by tosylation of the primary hydroxyl group followed by base-assisted ring closure with elimination of toluenesulfonic acid (28). The 7-substituted guanosine markers were prepared from guanosine and optically active epoxides (Scheme 1). Reactions in glacial acetic acid or 50% ethanol were known to favor attack by the guanosine ring nitrogen at either the R- (benzylic) or β-position of the epoxide, respectively (18). Reactions in glacial acetic acid pro-

Mechanisms of Guanosine Aralkylation Chart 1. Diastereomeric Pairs of Products Anticipated from Reaction of p-Methyl- and p-Bromostyrene Oxides with Guanosine under Neutral Aqueous Conditions

Scheme 1. Synthesis of 7-Substituted Styrene Oxide-Guanosine Adducts

Chem. Res. Toxicol., Vol. 11, No. 1, 1998 49 Scheme 2. Synthesis of O6-Substituted Styrene Oxide-Guanosine Adducts

R- and β-carbons in 1H NMR spectra. The positive charge on the ring nitrogen resulted in a downfield shift of the adjacent alkyl protons, such that the proton on the R-carbon shifted from around 5.1 ppm in the β-substituted compounds to around 6.5 ppm in the R-substituted compounds. This change was also apparent, but less dramatic, for the protons on the β-carbon that typically shifted from between 4 and 3.5 ppm in the R-substituted compounds to between 4.8 and 4.2 ppm in the β-substituted compounds. The finding that optically active R-substituted products were formed in each case is consistent with a bimolecular reaction mechanism which gives rise to a product with inversion of stereochemistry at the R-carbon. The alternative explanation that substitution at the chiral center occurred with complete retention of configuration seems unlikely, and the R-substituted products were therefore assigned the inverse configuration of the starting epoxide. Since formation of the β-substituted products does not involve any bond-breaking or bond-making steps at the chiral carbon, the products at this position must retain the stereochemistry of the starting epoxide.

duced optically active R-substituted products from both the p-methyl- and p-bromostyrene oxides. In 50% ethanol, however, mixtures of R- and β-products were found although each product constituted a single diastereomer. β-Substituted products predominated, especially when p-bromostyrene oxide was the substrate. The R- and β-substituted regioisomers were readily distinguished by the chemical shifts of the protons at the

Synthesis of the O6-substituted markers was achieved by displacement of chloride from 2-amino-6-chloropurine riboside with alkoxides prepared from addition of sodium to the optically active p-substituted diols. As formation of the alkoxides and the subsequent reaction with the chloropurine riboside do not involve any changes in bonding at the chiral carbon of the diols, the configuration of the starting diol is conferred upon the products. This method produced markers of the RO6- and βO6-substituted products that would have arisen from aralkylation of guanosine by p-substituted styrene oxides (Scheme 2). It has been reported that these O6-substituted guanosine-styrene oxide derivatives isomerize in a basecatalyzed rearrangement (27), and as anticipated, the analogous reaction products in this study also behaved similarly. When a single isomer was treated with base, an equilibrium mixture of two isomers was produced. The

50 Chem. Res. Toxicol., Vol. 11, No. 1, 1998

Barlow and Dipple

Scheme 3. Synthesis of N2-Substituted Styrene Oxide-Guanosine and N2-Substituted Styrene Oxide-Guanine Adducts

equilibrium ratio of R:β O6-substituted products was found to be ∼1:1 for the p-methyl-substituted compounds and ∼1:2 for the p-bromo-substituted compounds. After separation by HPLC, the R- and β-substituted forms could easily be distinguished by examination of the chemical shifts of the protons at the R- and β-carbons in the 1H NMR spectra. These protons shifted in a manner similar to that seen for the 7-substituted guanosines. The electron-withdrawing effect of the oxygen conjugated with the purine ring shifted the proton at the R-position from around 5.0 ppm in the β-substituted compounds to around 6.3 ppm in the R-substituted compounds. Similarly, the protons on the β-carbon were shifted from around 3.8 ppm in the R-substituted compounds to around 4.4 ppm in the β-substituted compounds. N2-Substituted markers were prepared by reactions of racemic epoxides with guanosine under alkaline conditions that are known to increase the reactivity of the exocyclic nitrogen (16) (Scheme 3). The reactions with the racemic epoxides gave only two N2-substituted guanosines which, after purification and separation by HPLC, were identified as diastereomers by their 1H NMR spectra. The mechanism by which ionized guanosine undergoes alkylation at the N2-position is unclear, and therefore the regio- and stereochemistry of these products was not immediately obvious. Independent structure-defining syntheses of the corresponding RN2-substituted guanine enantiomers were achieved, however, by the displacement of chloride from 2-chloro-6-hydroxypurine by each optically active psubstituted phenylglycinol (Scheme 3). The necessary glycinols were synthesized via a route developed by Chang and Sharpless (30) in which optically active diols were converted to cyclic carbonates and subsequently ring-opened at the R-carbon, with inversion of stereochemistry, by azide. Reduction then gave a glycinol of known configuration. The CD spectra (Figure 1) and 1H NMR spectra of the guanine reference compounds prepared in this fashion were compared to those of N2substituted guanines prepared from the nucleosides by acid hydrolysis. This comparison allowed the marker nucleoside products to be identified as R-substituted compounds and their configurations to be assigned.

Thus, the R-diastereomer was identified as the product which eluted earlier than the S-diastereomer in the HPLC system used here. Once all the markers were prepared and characterized, HPLC conditions were developed to separate the markers and their order of elution was established (Figure 2). Separation of radioactive products [illustrated for (S)-pmethylstyrene oxide-guanosine products in Figure 2C] allowed the product stereochemistry and yields to be evaluated in reactions of optically active p-substituted styrene oxides and [14C]guanosine in aqueous solution. The results from triplicate analyses with both the pmethyl- and the p-bromostyrene oxides are summarized in Table 1 [previously published data for reactions with styrene oxide are included to aid comparison (18)]. The overall yield of products from both epoxides was low. The p-bromostyrene oxides converted approximately 4% of the guanosine to products, whereas the p-methylstyrene oxides reacted more extensively, converting approximately 8% of the guanosine to products. Examination of the products formed in each reaction indicated that the para-substituent had a marked effect not only on the product yields but also on the distribution of products at the 7-, N2-, and O6-positions of guanosine. N2-Substituted adducts accounted for around 60% of the total products formed in reactions involving the pmethylstyrene oxides with the remainder mostly being 7-substituted compounds. This selectivity was reversed in reactions involving the p-bromostyrene oxides where nearly 85% of the total products formed were 7-substituted compounds and the remainder almost all N2substituted adducts. Neither the p-methyl- nor the p-bromo-substituted epoxides formed substantial quantities of O6-substituted products. The chirality of the epoxide did not display any appreciable effect on either the product yields or the distributions. The stereochemical differences in products at the 7-, N2-, and O6-positions indicated that these sites reacted with the epoxide in different ways. Reactions at the 7-position for p-substituted styrene oxides gave products with predominantly inverted stereochemistry. In contrast, reactions at the N2- and O6-positions gave products of both inverted and retained stereochemistry, with the

Mechanisms of Guanosine Aralkylation

Figure 1. CD spectra of N2-[2-hydroxy-1-(para-substituted phenyl)ethyl]guanines prepared by acid hydrolysis of N2-[2hydroxy-1-(para-substituted phenyl)ethyl]guanosines overlayed on the CD spectra of N2-[2-hydroxy-1-(para-substituted phenyl)ethyl]guanines prepared by displacement of chloride from 2-chloro-6-hydroxypurine with optically active para-substituted phenylglycinols: panel A, N2-[2-hydroxy-1-(p-methylphenyl)ethyl]guanines; panel B, N2-[2-hydroxy-1-(p-bromophenyl)ethyl]guanines.

O6-substituted products undergoing more extensive racemization than the N2-substituted products. The para-substituents of the epoxides influenced the site of ring opening by the 7-position. The p-methylstyrene oxide was opened by the ring nitrogen to similar extents at the R- and β-carbons, whereas the p-bromo substituent seemed to direct attack preferentially to the β-carbon (almost 4-fold). All the p-substituted epoxides were ring-opened by the exocyclic sites only at the R-carbon. However, the para substituents did influence the stereochemistry of products at the N2- and O6positions. The p-methyl substituent increased the proportion of retained to inverted stereochemistry in the products relative to the p-bromo substituent. Complete racemization was found in the O6-substituted products from the reactions with p-methylstyrene oxides, but a ratio of between 1:3 and 1:2 retained-to-inverted stereochemistry was found in products at the N2-position. This contrasted with that found in reactions involving the p-bromostyrene oxides where the ratio of retained-to-

Chem. Res. Toxicol., Vol. 11, No. 1, 1998 51

Figure 2. HPLC separation of para-substituted styrene oxideguanosine adducts: panel A, separation of p-bromostyrene oxide-guanosine adducts; panel B, separation of p-methylstyrene oxide-guanosine adducts. Peaks: 1, guanosine; 2, SβN7XSOG; 3, RRN-7XSOG; 4, RβN-7XSOG; 5, RRN2XSOG; 6, SRN7XSOG; 7, SRN2XSOG; 8, SRO6XSOG; 9, SβO6XSOG; 10, RRO6XSOG; 11, RβO6XSOG. Panel C: Representative radiochromatogram separating the mixture of products formed in a reaction between [14C]guanosine and (S)-p-methylstyrene oxide.

inverted stereochemistry was 1:3 in O6-substituted products and between 1:5 and 1:8 in N2-substituted products.

Discussion In this study, 10 p-methyl- and 10 p-bromostyrene oxide-guanosine markers were prepared and characterized spectroscopically. Stereochemical assignments of O6substituted diastereomeric pairs were straightforward since each was made from an optically active diol in a reaction that did not involve the chiral center of the epoxide. Assignment of the N2-substituted diastereomers was more complex since these were derived from racemic epoxides. Assignment was achieved, however, by converting these N2-substituted nucleosides, by acid hydrolysis, to N2-substituted guanines of known configuration. Reactions of guanosine with optically active epoxides gave optically active 7-substituted products that were presumed to have arisen with inversion of configuration. With the aid of these markers, the effect of the para substituent on configurations and relative proportions of products formed in reactions of guanosine with p-substituted styrene oxides in neutral aqueous media could be readily determined (Table 1).

52 Chem. Res. Toxicol., Vol. 11, No. 1, 1998

Barlow and Dipple

Table 1. Proportion of Total Products Formed from Reactions of Optically Active Para-Substituted Styrene Oxides and Guanosine Given as Ratios of Stereoisomers (S/R)a epoxide

βN-7XSOGb

RN-7XSOGb

RN2XSOGb

O6(R+β)XSOGc

(R)-MeSO