Deoxyguanosine and Solvent on Its Aralkylation by Benzyl Bromide

In striking contrast, the reaction of the anion of 2'-deoxyguanosine with benzyl bromide in TFE produced N2-benzyl-2'-deoxyguanosine in significant yi...
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Chem. Res. Toxicol. 1998, 11, 696-702

Effect of Ionic State of 2′-Deoxyguanosine and Solvent on Its Aralkylation by Benzyl Bromide Ki-Young Moon and Robert C. Moschel* Chemistry of Carcinogenesis Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, P.O. Box B, Frederick, Maryland 21702 Received January 15, 1998

To extend studies of the aralkylation of nucleic acid components under a variety of solvent conditions, we determined product distributions from the reactions of benzyl bromide with 2′-deoxyguanosine and the anion of 2′-deoxyguanosine in 2,2,2-trifluoroethanol (TFE) and compared these distributions with those from the reaction of the anion with benzyl bromide in N,N-dimethylacetamide (DMA). 7-Benzylguanine was the only benzylated product detected in the reaction with the neutral nucleoside in TFE. In striking contrast, the reaction of the anion of 2′-deoxyguanosine with benzyl bromide in TFE produced N2-benzyl-2′-deoxyguanosine in significant yield and with high selectivity. The reaction of the anion of 2′-deoxyguanosine with benzyl bromide in DMA produced products derived only from reaction at the 1- and/or 7-position of the nucleoside. The weakly nucleophilic but protic polar solvent TFE and the iminolate tautomeric form of the 2′-deoxyguanosine anion appear to be essential for benzylation at the exocyclic N2-position.

Introduction Several studies of the reaction of benzylic electrophiles with guanine and adenine nucleosides have been carried out in efforts to explain the site selectivity exhibited by a wide variety of chemical carcinogens in their reactions with DNA (1-9). These studies showed that product distributions were markedly dependent on the reaction solvent. For example, in dipolar aprotic solvents [e.g., N,N-dimethylacetamide (DMA)1 or N,N-dimethylformamide] reaction occurred primarily on ring nitrogen sites (i.e., the 7-position on guanine nucleosides and the 1-position in adenine nucleosides) (1, 2). In solvents of greater ionizing power (e.g., aqueous organic solvent mixtures or H2O) reaction at exocyclic sites (i.e., the N2and O6-positions on guanine nucleosides and the N6position on adenine nucleosides) occurred in addition to reaction at ring nitrogen sites (2). However, the preferred sites of benzylation of these nucleosides in aqueous solvents depended on the benzylating agents’ leaving group and/or para-substituent (1-6 ). Additionally, with guanine nucleosides, sites of reaction also depended on whether reaction occurred with the anionic or neutral form of the guanine residue (4-7). Surprisingly, benzylation at the N2-position on guanine nucleosides was shown to increase substantially in aqueous reactions involving the nucleoside anion compared to the neutral nucleoside even though the exocyclic amino group is not formally charged in the anion (4-7). Still, however, the total yield of the N2-substituted products was fairly low due to the high nucleophilicity of the aqueous solvents which led to preferential hydrolysis of the benzylating agents rather than to reaction with the nucleoside. To test whether solvents of high ionizing power but low nucleophilicity such as 2,2,2-trifluoroethanol (TFE) 1 Abbreviations: DMA, N,N-dimethylacetamide; TFE, 2,2,2-trifluoroethanol.

(10) could be used to produce improved yields for an N2substituted 2′-deoxyguanosine product, we examined the product distributions for reactions between 7-(bromomethyl)benz[a]anthracene and either the neutral or anionic form of 2′-deoxyguanosine in TFE (8). These reactions produced substantial amounts of N2-(benz[a]anthracen7-ylmethyl)-2′-deoxyguanosine which is formed in only very low yield under aqueous solvent conditions. These observations prompted us to examine reactions between the neutral (1) or anionic form (1-) of 2′-deoxyguanosine (Scheme 1) and the simplest benzylating agent, benzyl bromide in TFE and DMA, to determine which system might afford the highest yield of N2-benzyl-2′-deoxyguanosine. We also compared these product distributions with those obtained under similar conditions but in the presence of silver nitrate. Our intent was to determine if silver coordination with the departing bromide leaving group could increase the ionic character of the benzylating agent and alter product distributions observed in the absence of silver ions. Silver catalysis has been used previously to prepare O4-isopropyl- (11) and O4-benzylthymidine (12) in reactions between thymidine anion and isopropyl or benzyl bromide, respectively. In this report we describe product distributions resulting from a variety of reaction conditions and demonstrate that the reaction of 1- with benzyl bromide in TFE provides N2benzyl-2′-deoxyguanosine in high yield. The factors that contribute to the efficient formation of this product appear to be the polar but low nucleophilic character of the TFE solvent and the iminolate tautomeric form of 1 -.

Experimental Section Ultraviolet absorption spectra were determined on a Milton Roy SLM-AMINCO 3000 diode array spectrophotometer. 1H NMR spectra were recorded on a Varian VXR 500S spectrometer equipped with Sun 4/110 data stations or a Varian XL200

S0893-228x(98)00012-5 CCC: $15.00 © 1998 American Chemical Society Published on Web 05/13/1998

Benzylation of 2′-Deoxyguanosine

Chem. Res. Toxicol., Vol. 11, No. 6, 1998 697 Scheme 1

instrument interfaced to an Advanced data system. Samples were dissolved in dimethyl-d6 sulfoxide, and chemical shifts are reported as δ values (ppm) downfield from tetramethylsilane as an internal standard. Positive ion (+ve) fast atom bombardment (FAB) and high-resolution electron impact (HREI) MS were obtained with a reversed-geometry VG Micromass ZAB2F spectrometer interfaced to a VG 2035 data system. A mixture of dithiothreitol and dithioerythritol (1:1) was used as the FAB matrix. Elemental analyses were performed by Galbraith Laboratories, Inc., (Knoxville, TN). Thin-layer chromatography (TLC) was performed on Kodak silica gel (Eastman Kodak Co., Rochester, NY) adsorbent with fluorescent indicator (0.2-mm layer thickness, 2.5 × 7.5 cm), and visualization was accomplished by UV illumination. Sephadex LH-20 was purchased from Pharmacia, Biotechnology AB (Uppsala, Sweden). 2′-Deoxyguanosine was purchased from Sigma Chemical Co. (St. Louis, MO). O6-Benzyl-2′-deoxyguanosine (7) (13) was prepared as described previously (14). All other chemicals were from Aldrich Chemical Co. (Milwaukee, WI). Benzylation of 2′-Deoxyguanosine (1) in TFE. To a stirred suspension of 2′-deoxyguanosine (0.65 g, 2.43 mmol) in 13 mL of TFE was added benzyl bromide (0.28 mL, 2.32 mmol) over a 2-min period. The reaction mixture was stirred for 72 h at room temperature. The resulting white suspension was diluted with 120 mL of MeOH/H2O (1:9), was stirred for 30 min at 55 °C to dissolve nearly all suspended solids, and was allowed to cool to room temperature. The precipitated solid that formed was filtered. Crystallization of the filtered solid from 1 N HCl afforded the hydrochloride salt of 7-benzylguanine (2) (15, 16) (145 mg, 21%): UV pH 1 λmax 252 nm, 272 nm (sh), pH 6.9 244

nm (sh), λmin 262 nm, λmax 285 nm, pH 13 λmin 259 nm, λmax 282 nm; 1H NMR (of the hydrochloride salt) δ 12.10 (1H, s, 1-NH, exchanges with D2O), 8.95 (1H, s, 8-H), 7.64 (2H, s, NH2, exchange with D2O), 7.32-7.43 (5H, m, C6H5), 5.53 (2H, s, C6H5CH2). The pH of the filtrate was increased to 6.5 by the addition of 0.1 N NaOH which led to the precipitation of guanine (70 mg, 19%) which was collected by filtration. The identification of guanine was confirmed by comparison of its UV absorption characteristics with those of an authentic sample. The filtrate was loaded on a Sephadex LH-20 column (2.8 × 78 cm) eluted with MeOH/H2O (1:9) at 1 mL/min. UV absorption was continuously monitored at 280 nm and fractions (10 mL) were collected. Unreacted 2′-deoxyguanosine eluted in fractions 3137. Fractions 38-61 were pooled and evaporated to dryness. The resulting solid was dissolved in 25 mL of MeOH/H2O (5: 95) and was loaded on a 2.8- × 78-cm Sephadex LH-20 column eluted with MeOH/H2O (5:95) at 0.8 mL/min. Under these conditions, unreacted 2′-deoxyguanosine eluted in fractions 5468. Guanine (6 mg) eluted in fractions 74-88. No other products were observed. Benzylation of 2′-Deoxyguanosine (1) in TFE Containing AgNO3. To a stirred suspension of 2′-deoxyguanosine (0.65 g, 2.43 mmol) in 13 mL of TFE was added ground AgNO3 (0.4 g, 2.4 mmol) followed by the dropwise addition of benzyl bromide (0.28 mL, 2.3 mmol) over a 2-min period. The reaction mixture was stirred at room temperature for 72 h. The resulting purple suspension was diluted with 120 mL of MeOH/H2O (1:9) and was stirred for 30 min at 55 °C to dissolve suspended solids other than AgBr. The suspended AgBr was filtered (405 mg).

698 Chem. Res. Toxicol., Vol. 11, No. 6, 1998 On cooling to room temperature crude 7-benzylguanine (2) precipitated. This was filtered and crystallized from 1 N HCl to afford the hydrochloride salt of 7-benzylguanine (75 mg, 11%). Increasing the pH of the filtrate to pH 6.0 with 0.1 N NaOH caused precipitation of guanine (160 mg, 43%) which was collected by filtration. Chromatography of the filtrate on a Sephadex LH-20 column (2.8 × 78 cm) eluted with MeOH/H2O (1:9) at 0.8 mL/min showed that the filtrate contained only unreacted 2′-deoxyguanosine which eluted in fractions (10 mL) 44-65 and a trace of guanine in fractions 71-81. Benzylation of 2′-Deoxyguanosine (1) in TFE Containing NaOH. To a stirred suspension of 2′-deoxyguanosine (1.3 g, 4.87 mmol) in 25 mL of TFE was added 0.23 g of NaOH (5.75 mmol) at room temperature. After stirring for 30 min, benzyl bromide (0.55 mL, 4.63 mmol) was added dropwise over 3 min. The reaction mixture was stirred at room temperature for 72 h. The resulting white suspension was evaporated in vacuo to give a crude solid. This solid was dissolved in 150 mL of MeOH/ H2O/NH4OH (50:50:3) and was loaded on a Sephadex LH-20 column (2.8 × 78 cm) eluted with MeOH/H2O/NH4OH (50:50:3) at 1 mL/min. UV absorption was monitored continuously at 280 nm, and fractions (10 mL) were collected. Fractions 30-62 contained a mixture of 2′-deoxyguanosine, 3-benzylguanine, N2benzyl-2′-deoxyguanosine, and N2,N2-dibenzyl-2′-deoxyguanosine. These fractions were pooled, evaporated, and rechromatographed (see below). 1,N2-Dibenzyl-2′-deoxyguanosine (6) eluted in MeOH/H2O/ NH4OH (50:50:3) fractions 75-91: UV pH 1 λmin 237 nm, λmax 263 nm, 283 nm (sh), pH 6.9 λmax 258 nm, 279 nm (sh), pH 13 λmax 259 nm, 279 nm (sh); 1H NMR δ 7.94 (1H, s, 8-H), 7.65 (1H, t, JCHNH ) 5.4 Hz, C6H5CH2NH, exchanges with D2O), 7.00-7.42 (10H, m, 2C6H5), 6.11 (1H, t, J ) 6.6 Hz, H-1′), 5.34 (2H, br s, 1-C6H5CH2N) 5.26 (1H, br s, 3′-OH, exchanges with D2O), 4.87 (1H, br s, 5′-OH, exchanges with D2O), 4.51 (2H, d, JCHNH ) 5.2 Hz, C6H5CH2NH, changes to a singlet on addition of D2O), 4.29 (1H, br s, H-3′), 3.79 (1H, m, H-4′), 3.48 (2H, m, H-5′), 2.53 (1H, m, H-2′b), 2.11 (1H, m, H-2′a); EI-MS m/z 447 ([C24H25N5O4]+), 331 ([C19H16N5O]+); HREI-MS calcd for C24H25N5O4 447.1906, found 447.1956. O6-Benzyl-2′-deoxyguanosine (7) (16) eluted in fractions 98132. N2,O6-Dibenzyl-2′-deoxyguanosine (5) eluted in fractions 181-220: TLC Rf ) 0.83 in CHCl3: UV pH 1 λmax 250 nm, λmin 272 nm, λmax 300 nm, pH 6.9 λmax 254 nm, λmin 271 nm, λmax 290 nm, pH 13 λmax 254 nm, λmin 271 nm, λmax 290 nm; 1H NMR δ 8.07 (1H, s, 8-H), 7.61 (1H, t, JCHNH ) 6.2 Hz, C6H5CH2NH, exchanges with D2O), 7.14-7.45 (10H, m, 2 C6H5), 6.21 (1H, t, J ) 6.5 Hz, H-1′), 5.48 (2H, s, C6H5CH2O), 5.31 (1H, s, OH-3′, exchanges with D2O), 4.93 (1H, br s, OH-5′, exchanges with D2O), 4.51 (2H, d, JCHNH ) 6.4 Hz, C6H5CH2NH, changes to a singlet on addition of D2O), 4.35 (1H, br s, H-3′), 3.81 (1H, m, H-4′), 3.52 (2H, m, H-5′), 2.63 (1H, m, H-2′b), 2.18 (1H, m, H-2′a); EI-MS m/z 447 ([C24H25N5O4]+), 331 ([C19H16N5O]+); HREI-MS calcd for C24H25N5O4 447.1906, found 447.1892. The white solid recovered from evaporation of MeOH/H2O/ NH4OH (50:50:3) fractions 30-62 (see above) was dissolved in 40 mL of MeOH/H2O (3:7) and was loaded on a 2.8- × 78-cm Sephadex LH-20 column eluted with MeOH/H2O (3:7) at 1 mL/ min. Under these chromatographic conditions, unreacted 2′deoxyguanosine (1) eluted in fractions 37-55. 3-Benzylguanine (8) eluted in fractions 57-75: UV pH 1 λmin 235 nm, λmax 263 nm, pH 6.9 234 nm (sh), λmin 249 nm, λmax 270 nm, pH 13 λmin 248 nm, λmax 274 nm; 1H NMR δ 7.84 (s, 1H, 8-H), 7.20-7.38 (m, 5H, C6H5), 6.92 (s, 2H, NH2, exchange with D2O), 5.34 (s, 2H, C6H5CH2); FAB+-MS m/z 242 ([C12H11N5O + H]+), 152 ([C5H5N5O + H]+). N2-Benzyl-2′-deoxyguanosine (3) eluted in fractions 83-111: mp 219-220 °C; TLC Rf ) 0.15 in MeOH/CHCl3 (2:8): UV pH 1 λmin 234 nm ( ) 0.468 × 104), λmax 261 nm ( ) 1.31 × 104), 281 nm (sh) ( ) 0.825 × 104), pH 6.9 λmin 229 nm ( ) 0.513 × 104), λmax 256 nm ( ) 1.315 × 104), 276 nm (sh) ( ) 0.950 × 104), pH 13 λmin 239 nm ( ) 0.673 × 104), λmax 261 nm ( )

Moon and Moschel 1.168 × 104); 1H NMR δ 10.61 (1H, s, 1-H, exchanges with D2O), 7.90 (1H, s, 8-H), 7.20-7.40 (5H, m, C6H5), 6.88 (1H, t, JCHNH ) 5.52 Hz, C6H5CH2NH, exchanges with D2O), 6.14 (1H, t, J ) 6.94 Hz, H-1′), 5.26 (1H, br s, OH-3′, exchanges with D2O), 4.86 (1H, br s, OH-5′, exchanges with D2O), 4.50 (2H, d, JCHNH ) 5.67 Hz, C6H5CH2NH changes to a singlet on addition of D2O), 4.33 (1H, br s, H-3′), 3.80 (1H, m, H-4′), 3.50 (2H, m, H-5′), 2.57 (1H, m, H-2′b), 2.16 (1H, m, H-2′a); FAB+-MS m/z 358 ([C17H19N5O4 + H]+), 242 ([C12H11N5O + H]+). Anal. Calcd for C17H19N5O4: C, H, N. N2,N2-Dibenzyl-2′-deoxyguanosine (4) eluted in fractions 115-170: TLC Rf ) 0.59 in MeOH/CHCl3 (2:8): UV pH 1 λmin 237 nm, λmax 266 nm, 289 nm (sh), pH 6.9 λmin 231 nm, λmax 263 nm, 281 nm (sh), pH 13 λmin 243 nm, λmax 268 nm; 1H NMR δ 10.98 (1H, s, 1-H, exchanges with D2O), 7.96 (1H, s, 8-H), 7.237.38 (10H, m, 2C6H5), 6.16 (1H, t, J ) 6.9 Hz, H-1′), 5.26 (1H, br s, OH-3′, exchanges with D2O), 4.88 (1H, br s, OH-5′, exchanges with D2O), 4.86, 4.83, 4.81, and 4.78 (4H, ab, Jab ) 16.5 Hz, 2C6H5CH2), 4.29 (1H, br s, H-3′), 3.79 (1H, m, H-4′), 3.49 (2H, m, H-5′), 2.55 (1H, m, H-2′b), 2.16 (1H, m, H-2′a); FAB+-MS m/z 448 ([C24H25N5O4 + H]+), 332 ([C19H16N5O + H]+). Anal. Calcd for C24H25N5O4‚0.5H2O: C, H, N. Benzylation of 2′-Deoxyguanosine (1) in TFE Containing NaOH and AgNO3. To a stirred suspension of 2′-deoxyguanosine (1.3 g, 4.87 mmol) in 25 mL of TFE was added 0.23 g of NaOH (5.75 mmol) at room temperature. After the mixture stirred for 30 min, ground AgNO3 (0.78 g, 4.6 mmol) was added followed by the dropwise addition of benzyl bromide (0.55 mL, 4.63 mmol) over 3 min. The reaction mixture was stirred at room temperature for 72 h. The resulting pale-purple solution was evaporated to dryness. The crude solid was stirred in 50 mL of MeOH/H2O/NH4OH (50:50:3) at 55 °C and was filtered to remove AgBr. This process was repeated a second time. The pooled filtrates were then chromatographed as described above for the separation of products derived from the reaction of 1with benzyl bromide in the absence of AgNO3. The product distribution and yields were essentially the same as those observed for reactions in the absence of AgNO3. Benzylation of 2′-Deoxyguanosine (1) in DMA Containing NaOH. To a stirred solution of 2′-deoxyguanosine (0.65 g, 2.43 mmol) in 13 mL of DMA was added NaOH (0.13 g, 3.2 mmol). When all sodium hydroxide had dissolved, benzyl bromide (0.28 mL, 2.32 mmol) was added dropwise over a 2-min period. The reaction mixture was stirred at room temperature for 72 h. The resulting solution was diluted with MeOH/H2O (1:1) to a final volume of 100 mL and was chromatographed on a Sephadex LH-20 column (2.8 × 78 cm). The column was eluted with MeOH/H2O (1:1) at 1 mL/min. Fractions 19-60 contained a mixture of imidazole ring-opened 7-benzyl-2′deoxyguanosine (12), unreacted 2′-deoxyguanosine, and 1-benzyl-2′-deoxyguanosine (10). These fractions were pooled, evaporated to dryness, and rechromatographed (see below). 1,7-Dibenzylguanine (11) eluted in MeOH/H2O (1:1) fractions 65-100 (115 mg, 14.3%): UV pH 1 λmin 237 nm, λmax 255 nm, 277 nm (sh), pH 6.9 246 nm (sh), λmin 265 nm, λmax 287 nm, pH 13 246 nm (sh), λmin 265 nm, λmax 286 nm; 1H NMR δ 8.14 (1H, s, 8-H), 7.10-7.59 (10H, m, 2C6H5), 6.70 (2H, s, NH2, exchange with D2O), 5.45 (2H, s, 7-C6H5CH2), 5.22 (2H, s, 1-C6H5CH2); FAB+-MS m/z 332 ([C19H18N5O + H]+), 242 ([C12H11N5O + H]+); HRFAB+-MS calcd for C19H18N5O 332.1511, found 332.1550. Rechromatography of pooled MeOH/H2O (1:1) fractions 1960 (see above) was on the 2.8 × 78-cm Sephadex LH-20 column eluted with MeOH/H2O (1:9) at 1 mL/min. The imidazole ringopened 7-benzyl-2′-deoxyguanosine (12) eluted in fractions (10 mL) 20-33 (14 mg, 1.5%): UV pH 1, 6.9 λmin 247, λmax 272 nm, pH 13 λmin 247 nm, λmax 265 nm. Unreacted 2′-deoxyguanosine (1) eluted in fractions 36-56. 1-Benzyl-2′-deoxyguanosine (10) eluted in fractions 70-114 (50 mg, 5.8%): UV pH 1 λmin 234 nm, λmax 260 nm, 279 nm (sh), pH 6.9 λmin 231 nm, λmax 257 nm, 272 nm (sh), pH 13 λmin 231 nm, λmax 257 nm, 271 nm (sh); 1H NMR δ 7.98 (1H, s, 8-H), 7.177.34 (5H, m, C6H5), 7.02 (2H, s, NH2, exchange with D2O), 6.14

Benzylation of 2′-Deoxyguanosine (1H, t, J ) 6.04 Hz, H-1′), 5.24 (2H, s, 1-C6H5CH2N), 5.10 (1H, br s, OH-3′, exchanges with D2O), 4.97 (1H, br s, OH-5′, exchanges with D2O), 4.35 (1H, m, H-3′), 3.82 (1H, m, H-4′), 3.54 (2H, m, H-5′), 2.54 (1H, m, H-2′b), 2.22 (1H, m, H-2′a); FAB+-MS m/z 358 ([C17H19N5O4 + H]+), 242 ([C12H11N5O + H]+); HRFAB+-MS calcd for C17H20N5O4 358.1514, found 358.1500. Benzylation of 2′-Deoxyguanosine (1) in DMA Containing NaOH and AgNO3. To a stirred solution of 2′-deoxyguanosine (0.65 g, 2.43 mmol) in 13 mL of DMA was added 0.13 g of NaOH (3.2 mmol). When all the NaOH had dissolved, ground silver nitrate (0.4 g, 2.4 mmol) was added followed by the dropwise addition of benzyl bromide (0.28 mL, 2.32 mmol) over a 2-min period. The reaction mixture was stirred at room temperature for 72 h. The resulting solution was evaporated to dryness to produce a crude solid. This solid was stirred twice in 50 mL of MeOH/H2O (1:1) at 50 °C and was filtered to remove undissolved AgBr. The combined filtrates were chromatographed under the same conditions as described for the benzylation of 2′-deoxyguanosine in DMA containing NaOH. The product distribution was the same as that observed in the absence of AgNO3. Benzylation of O6-Benzyl-2′-deoxyguanosine (7) in TFE Containing NaOH or Triethylamine. To a stirred solution of 7 (178.57 mg, 0.5 mmol) in 3 mL of TFE was added 25 mg of NaOH (0.6 mmol) or triethylamine (0.084 mL, 0.6 mmol) at room temperature. Benzyl bromide (0.06 mL, 0.5 mmol) was added dropwise to the reaction mixture which was stirred at room temperature in the dark for 72 h. The resulting solution was evaporated to a yellow solid which was redissolved in 15 mL of MeOH/H2O (1:1) and was loaded on a Sephadex LH-20 column (2.8 × 78 cm) eluted with MeOH/H2O (1:1) at 1 mL/min. UV absorption was monitored at 280 nm, and fractions (10 mL) were collected. Unreacted O6-benzyl-2′-deoxyguanosine (7) eluted in fractions 76-96. O6,7-Dibenzylguanine (13) eluted in fractions 101-121 (17 mg, 10%): UV pH 1 λmin 259 nm, λmax 290 nm, pH 6.9 239 nm (sh),_λmin 260 nm, λmax 286 nm, pH 13 239 nm (sh), λmin 260 nm, λmax 287 nm; 1H NMR δ 8.29 (1H, s, 8-H), 7.24-7.51 (10H, m, 2C6H5), 6.17 (2H, s, NH2, exchange with D2O), 5.42 (2H, s, C6H5CH2O), 5.37 (2H, s, 7-C6H5CH2); FAB+-MS m/z 332 ([C19H17N5O + H]+), 242 ([C12H11N5O + H]+); HRFAB+-MS calcd for C19H18N5O 332.1511, found 332.1503. Hydrolysis of this compound in 0.1 N HCl at 70 °C for 1 h afforded a product with the same UV absorption characteristics as 7-benzylguanine (2) (15). N2,O6-Dibenzyl-2′-deoxyguanosine (5) eluted in fractions 136-163 (30 mg, 13%). Benzylation of 1-Benzyl-2′-deoxyguanosine (10) in TFE Containing NaOH. Benzylation of 10 and column chromatography were carried out exactly as described for the benzylation of 7 (see above). Fractions 35-70 contained a mixture of imidazole ring-opened 1,7-dibenzyl-2′-deoxyguanosine (14) and unreacted 10. These fractions were pooled and rechromatographed (see below). 1,7-Dibenzylguanine (11) eluted in fractions 87-103 (10 mg, 6%). Rechromatography of pooled fractions 35-70 on the Sephadex LH-20 column eluted with MeOH/H2O (1:9) provided the imidazole ring-opened 1,7-dibenzyl-2′-deoxyguanosine (14) in fractions 41-60 (9 mg, 4%): UV pH 1, 6.9, 13 λmin 250 nm, λmax 276 nm. Unreacted 1-benzyl2′-deoxyguanosine (10) eluted in fractions 70-115.

Results and Discussion The reaction of neutral 2′-deoxyguanosine (1) in TFE produced only 7-benzylguanine (2, 21% yield; Scheme 1) which precipitated during the course of the reaction together with guanine resulting from acid hydrolysis of 1. Compound 2 was crystallized as the hydrochloride from dilute hydrochloric acid solution. Its chromatographic and spectroscopic properties were identical to those of authentic samples of 7-benzylguanine prepared previously (15, 16). Chromatographic analysis (Experi-

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mental Section) of the TFE reaction filtrate revealed the presence of only unreacted 2′-deoxyguanosine and a trace amount of guanine. No other products were present. In contrast to these data, chromatographic analyses of product mixtures derived from the reaction of 1- (produced from 1 in TFE by the addition of NaOH) with benzyl bromide indicated formation of N2-benzyl-2′deoxyguanosine (3) in significant yield (45%) accompanied by much lower yields of N2,N2-dibenzyl-2′-deoxyguanosine (4), N2,O6-dibenzyl-2′-deoxyguanosine (5), 1,N2dibenzyl-2′-deoxyguanosine (6), O6-benzyl-2′-deoxyguanosine (7), 3-benzylguanine (8), and 4-benzyl-5-guanidino1-(β-D-2′-deoxyribofuranosyl)imidazole (9). No 7-benzylguanine derivatives were detected in these reactions. The structure of N2-benzyl-2′-deoxyguanosine (3) (Scheme 1) was established by comparison of its mass, UV, and 1H NMR spectral characteristics with those of the ribonucleoside, N2-benzylguanosine (1). The 1H NMR spectrum of 3 in DMSO-d6 showed an exchangeable oneproton triplet at δ 6.88 coupled to a two-proton doublet for the benzylic hydrogens at δ 4.50. On addition of D2O, the triplet disappeared and the benzylic hydrogens appeared as a singlet. These findings were consistent with attachment of the benzyl group through the benzylic carbon to the exocyclic N2-position on 2′-deoxyguanosine. The mass spectral properties of products 4-6 indicated that each was a dibenzylated nucleoside exhibiting a molecular ion at m/z 477. The UV absorption spectrum of 4 showed that the molecule could dissociate in alkaline solution suggesting that the 1-position was unsubstituted. Additionally, the spectral characteristics were not consistent with substitution at either the O6- or 7-position. This suggested that both benzyl residues in the product were attached to the exocyclic amino group of the 2′-deoxyguanosine. The 1H NMR spectrum of 4 was consistent with this assignment since it showed the presence of a normal 2-deoxyribose residue but failed to exhibit any peaks for an exchangeable exocyclic amino proton. Instead, it showed superimposed two-proton ab quartets with resonances at δ 4.86, 4.83, 4.81, and 4.78 (Jab ) 16.5 Hz) for four benzylic protons. These data are consistent with a structure having two benzyl groups attached to the exocyclic N2-nitrogen of 2′-deoxyguanosine. The UV absorption spectrum for compound 5 resembled that for an O6-substituted 2′-deoxyguanosine although the various maxima appeared at longer wavelength than those for 7. Overall, the UV spectral characteristics for 5 most closely resembled those for N2,O6-dimethyl-2′-deoxyguanosine and N2,O6-dimethylguanosine published previously (17, 18). No spectral shifts in wavelength under alkaline conditions were observed with 5 suggesting that the dissociable proton at the 1-position was absent in the structure. The 1H NMR spectrum for 5 was confirmatory of structure since it showed a singlet for one pair of benzylic hydrogens at δ 5.48 (assignable to attachment of the benzyl carbon to the O6-position) and a doublet (δ 4.51) for benzylic protons coupled to a single exchangeable N2-hydrogen (δ 7.61) which appeared as a triplet (JCHNH ) 6.4 Hz). The UV absorption spectra of 6 resembled that for N2-benzyl-2′deoxyguanosine under acidic and neutral aqueous conditions, although the spectrum for 6 in alkali was not changed significantly from that in neutral solution. This suggested that the two benzyl groups were attached to the 1- and N2-positions on 2′-deoxyguanosine. 1H NMR data showed a singlet for one pair of benzylic hydrogens

700 Chem. Res. Toxicol., Vol. 11, No. 6, 1998

at δ 5.34 (assignable to benzylic attachment to the 1-position) and a doublet (δ 4.51) for the second pair of benzylic protons coupled to a single exchangeable N2proton which appeared as a triplet at δ 7.65 (JCHNH ) 5.2 Hz). The chromatographic and spectroscopic properties of O6-benzyl-2′-deoxyguanosine (7) were identical to those of an authentic sample (13, 14). Compound 8 was concluded to be 3-benzylguanine on the basis of its 1H NMR and UV spectra. The 1H NMR spectrum showed a two-proton singlet for the benzylic hydrogens at δ 5.34 and no protons assignable to a 2-deoxyribofuranosyl residue. The UV absorption spectra agreed well with those reported for 3-methylguanine (19) and 3-benzylguanine described previously (20). Compound 9, 4-benzyl-5-guanidino-1-(β-D-2′-deoxyribofuranosyl)imidazole, could be detected by thin-layer chromatography after its color development with a nitroprusside-ferricyanide-hydroxide spray reagent (3 ,4, 6, 7). However, its very low yield did not permit a detailed spectroscopic characterization. Clearly, benzylation of 1- in TFE directs reaction away from the 7-position and produces N2-benzylguanine in significant yield together with lesser amounts of a variety of other products (many of which are derived from N2benzylation), while the benzylation of 1 in TFE leads only to reaction at the 7-position. This site was previously shown to be the major site of reaction of guanosine with benzyl bromide in DMA (15). In contrast, the reaction of 1- with benzyl bromide in DMA (Scheme 1) produced products derived from reaction at the 1-position [i.e., 1-benzyl-2′-deoxyguanonsine (10)], both the 1- and 7-positions [1,7-dibenzylguanine (11)], and the 7-position [e.g., an imidazole ring-opened form of 7-benzyl-2′-deoxyguanosine (12)]. The mass spectrum for 10 indicated the product was a monobenzylated nucleoside. Its UV spectra in neutral and alkaline aqueous solvents were very similar indicating that the purine portion of the nucleoside did not ionize in alkali which was consistent with substitution at the 1-position. The 1H NMR data for this compound showed a singlet for the benzylic hydrogens of 10 at δ 5.24 and no resonance for an exchangeable 1-proton in the δ 10-11 range where the resonance for the 1-proton on 2′-deoxyguanosine or on N2-substituted products appears (see Experimental Section). These data were consistent with the 1-benzyl-2′-deoxyguanosine (10) assignment and were consistent with the spectral properties exhibited by the ribonucleoside, 1-benzylguanosine (1, 2). The mass spectrum for compound 11 suggested that the molecule was a dibenzylated guanine derivative. The UV spectra for compound 11 under both neutral and alkaline conditions resembled the spectrum for neutral 7-benzylguanine (2) suggesting that no alkali-dissociable protons were present in the molecule. The similarity of the UV absorption spectra for neutral 11 and neutral 2 suggested that one benzyl group was attached to the 7-position. The lack of a spectral change in alkaline solution would be consistent with a second benzyl group being attached to the 1-position in place of the dissociable 1-proton in a molecule such as 2. The 1H NMR data for 11 were consistent with this assignment since no peaks for protons on a 2-deoxyribose residue were present. In addition the spectra showed a peak for one set of benzylic hydrogens at δ 5.45 assignable to benzyl group attachment at the 7-position and a second set of benzylic hydrogens at δ 5.22 resulting from attachment of the benzyl group to the 1-position. The analogous resonances

Moon and Moschel Scheme 2

for the benzylic hydrogens in the neutral form of 2 and 10 appear at δ 5.41 and 5.24, respectively. The structural assignment for compound 12 was based on its UV absorption characteristics which were the same as those reported for the imidazole ring-opened form of other 7-substituted guanine nucleosides (17, 18). The presence of silver nitrate in these reaction solutions had no effect on the product distributions in either TFE or DMA. Thus, the major determinants of product distributions were the ionic form of 2′-deoxyguanosine and the reaction solvent. However, while the reaction of 1- in DMA resulted in formation of products derived from reaction at the 1- and/or 7-position, the reaction of 1- in TFE led primarily to reaction at the exocyclic amino group. Since the iminolate tautomeric form of 1- (Scheme 1) resembles the structure of O6-benzyl-2′-deoxyguanine (7) (21), we examined the reaction of 7 with benzyl bromide in alkaline TFE solution (Scheme 2) and compared the products formed with those produced in the reaction of 1-benzyl-2′-deoxyguanosine (10) with benzyl bromide under the same solvent conditions (Scheme 2). The benzyl group at the 1-position in 10 locks the guanine moiety in the predominant tautomeric form of the neutral nucleoside, while the benzyl group in 7 locks the nucleoside in the iminol tautomeric form (21). Significantly, benzylation of 7 led to roughly equal amounts of N2substituted and 7-substituted O6-benzylguanine products (i.e., 5 and 13), while the benzylation of 10 led only to products derived from reaction at the 7-position (i.e., 11 and 14). The structures for 13 and 14 were assigned on the basis of their 1H NMR and/or UV absorption properties. The spectroscopic properties for 13 were consistent with data for several 7-substituted O6-benzylguanine derivatives (22, 23). Thus, the product distribution observed in the benzylation of 7 (Scheme 2) clearly indicates that the exocyclic amino group of O6-benzyl-2′deoxyguanosine is more reactive than the amino group of 1-benzyl-2′-deoxyguanosine toward benzyl bromide and suggests that the iminolate anionic form of 1- probably contributes to the enhanced nucleophilicity of the amino group in 1- compared to that in 1. We previously showed that the neutral form of 2′deoxyguanosine reacts with 7-(bromomethyl)benz[a]anthracene in TFE to produce N2-(benz[a]anthracen-7ylmethyl)-2′-deoxyguanosine in reasonable quantity. We interpreted this to be a result of the solvent’s ionizing power which favored formation of a soft, stabilized ionic

Benzylation of 2′-Deoxyguanosine

intermediate from 7-(bromomethyl)benz[a]anthracene that reacted preferentially with the exocyclic amino group on 2′-deoxyguanosine (8). Since benzyl bromide is not able to ionize as readily as 7-(bromomethyl)benz[a]anthracene in TFE, its preferential reaction at the 7-position of neutral 2′-deoxyguanosine is not unexpected since 7-substitution is favored with agents that react by an SN2 mechanism, such as simple methylating agents (e.g., methyl iodide, dimethyl sulfate, or methyl methanesulfonate) (24). Reaction at the 7-position of guanine nucleosides is also favored in dipolar aprotic solvents (e.g., DMA) with both benzyl bromide and 7-(bromomethyl)benz[a]anthracene (25) since these solvents enhance rates for SN2-type reactions and do not favor aralkyl halide ionization. With 2′-deoxyguanosine anion (1-) in DMA, benzylation at the 1-position accompanies reaction at the 7-position since the nucleophilicity of the 1-position in 1- is greatly enhanced compared to the same site in the neutral nucleoside. On the basis of these data, one might then expect that the benzylation of 1- in TFE would also produce 1- and 7-benzyl-2′-deoxyguanosine products (and perhaps some minor products) since the ionizing power of the TFE solvent is probably not adequate to produce an ionic intermediate from benzyl bromide. Surprisingly, however, the major site of benzylation of the 1- in TFE was the N2-position. Only a trace amount of a 1-substituted product and no 7-substituted products were detected (Scheme 1). Data for the reaction of 7-(bromomethyl)benz[a]anthracene with 1- in TFE was similar to that for the benzyl bromide reaction since N2-(benz[a]anthracen-7-ylmethyl)-2′-deoxyguanosine was the major product, although reaction at the 1-position was more readily detected in the 7-(bromomethyl)benz[a]anthracene reactions (8). It is interesting to compare our data with those for the methylation of 1 by N-methyl-N-nitrosourea in aqueous solutions as a function of increasing pH (26). Under neutral aqueous conditions, methylation occurred primarily at the 7-position to produce 7-methyl-2′-deoxyguanosine and 7-methylguanine accompanied by lesser amounts of 1- and O6-methyl-2′-deoxyguanosine (26). However, reactions under more alkaline conditions with 1- produced substantial increases in the amounts of 1and O6-methyl-2′-deoxyguanosine relative to 7-substituted products (26). Benzylation of anionic guanosine in water was shown to favor reaction primarily at the N2position, although lower amounts of products derived from reaction at the 7-, 1-, and O6-positions were detected (3-6). Thus, these data emphasize major differences between nucleoside aralkylation and alkylation as exemplified by methylation. Although details of the mechanistic differences between benzylation and methylation of 1- are not completely understood, they reflect the reactivity differences exhibited by alkylating (24, 27) versus aralkylating carcinogens (9, 28) in neutral aqueous DNA reactions. It is well-known that the alkylating agents react with DNA guanine residues at the 7- and O6-positions (24, 26, 27), while the aralkylating carcinogens primarily react at the N2-position (9, 28). It remains to be determined if the reaction between 2′-deoxyguanosine anion and more complex alkylating agents (e.g., isopropylating or tert-butylating agents) in TFE provides a general route to N2-alkylated products. This could be very useful since N2-alkylated guanine nucleosides are not available by alkylation of the neutral nucleoside.

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Acknowledgment. This research was supported by the National Cancer Institute, DHHS, under contract with ABL.

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Moon and Moschel acids, polynucleotides, and nucleosides. Biochemistry 10, 43234330. (26) Golding, B. T., Bleasdale, C., McGinnis, J., Mu¨ller, S., Rees, H. T., Rees, N. H., Farmer, P. B., and Watson, W. P. (1997) The mechanism of decomposition of N-methyl-N-nitrosourea (MNU) in water and a study of its reactions with 2′-deoxyguanosine, 2′deoxyguanosine 5′-monophosphate and d(GTGCAC). Tetrahedron 53, 4063-4082. (27) Shuker, D. E. G., and Bartsch, H. (1994) DNA adducts of nitrosamines. In DNA Adducts: Identification and Biological Significance (Hemminki, K., Dipple, A., Shuker, D. E. G., Kadlubar, F. F., Segerba¨ck, and Bartsch, H., Eds.) IARC Scientific Publication No. 128, pp 73-89, IARC, Lyon, France. (28) Dipple, A. (1994) Reactions of polycyclic aromatic hydrocarbons with DNA. In DNA Adducts: Identification and Biological Significance (Hemminki, K., Dipple, A., Shuker, D. E. G., Kadlubar, F. F., Segerba¨ck, and Bartsch, H., Eds.) IARC Scientific Publication No. 128, pp 107-129, IARC, Lyon, France.

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