Coupling Products of Nucleosides with the Glyoxal Adduct of

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Chem. Res. Toxicol. 2004, 17, 1047-1056

1047

Coupling Products of Nucleosides with the Glyoxal Adduct of Deoxyguanosine Angela K. Brock, Ivan D. Kozekov, Carmelo J. Rizzo, and Thomas M. Harris* Department of Chemistry, Center in Molecular Toxicology and Institute of Chemical Biology, Vanderbilt University, Box B-1715, Vanderbilt University, Nashville, Tennessee 37235 Received March 25, 2004

Glyoxal is a widely dispersed environmental mutagen that reacts with DNA and deoxyguanosine to give primarily the 1,N2-guanine adduct, i.e., 3-(2′-deoxy-β-D-erythro-pentofuranosyl)5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purin-9-one. Kasai et al. have reported [Kasai, et al. (1998) Carcinogenesis 19, 1459-1465] additional minor reactions of glyoxal to give bisnucleosides of unknown structure involving glyoxal conjugation of dG with dA, dC, and dG itself. Reaction conditions have been modified to give large increases in the yields of the adducts, which has permitted structural characterization utilizing chemical and spectroscopic techniques. The glyoxal conjugates of dG with dA and dC are imidazo[1,2-a]purines involving displacement of the 6-hydroxyl group of the dG conjugate by the exocyclic amino groups of dA and dC. The dG conjugate is a symmetrical fusion of two imidazo[1,2-a]purines in which both the 6- and the 7-hydroxyl groups of the dG conjugate have been replaced. The glyoxal conjugates are formed as pairs of diastereomers. The dC and dA have a trans orientation of substituents at C6 and C7; the adduct of dG has a cis orientation. The absolute configurations of the individual diastereomers have been tentatively assigned based on comparison of their CD spectra with configurationally assigned diastereomers of the crotonaldehyde adduct of deoxyguanosine.

Introduction Glyoxal (gx) is a widely dispersed environmental mutagen that is found in foods, beverages, and cigarette smoke (1, 2). It also arises endogenously by oxidation of nucleic acids (3, 4) and lipids (5, 6) and by metabolism of a number of nitrosamines (7). The reactions of gx with DNA and with nucleosides occur preferentially with guanine to form a five-membered ring fused between N1 and N2 and bearing a pair of vicinal hydroxyl groups (Scheme 1, gx-dG, 1) (8, 9).1 Kasai and co-workers have reported the formation of bis-nucleosides by the action of gx on nucleosides and DNA (10). They observed coupling products of dA, dC, and dG with dG and gx, but low yields precluded the assignment of structures; characterization was limited to UV and mass spectra. We were interested in establishing the structures of these conjugates so that we might then evaluate the possibility that they create cross-links in DNA. Kasai detected dC-gx-dG and dA-gx-dG in enzymatic hydrolysates of gx-treated ssDNA but not in correspondingly treated dsDNA (10). However, their experiment in which bis-nucleosides were detected in ssDNA was conducted with a much higher concentration of gx and a longer reaction period than the dsDNA experiment. Comet assays of gx-treated DNA have suggested the formation of interchain cross-links (11). A complication in the detection of interchain cross-links is that dsDNA contain* To whom correspondence should be addressed. Tel: 615-322-2861. Fax: 615-322-4936. E-mail: [email protected]. 1 Abbreviations: gx-dG, 3-(2′-deoxy-β-D-erythro-pentofuranosyl)5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purin-9-one. COSY and HMBC refer to commonly used pulse programs to create two-dimensional NMR spectra that correlate the signals of neighboring protons and of protons with neighboring carbon atoms, respectively.

Scheme 1

ing these cross-links may be resistant to enzymatic degradation. The slow reactions and low yields of bisnucleosides observed in Kasai’s studies made it impractical to produce the quantities of bis-nucleosides that would be needed for structural characterization. Consequently, we initially sought to improve upon the efficiency of the reactions. These efforts have been successful, and as a result, we have been able to obtain sufficient material to elucidate the structures of conjugates 2, 3, and 4 derived from the reactions of gx-dG with dA, dC, and dG, respectively (Scheme 2).

Materials and Methods General Methods. The reagents were purchased from Aldrich Chemical Co. unless otherwise noted and used without further purification. NMR spectra were recorded using solutions in DMSO-d6. 1H NMR spectra were recorded at 400 and 500 MHz. Analyses of reaction mixtures were performed on a Beckman HPLC system with a diode array UV detector monitoring at 254 nm using a 250 mm × 4.6 mm i.d. reverse phase column (YMC ODS-AQ column) at a flow rate of 1.5 mL/min using gradient 1. Preparative separations were carried out with a 250 mm × 10 mm i.d. reverse phase column (YMC ODS-AQ column) at a flow rate of 5 mL/min using gradients 1-5. In both cases, H2O (A) and CH3CN (B) mixtures were used. Gradient 1: 1% B initial mixture, 15 min linear gradient to 10% B, 5

10.1021/tx049906z CCC: $27.50 © 2004 American Chemical Society Published on Web 07/13/2004

1048 Chem. Res. Toxicol., Vol. 17, No. 8, 2004 Scheme 2

min linear gradient to 20% B, 5 min at 20% B, 3 min linear gradient to 100% B, 2 min at 100% B, followed by 3 min linear gradient to the initial conditions. Gradient 2: 1% B initial mixture, 15 min linear gradient to 10% B, 5 min linear gradient to 20% B, 2 min linear gradient to 100% B, 1 min at 100% B, followed by 2 min linear gradient to the initial conditions. Gradient 3: 1% B initial mixture, 2 min linear gradient to 6.5% B, 43 min at 6.5% B, 2 min linear gradient to 80% B, 1 min at 80% B, 2 min linear gradient back to initial conditions. Gradient 4: 1% B initial mixture, 2 min linear gradient to 6% B, 30 min linear gradient to 9% B, 2 min linear gradient to 80% B, 1 min at 80% B, 2 min linear gradient to initial conditions. Gradient 5: 1% B initial mixture, 15 min linear gradient to 10% B, 5 min linear gradient to 20% B, 3 min linear gradient to 100% B, 2 min at 100% B, 3 min linear gradient to 0% B, 6 min at 0% B, followed by 1 min linear gradient to initial conditions. Preparation of gx-dG (1) (3, 7, 12). A mixture of dG‚H2O (500 mg, 1.8 mmol) and gx (40% solution in water, 300 µL, 151 mg, 2.6 mmol) in water (5 mL) was stirred for 3 days at room temperature. The solution was evaporated in vacuo; the residue was purified by flash column chromatography (9:1 CH2Cl2: MeOH) to give 508 mg of 1 (89%). 1H NMR (DMSO-d6): δ 8.81 (bs, 1H, 5-NH), 7.96 (s, 1H, H2), 7.21 (d, 1H, 7-OH, J ) 6.4 Hz), 6.48 (d, 1H, 6-OH, J ) 7.3 Hz), 6.13 (t, 1H, H1′, J ) 6.7 Hz), 5.48 (d, 1H, H7, J ) 3.8 Hz), 5.28 (d, 1H, 3′-OH, J ) 2.5 Hz), 4.93 (t, 1H, 5′-OH, J ) 5.4 Hz), 4.87 (d, 1H, H6, J ) 4.8 Hz), 4.34 (m, 1H, H3′), 3.83 (m, 1H, H4′), 3.63-3.46 (m, 2H, H5′, H5′′), 2.53 (m, 1H, H2′), 2.22 (m, 1H, H2′′).2,3 Independent Syntheses of N2-(2-Hydroxyethyl)-2′-deoxyguanosine (7). 2-Aminoethanol (5 mg, 0.08 mmol) was added to a mixture of 2-fluoro-O6-[2-(trimethylsilyl)ethyl]-2′-deoxyguanosine (15 mg, 0.04 mmol), DMSO (150 µL), and diisopropylethylamine (50 µL) (13). The mixture was stirred at 65 °C for 18 h. The reaction was stopped, and the solvents were evaporated under vacuum. The residue was dissolved in 5% acetic acid (0.5 mL) and stirred at room temperature for 1 h. HPLC purification (gradient 1) of the reaction mixture gave 11.5 mg (91%) of 7. 1H NMR (DMSO-d6): δ 10.53 (bs, 1H, 1-NH), 7.90 2 Note that the carbon atoms derived from gx are C6 and C7 in the imidazo[1,2-a]purine numbering system. 3 The NMR assignments differ from those reported by Loeppky (12). The present assignments are derived from a COSY spectrum, which showed the signal assigned as the 5-NH having coupling to both H6 and H7. The latter was differentiated on the basis of stronger coupling of the 5-NH to H6 than to H7. The 6-OH and 7-OH signals showed coupling to the vicinal carbon-bound protons. Prof. Loeppky has informed us that the hydroxyl assignments depicted in the figure on p 156 of ref 12 were inadvertently interchanged (personal communication).

Brock et al. (s, 1H, H8), 6.51 (m, 1H, 2-NH), 6.14 (dd, 1H, H1′, J ) 6.2, 6.4 Hz), 5.28 (d, 1H, 3′-OH, J ) 4.0 Hz), 4.87 (t, 2H, 5′-OH, CH2OH, J ) 5.5 Hz) 4.35 (m, 1H, H3′), 3.80 (m, 1H, H4′), 3.54 (m, 3H, H5′, CH2OH), 3.49 (m, 1H, H5′′), 3.35 (m, 2H, CH2NH), 2.60 (m, 1H, H2′), 2.24 (m, 1H, H2′′). HRMS-FAB C12H18N5O5 [M + H+] calcd, 312.1308; found, 312.1322. Compound 7 was prepared previously by a similar route (14). Independent Syntheses of N6-(2-Hydroxyethyl)-2′-deoxyadenosine (8). 2-Aminoethanol (4.5 mg, 0.074 mmol, 4.4 µL) was added to a mixture of 6-chloropurine 9-(2′-deoxyriboside) (10 mg, 0.037 mmol), DMSO (150 µL), and diisopropylethylamine (50 µL) (15). The mixture was stirred at 65 °C for 18 h. HPLC analysis showed complete disappearance of the starting material. The mixture was cooled, and the solvents were evaporated under vacuum. The residue was purified by HPLC (gradient 1) to give 9.5 mg (87%) of 8. 1H NMR (DMSO-d6): δ 8.27 (s, 1H, H8), 8.14 (s, 1H, H2), 7.59 (bs, 1H, NH), 6.28 (t, 1H, H1′, J ) 7.0 Hz), 5.24 (d, 1H, 3′-OH, J ) 3.5 Hz), 5.16 (t, 1H, 5′-OH, J ) 5.5 Hz), 4.69 (m, 1H, CH2OH), 4.34 (m, 1H, H3′), 3.81 (m, 1H, H4′), 3.49 (m, 6H, H5′, H5′′, NHCH2CH2OH), 2.65 (m, 1H, H2′), 2.19 (m, 1H, H2′′). HRMS-FAB C12H18N5O4 [M + H+] calcd, 296.1359; found, 296.1338. Compound 8 was prepared previously by another route (16). Independent Syntheses of N4-(2-Hydroxyethyl)-2′-deoxycytidine (9). 2-Aminoethanol (6.5 mg, 10.7 mmol, 6.5 µL) was added to a mixture of 4-(1-triazolyl)-2′-deoxyuridine (15 mg, 0.054 mmol) and pyridine (200 µL) (17, 18). The mixture was stirred at 65 °C for 10 min. The reaction was stopped, and the solvents were evaporated in vacuo. The residue was purified by HPLC (gradient 1) to give 12.5 mg (85%) of 9. 1H NMR (DMSO-d6): δ 7.75 (bs, 1H, NHCH2), 7.73 (d, 1H, H6, J ) 7.63 Hz), 6.16 (dd, 1H, H1′, J ) 6.2, 7.5 Hz), 5.79 (d, 1H, H5, J ) 7.63 Hz), 5.18 (bs, 1H, 3′-OH), 4.95 (bs, 1H, 5′-OH), 4.80 (bs, 1H, CH2OH), 4.19 (m, 1H, H3′), 3.75 (m, 1H, H4′), 3.54 (m, 2H, H5′, H5′′), 3.48 (m, 2H, CH2OH), 3.30 (m, 2H, CH2NH), 2.10 (m, 1H, H2′), 1.93 (m, 1H, H2′′). HRMS-FAB C11H18N3O5 [M + H+] calcd, 272.1246; found, 272.1233. Compound 9 was prepared previously (19). Independent Synthesis of 1,2-Bis(2′-deoxyguanosin-N2yl)ethane (11). Using a variation of the method of Kowalczyk et al., a mixture of 1.9 mg (0.032 mmol) of ethylenediamine, 35.5 mg (0.096 mmol) of 2-fluoro-O6-TMSE-2′-deoxyinosine, and 10.9 mg (0.08 mmol) of diisopropylethylamine in DMSO (100 µL) was heated at 65 °C for 18 h to afford after HPLC purification (gradient 1) 14.8 mg (82%) of 11 (20); elution time, 10-11 min. 1H NMR (DMSO-d6, 35 °C): δ 7.87 (s, 2H, 2 × H8), 6.11 (m, 2H, 2 × H1′), 4.32 (m, 2H, 2 × H3′), 3.79 (m, 2H, 2 × H4′), 3.51 (m, 8H, 2 × H5′, 2 × H5′′, 2 × NHCH2), 2.54 (m, 2H, 2 × H2′′), 2.18 (m, 2H, 2 × H2′). HRMS-FAB C22H29N10O8 [M + H+] calcd, 561.2169; found, 561.2173. Formation of Conjugate 2 of dA with gx-dG. A solution of gx-dG (25 mg, 0.077 mmol) and dA‚H2O (105 mg, 0.39 mmol) in 150 µL of DMSO was heated for 4 days at 60 °C. The progress of the reaction was checked periodically by HPLC. HPLC purification (gradient 3) gave equal quantities of the diastereomers of dA-gx-dG (2, 26.3 mg, 61%). No interconversion of diastereomers was observed on standing in H2O or DMSO. Diastereomer 2a: elution time, 38 min; UV, 268 nm. 1H NMR (DMSO-d6): δ 8.78 (br, 1H, 6-NH of dA), 8.73 (s, 1H, 5-NH), 8.44 (s, 1H, H8 of dA), 8.30 (s, 1H, H2 of dA), 7.96 (s, 1H, H2 of gx-dG), 7.43 (d, 1H, CHOH, J ) 6.7 Hz), 6.38 (t, 1H, J ) 6.7 Hz, H1′), 6.12 (t, 1H, J ) 6.7 Hz, H1′), 5.90 (br, 1H, NHCH), 5.81 (br, 1H, CHOH), 5.32 (d, 1H, J ) 4.1 Hz, 3′-OH), 5.28 (d, 1H, J ) 4.1 Hz, 3′-OH), 5.12 (t, 1H, J ) 5.4 Hz, 5′-OH), 4.93 (t, 1H, J ) 5.4 Hz, 5′-OH), 4.41 (m, 1H, H3′), 4.33 (m, 1H, H3′), 3.88 (m, 1H, H4′), 3.82 (m, 1H, H4′), 3.65-3.46 (m, 4H, 2 × H5′, 2 × H5′′), 2.71 (m, 1H, H2′), 2.53 (m, 1H, H2′), 2.28 (m, 1H, H2′′), 2.19 (m, 1H, H2′′). HRMS-FAB C22H27N10O8 [M + H+] calcd, 559.2013; found, 559.2038. Diastereomer 2b: elution time, 41 min; UV, 268 nm. 1H NMR (DMSO-d6): δ 8.80 (br, 1H, 6-NH of dA), 8.73 (s, 1H, 5-NH), 8.44 (s, 1H, H8 of dA), 8.30 (s, 1H, H2 of dA), 7.96 (s, 1H, H2 of gx-dG), 7.42 (d, 1H, CHOH, J )

Nucleoside Conjugates of Glyoxal-Deoxyguanosine 6.7 Hz), 6.38 (t, 1H, J ) 6.7 Hz, H1′), 6.12 (t, 1H, J ) 6.7 Hz, H1′), 5.90 (br, 1H, NHCH), 5.82 (br, 1H, CHOH), 5.32 (d, 1H, J ) 3.8 Hz, 3′-OH), 5.28 (d, 1H, J ) 3.8 Hz, 3′-OH), 5.13 (t, 1H, J ) 5.4 Hz, 5′-OH), 4.94 (t, 1H, J ) 5.4 Hz, 5′-OH), 4.41 (m, 1H, H3′), 4.33 (m, 1H, H3′), 3.88 (m, 1H, H4′), 3.81 (m, 1H, H4′), 3.66-3.46 (m, 4H, 2 × H5′, 2 × H5′′), 2.73 (m, 1H, H2′), 2.49 (m, 1H, H2′), 2.28 (m, 1H, H2′′), 2.22 (m, 1H, H2′′). HRMS-FAB C22H27N10O8 [M + H+] calcd, 559.2013; found, 559.2032. Reduction of 2ab with NaBH4. 1. Reaction at Room Temperature. Adduct 2ab (3.5 mg, 0.0063 mmol) in 1 mL of 50% aqueous MeOH was treated with powdered NaBH4 (12.1 mg, 0.32 mmol) for 10 min at ambient temperature. HPLC analysis of an acidified aliquot indicated that the reaction was complete. After the reaction mixture was neutralized with 500 µL of 5% acetic acid, HPLC purification (gradient 2) gave 2.36 mg (68%) of the two diastereomers of 5. Compound 5a: elution time, 19.0 min; UV, 270 nm (sh 255 nm). 1H NMR (DMSO-d6): δ 10.85 (br, 1H, 1-NH of dG), 8.37 (s, 1H, H8 of dA), 8.29 (s, 1H, H2 of dA), 7.93 (br, 6-NH of dA), 7.88 (s, 1H, H8 of dG), 6.82 (d, 1H, 2-NH of dG, J ) 6.4 Hz), 6.35 (t, 1H, J ) 6.4 Hz, H1′), 6.22 (br, 1H, NHCHNH), 6.04 (br, 1H, H1′), 5.29 (d, 1H, J ) 4.1 Hz, 3′-OH), 5.23 (d, 1H, J ) 3.5 Hz, 3′-OH), 5.16 (t, 1H, J ) 6.4 Hz, CH2OH), 5.15 (t, 1H, J ) 5.0 Hz, 5′-OH), 4.84 (t, 1H, J ) 5.0 Hz, 5′-OH), 4.40 (m, 1H, H3′), 4.26 (m, 1H, H3′), 3.86 (m, 1H, H4′), 3.76 (m, 1H, H4′), 3.72 (t, 2H, J ) 5.4 Hz, CH2OH), 3.61 (m, 1H, H5′), 3.56-3.44 (m, 3H, H5′, 2 × H5′′), 2.70 (m, 1H, H2′), 2.51 (m, 1H, H2′), 2.26 (m, 1H, H2′′), 2.09 (m, 1H, H2′′). LC-MS [M + H+] 561. HRMS-FAB [M + H+] C22H29N10O8 calcd, 561.2170; found, 561.2202. Compound 5b: elution time, 19.5 min; UV, 270 nm (sh 255 nm). 1H NMR (DMSO-d6): δ 10.86 (br, 1H, 1-NH of dG), 8.38 (s, 1H, H8 of dA), 8.28 (s, 1H, H2 of dA), 7.95 (br, 6-NH of dA), 7.90 (s, 1H, H8 of dG), 6.82 (d, 1H, 2-NH of dG, J ) 7.0 Hz), 6.35 (t, 1H, J ) 6.4 Hz, H1′), 6.23 (br, 1H, NHCHNH), 6.08 (br, 1H, H1′), 5.29 (d, 1H, J ) 4.1 Hz, 3′OH), 5.23 (d, 1H, J ) 3.8 Hz, 3′-OH), 5.15 (t, 1H, J ) 6.4 Hz, CH2OH), 5.13 (t, 1H, J ) 5.1 Hz, 5′-OH), 4.85 (t, 1H, J ) 5.1 Hz, 5′-OH), 4.40 (m, 1H, H3′), 4.26 (m, 1H, H3′), 3.87 (m, 1H, H4′), 3.77 (m, 1H, H4′), 3.72 (t, 2H, J ) 5.4 Hz, CH2OH), 3.60 (m, 1H, H5′), 3.54-3.37 (m, 3H, H5′, 2 × H5′′), 2.72 (m, 1H, H2′), 2.53 (m, 1H, H2′), 2.26 (m, 1H, H2′′), 2.05 (m, 1H, H2′′). HRMS-FAB [M + H+] C22H29N10O8 calcd, 561.2170; found, 561.2189. 2. Reaction at 60 °C. Compound 2ab (11 mg, 0.020 mmol) in 1 mL of 0.1 M NaOH was treated with powdered NaBH4 (378 mg, 10 mmol) for 6 h at 60 °C. HPLC analysis of an acidified aliquot indicated that the reaction was complete. The reaction mixture was neutralized with 1 mL of 20% acetic acid. Purification by HPLC (gradient 4) gave four identifiable products: N2hydroxyethyl-dG [7, 1.50 mg, 24%; elution time, 13 min; UV, 255 nm (sh 280 nm); LC-ES-MS [M + H+] 312], N6-hydroxyethyl-dA [8, 1.75 mg, 30%; elution time, 20.4 min; UV, 268 nm; LC-ES-MS [M + H+] 296], dG [2.78 mg, 52%; elution time, 9.1 min; UV, 251 nm (sh 274 nm)], and dA [0.92 mg, 18%; elution time, 14.3 min; UV, 261 nm]. Structural assignments were made by coinjection of authentic samples with comparison of retention times and UV spectra. Assignments were confirmed by MS. Further Reduction of 5. Reduction product 5 (0.25 mg, 0.00045 mmol) in 100 µL of 0.1 M NaOH was treated with NaBH4 (2.5 M in 0.1 M NaOH, 92 µL) for 6 h at 60 °C. HPLC analysis after acidification showed that the reaction was complete giving 7 (12%), 8 (18%), dG (40%), and dA (28%). Structural assignments were made by coinjection of authentic samples with comparison of retention times and UV spectra. Assignments were confirmed by MS. Depurination of 2ab. Compounds 2a (0.22 mg, 0.00039 mmol) and 2b (0.35 mg, 0.00063 mmol) were individually treated with 0.10 M HCl (500 µL) for 1 h at 70 °C. The reactions were neutralized with 0.1 M NaOH, and the product mixtures were purified by HPLC (gradient 1). The resulting enantiomeric bases had identical elution times (14.2 min) and mass spectra (ES-MS [M + H+] 327). CD spectra (Figure 3B) were recorded using solutions in methanol.

Chem. Res. Toxicol., Vol. 17, No. 8, 2004 1049 Formation of Conjugate 3 of dC with gx-dG. A solution of gx-dG (25 mg, 0.077 mmol) and 2′-deoxycytidine (89 mg, 0.39 mmol) in 150 µL of DMSO was heated for 6 days at 60 °C, at which point HPLC analysis indicated that the reaction was nearly complete. HPLC purification (gradient 2) gave dC-gxdG (3ab) (71% total). The diastereomers did not interconvert on standing in H2O or DMSO. Compound 3a (14.9 mg): Elution time, 14.6 min; UV, 256 nm (sh 275 nm). 1H NMR (DMSO-d6): δ 8.77 (s, 1H, 5-NH), 8.43 (d, 1H, J ) 7.9 Hz, 4-NH of dC), 7.98 (s, 1H, H2), 7.90 (d, 1H, J ) 7.3 Hz, H6 of dC), 7.46 (d, 1H, J ) 6.4 Hz, CHOH), 6.18-6.07 (m, 2H, 2 × H1′), 5.70 (d, 1H, J ) 7.3 Hz, H5 of dC), 5.65 (d, 1H, J ) 6.4 Hz, CHOH), 5.57 (d, 1H, J ) 8.3 Hz, CHNH), 5.27 (d, 1H, J ) 4.1 Hz, 3′-OH), 5.19 (d, 1H, J ) 4.1 Hz, 3′-OH), 4.96 (t, 1H, J ) 5.4 Hz, 5′-OH), 4.91 (t, 1H, J ) 5.4 Hz, 5′-OH), 4.34 (m, 1H, H3′), 4.20 (m, 1H, H3′), 3.82 (m, 1H, H4′), 3.79 (m, 1H, H4′), 3.60-3.46 (m, 4H, 2 × H5′, 2 × H5′′), 2.52 (m, 1H, H2′), 2.22 (m, 1H, H2′), 2.16 (m, 1H, H2′′), 1.93 (m, 1H, H2′′). HRMS-FAB C21H27N8O9 [M + H+] calcd, 535.1901; found, 535.1886. Compound 3b (14.1 mg): Elution time, 15.5 min; UV, 256 nm (sh 275 nm). 1H NMR (DMSO-d6): δ 8.80 (s, 1H, 5-NH), 8.42 (d, 1H, J ) 8.3 Hz, 4-NH of dC), 7.97 (s, 1H, H2), 7.90 (d, 1H, J ) 7.3 Hz, H6 of dC), 7.46 (d, 1H, J ) 6.36 Hz, CHOH), 6.18-6.07 (m, 2H, 2 × H1′), 5.70 (d, 1H, J ) 7.3 Hz, H5 of dC), 5.64 (d, 1H, J ) 6.4 Hz, CHOH), 5.58 (d, 1H, J ) 8.3 Hz, CHNH), 5.28 (d, 1H, J ) 4.1 Hz, 3′OH), 5.19 (d, 1H, J ) 4.1 Hz, 3′-OH), 4.96 (t, 1H, J ) 5.4 Hz, 5′-OH), 4.92 (t, 1H, J ) 5.4 Hz, 5′-OH), 4.34 (m, 1H, H3′), 4.20 (m, 1H, H3′), 3.83 (m, 1H, H4′), 3.79 (m, 1H, H4′), 3.61-3.46 (m, 4H, 2 × H5′, 2 × H5′′), 2.52 (m, 1H, H2′), 2.20 (m, 1H, H2′), 2.16 (m, 1H, H2′′), 1.95 (m, 1H, H2′′). HRMS-FAB C21H27N8O9 [M + H+] calcd, 535.1901; found, 535.1920. Reduction of 3a with NaBH4. 1. Reaction at Room Temperature. A solution of 3a (2.3 mg, 0.0043 mmol) in 1 mL of 50% aqueous MeOH was treated with powdered NaBH4 (8.3 mg, 0.22 mmol) for 10 min at room temperature. HPLC analysis of an acidified aliquot indicated that the reaction was complete. The reaction mixture was quenched with 500 µL of 5% acetic acid. Purification by HPLC (gradient 2) gave 1.66 mg (72%) of 6: elution time, 14.4 min; UV, 276 nm (sh 257 nm). 1H NMR (DMSO-d6): 10.75 (br, 1H, 1-NH of dG), 8.10 (d, 1H, J ) 7.3 Hz, 4-NH of dC), 7.86 (s, 1H, H8 of dG), 7.84 (d, 1H, J ) 7.6 Hz, H6 of dC), 6.98 (d, 1H, J ) 7.0 Hz, 2-NH of dG), 6.18 (t, 1H, J ) 6.4 Hz, H1′), 6.06 (t, 1H, J ) 6.4 Hz, H1′), 5.92 (m, 1H, NHCHNH), 5.84 (d, 1H, J ) 7.3 Hz, H5 of dC), 5.38 (t, 1H, J ) 5.4 Hz, 5′-OH), 5.27 (d, 1H, J ) 3.5 Hz, 3′-OH), 5.21 (d, 1H, J ) 4.1 Hz, 3′-OH), 5.11 (t, 1H, J ) 6.0 Hz, CH2OH), 4.97 (t, 1H, J ) 5.4 Hz, 5′-OH), 4.28 (m, 1H, H3′), 4.21 (m, 1H, H3′), 3.78 (m, 3H, 2 × H4′, 1 × CH2OH), 3.69-3.49 (m, 4H, 2 × H5′, 2 × H5′′), 3.40 (m, 1H, 1 × CH2OH), 2.77 (m, 1H, H2′), 2.18 (m, 1H, H2′), 2.01-1.88 (m, 2H, 2 × H2′′). HRMS-FAB C21H29N8O9 [M + H+] calcd, 537.2058; found, 537.2028. 2. Reaction at 60 °C. A solution of 3a (3.2 mg, 0.006 mmol) in 250 µL of 0.1 M NaOH was treated with powdered NaBH4 (113 mg, 3 mmol) for 6 h at 60 °C. HPLC analysis of an acidified aliquot showed that the reaction was complete. The reaction mixture was neutralized with 1 mL of 20% acetic acid and purified by HPLC (gradient 2) to give three identifiable products: N4-hydroxyethyl-dC [9, 0.32 mg, 20%; elution time, 10 min; UV, 274 nm; LC-ES-MS [M + H+] 272], N2-hydroxyethyl-dG [7, 0.61 mg, 33%; elution time, 14.9 min; UV, 255 nm (sh 280 nm); LC-ES-MS 312 [M + H+]], and dG [0.64 mg, 40%; elution time, 12 min; UV, 251 nm (sh 274 nm)]. Structural assignments were made by coinjection of authentic samples with comparison of retention times and UV spectra. Assignments were confirmed by MS. Further Reduction of 6. Reduction product 6 (single diastereomer, 0.20 mg, 0.00037 mmol) in 100 µL of 0.1 M NaOH was treated with NaBH4 (2.5 M solution in 0.1 M NaOH, 74 µL) for 6 h at 60 °C. HPLC analysis after acidification showed that the reaction was complete giving N4-hydroxyethyl-dC (9, 6%), dG (38%), and N2-hydroxyethyl-dG (7, 30%). Structural assignments were made by coinjection of authentic samples with

1050 Chem. Res. Toxicol., Vol. 17, No. 8, 2004 comparison of retention times and UV spectra. Assignments were confirmed by MS. Depurination of 3. Compounds 3a (0.26 mg, 0.00049 mmol) and 3b (0.15 mg, 0.00028 mmol) of 3 were individually treated with 0.10 M HCl (500 µL) for 1 h at 70 °C. The reactions were neutralized with 0.1 M NaOH, and the products were purified by HPLC (gradient 1). The products had identical elution times (11.8 min) and mass spectra (ES-MS [M + H+] 419). The CD spectra (Figure 3C) were recorded using methanol solutions. Formation of Conjugate 4 of dG with gx-dG. 1. DMSO Solution. A solution of gx-dG (25 mg, 0.077 mmol) and dG‚ H2O (111 mg, 0.39 mmol) in 150 µL of DMSO was stirred for 6 days at 60 °C. Purification by HPLC (gradient 2) gave 0.93 and 1.18 mg (5%) of diastereomers 4ab of dG-gx-dG. Compound 4a: elution time, 18.8 min; UV, 252 nm (sh 278 nm). 1H NMR (DMSO-d6): δ 9.56 (br, 2H, 2 × 5-NH), 7.97 (s, 2H, 2 × H2), 6.34 (s, 2H, NHCHCHNH), 6.10 (t, 2H, J ) 6.7 Hz, 2 × H1′), 5.28 (br, 2H, 2 × 3′-OH), 4.98 (br, 2H, 2 × 5′-OH), 4.34 (m, 2H, 2 × H3′), 3.81 (m, 2H, 2 × H4′), 3.60-3.45 (m, 4H, 2 × H5′, 2 × H5′′), 2.52 (m, 2H, 2 × H2′), 2.19 (m, 2H, 2 × H2′′). HRMSFAB C22H25N10O8 [M + H+] calcd, 557.1857; found, 557.1896. Compound 4b: elution time, 19.7 min; UV, 252 nm (sh 278 nm). 1H NMR (DMSO-d ): δ 9.57 (br, 2H, 2 × 5-NH), 7.99 (s, 2H, 2 6 × H2), 6.37 (s, 2H, NHCHCHNH), 6.10 (t, 2H, J ) 6.7 Hz, 2 × H1′), 5.29 (br, 2H, 2 × 3′-OH), 4.97 (br, 2H, 2 × 5′-OH), 4.32 (m, 2H, 2 × H3′), 3.81 (m, 2H, 2 × H4′), 3.58-3.46 (m, 4H, 2 × H5′, 2 × H5′′), 2.50 (m, 2H, 2 × H2′), 2.18 (m, 2H, 2 × H2′′). HRMS-FAB C22H25N10O8 [M + H+] calcd, 557.1857; found, 557.1830. 2. Aqueous Solutions. A suspension of gx-dG (25 mg, 0.077 mmol) and dG‚H2O (43 mg, 0.15 mmol) in 500 µL of buffer, pH 7.0 (50 mM potassium phosphate), was stirred for 6 days at 60 °C. Unreacted dG was removed by filtration. Purification by HPLC (gradient 2) gave 0.70 and 1.12 mg (4%) of 4a and 4b. A similar reaction at pH 10.0 (50 mM potassium carbonate, potassium borate, and potassium hydroxide) gave 6.08 and 6.42 mg of 4a and 4b (28%). A suspension of gx-dG (25 mg, 0.077 mmol) and dG‚H2O (43 mg, 0.15 mmol) in 500 µL of 0.1 M NaOH was heated for 2 days at 60 °C to give 7.17 and 8.27 mg of 4a and 4b (35%) after HPLC purification (gradient 2). Reduction of 4 with NaBH4. 1. Reaction at Room Temperature. Compound 4ab (10 mg, 0.018 mmol) in 3 mL of 50% aqueous MeOH was treated with a large excess of powdered NaBH4 for 6 days at room temperature. On the first day, 17 mg (0.45 mmol) was added followed by an additional 34 mg (0.90 mmol) on each of the following days. Purification of the mixture by HPLC (gradient 5) gave four identifiable products: N2Hydroxyethyl-dG [7, 0.17 mg, 3%; elution time, 14.3 min; UV, 255 nm (sh 280 nm); ES-MS [M + H+] 312], 1,2-Bis(2′deoxyguanosin-N2-yl)ethane [11, 0.84 mg, 8%; elution time, 19.5 min; LC-ES-MS [M + H+] 561]. Singly reduced product 10ab [1.33 mg, 13%]. Diastereomer 10a: elution time, 14.8 min; UV, 255 nm (sh 283 nm). 1H NMR (DMSO-d6): δ 10.69 (br, 1H, 1-NH of dG), 8.16 (s, 1H, 5-NH of gx-dG), 7.97 (s, 1H, H8 of dG or H2 of gx-dG), 7.93 (s, 1H, H8 of dG or H2 of gx-dG), 7.57 (br, 1H, 2-NH of dG), 6.37 (m, 1H, CHNH), 6.13 (m, 2H, 2 × H1′), 5.28 (d, 1H, J ) 4.1 Hz, 3′-OH), 5.24 (d, 1H, J ) 4.1 Hz, 3′-OH), 4.96 (m, 2H, 2 × 5′-OH), 4.34 (m, 2H, 2 × H3′), 3.96 (m, 1H, NHCH2), 3.82 (m, 2H, H4′), 3.61-3.46 (m, 5H, 2 × H5′, 2 × H5′′, NHCH2), 2.54 (m, 1H, H2′), 2.21 (m, 1H, H2′′). LC-ES-MS [M + H+] 559. HRMS-FAB C22H27N10O8 [M + H+] calcd, 559.2013; found, 559.2015. Diastereomer 10b: elution time, 15.3 min; UV, 255 nm (sh 283 nm). 1H NMR (DMSO-d6): δ 10.67 (br, 1H, 1-NH of dG), 8.16 (s, 1H, 5-NH of gx-dG), 7.97 (s, 1H, H8 of dG or H2 of gx-dG), 7.93 (s, 1H, H8 of dG or H2 of gx-dG), 7.56 (br, 1H, 2-NH of dG), 6.36 (m, 1H, CHNH), 6.14 (m, 2H, 2 × H1′), 5.27 (d, 1H, J ) 4.1 Hz, 3′-OH), 5.23 (d, 1H, J ) 4.1 Hz, 3′-OH), 4.94 (m, 2H, 2 × 5′-OH), 4.34 (m, 2H, 2 × H3′), 3.96 (m, 1H, NHCH2), 3.81 (m, 2H, H4′), 3.61-3.46 (m, 5H, 2 × H5′, 2 × H5′′, NHCH2), 2.51 (m, 1H, H2′), 2.20 (m, 1H, H2′′). LC-ES-MS [M + H+] 559], dG [3.46 mg, 72%; elution time, 11.2 min; UV, 251 nm (sh 274 nm)], and recovered 4ab [1.29 mg, 13%; elution times, 18.8 and

Brock et al. 19.7 min; UV, 252 nm (sh 278 nm)]. Structural assignments were made by coinjection of authentic samples with comparison of retention times and UV spectra. In a second experiment, 4b (0.35 mg, 0.00063 mmol) in 100 µL of 50% aqueous MeOH was treated with 0.25 M aqueous NaBH4 at room temperature; ∼80 equiv was added per day for 10 days. After 3 days, analysis by HPLC (gradient 1) with monitoring by LC-ES-MS showed the following products: 10 (two diastereomers; 15.5 and 16 min; LC-ES-MS [M + H+] 559) and 11 (elution time, 19.5 min; LC-ES-MS [M + H+] 561), 13 (tentative assignment: elution time, 22 min; LC-ES-MS [M + H+] 557). Compound 13 disappeared as the reaction progressed. At the end of the reaction, three major products were present (relative peak areas): 10 (two diastereomers, 30%), 11 (40%), and dG (23%). The structure of 11 was confirmed by coinjection of an authentic sample. 2. Reaction at 60 °C. Compound 4a (0.45 mg, 0.00081 mmol) in 100 µL of 0.1 M NaOH was treated with NaBH4 as a 2.5 M solution in 0.1 M NaOH (163 µL, 15.5 mg, 0.41 mmol) for 6 h at 60 °C. HPLC analysis after quenching with 5% aqueous acetic acid showed that the reaction was complete giving the following products, which were identified by coinjection of authentic samples, UV spectra, and LC-ES-MS: 7 (12%, [M + H+] 312), 11 (11%, [M + H+] 561), and dG (52%, [M + H+] 268). The percentages represent relative peak areas. Further Reduction of 10. Reduction product 10 (0.65 mg, 0.00112 mmol) in 100 µL of 0.1 M NaOH was treated with 242 µL of 2.5 M NaBH4/0.1 M NaOH for 6 h at 60 °C, at which point HPLC indicated that the reaction was complete. HPLC analysis after quenching with 5% aqueous acetic acid showed the following products (relative peak areas): dG (13%, LC-ES-MS [M + H+] 268), 7 (12%, LC-ES-MS [M + H+] 312), and 11 (60%, LC-ES-MS [M + H+] 561). The products were identified on the basis of retention times, UV spectra, and coinjections with authentic samples. Depurination of dG-gx-dG. Diastereomers 4a (3.1 mg, 0.0056 mmol) and 4b (3.3 mg, 0.0059 mmol) were individually treated with 0.10 M HCl (500 µL) for 1 h at 70 °C. The reaction mixtures were neutralized with 0.1 M NaOH and purified by HPLC (gradient 1). The products had identical elution times (14.2 min) and mass spectra (ES-MS [M + H+] 325). The CD spectra (Figure 3D) were recorded using methanol solutions.

Results The reaction conditions used by Kasai et al. to form gx conjugates of dG with dC and dA utilized 1:1:1 mixtures of dG, gx, and the appropriate nucleoside with the reactions being carried out at room temperature in pH 7.4 buffer and with each component at a concentration of 16.7 mM (10). After 7 days, the reactions gave dA-gx-dG (2) and dC-gx-dG (3) in yields of 0.25 and 2.3%, respectively. In addition, the reaction with dA gave 1.3% of the dG conjugate dG-gx-dG (4). In each case, two diastereomers of the adduct were formed. Mass spectrometry showed that 2 and 3 were formed with the loss of one molecule of water while 4 arose by the loss of two molecules of water. Repetition of the gx condensation reactions using their reaction conditions confirmed the formation of conjugates 2-4 and that the reactions were slow and gave only meager yields (