838
Chem. Res. Toxicol. 1998, 11, 838-845
Deamination and Dimroth Rearrangement of Deoxyadenosine-Styrene Oxide Adducts in DNA Thomas Barlow,* Junko Takeshita, 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 February 27, 1998
In reactions between styrene oxide and the ring nitrogen at the 1-position of deoxyadenosine, the epoxide is opened at both the R- (benzylic) and β-carbons. The 1-substituted nucleosides formed are unstable and subsequently undergo either Dimroth rearrangement to give N6substituted deoxyadenosines or deamination to give 1-substituted deoxyinosines. RN6Substituted compounds are also formed from direct reaction at the exocyclic nitrogen. Kinetic experiments revealed that relative rates of deamination of 1-substituted deoxyadenosinestyrene oxides and 1-substituted adenosine-styrene oxides were similar. However, the rate of Dimroth rearrangement in β1-substituted adenosine-styrene oxides was ∼2.3-fold greater than that of β1-substituted deoxyadenosine-styrene oxides and ∼1.5-fold greater in R1substituted adenosine-styrene oxides relative to R1-substituted deoxyadenosine-styrene oxides. Analysis of the products formed from reactions of styrene oxide with [3H]deoxyadenosine and [3H]deoxyadenosine incorporated into native and denatured DNA showed that the doublehelical DNA structure reduced the levels of adducts formed 5-fold relative to denatured DNA but did not present a complete barrier to formation of either N6-substituted deoxyadenosineor 1-substituted deoxyinosine-styrene oxide adducts in native DNA. Additionally, in denatured and native DNA the product distributions were altered in favor of formation of β1-substituted deoxyinosine-styrene oxide adducts with respect to reactions of the nucleoside. The ratio of retained to inverted configuration of RN6-substituted products was higher in DNA than in nucleoside reactions. These experiments indicate that in addition to the N6-position, the ring nitrogen at the 1-position of deoxyadenosine is available, to some extent, for reaction in native DNA. In styrene oxide-DNA reactions, formation of 1-substituted adenines can lead to deaminated products where both Watson-Crick hydrogen-bonding sites are disrupted.
Introduction Styrene oxide, the primary metabolite of the major industrial chemical styrene (1) is mutagenic (2) and carcinogenic (3), and these properties have prompted investigations into styrene oxide reactions with DNA and nucleic acid constituents (4-13). Styrene oxide reacts with all four deoxyribonucleosides with relative yields of alkylated product: dGuo > dCyd > dAdo > dThd (7). However, only dGuo adducts have been identified as reaction products from in vitro incubations of styrene oxide and single- and double-stranded DNA (14), although dGuo-, dCyd-, dAdo-, and dThd-derived products have been identified from in vitro reactions of DNA with other epoxides such as propylene oxide (15), ethylene oxide (16), and cyanoethylene oxide (17). The N6-position of Ado, which opens the epoxide at the R-carbon, and the 1-position, which opens the epoxide at both the R- and β-carbons, have been identified as sites of initial product formation in styrene oxide-Ado reactions (10-12). The 1-substituted styrene oxide-Ado products are unstable and rapidly undergo either a Dimroth rearrangement (18) to give N6-substituted compounds or a deamination, especially for R-substituted products, to give 1-substituted Ino compounds (Scheme * Address for correspondence: Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, U.K.
1) (10, 13). Styrene oxide-induced deamination at adenine residues in DNA has not been reported. However, it has been shown that alkylation of DNA in vitro by propylene oxide (15), ethylene oxide (16), and cyanoethylene oxide (17) gives rise to deaminated dCyd residues. Deamination of 1-substituted dAdo alters both Watson-Crick hydrogen-bonding sites of dAdo, and therefore, formation of these products in DNA could conceivably play a role in styrene oxide-induced mutagenesis or carcinogenesis, especially in the light of previously expressed views that adduction at dAdo residues may be a particularly potent mutational event (19, 20). For this reason, we have investigated the formation of 1-substituted dIno residues on incubation of styrene oxide with dAdo or with native and denatured DNA. We report that DNA structure does not present a complete barrier to the formation of either N6-substituted dAdo- or 1-substituted dIno-styrene oxide adducts. Significant quantities of each are produced.
Experimental Section Caution: Styrene oxide is mutagenic and/or carcinogenic and should be handled with care. Chemicals were used as purchased from the manufacturer. Deoxyadenosine, deoxyinosine, and racemic and optically active styrene oxides were purchased from Aldrich Chemical Co. (Milwaukee, WI). Sephadex LH-20 and NAP-10 columns were
S0893-228x(98)00038-1 CCC: $15.00 © 1998 American Chemical Society Published on Web 07/20/1998
Deoxyadenosine-Styrene Oxide Adducts in DNA
Chem. Res. Toxicol., Vol. 11, No. 7, 1998 839
Scheme 1. Synthesis of dAdo- and Ado-Derived Styrene Oxide Adducts
purchased from Pharmacia Biotech Inc. (Piscataway, NJ). Deoxy[1′,2′,2,8-3H]adenosine 5′-triphosphate with a label distribution of 27.5%, 20.0%, 21.5%, and 31% at the 1′-, 2′-, 2-, and 8-positions, respectively, was purchased from Amersham Life Science (U.K.). Nick translation system was obtained from Bethesda Research Laboratories (Gaithersburg, MD). Calf thymus DNA and snake venom phosphodiesterase were purchased from Sigma Chemical Co. (St. Louis, MO). Calf intestine alkaline phosphatase and Quick Spin columns were purchased from Boehringer Mannheim (Indianapolis, IN). Ecoscint A scintillant was obtained from National Diagnostics (Atlanta, GA). HPLC was carried out on a Hewlett-Packard model 1090 high-pressure chromatograph equipped with a diode array detector and a YMC J′ sphere ODS-M80 250- × 4.6-mm column (Wilmington, NC). 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. Proton NMR spectra and COSY homonuclear 2D spectra were obtained using a Varian500S instrument. Samples were dissolved in DMSO-d6 with tetramethylsilane as an internal standard. Positive ion (+ve) fast atom bombardment (FAB)1 mass spectra (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. Synthesis of the Diastereomers of 1-(2-Hydroxy-2-phenylethyl)deoxyinosine [In1β(R) and In1β(S), 1 and 2]. 1 Abbreviations: BSA, bovine serum albumin; DMF, dimethyl formamide; FAB, fast atom bombardment; PMSF, phenylmethanesulfonyl fluoride.
Deoxyinosine (500 mg, 1.9 mmol, 1 equiv) and anhydrous potassium carbonate (400 mg, 2.8 mmol, 2 equiv) were suspended in dimethyl formamide (DMF) (15 mL) and stirred at 70 °C for 4 h. Racemic styrene oxide (878 µL, 7 mmol, 3.7 equiv) was added, and the mixture was stirred for 4 h at 70 °C before a further addition of racemic styrene oxide (353 µL, 3 mmol, 1.6 equiv). The reaction mixture was stirred for an additional 16 h and concentrated in vacuo. The resulting solid was suspended in 40% (v/v) methanol and filtered. The filtrate was applied to a Sephadex LH-20 column (2.8 × 80 cm) and eluted at a flow rate of 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 products eluted in fractions 5671 and were identified by comparison of the UV spectra with those of literature spectra for 1-substituted inosines (21), and those fractions were pooled. The products were further purified by reversed-phase HPLC eluting isocratically with 25% (v/v) methanol in water. Both diastereomers eluted at 44 min. The experimental procedures described above were repeated at 1/10 of the scale with optically active styrene oxides to identify the individual diastereomers. In1β(R) (1): UV λmax (methanol) 245 (shoulder), 252, 270 (shoulder) nm; 1H NMR (DMSO-d6) δ 8.32 (s, 1, H-8), 8.20 (s, 1, H-2), 7.46 (d, 2, Ar-H, ortho; J ) 7.2 Hz), 7.37 (t, 2, Ar-H, meta; J ) 7.3 Hz), 7.30 (t, 1, Ar-H, para; J ) 6.5 Hz), 6.42 (t, 1, H1′; J ) 6.5 Hz), 4.98 (dd, 1, R-CH; J ) 3.5 and 9.1 Hz), 4.564.54 (m, 1, H3′), 4.46 (dd, 1, β-CHa; J ) 3.7 and 13.6 Hz), 4.04 (dd, 1, H4′; J ) 3.6 and 6.8 Hz), 3.97 (dd, 1, β-CHb; J ) 9.1 and 13.6 Hz), 3.81 (dd, 1, H5′a; J ) 3.5 and 12.1 Hz), 3.73 (dd, 1, H5′b; J ) 3.8 and 12.0 Hz), 2.76-2.70 (m, 1, H2′a), 2.47-2.42 (m, 1, H2′b); +ve FAB MS m/z 373.1547 ([M + H]+; calcd for C18H21N4O5, 373.1512).
840 Chem. Res. Toxicol., Vol. 11, No. 7, 1998 In1β(S) (2): UV λmax (methanol) 245 (shoulder), 252, 270 (shoulder) nm; 1H NMR (DMSO-d6) δ 8.33 (s, 1, H-8), 8.26 (s, 1, H-2), 7.43-7.37 (m, 4, Ar-H, ortho and meta), 7.32-7.29 (m, 1, Ar-H, para), 6.31 (t, 1, H1′; J ) 6.7 Hz), 4.84-4.82 (dd, 1, R-CH), 4.39 (m, 1, H3′), 4.32-4.28 (dd, 1, β-CHa; J ) 3.7 and 13.6 Hz), 3.92-3.89 (m, 1, β-CHb), 3.88-3.85 (m, 1, H4′), 3.623.57 (m, 1, H5′a), 3.55-3.50 (m, 1, H5′b), 2.67-2.60 (m, 1, H2′a), 2.33-2.28 (m, 1, H2′b); +ve FAB MS m/z 373.1509 ([M + H]+; calcd for C18H21N4O5, 373.1512). Synthesis of 1-(2-Hydroxy-1-phenylethyl)deoxyinosine [In1r(R) and In1r(S), 3 and 4. Deoxyinosine (500 mg, 1.9 mmol, 1 equiv) and anhydrous potassium carbonate (400 mg, 2.8 mmol, 2 equiv) were suspended in trifluoroethanol (10 mL) and stirred under reflux for 4 h. Racemic styrene oxide (1.2 mL, 10 mmol, 5.3 equiv) was added in four portions over a period of 4 h. The reaction mixture was stirred under reflux for an additional 16 h. The reaction mixture was concentrated in vacuo and suspended in 40% (v/v) methanol and filtered. The filtrate was applied to a Sephadex LH-20 column (2.8 × 80 cm) and eluted at a flow rate of 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 products eluted in fractions 57-67 and were identified by comparison of the UV spectra with those of literature spectra for 1-substituted inosines (21), and those fractions were pooled. The products were further purified by reversed-phase HPLC eluting isocratically with 22% (v/v) methanol in water. The β-substituted diastereomers eluted at 60 min, and the two R-substituted diastereomers eluted at 69 and 83 min. The experimental procedures described above were repeated at 1/10 of the scale with optically active styrene oxides to identify the individual diastereomers. The earlier running R-substituted diastereomer was identified as In1R(R), and the later running R-substituted diastereomer was identified as In1R(S). In1r(R) (3): UV λmax (methanol) 246 (shoulder), 252, 270 (shoulder) nm; 1H NMR (DMSO-d6) δ 8.46 (s, 1, H-8), 8.33 (s, 1, H-2), 7.43-7.38 (m, 2, Ar-H, ortho), 7.37-7.36 (m, 2, Ar-H, meta), 7.33-7.29 (m, 1, Ar-H, para), 6.31 (t, 1, H1′; J ) 6.6 Hz), 6.05-6.02 (m, 1, R-CH), 4.84-4.82 (m, 1, H3′), 4.39 (m, 1, H4′), 4.32-4.27 (m, 1, β-CHa), 3.88-3.85 (m, 1, β-CHb), 3.613.57 (m, 1, H5′a), 3.53-3.49 (m, 1, H5′b), 2.68-2.60 (m, 1, H2′a), 2.33-2.27 (m, 1, H2′b); +ve FAB MS m/z 373.1621 ([M + H]+; calcd for C18H21N4O5, 373.1512). In1r(S) (4): UV λmax (methanol) 246 (shoulder), 252, 270 (shoulder) nm; 1H NMR (DMSO-d6) δ 8.45 (s, 1, H-8), 8.33 (s, 1, H-2), 7.38-7.36 (m, 4, Ar-H, ortho and meta), 7.32-7.29 (m, 1, Ar-H, para), 6.31 (t, 1, H1′; J ) 6.7 Hz), 6.04 (dd, 1, R-CH; J ) 5.4 and 8.4 Hz), 4.38 (m, 1, H3′), 4.29 (t, 1, β-CHa; J ) 10.2 Hz), 4.10 (m, 1, β-CHb), 3.86 (dd, 1, H4′; J ) 4.4 Hz), 3.60-3.50 (m, 1, H5′a), 3.35-3.29 (m, 1, H5′b), 2.66-2.61 (m, 1, H2′a), 2.322.27 (m, 1, H2′b); +ve FAB MS m/z 373.1480 ([M + H]+; calcd for C18H21N4O5, 373.1512). Synthesis of the Diastereomers of N6-(2-Hydroxy-1phenylethyl)deoxyadenosine [N6r(R) and N6r(S), 5 and 6] and N6-(2-Hydroxy-2-phenylethyl)deoxyadenosine [N6β(R) and N6β(S), 7 and 8]. Deoxyadenosine (500 mg, 1.86 mmol, 1 equiv) and ammonium acetate (100 mg) were dissolved in 50% (v/v) ethanol (70 mL) on warming. (R)- or (S)-Styrene oxide (1.8 mL, 15 mmol, 8 equiv) was added, and the reaction mixture was stirred at room temperature for 96 h. The reaction mixture was concentrated in vacuo, and the residue was washed with diethyl ether (50 mL). The suspension was filtered, and the solid was resuspended in 40% (v/v) methanol (20 mL). The suspension was filtered, and the filtrate was applied to a Sephadex LH-20 column (2.8 × 80 cm) and eluted at a flow rate of 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 products eluted in fractions 83-106 and were identified by comparison of the UV spectra with those of literature spectra for N6-substituted adenosines (21), and those fractions were pooled. The products were further purified by reversed-phase HPLC eluting isocratically with 33% (v/v)
Barlow et al. methanol in water. The stereochemistry of the products was assigned by comparison of the CD spectra of 1-substituted adenosine-styrene oxides of known configuration which had been synthesized previously (10). The N6R(S) product eluted at 31 min, the N6R(R) product eluted at 38 min, the N6β(R) product eluted at 42 min, and the N6β(S) product eluted at 53 min. N6r(R) (5): UV λmax (methanol) 269 nm; 1H NMR (DMSOd6) δ 8.38 (s, 1, H-8), 8.14 (s, 1, H-2), 7.42 (d, 2, Ar-H, ortho; J ) 7.4 Hz), 7.30 (t, 2, Ar-H, meta; J ) 7.4 Hz), 7.20 (t, 1, Ar-H, para; J ) 7.1 Hz), 6.34 (t, 1, H1′; J ) 6.1 Hz), 5.37 (br s, 1, R-CH), 4.56 (d, 1, H3′; J ) 5.0 Hz), 4.04 (d, 1, H4′; J ) 2.4 Hz), 3.803.73 (m, 2, β-CHa,b), 3.62-3.59 (m, 1, H5′a), 3.52-3.49 (m, 1, H5′b), 2.72-2.69 (m, 1, H2′a), 2.27-2.22 (m, 1, H2′b); +ve FAB MS m/z 372.1625 ([M + H]+; calcd for C18H22N5O4, 372.1671). N6r(S) (6): UV λmax (methanol) 269 nm; 1H NMR (DMSOd6) δ 8.30 (s, 1, H-8), 8.18 (s, 1, H-2), 7.44 (d, 2, Ar-H, ortho; J ) 7.4 Hz), 7.32 (t, 2, Ar-H, meta; J ) 7.4 Hz), 7.24 (t, 1, Ar-H, para; J ) 7.3 Hz), 6.42 (t, 1, H1′; J ) 6.2 Hz), 5.48 (br s, 1, R-CH), 4.58-4.56 (d, 1, H3′; J ) 5.0 Hz), 4.06 (dd, 1, H4′; J ) 3.0 and 5.6 Hz), 3.94-3.86 (m, 2, β-CHa,b), 3.82 (dd, 1, H5′a; J ) 2.9 and 12.3 Hz), 3.73 (dd, 1, H5′b; J ) 3.3 and 12.3 Hz), 2.82-2.76 (m, 1, H2′a), 2.42-2.36 (m, 1, H2′b); +ve FAB MS m/z 372.1665 ([M + H]+; calcd for C18H22N5O4, 372.1671). N6β(R) (7): UV λmax (methanol) 269 nm; 1H NMR (DMSOd6) δ 8.28 (s, 1, H-8), 8.18 (s, 1, H-2), 7.22-7.19 (m, 2, Ar-H, ortho), 7.15-7.14 (m, 2, Ar-H, meta), 7.12-7.09 (m, 1, Ar-H, para), 6.42 (t, 1, H1′; J ) 6.2 Hz), 4.95 (br s, 1, R-CH), 4.584.56 (m, 1, H3′), 4.08-4.06 (m, 1, H4′), 3.93-3.89 (m, 1, β-CHa), 3.86-3.83 (m, 1, H5′a), 3.75-3.72 (m, 2, β-CHb and H5′b), 2.832.78 (m, 1, H2′a), 2.42-2.38 (m, 1, H2′b); +ve FAB MS m/z 372.1665 ([M + H]+; calcd for C18H22N5O4, 372.1671). N6β(S) (8): UV λmax (methanol) 269 nm; 1H NMR (DMSO-d6) δ 8.34 (s, 1, H-8), 8.23 (s, 1, H-2), 7.38-7.36 (m, 2, Ar-H, ortho), 7.32 (t, 2, Ar-H, meta; J ) 7.4 Hz), 7.24 (d, 1, Ar-H, para; J ) 7.3 Hz), 6.35 (t, 1, H1′; J ) 6.3 Hz), 4.88 (br s, 1, R-CH), 4.41 (m, 1, H3′; J ) 2.7 Hz), 3.89 (dd, 1, H4′; J ) 4.3 and 6.8 Hz), 3.72 (m, 1, β-CHa), 3.63-3.51 (m, 3, β-CHb and H5′a,b), 2.75-2.70 (m, 1, H2′a), 2.28-2.24 (m, 1, H2′b); +ve FAB MS m/z 372.1663 ([M + H]+; calcd for C18H22N5O4, 372.1671). Synthesis of the Diastereomers of 1-(2-Hydroxy-1-phenylethyl)adenosine [Ado1r(R) and Ado1r(S), 9 and 10] and 1-(2-Hydroxy-2-phenylethyl)adenosine [Ado1β(R) and Ado1β(S), 11 and 12]. Adenosine (300 mg, 1.12 mmol, 1 equiv) and ammonium formate (50 mg) were dissolved in water (20 mL), and optically active styrene oxide (1 mL, 8.8 mmol, 7 equiv) was added. The reaction mixture was stirred at 55 °C for 5 h and then cooled to room temperature. The reaction mixture was washed with diethyl ether (2 × 30 mL), and the aqueous phase was applied to a Sephadex LH-20 column (2.8 × 80 cm) and eluted at a flow rate of 1 mL/min with 30% (v/v) methanol. Absorption of the eluate was monitored continuously at 254 nm, and 8-mL fractions were collected. The products eluted in fractions 35-46, before unreacted adenosine (fractions 58-82) and N6-substituted compounds (fractions 96-113), and were identified by comparison of the UV spectra with those of literature spectra for 1-substituted adenosines (21), and those fractions were pooled. The products were further purified by reversed-phase HPLC eluting isocratically with 15% (v/v) methanol in 50 mM ammonium formate at pH 6.0. Ado1β(R) eluted at 18 min, Ado1β(S) eluted at 20 min, Ado1R(S) eluted at 45 min, and Ado1R(R) eluted at 54 min. Their identities were verified by comparison of UV and 1H NMR spectra with those published (10). Synthesis of the Diastereomers of 1-(2-Hydroxy-1-phenylethyl)deoxyadenosine [dAdo1r(R) and dAdo1r(S), 13 and 14] and 1-(2-Hydroxy-2-phenylethyl)deoxyadenosine [dAdo1β(R) and dAdo1β(S), 15 and 16]. As described above for the preparation of 1-substituted adenosine-styrene oxides except on the Sephadex LH-20 column (2.8 × 80 cm), the products eluted in fractions 45-54, before unreacted deoxyadenosine (fractions 61-79) and N6-substituted compounds (frac-
Deoxyadenosine-Styrene Oxide Adducts in DNA tions 105-114). The products were further purified on reversedphase HPLC eluting isocratically with 18% (v/v) methanol in 50 mM ammonium formate at pH 6.0. dAdo1β(R) eluted at 16 min, dAdo1β(S) eluted at 17 min, dAdo1R(S) eluted at 46 min, and dAdo1R(R) eluted at 54 min. dAdo1r(R) (13): UV λmax (methanol) 259 nm; 1H NMR (CD3OD) δ 8.18 (s, 1, H-2), 8.11 (s, 1, H-8), 7.38-7.26 (m, 5, Ar-H), 6.35 (t, 1, H1′; J ) 6.0 Hz), 6.31 (s, 1, R-CH), 4.55 (s, 1, H3′), 4.24-4.08 (m, 3, H4′ and β-CHa,b), 3.83-3.75 (m, 2, H5′), 2.802.71 (m, 2, H2′); +ve FAB MS m/z 372.1635 (M+; calcd for C18H22N5O4, 372.1670). dAdo1r(S) (14): UV λmax (methanol) 259 nm; 1H NMR (CD3OD) δ 8.16 (s, 1, H-2), 8.09 (s, 1, H-8), 7.39-7.29 (m, 5, Ar-H), 6.42 (t, 1, H1′; J ) 6.1 Hz), 6.29 (s, 1, R-CH), 4.56 (s, 1, H3′), 4.27-4.09 (m, 3, H4′ and β-CHa,b), 3.78-3.62 (m, 2, H5′), 2.822.76 (m, 2, H2′); +ve FAB MS m/z 372.1703 (M+; calcd for C18H22N5O4, 372.1670). dAdo1β(R) (15): UV λmax (methanol) 259 nm; 1H NMR (CD3OD) δ 8.24 (s, 1, H-2), 7.99 (s, 1, H-8), 7.38-7.29 (m, 5, Ar-H), 6.42 (t, 1, H1′; J ) 6.2 Hz), 4.99 (s, 1, R-CH), 4.55 (s, 1, H3′), 4.42-4.38 (m, 1, β-CHa), 4.02-3.93 (m, 2, β-CHb and H4′), 3.803.75 (m, 2, H5′), 2.82-2.70 (m, 2, H2′); +ve FAB MS m/z 372.1655 (M+; calcd for C18H22N5O4, 372.1670). dAdo1β(S) (16): UV λmax (methanol) 259 nm; 1H NMR (CD3OD) δ 8.23 (s, 1, H-2), 8.01 (s, 1, H-8), 7.36-7.28 (m, 5, Ar-H), 6.40 (s, 1, H1′), 5.02 (s, 1, R-CH), 4.56 (s, 1, H3′), 4.42-4.37 (m, 1, β-CHa), 4.00-3.93 (m, 2, β-CHb and H4′), 3.82-3.75 (m, 2, H5′), 2.80-2.69 (m, 2, H2′); +ve FAB MS m/z 372.1697 (M+; calcd for C18H22N5O4, 372.1670). Kinetics of Deamination and Dimroth Rearrangement. Samples of 1-substituted adenosines and deoxyadenosines (∼5 A260) were incubated in 50 mM Tris/HCl at pH 7.0 (0.2 mL) at 37 °C, and aliquots (20 µL) were analyzed at various times by reversed-phase HPLC. Elution was at 1 mL/min using the following solvents: A, 50 mM ammonium formate (pH 6.0); B, methanol in conjunction with the following gradient of B in A, 0 min, 15%; 20 min, 15%; 40 min, 35%; 100 min, 35%. Products were identified by their characteristic UV spectra [UV λmax of 259 nm for 1-substituted (deoxy)adenosines, UV λmax of 252 nm and a shoulder at 270 nm for 1-substituted (deoxy)inosines, and UV λmax of 268 nm for N6-substituted (deoxy)adenosines] and were quantified by integration of the peaks. Retention times were 27 min for β1-substituted adenosines, 30 and 32 min for R1-substituted adenosines, 41 min for β1-substituted inosines, 44 and 47 min for R1-substituted inosines, 69 and 71 min for RN6-substituted adenosines, 75 and 77 min for βN6-substituted adenosines, 38 and 39 min for β1-substituted deoxyadenosines, 45 and 48 min for R1-substituted deoxyadenosines, 51 min for β1-substituted deoxyinosines, 53 and 55 min for R1-substituted deoxyinosines, 75 and 78 min for RN6-substituted deoxyadenosines, and 81 and 83 min for βN6-substituted deoxyadenosines. Preparation of DNA Specifically Radiolabeled at Adenine Residues. [3H]Deoxyadenosine triphosphate (5 µL, 5 µCi) was aspirated to dryness, and calf thymus DNA (50 µL, 50 µg in water) was added with the following components from a nick translation kit from Bethesda Research Laboratories (Gaithersburg, MD): deoxyguanosine triphosphate, deoxycytidine triphosphate, and deoxythymidine triphosphate (0.2 mM of each triphosphate in 25 µL of buffer containing 0.5 M Tris/ HCl, 50 mM magnesium chloride, and 0.1 M 2-mercaptoethanol at pH 7.8) and Pol I/DNase I mix [25 µL of 0.5 U/µL DNA polymerase I, 0.4 mU/µL DNase I in 50 mM Tris‚HCl, 5 mM magnesium acetate, 0.1 mM phenylmethanesulfonyl fluoride (PMSF), 50% (v/v) glycerol, and 100 µg/mL nuclease-free bovine serum albumin (BSA) at pH 7.5]. The mixture was vortexed for 5 s and incubated at 15 °C for 1 h before addition of stop buffer (25 µL of 0.5 M EDTA at pH 8.0). The DNA was purified with a Quick Spin Sephadex G-50 column equilibrated in 50 mM Tris/HCl at pH 7.0. The DNA was collected in 50 mM Tris/ HCl (100 µL), and the remaining impurities on the column were also collected in 50 mM Tris/HCl (100 µL). The radioactivity of aliquots (5 µL) of both solutions was determined by liquid
Chem. Res. Toxicol., Vol. 11, No. 7, 1998 841 scintillation counting to estimate the incorporation of [3H]deoxyadenosine triphosphate into the calf thymus DNA. Reactions of [3H]Deoxyadenosine with Optically Active Styrene Oxide. To 50 mM Tris/HCl at pH 7.0 (1 mL) were added [3H]deoxyadenosine triphosphate (1 µL, 1 µCi), calf thymus alkaline phosphatase2 (15 U), and snake venom phosphodiesterase (0.05 U), and the mixture was stirred at 37 °C for 24 h. (R)- or (S)-Styrene oxide (5 µL) was added, and the reaction mixture was incubated at 37 °C for a further 48 h. Aliquots (250 µL) were used to dissolve a mixture containing deoxyadenosine, In1β(R), In1β(S), In1R(R), In1R(S), N6R(R), N6R(S), N6β(R), and N6β(S). The mixture was separated by reversed-phase HPLC eluting at 1 mL/min using the following solvents: A, 50 mM ammonium formate (pH 6.0); and B, methanol in conjunction with the following gradient of B in A, 0 min, 20%; 70 min, 28%; 150 min, 28%. Typical retention times for the individual products were as follows: deoxyadenosine, 9 min; In1β(R), 56 min; In1β(S), 57 min; In1R(R), 60 min; In1R(S), 63 min; N6R(S), 94 min; N6R(R), 109 min; N6β(R), 122 min; and N6β(S), 126 min. Fractions (1 mL) were collected throughout the elution, and the radioactivity in each fraction was determined by liquid scintillation counting. Reactions of [3H]Deoxyadenosine Labeled Calf Thymus DNA with Optically Active Styrene Oxides. The procedure used above for reaction with [3H]dAdo was repeated using [3H]dAdo-labeled calf thymus DNA (10 µg, ∼1 µCi). For reactions with denatured calf thymus DNA, the solutions were incubated at >90 °C for 5 min and cooled to room temperature prior to addition of the styrene oxide. The DNA was purified with a NAP-10 Sephadex G 25 column, equilibrated in 50 mM Tris/ HCl at pH 7.0, to remove unreacted styrene oxide. Calf thymus alkaline phosphatase (15 U) and snake venom phosphodiesterase (0.05 U) were added, and the reaction mixture was incubated at 37 °C for 48 h (the DNA was found to be completely digested under these conditions).
Results and Discussion To determine whether the deamination and Dimroth rearrangement previously observed for 1-substituted Ado-styrene oxide adducts (10) could arise in DNA, markers for the expected deoxyribonucleoside adducts were prepared, the rates of these reactions in 1-substituted ribosides and deoxyribosides were compared, and radioactively labeled products were quantified in denatured and native DNA. To facilitate these investigations, markers of four pairs of diastereomers, i.e., two pairs of R- and βN6-substituted dAdo diastereomers and two pairs of R- and β1-substituted dIno diastereomers, anticipated as products in the various reactions, were prepared (Scheme 1). Preparation of Marker Compounds. The N6substituted dAdo markers (5-8) were prepared from reactions of optically active styrene oxide and dAdo in 50% ethanol. Each reaction gave two major products in comparable yields. These were separated by HPLC and identified as R- and βN6-substituted products by UV, 1H NMR, and mass spectroscopies. The regiochemistry of epoxide ring opening was assigned on the basis of the chemical shift of the R-methine proton that appeared at lower field (∼5.43 ppm) in RN6-substituted products (5 and 6) than in βN6-substituted products (7 and 8) (∼4.91 ppm) due to the electron-withdrawing effect of the adjacent aromatic amino group. The resonances of the β-methylene protons also shifted in a similar manner, 2 The alkaline phosphatase used in the enzymatic digestions contains a small amount of deaminase activity. It was found that this did not produce detectable quantities of deaminated products in the time scale of the experiments.
842 Chem. Res. Toxicol., Vol. 11, No. 7, 1998
Barlow et al. Table 1. Rates of Deamination and Dimroth Rearrangement and pKa of 1-Substituted Ado- and dAdo-Styrene Oxides compda
rate of deamination (h-1)
rate of dimroth rearrangement (h-1)
pKab
Ado1β Ado1R dAdo1β dAdo1R
NDc 0.036 0.002 0.033
0.030 0.022 0.013 0.015
7.95 8.34 8.52 8.45
a Values were calculated for each diastereomer, but the chirality of the alkyl chain did not seem to have an appreciable effect on the rate of deamination or Dimroth rearrangement or on pKa. b The pK values were determined spectrophotometrically. c The a rate could not be calculated as the quantities of products were too small to be determined.
Figure 1. CD spectra of N6-substituted adenosine-styrene oxide adducts overlayed on the CD spectra of N6-substituted deoxyadenosine-styrene oxide adducts. Panel A: RN6-Substituted adenosine- and deoxyadenosine-styrene oxide adducts. Panel B: βN6-Substituted adenosine- and deoxyadenosinestyrene oxide adducts.
from ∼3.73 ppm in RN6-substituted compounds to ∼3.83 ppm in βN6-substituted compounds. The configurations of each compound were assigned by comparison of CD spectra of previously synthesized RN6- and βN6-substituted styrene oxide-Ado adducts of known configuration (Figure 1) (10). As expected, the β-substituted products had the same configuration as the starting epoxide, and the configuration of the major R-substituted products was the inverse of that in the starting epoxide (10). The 1-substituted styrene oxide-dIno markers (1-4) were prepared in reactions of racemic styrene oxide with dIno in the presence of base, which converts the dIno into a more reactive anionic form. In either DMF or trifluoroethanol, the reactions yielded predominantly β1substituted products (1 and 2), with ratios of R1- to β1substituted product of ∼15:85 in DMF and ∼40:60 in trifluoroethanol. The pairs of diastereomeric R1-substituted products (3 and 4) were separated by HPLC, but separation of the β1-substituted diastereomers proved very difficult. The β-substituted diastereomers were characterized, therefore, from small-scale reactions using optically active styrene oxides. These products were characterized by UV, 1H NMR, and mass spectroscopies, and the position of epoxide substitution was assigned using the R-methine proton resonances in the 1H NMR spectra which shifted with the proximity of the electronwithdrawing aromatic ring nitrogen to the R-methine proton. The R-methine resonance shifted from ∼6.03 ppm in R1-substituted dIno diastereomers to ∼4.90 ppm in the β1-substituted compounds; however, the β-methylene proton resonances did not appreciably shift in R1substituted dIno compounds to β1-substituted dIno compounds. The configurations of the R1-substituted diastereomers were assigned by comparison of the HPLC retention times with those of products from small-scale reactions using optically active styrene oxides, knowing from previous findings (9, 10, 15, 17, 22) that reactions of epoxides at the ring nitrogen of nucleosides proceed with inversion of configuration of the starting epoxide. As formation of the β1-substituted dIno compounds does
not disrupt bonding at the chiral center, these products could be assigned the configuration of the starting epoxide. R- and β1-Substituted adducts of Ado (9-12) and dAdo (13-16) were prepared from reactions of styrene oxide and Ado or dAdo in water (Scheme 1). Reaction times of 5 h at temperatures of ∼50 °C favored greater yields of product than reactions conducted at room temperature over 16 h. Both R- and β1-substituted dAdo- and Adostyrene oxides were formed in approximately equal quantities, and their identities were established by UV, 1 H NMR, and mass spectroscopic techniques. Spectra for the ribosides were the same as those previously published (10). The stereochemical assignments for the diastereomers of the R- and β1-substituted dAdo-styrene oxide adducts were made based on the assumption that these eluted from HPLC in the same sequence as in the case of the ribose analogues described previously (10). Kinetics of Deamination and Dimroth Rearrangement. To examine any contribution of the 2′-hydroxyl group to the kinetics of deamination and Dimroth rearrangement of R- and β1-substituted styrene oxide adducts, 1-substituted Ado- and dAdo-styrene oxides were incubated at 37 °C and pH 7.0 (Table 1) and the relative proportions of deaminated and Dimroth rearrangement products formed over time were monitored by HPLC. The stereochemistry of the alkyl side chain had little effect on either the deamination or the Dimroth rearrangement in diastereomerically related pairs of compounds, and the kinetic experiments (Table 1) with 1-substituted Adostyrene oxides mirrored those previously published by Qian and Dipple (10). However, kinetic experiments involving the 1-substituted dAdo-styrene oxides revealed that, although deamination of both R- and β1-substituted dAdo-styrene oxides was unaffected by the absence of the 2′-hydroxyl group, the rate of Dimroth rearrangement was reduced in 1-substituted dAdo-styrene oxides relative to the ribofuranosyl analogues (Table 1). Also, in concert with previous suggestions by Fujii et al. (23), the rate of Dimroth rearrangement of the 1-substituted adenine derivatives correlated with pKa (Table 1). Thus, the electron-withdrawing effect of the 2′-hydroxyl group promotes attack of hydroxide on the positively charged purine ring, thereby enhancing the rate of the Dimroth rearrangement. Deamination and Dimroth Rearrangement in DNA. Since it is not possible to prepare DNA containing 1-substituted adenine residues and examine the kinetics of deamination or Dimroth rearrangement, experiments to measure these reactions in DNA were limited to product ratio determinations of the stable reaction prod-
Deoxyadenosine-Styrene Oxide Adducts in DNA
Figure 2. HPLC separation of stable dAdo-derived styrene oxide adducts. Panel A: Chromatogram separating a mixture of the synthesized marker compounds. Panel B: Radiochromatogram separating the mixture of products formed in a reaction between (R)-styrene oxide and [3H]dAdo. Panel C: Radiochromatogram separating the mixture of products formed in a reaction between (S)-styrene oxide and [3H]dAdo incorporated into native DNA. Peaks: 1, dAdo; 2, In1β(R) or In1β(S); 3, In1β(R) or In1β(S); 4, In1R(R); 5, In1R(S); 6, N6R(S); 7, N6R(R); 8, N6β(R); 9, N6β(S).
ucts formed on incubation of DNA with styrene oxide. Since styrene oxide is known to form products with all four nucleosides (7), reactions were undertaken with DNA radiolabeled specifically at Ade residues. Thus, simultaneous reactions were carried out with [3H]dAdo and with quantities of native and denatured DNA containing quantities of [3H]dAdo approximately equal to that used in nucleoside reactions. Optically active styrene oxides were used in all the radioisotopic reactions to identify any stereochemical differences in the reactions. After hydrolysis of the nucleic acid reaction products to nucleosides, the radioactive products were separated by HPLC using conditions that separated all the markers (Figure 2A) (the peaks corresponding to the β1-substituted dIno and βN6-substituted dAdo compounds that were not well-resolved by HPLC could be unambiguously assigned as optically active styrene oxides had been used). Prior to enzymatic digestion the DNA was purified to remove styrene-1,2-diol and excess styrene oxide. It may be expected that styrene oxide would produce adducts at the 3-position of adenine residues in DNA as reported for epoxides such as butadiene mono- and dioxides (24, 25). However, due to the
Chem. Res. Toxicol., Vol. 11, No. 7, 1998 843
extreme lability of the glycosidic bond in 3-substituted dAdo, these rapidly depurinate (26) and would be removed during the DNA purification step. Separation of the products formed from radiolabeled dAdo allowed the product yields to be determined from reactions between (R)- and (S)-styrene oxides and [3H]dAdo [illustrated for (R)-styrene oxide-dAdo products in Figure 2B] and also, after digestion to nucleosides, from reactions of [3H]dAdo when incorporated within denatured and native DNA2 [illustrated for (S)-styrene oxide-dAdo products in Figure 2C]. The results from triplicate analyses of reactions of (R)and (S)-styrene oxide are summarized in Table 2. The overall yields of dAdo converted to products were low and decreased with greater structural complexity such that 0.6% of [3H]dAdo was converted to products in native DNA whereas 3% of [3H]dAdo was converted to products in denatured DNA (the reaction times and concentrations of dAdo in the nucleoside reactions differed from those in the DNA reactions and are not, therefore, directly comparable to those in DNA). Both the 1- and N6positions of dAdo are somewhat hindered in denatured DNA by the phosphate backbone and by the 3′- and 5′flanking nucleosides, but in native DNA these sites are internalized due to their participation in Watson-Crick hydrogen bond base pairing with dThd residues. The accessibility of these reaction sites may determine their relative reactivities in native and denatured DNA. The environment of the dAdo residues also affected the relative yields of the four pairs of reaction products. It has been suggested previously that the adenine 1-position is protected from alkylation in native DNA by steric protection from the double-helical structure and because the lone pair electrons on the ring nitrogen are involved in hydrogen bonding and are therefore less nucleophilic (27). However, the results from this study indicate that at least half of the styrene oxide adducts derived from dAdo in native DNA result from initial reaction at the ring nitrogen (calculated as the sum of 1-substituted dIno and βN6-substituted dAdo) [it has been demonstrated that βN6-substituted dAdo-styrene oxide adducts can only form from initial reaction at the 1-position followed by Dimroth rearrangement (10-12)], irrespective of whether dAdo was a nucleoside or incorporated into DNA. This can perhaps be rationalized by DNA “breathing”, where the double-helical structure relaxes and opens to expose the hydrogen-bonding sites (28). This could allow reaction at these positions. The degree of racemization of RN6-substituted products in DNA was greater than that found with the nucleoside. The ratios of inverted to retained products ranged from 5.25-4.5:1 in reactions of dAdo nucleoside to 3.4-2.6:1 in reactions of denatured and native DNA. Direct alkylation at the N6-position occurs with a substrate of considerable ionic character, leading to some product racemization (10). It has been suggested that DNA structure can promote the ionization of epoxides due to a local pH drop around both denatured and native DNA arising from the higher concentrations of hydronium counterions around the phosphate backbone (29-31). This localized field of hydronium ions is thought to promote epoxide ring opening by protonation of the substrate and thereby favoring reaction at the exocyclic amino group (29-31). The ratio of β- to R-substituted products increased in the DNA reactions relative to the nucleoside reactions
844 Chem. Res. Toxicol., Vol. 11, No. 7, 1998
Barlow et al.
Table 2. Products as a Fraction of Total Products Given as Ratios of Diastereomers (S/R)a epoxide dAdo
R
ssDNA
R
dsDNA
R
dAdo
S
ssDNA
S
dsDNA
S
β1-dIno 0/0.06 0/((0.002) 0/0.11 0/((0.002) 0/0.18 0/((0.005) 0.04/0 ((0.001)/0 0.11/0 ((0.014)/0 0.21/0 ((0.018)/0
R1-dIno 0.13/0 ((0.005)/0 0.13/0 ((0.005)/0 0.11/0 ((0.011)/0 0/0.13 0/((0.005) 0/0.17 0/((0.007) 0/0.14 0/((0.011)
RN6-dAdo 0.42/0.08 ((0.007)/((0.003) 0.34/0 ((0.005)/((0.005) 0.34/0.13 ((0.017)/((0.010) 0.10/0.45 ((0.004/(0.004) 0.11/0.33 ((0.012)/((0.006) 0.11/0.31 ((0.011)/((0.008)
βN6-dAdo 0/0.31 0/((0.001) 0/0.32 0/((0.006) 0/0.24 0/((0.02) 0.28/0 ((0.005)/0 0.28/0 ((0.004)/0 0.23/0 ((0.023)/0
a Conversion of dAdo to products averaged ∼8% in reactions with dAdo, ∼3% in reactions with dAdo incorporated into denatured DNA, and ∼0.6% in reactions with dAdo incorporated into native DNA. Results are given as the mean of three determinations, and standard deviations of the mean values are given in parentheses.
although the relative levels of βN6-substituted products, which can only be formed from Dimroth rearrangement from β1-substituted products (10-12), decreased in reactions with native DNA. However, the relative proportions of β1-substituted dIno products increased with structural complexity. The β1-substituted dIno products comprised ∼5% of total products in nucleoside reactions, ∼10% of total products in reactions with denatured DNA, and ∼20% of total products in reactions with native DNA (Table 2). This observation that the proportion of deaminated products increased when dAdo was incorporated into DNA implies that DNA structure somehow favors this reaction (Table 2). It has been suggested that deamination in such 1-substituted hydroxyalkyladenines is facilitated by an intramolecular cyclization where the hydroxyl group displaces the amino group from the purine 6-position (13, 23). If this were the case, it is conceivable that the structure of both denatured and native DNA may promote cyclization of β1-substituted dAdo-styrene oxide adducts by orientating the alkyl side chain to place the hydroxyl group in a favorable position to displace the amino group from the 6-carbon of the purine. It is also possible that the competing Dimroth rearrangement reaction is impeded by the 3′- and 5′flanking nucleosides, as rotation around the C5-C6 bond of the purine, which is required for Dimroth rearrangement, may be hindered.
Summary In conclusion, the findings of this study have demonstrated that the rate of Dimroth rearrangement of 1-substituted dAdo-styrene oxide adducts is slower than that of the riboside analogues resulting in greater yields of deaminated compounds than found from 1-substituted Ado-styrene oxides. In reactions of styrene oxide with adenine residues in DNA, both R- and βN6-substituted dAdo and 1-substituted dIno adducts are formed, and the relative proportions of products differed depending upon the structural complexity of the system under study. Denatured and native DNA favored formation of β1substituted dIno products relative to the other products and also increased the proportion of retained to inverted configuration of RN6-substituted products. RN6-Substituted dAdo-styrene oxide adducts have been shown to possess little miscoding potential in vivo (32), possibly because these adducts retain the ability to hydrogen-bond base pair with dThd. Although, 1-substituted dIno-styrene oxide adducts are formed at lower
levels than N6-substituted dAdo-styrene oxide adducts in styrene oxide reactions with DNA, they may greatly contribute to the mutagenic properties of styrene oxide as both hydrogen-bonding sites of dAdo are disrupted.
Acknowledgment. We thank J. Klose and M. Maguire for NMR studies and Dr. C. J. Metral for MS studies. Research was sponsored by the National Cancer Institute DHHS, under contract with ABL.
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