Chem. Res. Toxicol. 1999, 12, 883-886
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Formation of Deaminated Products in Styrene Oxide Reactions with Deoxycytidine Thomas Barlow* and Anthony Dipple† Chemistry of Carcinogenesis Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, P.O. Box B, Frederick, Maryland 21702 Received April 23, 1999
The reaction of racemic styrene oxide with deoxycytidine under aqueous conditions was studied. The four principal products isolated were a pair of diastereomeric N4-(2-hydroxy-1phenylethyl)deoxycytidines (∼20% of the products) and a pair of diastereomeric 3-(2-hydroxy2-phenylethyl)deoxyuridines (∼80% of the products). Reactions with optically active styrene oxides allowed the configurations of the 3-(2-hydroxy-2-phenylethyl)deoxyuridines to be assigned, and these structures were confirmed by an independent synthesis from deoxyuridine. Also, it was possible to tentatively assign the configurations of the N4-(2-hydroxy-1-phenylethyl)deoxycytidines that had undergone some racemization during the reaction (the ratio of the retained to inverted configuration of the products was ∼1:7).
Introduction Styrene oxide, the principal metabolite of the industrial chemical styrene (1), has been reported to be mutagenic (2) and carcinogenic (3). This biological activity has prompted studies of styrene oxide binding to nucleic acids (4-19). Savela et al. (9, 13) have reported that reaction of deoxycytidine with styrene oxide gave N4-, 3-, and O2substituted styrene oxide adducts of deoxycytidine. However, these products were characterized solely on the basis of their pKa values and their UV spectra. Similar distributions of products were reported for reactions of deoxycytidine with other alkyl epoxides such as propylene oxide (20), ethylene oxide (21), cyanoethylene oxide (22), and butadiene monoepoxide (23); however, in addition to N4-, 3-, and O2-substituted deoxycytidine products, the 3-substituted products were found to subsequently undergo deamination to give rise to 3-substituted deoxyuridines. An analogous deamination of 1-substituted styrene oxide-adenosine (17) and -deoxyadenosine (16) adducts has been elucidated. To determine whether styrene oxide can induce a deamination of deoxycytidine, similar to that found for the alkyl epoxides (20-23), its reaction with deoxycytidine under physiological conditions was reinvestigated. Predominantly, the reaction yielded deaminated products characterized as β3-substituted deoxyuridines, as well as minor quantities of RN4-substituted deoxycytidines (Figure 1). The 3-substituted deoxyuridine-styrene oxide adducts, if found in DNA, are potentially promutagenic since two of the three Watson-Crick hydrogen bonding sites of deoxycytidine have been disrupted.
Experimental Procedures Caution: Styrene oxide is mutagenic and/or carcinogenic and should be handled with care. * To whom correspondence should be addressed: Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, U.K. † Deceased May 26, 1999.
Figure 1. Reactions of deoxycytidine with styrene oxide. Chemicals were used as purchased from the manufacturer. Deoxycytidine, deoxyuridine, and racemic and optically active styrene oxides were purchased from Aldrich Chemical Co. (Milwaukee, WI). Sephadex LH-20 was purchased from Pharmacia Biotech Inc. (Piscataway, NJ). 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 mm × 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 two-dimensional spectra were obtained using a Varian-500S instrument. Positive ion (+ve) fast atom bombardment (FAB)1 mass spectra (MS) were obtained with a reverse geometry VG Micromass ZAB-2F spectrometer interfaced with a VG 203S data system using glycerol as the FAB matrix. Reaction of Deoxycytidine with Racemic and Optically Active Styrene Oxide. Deoxycytidine monohydrate (200 mg, 0.82 mmol, 1 equiv) was dissolved in 50 mM Tris-HCl at pH 1
Abbreviation: FAB, fast atom bombardment.
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7.0 (20 mL), and racemic styrene oxide (1 mL, 8.8 mmol, 11 equiv) was added. The reaction mixture was stirred at 37 °C for 72 h and cooled to room temperature. The reaction mixture was washed with diethyl ether (2 × 20 mL), and the aqueous phase was applied to a Sephadex LH-20 column (2.8 cm × 80 cm) and eluted at a flow rate of 1 mL/min with 40% (v/v) methanol/water. [A small sample was retained for direct analysis by reversed phase HPLC eluting isocratically with 30% (v/v) methanol in 50 mM ammonium formate (pH 6).] Absorption of the eluate was monitored continuously at 254 nm, and 8 mL fractions were collected. The reaction products eluted after unreacted deoxycytidine (fractions 34-46) and were identified by comparison of their UV spectra under neutral, basic, and acidic conditions with those of known cytidine and uridine adducts (24). N4-substituted deoxycytidines eluted in fractions 48-53, and 3-substituted deoxyuridines eluted in fractions 5473. After appropriate fractions had been pooled, the products were further purified by reversed phase HPLC. With isocratic elution using 20% (v/v) methanol/water, two deoxycytidine adducts eluted at 34 (dCydI) and 36 min (dCydII). With isocratic elution with 30% (v/v) methanol/water, two deoxyuridine adducts eluted at 27 (dUrdI) and 29 min (dUrdII). The reactions were repeated at one-tenth of this scale with optically active styrene oxides and analyzed directly by reversed phase HPLC eluting isocratically with 30% (v/v) methanol/50 mM ammonium formate (pH 6). dCydI: UV λmax [40% (v/v) methanol/water] 241, 274 (pH 7), 274 (pH 13), 287 nm (pH 1); 1H NMR (acetone-d6) δ 7.85 (d, 1, H-6, J ) 7.4 Hz), 7.42-7.22 (m, 6, Ar-H and NH which exchanges with D2O), 6.22 (t, 1, H1′, J ) 6.9 Hz), 5.95 (d, 1, H-5, J ) 7.5 Hz), 5.32 (d, 1, R-CH, J ) 5.9 Hz), 4.44 (s, 1, H3′), 4.40 (s, 1, OH which exchanges with D2O), 4.34 (s, 1, OH which exchanges with D2O), 4.28 (s, 1, OH which exchanges with D2O), 3.91 (s, 1, H4′), 3.83-3.73 (m, 4, β-CH, H5′), 2.26-2.15 (m, 2, H2′); +ve FAB MS (relative intensity) m/z 348 ([M + H]+, 100), 232 ([M + H]+ - ribose, 30). dCydII: UV λmax [40% (v/v) methanol/water] 241, 273 (pH 7), 274 (pH 13), 287 nm (pH 1); 1H NMR (acetone-d6) δ 7.85 (d, 1, H-6, J ) 7.4 Hz), 7.42-7.23 (m, 6, Ar-H and NH which exchanges with D2O), 6.20 (t, 1, H1′, J ) 6.6 Hz), 5.95 (d, 1, H-5, J ) 7.2 Hz), 5.32 (d, 1, R-CH, J ) 5.5 Hz), 4.45 (s, 1, H3′), 4.40 (s, 1, OH which exchanges with D2O), 4.34 (s, 1, OH which exchanges with D2O), 4.28 (s, 1, OH which exchanges with D2O), 3.91 (s, 1, H4′, J ) 3.1 and 6.4 Hz), 3.82-3.70 (m, 4, β-CH, H5′), 2.28-2.16 (m, 2, H2′); +ve FAB MS (relative intensity) m/z 348 ([M + H]+, 100), 232 ([M + H]+ - ribose, 20). dUrdI: UV λmax [40% (v/v) methanol/water] 263 (pH 7), 264 (pH 13), 262 nm (pH 1); 1H NMR (acetone-d6) δ 8.01 (d, 1, H-6, J ) 8.1 Hz), 7.45-7.24 (m, 5, Ar-H), 6.28 (t, 1, H1′, J ) 6.7 Hz), 5.68 (d, 1, H-5, J ) 7.5 Hz), 5.06 (d, 1, R-CH, J ) 5.9 Hz), 4.52 (s, 1, OH which exchanges with D2O), 4.49 (s, 1, H3′, J ) 3.0 Hz), 4.48 (s, 1, OH which exchanges with D2O), 4.47 (s, 1, OH which exchanges with D2O), 4.25 (dd, 1, β-CH, J ) 8.8 and 13.2 Hz), 4.10 (dd, 1, β-CH, J ) 8.7 and 13.2 Hz), 3.96 (d, 1, H4′, J ) 3.2 Hz), 3.79 (dd, 2, H5′, J ) 3.5 and 4.6 Hz), 2.30-2.15 (m, 2, H2′); +ve FAB MS (relative intensity) m/z 349 ([M + H]+, 25), 233 ([M + H]+ - ribose, 21). dUrdII: UV λmax [40% (v/v) methanol/water] 263 (pH 7), 264 (pH 13), 262 nm (pH 1); 1H NMR (acetone-d6) δ 8.01 (d, 1, H-6, J ) 8.1 Hz), 7.45-7.24 (m, 5, Ar-H), 6.28 (dd, 1, H1′, J ) 6.6 and 11.6 Hz), 5.68 (d, 1, H-5, J ) 7.5 Hz), 5.06 (s, 1, R-CH), 4.54 (s, 1, OH which exchanges with D2O), 4.49 (s, 1, H3′, J ) 3.2 Hz), 4.47 (s, 1, OH which exchanges with D2O), 4.43 (s, 1, OH which exchanges with D2O), 4.25-4.21 (m, 1, β-CH), 4.124.10 (m, 1, β-CH), 3.98 (d, 1, H4′, J ) 3.4 Hz), 3.79-3.75 (m, 2, H5′), 2.32-2.16 (m, 2, H2′); +ve FAB MS (relative intensity) m/z 349 ([M + H]+, 50), 233 ([M + H]+ - ribose, 27). Reaction of Deoxyuridine with Racemic Styrene Oxide. Deoxyuridine (200 mg, 0.88 mmol, 1 equiv) and ammonium formate (50 mg) were dissolved in water (20 mL), and racemic styrene oxide (1 mL, 8.8 mmol, 10 equiv) was added. The reaction mixture was stirred at 37 °C for 72 h and cooled to
Barlow and Dipple
Figure 2. HPLC separation of deoxycytidine-derived styrene oxide adducts: (A) reaction with (R)-styrene oxide, (B) reaction with (S)-styrene oxide, and (C) reaction with racemic styrene oxide. room temperature. The reaction mixture was washed with diethyl ether (2 × 20 mL), and the aqueous phase was applied to a Sephadex LH-20 column (2.8 cm × 80 cm) and eluted at a flow rate of 1 mL/min with 40% (v/v) methanol/water. Absorption of the eluate was monitored continuously at 254 nm, and 8 mL fractions were collected. The products eluted after unreacted deoxyuridine (fractions 39-49). Products were identified by comparison of the UV spectra under neutral, basic, and acidic conditions with those of known uridine adducts (24). 3-Substituted deoxyuridines eluted in fractions 55-71. After pooling, the products were further purified by reversed phase HPLC eluting isocratically with 30% (v/v) methanol/water. dUrdI eluted at 27 min, and dUrdII eluted at 29 min. The products had UV, 1H NMR, and MS spectra consistent with structures previously assigned to dUrdI and dUrdII (see above).
Results To determine if styrene oxide can generate deoxyuridine adducts from reaction with deoxycytidine, as reported for several alkyl epoxides (20-23), deoxycytidine was allowed to react with optically active and racemic styrene oxide under aqueous conditions at pH 7 and 37 °C. The resulting products were separated by reversed phase HPLC and characterized by spectral methods. The HPLC profiles resulting from analyses of reactions of deoxycytidine with (R)-, (S)-, and racemic styrene oxide are shown in panels A-C of Figure 2, respectively. The racemic epoxide gave two deoxycytidine and two deoxyuridine products (Figure 2C). However, each optically active epoxide (panels A and B of Figure 2) gave largely only one of each of these products. This finding suggests that the products at 28 and 29 min, i.e., dCydI and dCydII, are diastereomers and that the products at 49 and 54 min, i.e., dUrdI and dUrdII, are also diastereomers. Each of these four products was isolated from the
Styrene Oxide-Deoxycytidine Adducts
Figure 3. CD spectra of deoxycytidine-derived styrene oxide adducts: (A) RN4-substituted deoxycytidine adducts and (B) β3substituted deoxyuridine adducts.
reaction with racemic styrene oxide by Sephadex LH-20 chromatography followed by HPLC, and each was characterized by 1H NMR, MS, UV, and CD spectroscopy. For dCydI and dCydII, UV spectra were identical and 1H NMR data were similar. Likewise, dUrdI and dUrdII exhibited identical UV spectra and similar 1H NMR data. These similarities suggested that each member of the pair was a diastereomer of the other member of the pair, and this suggestion was solidified when each member of a pair was found to have a circular dichroism spectrum that was equal in intensity to but opposite in sign from the other. These spectra for dCydI and dCydII, and for dUrdI and dUrdII, are shown in Figure 3. The earlier running pair of diastereomers, dCydI and dCydII, which constituted 18% of the total products that were formed, were tentatively characterized as N4substituted deoxycytidine products by comparison of their UV spectra under neutral, basic, and acidic conditions with those previously published (24). This assignment was confirmed by the presence of a single aromatic amino proton resonance in the 1H NMR spectra and a protonated molecular ion of m/z 348 in the mass spectra. Additionally, COSY 1H NMR experiments revealed crosspeaks between the hydroxyl group, formed from the epoxide ring opening, and the β-alkyl protons, but not to the R-alkyl protons, indicating that the β-protons and the hydroxyl proton were coupled. Such coupling should be possible only in products substituted at the R-carbon. Thus, the regiochemistry of epoxide substitution of the N4-substituted products was assigned at the R-carbon. The configurational assignment of the N4-substituted products was made from the observation that although some racemization had occurred during the reactions, the products were not completely racemized (optical isomer ratios between 1:6 and 1:8 were found). It would be expected that these nucleophilic reactions would proceed predominantly with inversion of configuration. Indeed, it is difficult to imagine a chemical mechanism whereby retention of configuration would be favored. Therefore, the reaction products were assigned configurations that were the inverse of that of the starting epoxide. Thus, dCydI was assigned the (R)-configuration and dCydII was assigned the (S)-configuration. The second pair of diastereomers isolated (dUrdI and dUrdII) constituted 82% of the total products that were formed. These were tentatively characterized as 3-substituted deoxyuridines, on the basis of the relationship of their UV spectra under neutral, basic, and acidic conditions to those previously published (24). The protonated molecular ion of m/z 349 was consistent with this characterization, as was the absence of any amino
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protons in the 1H NMR spectra. In a manner analogous to that found for dCydI and dCydII, the COSY 1H NMR spectra also revealed cross-peaks between the alkyl and hydroxyl protons. However, in the deaminated compounds, it was the R-proton and the hydroxyl proton that were coupled, indicating that the site of substitution in these products was the β-carbon of the epoxide. These structural assignments were confirmed by the synthesis of 3-substituted deoxyuridines by reaction of racemic styrene oxide with deoxyuridine (Figure 1). The resulting compounds had UV, CD, 1H NMR, and mass spectra that were identical with those of dUrdI and dUrdII. The reactions of deoxycytidine and optically active styrene oxides allowed the configurations of UrdI and UrdII to be assigned. As these products are substituted at the β-carbon of the epoxide and not at the chiral R-carbon, the configuration at the chiral carbon is undisturbed during the reaction. Therefore, these products were assigned the configurations of the starting epoxides. Thus, dUrdI was assigned the (R)-configuration and dUrdII was assigned the (S)-configuration.
Discussion The pathway from deoxycytidine to the products, dUrdI and dUrdII, must first involve alkylation of the ring nitrogen of deoxycytidine and then a rapid deamination of this unstable charged pyrimidine ring system (Figure 1). As with previously reported epoxide-induced deamination of deoxycytidine (20-23), we were unable to observe the charged intermediate, presumably due to its instability. However, Selzer and Elfarra (23) have reported isolation of β3-substituted deoxycytidine-butadiene monoxide adducts prior to deamination. It has been suggested that this deamination reaction is facilitated by the hydroxyl group formed from the oxirane ring opening (15-17, 20-23, 25). Selzer and Elfarra (23) also found R3-substituted deoxyuridine-butadiene monoxide adducts but were unable to isolate their R3-substituted precursors. In contrast, no R1-substituted deoxyuridinestyrene oxide adducts were observed in this study. Ring opening at the β-carbon of the epoxide by the ring nitrogen of deoxycytidine is consistent with previous findings that reactions at ring nitrogen positions proceed via an SN2 mechanism that favors reaction at the less sterically hindered β-carbon (4, 17, 26). Both R- and β-substituted styrene oxide products have been found at the 7-position of guanosine (4) and the 1-position of adenosine (17). However, it is conceivable that the oxo substituent at the 2-position of deoxycytidine could sterically prevent nucleophilic attack (by the ring nitrogen at the 3-position of deoxycytidine) at the R-carbon of styrene oxide because this position is more hindered than the R-carbon of butadiene monoxide. As substitution at the 3-position destabilizes the glycosidyl bond, it may have been anticipated that 3-substituted cytosine products would also have been derived from the 3-substituted deoxycytidine-styrene oxide adducts; however, no additional compounds other than those described were detected during either the Sephadex LH-20 or HPLC analytical or purification steps. Thus, consistent with previous studies (20-23), it appears that substitution of the amino at the 4-position by water rather than hydrolysis of the glycosidyl bond is favored by the 3-substituted deoxycytidine adducts formed by alkyl epoxides.
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The regiochemistry of epoxide ring opening for reactions at the 3-position of deoxycytidine was reversed in reactions at the exocyclic amino group. In this latter case, the R-carbon of the epoxide was the site of ring opening and formation of βN4-substituted products was not detected. In reactions with optically active styrene oxides, RN4-substituted products with both retained and inverted stereochemistry were found. The ratios of retained to inverted stereochemistry, ∼1:7, indicated that a somewhat ionized substrate was involved in reaction at the exocyclic site. Similar findings have been reported previously for reactions of guanosine (4) and adenosine (17) with optically active styrene oxides, where the exocyclic amino groups opened the epoxide at the R-carbon to give products with ratios of retained to inverted stereochemistry of ∼1:4 and ∼1:6, respectively. Thus, it would appear that reactions at the exocyclic amino groups of nucleosides involve substrate ionization that decreases in the order guanine > adenine > cytosine, correlating inversely with the pKa values of those nucleic acid bases. In conclusion, these findings have demonstrated that in reactions of styrene oxide and deoxycytidine, the exocyclic amino group opens the epoxide exclusively at the R-carbon with some small degree of racemization involved in the reaction. However, the majority of the products that are formed result from reaction at the ring nitrogen at the 3-position which opens the epoxide at the β-carbon. The β3-substituted deoxycytidine products are unstable and subsequently undergo a rapid deamination reaction to give β3-substituted deoxyuridine adducts. The relative proportions of N4- and 3-substituted deoxycytidine-styrene oxide products would be expected to be altered in styrene oxide reactions with double-stranded DNA with formation of N4-substituted products favored, due to the decreased reactivity of the 3-position, which is internalized within the double helix (the N4-position can still be accessed from the major groove in doublestranded DNA). However, it has been shown in previous studies of styrene oxide reactions with deoxyadenosine residues in DNA (16) that significant quantities of products arise at positions that are internalized within the double helix, and although the possibility for Watson-Crick hydrogen bonding is preserved in the RN4substituted styrene oxide-deoxycytidine adducts, the β3substituted deaminated products could lead to misinstructional events during replication because two of the three Watson-Crick hydrogen bonding sites of deoxycytidine have been disrupted.
Acknowledgment. We thank J. Klose and M. Maguire for NMR studies and Dr. C. J. Metral for mass spectrometric studies. This research was sponsored by the National Cancer Institute DHHS, under contract with ABL.
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