Synthesis and Characterization of Nucleosides and Oligonucleotides

Mar 14, 2001 - Danaè Quirk Dorr , Kristopher Murphy , Natalia Tretyakova ... Ewan D Booth , Joanne D Kilgour , Stephen A Robinson , William P Watson...
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Chem. Res. Toxicol. 2001, 14, 379-388

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Synthesis and Characterization of Nucleosides and Oligonucleotides Bearing Adducts of Butadiene Epoxides on Adenine N6 and Guanine N2 Lubomir V. Nechev, Mingzhu Zhang,† Dimitrios Tsarouhtsis,‡ Pamela J. Tamura, Amanda S. Wilkinson, Constance M. Harris, and Thomas M. Harris* Chemistry Department and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235 Received November 24, 2000

Butadiene is a major industrial chemical whose genotoxic effects are attributed to the reaction of its oxidized metabolites, butadiene monoepoxide (BDO) and butadiene diepoxide (BDO2), with DNA. Nucleosides and oligonucleotides containing regio- and stereochemically specific adducts of BDO and the BDO2-related compound, butene 3,4-diol 1,2-epoxide (BDE), on guanine [(2R)- and (2S)-N2-(1-hydroxy-3-buten-2-yl) and (2R,3R)- and (2S,3S)-N2-(2,3,4-trihydroxybut1-yl), respectively] and on adenine [(2R)- and (2S)-N6-(1-hydroxy-3-buten-2-yl) and (2R,3R)and (2S,3S)-N6-(2,3,4-trihydroxybut-1-yl), respectively] have been prepared by nonbiomimetic routes. For guanine adducts, 2-fluoro-O6-(trimethylsilylethyl)-2′-deoxyinosine was treated with (2R)- and (2S)-2-amino-3-buten-1-ol to give the BDO adducts and with (2R,3R)- and (2S,3S)1-amino-2,3,4-butanetriol to produce the BDE adducts; the adducted oligonucleotides were prepared from 11-mer oligonucleotides containing the halopurine. Adenine adducts were prepared in a similar fashion using 6-chloropurine 2′-deoxyriboside as the reactive purine component.

Introduction Butadiene (1) (Scheme 1) is a major industrial chemical used widely in the production of polymeric materials such as synthetic rubber (styrene-butadiene) and ABS (acrylonitrile-butadiene-styrene) plastics. It is also a combustion product, found in cigarette smoke and automotive emissions. Butadiene is a gas (bp -4.4 °C), leading to problems of containment and increasing the risk of exposure for those involved in its manufacture, transport, and use. It is carcinogenic in mice, although less so in rats, and is listed as a human carcinogen in the latest report from the National Toxicology Program (1). Butadiene causes chromosomal abnormalities and increased levels of lymphatic and hemopoietic cancers in workers with long-term exposure in the workplace. The toxicology and epidemiology of butadiene have recently been reviewed by Himmelstein et al. (2). The genotoxic effects of butadiene are attributed to its metabolic activation, in a non-stereospecific manner, by P450s (primarily 2A6 and 2E1) to the (R)- and (S)-monoepoxides (2, BDO)1 which can undergo diverse reactions, including hydrolysis, conjugation with glutathione, reaction with DNA, and further oxidation by P450 to (R,R)-, (S,S)-, or (R,S)diepoxide (3, BDO2) (Scheme 1). The monoepoxide can react either at C1 by an SN2 mechanism or at C2 by a route with more SN1 character (Scheme 2), although inversion products predominate at C2 also, at least when the nucleophile is N7 of guanine (3).2 This diverse * To whom correspondence should be addressed: Department of Chemistry, Box 351822, Station B, Vanderbilt University, Nashville, TN 37235. E-mail: [email protected]. † Current address: DuPont Pharmaceuticals Co., Wilmington, DE 19880. ‡ Current address: Abbott Laboratories, Abbott Park, IL 60064.

reactivity is seen in the variety of products thus far isolated from the reaction of BDO with purines, nucleosides, and DNA. Guanine adducts that have been identified include regioisomeric N7 adducts (3-7) and N1- and N2-(1-hydroxy-3-buten-2-yl) compounds (8). Adenine adducts include regioisomeric N1 adducts, N6-(2-hydroxy3-buten-yl) adducts, and an N1-(1-hydroxy-3-buten-2-yl) inosine derivative (9, 10). The N6 adduct was postulated to arise from the N1-(2-hydroxy-3-buten-1-yl) compound by Dimroth rearrangement and the N1 inosine compound from the N1-(1-hydroxy-3-buten-2-yl) adduct by deamination catalyzed by the primary hydroxyl group; analogous reactions have been described for the reaction of styrene oxide with adenine (11, 12). In addition, N3 adducts have been detected by Tretyakova et al. in vitro and in vivo (7) and by Selzer and Elfarra (13). 1 Abbreviations: BD, butadiene; BDO, butadiene monoepoxide; BDO2, butadiene diepoxide; BDE, butene 3,4-diol 1,2-epoxide; R-BDOdAdo, (R)-N6-(1-hydroxy-3-buten-2-yl)deoxyadenosine; S-BDO-dAdo, (S)-N6-(1-hydroxy-3-buten-2-yl)deoxyadenosine; R,R-BDE-dAdo, (R,R)N6-(2,3,4-trihydroxybut-1-yl)deoxyadenosine; S,S-BDE-dAdo, (S,S)-N6(2,3,4-trihydroxybut-1-yl)deoxyadenosine; R-BDO-dGuo, (R)-N2-(1hydroxy-3-buten-2-yl)deoxyguanosine; S-BDO-dGuo, (S)-N2-(1-hydroxy3-buten-2-yl)deoxyguanosine; R,R-BDE-dGuo, (R,R)-N2-(2,3,4-trihydroxybut-1-yl)deoxyguanosine; S,S-BDE-dGuo, (S,S)-N2-(2,3,4-trihydroxybut-1-yl)deoxyguanosine; TMSE, trimethylsilylethyl; DIPEA, diisopropylethylamine; MALDI, matrix-assisted laser desorption ionization mass spectrometry; FAB, fast atom bombardment mass spectrometry; NBA, 3-nitrobenzyl alcohol; CGE, capillary gel electrophoresis; PAGE, polyacrylamide gel electrophoresis. 2 The adducts of BDO at C2 can form by either an S 2 or S 1 N N process; the former gives adducts with the configuration that is the opposite of that of the parent epoxide [(R)-adduct from the (S)-epoxide], whereas the latter gives adducts of both retained and inverted configurations. Adducts at C1 of BDO have the same configuration as the epoxide since the reaction does not involve the chiral center. Similarly, BDE and BDO2 give adducts of the same configuration as the parent compound because the reaction occurs at the terminal position [R,R-BDE or -BDO2 gives (R,R)-adducts].

10.1021/tx000241k CCC: $20.00 © 2001 American Chemical Society Published on Web 03/14/2001

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Nechev et al.

Scheme 1

Scheme 2

Butadiene diepoxide (3, BDO2) is formed in vivo in much smaller quantities than the monoepoxide, but is more genotoxic. The diepoxide can react at one or both epoxides. Reaction at one epoxide followed by hydrolysis of the second can lead to trihydroxy adducts. However, recent papers (14, 15) present evidence that trihydroxy adducts arise largely from butene 3,4-diol 1,2-epoxide (4, BDE) formed by rapid hydrolysis of BDO2 by epoxide hydrolase. BDO2 adducts of guanine that have been identified include N7-(2,3,4-trihydroxybut-1-yl)guanine (7, 16, 17) and a bis N7 adduct [1,4-bis(guanin-7-yl)butane-2,3-diol] isolated by Lawley and Brookes (16, 18) from salmon sperm DNA treated with the diepoxide. Adenine adducts of BDO2 that have been isolated include N3- and N6-(2,3,4-trihydroxybut-1-yl)adenine (7, 19). A 32P-postlabeling study of DNA adducts formed in vivo and in vitro from BDO2 showed that the predominant bands corresponded to adenine derivatives, although they were not specifically identified (20). Leuratti et al. (21, 22), using an HPLC/32P-postlabeling method, detected an adenine N6 adduct from treatment of CT DNA or Chinese hamster ovary (CHO) cells with BDO2; guanine-BDO2 adducts were also detected in these studies but at much lower levels. No cross-linked adducts of BDO2 have yet been isolated from in vivo sources. As investigations into the isolation and identification of DNA-bound metabolites of BDO and BDO2 have progressed, other research groups have examined the mutations induced by exposure to BD or its epoxides. In 1990, Goodrow et al. (23) examined NIH 3T3 cells transformed by DNA from lymphomas and lung and liver tumors induced in B6C3F1 mice by long-term exposure to butadiene. The most common mutations found in transformed cells were G to C transversions in codon 13 of the K-ras protooncogene. Studies by Cochrane and Skopek (24-26) of hprt mutations in TK6 human lymphoblastoid cells and splenic T cells exposed to BDO, BDO2, or BDE showed that BDO2 is approximately 100 times more mutagenic than the monoepoxide and BDE was the least mutagenic of the three. Transitions and transversions at both GC and AT sites as well as frameshifts were detected. The BDO2-treated cells showed large deletions, seen previously, for example, in Drosophila melanogaster exposed to diepoxide (27, 28). Extensive in vivo mutagenicity studies in B6C3F1 lacI trans-

genic mice have been carried out by Recio (29-35). Mutations in the lacI gene from exposure to BD were primarily point mutations (61% at GC and 20% at AT) with the level of AT mutations being significantly higher than in the controls (32). When TK6 cells were exposed to butadiene monoepoxide, most (78%) of the mutations observed in the hprt gene were single-base substitutions, with a significant increase again being observed in the level of A‚T f T‚A transversions (36). A similar study with the diepoxide (37) also showed a significant increase in the level of A‚T f T‚A transversions together with increased numbers of deletions. These results were in general agreement with those reported by Cochrane and Skopek. When Rat2 lacI transgenic fibroblasts were treated with monoepoxide, the mutations seen were primarily base substitutions (60% at GC and 31% at AT) (30), whereas the diepoxide in the same system induced micronuclei but few mutations (38). The authors of the latter studies suggest that BDO is largely responsible for

BD Epoxide Adducts at Adenine N6 and Guanine N2

base substitutions whereas BDO2 exposure leads to deletions which are not easily detected in the Rat2 cell assay. To gain more insight into the source of mutations arising from exposure to butadiene or its metabolites, we have undertaken the synthesis of a series of oligonucleotides containing regio- and stereospecific adducts of (R)and (S)-BDO and (R,R) and (S,S)-BDE on the exocyclic amino groups of guanine and adenine. The other major adducts thus far identified, namely, the N7 guanine and N1 and N3 adenine adducts, are unstable and not readily amenable to oligonucleotide synthesis. Presumably, they would not survive very long in vivo but could, however, be mutagenic. The target adducts in this study are those arising from attack at the C2 position of BDO and the C1 position of BDO2 and BDE (Scheme 2).

Experimental Section Materials. Methylene chloride, pyridine, triethylamine, and DIPEA were distilled from calcium hydride under a nitrogen atmosphere. 1,4-Dioxane was distilled from sodium under a nitrogen atmosphere. THF was distilled from a sodium/potassium alloy with benzophenone ketyl as an indicator. Methanol was distilled from sodium methoxide (5 mol %) under a nitrogen atmosphere. Other chemicals were used as purchased without further purification. Melting points are uncorrected. Chromatography. Thin-layer chromatography was performed with silica gel F254 (Merck) as the adsorbent on glass plates. The chromatograms were visualized under UV (254 nm) or by staining with an anisaldehyde/sulfuric acid solution, followed by heating. Column chromatography was performed using silica gel 60, 70-230 mesh (E. Merck). Oligonucleotides were desalted via a Sephadex G-25 column using a Bio-Rad Biologic medium-pressure chromatography system. Vacuum centrifugation was performed on a Jouan RC10.22 instrument equipped with a Titan vapor trap. HPLC analyses and purifications were carried out on a gradient HPLC (Beckman Instruments; System Gold software) equipped with pump module 125 and photodiode array detector module 168. NMR. 1H NMR spectra were recorded on a Bruker AC 300 or AM 400 NMR spectrometer with DMSO-d6, CD3CN, CDCl3, or MeOH-d4 as the solvent. Mass Spectrometry. Low- and high-resolution FAB mass spectra were obtained at the Mass Spectrometry Facility at the University of Notre Dame (Notre Dame, IN). Mass spectra (MALDI) of oligonucleotides were obtained using a Voyager Elite DE instrument (PerSeptive Biosystems). The system was operated in the negative ion mode using a matrix mixture of 2′,4′,6′trihydroxyacetophenone monohydrate and ammonium hydrogen citrate or ammonium tartrate. Oligonucleotides. Oligodeoxynucleotides were synthesized on an Expedite 8909 DNA synthesizer (PerSeptive Biosystems) on a 1 µmol scale using the manufacturer’s 4-tert-butylphenoxyacetyl (tBPA)-protected phosphoramidites and standard synthesis protocol. CD Spectroscopy. CD spectra were recorded in methanol at 25 °C on a JASCO J-700 spectropolarimeter. CGE. Capillary gel electrophoresis was performed on a Beckman P/ACE 5000 instrument using the manufacturer’s ssDNA 100-R gel capillary and Tris-borate-urea buffer. Samples were applied at -10 kV and run at -10 kV at 30 °C. Thermal Stability Studies. Adducted oligonucleotides and their complements (∼0.5 A260 units of each) were dissolved in buffer [a 10 mM Na2HPO4/NaH2PO4 mixture containing 1.0 M NaCl and 50 µM EDTA (pH 7.0)]. The sample vials were placed in a water bath at 85 °C; heating was discontinued, and the samples were allowed to cool to ambient temperature and stored at 4 °C overnight. Melting studies were performed using a Varian Cary 04E spectrophotometer. UV measurements were

Chem. Res. Toxicol., Vol. 14, No. 4, 2001 381 taken at 1 min intervals with a 1 °C/min temperature gradient. The temperature was increased from 5 to 90 °C, and then lowered to 5 °C. Unmodified and modified samples were run simultaneously to ensure identical reaction conditions. Enzymatic Digestions. Enzymatic digestions of adducted dGuo oligonucleotides 19a-d were carried out under conditions described by Borowy-Borowski et al. (39). The oligonucleotide (0.2-2.0 A260 units), 20 µL of buffer [0.1 M Tris-HCl and 10 mM MgCl2 (pH 9)], 0.04 unit of snake venom phosphodiesterase (VIII-S from Crotalus durissus terrificus, Sigma), and 0.40 unit of alkaline phosphatase (Type III, Escherichia coli, Sigma) were mixed and incubated at 37 °C for 6-18 h. The reaction was stopped by heating at 75 °C for 1 min. The digest was diluted with H2O, filtered through a centrifugal filter, and analyzed by HPLC [YMC-ODS-AQ column (4.6 mm × 250 mm) with the following gradients: (A) 0.1 M ammonium formate at pH 6.4 and (B) from 1 to 10% B (CH3CN) over the course of 15 min and from 10 to 20% B over the course of 5 min, at a flow rate of 1.5 mL/min]. The adducted dAdo oligonucleotides 17a-d were digested according to a two-step protocol utilizing nuclease P1, snake venom phosphodiesterase, and alkaline phosphatase (40). Syntheses. (1) (2R)- and (2S)-2-Amino-3-buten-1-ol (5a and 5b, respectively). (2R)- and (2S)-2-amino-3-buten-1-ol were prepared from D- and L-N-Boc-protected methionine (Aldrich Chemical Co.) by a published procedure (41). (2) (2R,3R)- and (2S,3S)-1-Amino-2,3,4-butanetriol (5c and 5d, respectively, Scheme 3). Methyl (2R,3R)-2,3-OBenzylidenetartramide (7). Dimethyl 2,3-O-benzylidene-Dtartrate (6, 1 g, 3.8 mmol) was dissolved in methanol (6 mL). Ammonia (2 M in methanol, 1.9 mL, 3.8 mmol) was added to the solution, and the mixture was stirred at room temperature for 24 h. Solvents were evaporated, and the crude mixture was purified by column chromatography on silica gel (1:1 ethyl acetate/hexane mixture) to give 0.65 g (70%) of the desired product 7. About 19% of the unreacted starting material was isolated as well as 5% of the diamide. Compound 7 is a mixture of diastereoisomers (R,R,R and S,R,R) because of the chiral center in the benzylic position (R or S): 1H NMR of the first isomer (DMSO-d6) δ 3.61 (s, 3H, CH3O), 4.80 (d, 1H, CHCONH2), 4.84 (d, 1H, CHCOOCH3), 5.96 (s, 1H, benzylic), 7.43 (m, 3H, aromatic), 7.56 (m, 2H, aromatic); 1H NMR of the second isomer (DMSO-d6) δ 3.75 (s, 3H, CH3O), 4.65 (d, 1H, CHCONH2), 4.89 (d, 1H, CHCOOCH3), 5.99 (s, 1H, benzylic), 7.43 (m, 3H, aromatic), 7.56 (m, 2H, aromatic); HRMS-FAB (NBA matrix) calcd for C12H14NO5 [M + H]+ 252.0872, found 252.0887. (3) (2R,3R)-2,3-O-Benzylidene-1-amino-2,3,4-butanetriol (8). Ester amide 7 from the previous reaction (0.45 g, 1.8 mmol) was dissolved in 15 mL of anhydrous THF. The solution was added dropwise (10 min) under argon to a stirring mixture of LiAlH4 (0.6 g, 15 mmol) and anhydrous THF (30 mL). The mixture was stirred at room temperature for 1 h and heated under reflux for an additional 5 h. The reaction was followed by TLC (85:8:7 CH3CN/H2O/NH4OH mixture). Water was carefully added (ice/water bath), and the resulting mixture was filtered. The filtrate was diluted with ether and dried over sodium sulfate. Solvents were removed, and the crude product was purified by silica gel column chromatography (the same solvent system as for TLC) to give 0.340 g (90%) of protected aminotriol 8: 1H NMR (DMSO-d6 with D2O) δ 2.75 (m, 2H, CH2NH2), 3.55 (m, 2H, CH2OH), 3.87 (m, 2H, CHCH2NH2 and CHCH2OH), 5.82, 5.83 (two s, 1H, benzylic R and S), 7.36 (m, 3H, aromatic), 7.43 (m, 2H, aromatic); HRMS-FAB (NBA matrix) calcd for C11H16NO3 [M + H]+ 210.1130, found 210.1135. (4) (2R,3R)-1-Amino-2,3,4-butanetriol (5c). The protected aminotriol (8, 0.3 g, 1.4 mmol) was neutralized with 1 M HCl to pH 6.0. The solution was evaporated to dryness (rotary evaporator, 40 °C). Sulfuric acid (0.01 M, 15 mL) was added, and the mixture was stirred at 100 °C for 3 h. The solution was evaporated to dryness; water was added and evaporated three times (to remove the PhCHO). The product was dissolved in a small volume of water, and the pH was adjusted (1 M NaOH)

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to 12.0. The solution was evaporated to dryness. The mixture was dissolved in a small volume of boiling 80% ethanol. The insoluble inorganic salts were filtered out, and the filtrate was kept at -20 °C for 1 h. The additional amounts of inorganic salts which crystallized were filtered out again, and the solvents were evaporated to give 0.174 g (81%) of 5c as a clear oil: (R,R) 1 [R]20 D 8.5° (c 2, water); H NMR (MeOH-d4) δ 2.62 (m, 2H, CH2NH2), 3.48 (m, 4H, 2× CHOH and CH2OH); HRMS-FAB (NBA matrix) calcd for C4H12NO3 [M + H]+ 122.0817, found 122.0809. The (2S, 3S) isomer could have been prepared similarly from the corresponding L-tartrate, but a better route was available from the commercially available methyl 3,4-O-isopropylideneL-threonate (the corresponding D isomer of the threonate is not commercially available). (5) Amide of (2S,3R)-3,4-O-Isopropylidene-2,3,4-trihydroxybutyric Acid (10). Methyl 3,4-O-isopropylidene-L-threonate (9, 1.3 g, 6.8 mmol) was dissolved in 30 mL of methanol. A slow stream of ammonia (gas) was bubbled through the solution for 4 h. The reaction was followed by TLC (2:1 ethyl acetate/hexane mixture). Evaporation of the solvents yielded 10 as a clear oil (1.15 g, 97%) which crystallized in ∼10 min: 1H NMR (DMSO-d6) δ 1.25 (s, 3H, CH3), 1.32 (s, 3H, CH3), 3.79 (m, 2H, R-CHOH and 1H from CH2O), 3.94 (m, 1H, from CH2O), 4.20 (m, 1H, CHOC), 5.51 (d, 1H, OH, J ) 6.4 Hz), 7.22 (bs, 2H, CONH2); HRMS-FAB (NBA matrix) calcd for C7H14NO4 [M + H]+ 176.0923, found 176.0902. (6) (2S,3R)-3,4-Isopropylidene-1-amino-2,3,4-butanetriol (11). Amide 10 from the previous reaction (1.15 g, 6.6 mmol) was dissolved in 25 mL of anhydrous THF. The solution was added dropwise (10 min) under argon to a stirring mixture of LiAlH4 (1.25 g, 33 mmol) and 150 mL of anhydrous THF. The mixture was stirred at room temperature for 1 h and refluxed for an additional 5 h. The reaction was followed by TLC (85:8:7 CH3CN/H2O/NH4OH mixture). Water was carefully added (ice/ water bath), and the resulting mixture was filtered. The filtrate was diluted with ether and dried over sodium sulfate. Solvents were removed, and the crude product was purified by silica gel column chromatography (the same solvent system as for TLC) to give 0.93 g (89%) of protected aminotriol 11: 1H NMR (DMSO-d6) δ 1.27 (s, 3H, CH3), 1.33 (s, 3H, CH3), 2.52 (m, 2H, CH2NH2), 3.36 (m, 1H, CHOH) 3.67 (m, 1H, from CH2O), 3.90 (m, 1H, from CH2O), 3.99 (m, 1H, CHOC); HRMS-FAB (NBA matrix) calcd for C7H16NO3 [M + H]+ 162.1130, found 162.1110. (7) (2S,3S)-1-Amino-2,3,4-butanetriol (5d). Isopropylideneprotected aminotriol 11 from the previous step was dissolved in THF (10 mL). Aqueous 1 M HCl (10 mL) was added to the solution which was stirred at room temperature for 20 h. The solvents were evaporated, and the residue was dissolved in a small volume of water. The pH of the solution was adjusted to 11.0-12.0, and the mixture was evaporated to dryness. The crude product was dissolved in a small amount of a boiling mixture of ethanol and methanol (1:1) and filtered while hot. The filtrate was kept for 1 h at -20 °C. Additional amounts of crystallized inorganic salts were filtered out, and the solvents were evaporated to give 0.61 g (90%) of product 5d as a clear 1 oil: (S,S) [R]20 D -9.4° (c 2, water); H NMR (DMSO-d6 with D2O) δ 2.82 (m, 2H, CH2NH2), 3.39 (m, 4H, 2× CHOH, CH2OH); HRMS-FAB (NBA matrix) calcd for C4H12NO3 [M + H]+ 122.0817, found 122.0811. Synthesis of Nucleoside Adducts (Scheme 4). Designation of atoms in the butadiene epoxide side chains is shown below:

(1) (2R)-2′-Deoxy-N6-(1-hydroxy-3-buten-2-yl)adenosine (13a, R-BDO-dAdo). 6-Chloropurine 2′-deoxyriboside (12, 20 mg, 0.08 mmol), amino alcohol 5a (22 mg, 0.25 mmol), DIPEA

Nechev et al. (15 µL, 0.09 mmol), and 300 µL of dry DMF were mixed in a small glass test tube. The tube was flushed with nitrogen and closed, and the contents were stirred at 55 °C for 20 h. The reaction was followed by TLC. The solvents were evaporated to dryness (vacuum centrifuge), and the mixture was purified on a silica gel column (86:12:2 CH3CN/H2O/concentrated NH4OH mixture) to yield 20 mg (85%) of 13a: 1H NMR (DMSO-d6, 45 °C) δ 2.27 (m, 1H, H-2′′), 2.71 (m, 1H, H-2′), 3.63-3.52 (m, 5H, H-5′, H-5′′, β, β′, R), 3.88 (m, 1H, H-4′), 4.41 (m, 1H, H-3′), 4.71 (t, 1H, β-OH, J ) 4.7 Hz), 5.03 (t, 1H, 5′-OH, J ) 5.5 Hz), 5.07 (ddd, 1H, γ′, J1 ) 10.4 Hz, J2 ) J3 ) 1.6 Hz), 5.17 (d, 1H, 3′OH, J ) 4.1 Hz), 5.18 (ddd, 1H, γ, J1 ) 17.3 Hz, J2 ) J3 ) 1.6 Hz), 5.96 (m, 1H, β′′), 6.34 (dd, 1H, H-1′, J1 ) J2 ) 6.2 Hz), 7.29 (d, 1H, NH, J ) 8.7 Hz), 8.18 (s, 1H, H-8), 8.31 (s, 1H, H-2); HRMS-FAB (NBA matrix) calcd for C14H20N5O4 [M + H]+ 322.1515, found 322.1510. (2) (2S)-2′-Deoxy-N6-(1-hydroxy-3-buten-2-yl)adenosine (13b, S-BDO-dAdo). Following the procedure for the synthesis of 13a using amino alcohol 5b instead of 5a yielded 21 mg (88%) of the title compound: 1H NMR (DMSO-d6, 45 °C) δ 2.26 (m, 1H, H-2′′), 2.71 (m, 1H, H-2′), 3.61-3.52 (m, 5H, H-5′, H-5′′, β, β′, R), 3.87 (m, 1H, H-4′), 4.40 (m, 1H, H-3′), 4.76 (t, 1H, β-OH, J ) 4.6 Hz), 5.07 (ddd, 1H, γ′, J1 ) 10.4 Hz, J2 ) J3 ) 1.6 Hz), 5.11 (t, 1H, 5′-OH, J ) 5.7 Hz), 5.17 (ddd, 1H, γ, J1 ) 17.3 Hz, J2 ) J3 ) 1.6 Hz), 5.23 (d, 1H, 3′-OH, J ) 4.1 Hz), 5.96 (m, 1H, β′′), 6.34 (dd, 1H, H-1′, J1 ) J2 ) 6.1 Hz), 7.39 (d, 1H, NH, J ) 8.7 Hz), 8.18 (s, 1H, H-8), 8.33 (s, 1H, H-2); HRMS-FAB (NBA matrix) calcd for C14H20N5O4 [M + H]+ 322.1515, found 322.1512. (3) (2R,3R)-2′-Deoxy-N6-(2,3,4-trihydroxybut-1-yl)adenosine (13c, R,R-BDE-dAdo). 6-Chloropurine 2′-deoxyriboside (12, 10 mg, 0.04 mmol), the corresponding aminotriol (5c, 10 mg, 0.08 mmol), DIPEA (20 µL), and 100 µL of dry DMSO were mixed in a small glass test tube. The tube was flushed with nitrogen and closed, and the contents were stirred at 60 °C for 15 h. The reaction was followed by TLC. The solvents were evaporated to dryness (vacuum centrifuge), and the mixture was purified on a silica gel column (85:8:7 CH3CN/H2O/NH4OH) to yield 10.6 mg (81%) of 13c: 1H NMR (DMSO-d6, 45 °C) δ 2.26 (m, 1H, H-2′′), 2.70 (m, 1H, H-2′), 3.39-3.45 (m, 3H, β or γ, δ, δ′), 3.50-3.64 (m, 4H, H-5′, H-5′′, R, R′), 3.75 (m, 1H, γ or β), 3.88 (m, 1H, H-4′), 4.27 (bs, 1H, β- or γ-OH), 4.40 (m, 2H, H-3′, δ-OH), 4.45 (bs, 1H, γ- or β-OH), 5.00 (t, 1H, 5′-OH, J ) 5.4 Hz), 5.15 (d, 1H, 3′-OH, J ) 4.0 Hz), 6.34 (dd, 1H, H-1′, J1 ) J2 ) 6.8 Hz), 7.26 (bs, 1H, NH), 8.19 (s, 1H, H-8), 8.29 (s, 1H, H-2); HRMS-FAB (NBA matrix) calcd for C14H22N5O6 [M + H]+ 356.1570, found 356.1585. (4) (2S,3S)-2′-Deoxy-N6-(2,3,4-trihydroxybut-1-yl)adenosine (13d, S,S-BDE-dAdo). The same procedure that was used for 13c using aminotriol 5d led to 10.9 mg (83%) of the title compound: 1H NMR (DMSO-d6, 45 °C) δ 2.26 (m, 1H, H-2′′), 2.72 (m, 1H, H-2′), 3.38-3.50 (m, 3H, β or γ, δ, δ′), 3.53-3.63 (m, 4H, H-5′, H-5′′, R, R′), 3.75 (m, 1H, γ or β), 3.88 (m, 1H, H-4′), 4.29 (bs, 1H, β- or γ-OH), 4.41 (m, 2H, H-3′, δ-OH), 4.46 (d, 1H, γ- or β-OH, J ) 5.6 Hz), 5.02 (t, 1H, 5′-OH, J ) 5.0 Hz), 5.15 (d, 1H, 3′-OH, J ) 3.9 Hz), 6.35 (dd, 1H, H-1′, J1 ) J2 ) 6.9 Hz), 7.27 (bs, 1H, NH), 8.19 (s, 1H, H-8), 8.30 (s, 1H, H-2); HRMS-FAB (NBA matrix) calcd for C14H22N5O6 [M + H]+ 356.1570, found 356.1579. (5) (2R)-2′-Deoxy-N2-(1-hydroxy-3-buten-2-yl)guanosine (15a, R-BDO-dGuo). 2-Fluoro-O6-(trimethylsilylethyl)-2′-deoxyinosine (14, 7 mg, 0.02 mmol), contained in a small glass test tube equipped with a magnetic stirring bar, was dried overnight under vacuum. (2R)-2-Amino-3-buten-1-ol (5a, 2.5 mg, 0.03 mmol, 1.5 equiv), DMSO (40 µL), and DIPEA (10 µL, 0.06 mmol, 3 equiv) were added. The mixture was stirred at 60 °C and monitored by HPLC, using a YMC-ODS-AQ-C18 or Phenomenex C-8 Luna 2 column (250 mm × 4.6 mm) at a flow rate of 1.5 mL/min. Solvent A was 0.1 M ammonium formate and solvent B acetonitrile. The gradient for monitoring the reaction was from 10 to 90% B over the course of 25 min and the gradient for purity checking from 1 to 10% B over the course of 15 min, followed by 10 to 20% B over the course of 5 min. The reaction mixture

BD Epoxide Adducts at Adenine N6 and Guanine N2 was stirred for 20 h. Two product peaks were observed at 5.3 (15a) and 15.4 min (O6-protected 15a) in a ratio of 3.7:1. The solvent was removed by vacuum centrifugation. Water was added, and the reaction mixture was lyophilized. Purification was carried out on a YMC-ODS-AQ-C18 column (250 mm × 10 mm) at a flow rate of 5 mL/min. Solvent A was 0.1 M ammonium formate and solvent B MeOH. The gradient was from 10 to 90% B over the course of 25 min. After HPLC purification (retention time for 15a of 10 min), 3.9 mg (61%) of 15a was obtained: 1H NMR (CD3OD) δ 2.34 (m, 1H, H-2′′), 2.72 (m, 1H, H-2′), 3.603.80 (m, 4H, H-5′, H-5′′, β, β′), 3.95 (m, 1H, H-4′), 4.53 (m, 1H, H-3′), 4.65 (m, 1H, R), 5.20 (ddd, 1H, γ′, J1 ) 10.5 Hz, J2 ) J3 ) 1.5 Hz), 5.30 (ddd, 1H, γ, J1 ) 17.2 Hz, J2 ) J3 ) 1.5 Hz), 5.94 (ddd, 1H, β′′, J1 ) 17.6 Hz, J2 ) 10.5 Hz, J3 ) 5.4 Hz), 6.29 (dd, 1H, H-1′, J1 ) J2 ) 6.3 Hz), 7.96 (s, 1H, H-8); HRMS-FAB (NBA matrix) calcd for C14H19N5O5 [M + H]+ 338.1464, found 338.1471. (6) (2S)-2′-Deoxy-N2-(1-hydroxy-3-buten-2-yl)guanosine (15b, S-BDO-dGuo). 2-Fluoro-O6-TMSE-2′-deoxyinosine (14, 10 mg, 0.03 mmol), contained in a small glass test tube equipped with a magnetic stirring bar, was dried overnight in vacuo. (2S)2-Amino-3-buten-1-ol (5b, 3.5 mg, 0.04 mmol, 1.5 equiv), DMSO (50 µL), and DIPEA (10 µL, 0.06 mmol, 2 equiv) were added. The mixture was stirred at 60 °C and monitored as described above for the (R) isomer. The reaction appeared to be essentially finished within 6 h, but stirring was continued for an additional 14 h. Two product peaks were observed at 5.6 and 15.4 min in a ratio of 3.7:1. The bulk of the reaction was worked up by removing the solvent by vacuum centrifugation, addition of water, and lyophilization. The product was purified on a YMCODS-AQ-C18 column (10 mm × 250 mm) at a flow rate of 5 mL/min. Solvent A was 0.1 M ammonium formate and solvent B MeOH. The gradient was from 10 to 90% B over the course of 25 min. Two peaks were collected. Peak 1 (retention time of 12 min) was compound 15b (6 mg), the TMSE protecting group having been lost. Peak 2 (retention time of 26 min), which proved to be 15b (2 mg) with the TMSE group intact, was converted to peak 1 by treatment with 5% acetic acid (1 h at room temperature). The total yield of 15b was 84%: 1H NMR (CD3OD) δ 2.34 (m, 1H, H-2′′), 2.74 (m, 1H, H-2′), 3.60-3.80 (m, 4H, H-5′, H-5′′, β, β′), 3.95 (m, 1H, H-4′), 4.53 (m, 1H, H-3′), 4.63 (m, 1H, R), 5.21 (ddd, 1H, γ′, J1 ) 10.5 Hz, J2 ) J3 ) 1.5 Hz), 5.29 (ddd, 1H, γ, J1 ) 17.2 Hz, J2 ) J3 ) 1.5 Hz), 5.94 (ddd, 1H, β′′, J1 ) 17.6 Hz, J2 ) 10.5 Hz, J3 ) 5.4 Hz), 6.29 (dd, 1H, H-1′, J1 ) J2 ) 6.3 Hz), 7.96 (s, 1H, H-8); HRMS-FAB (NBA matrix) calcd for C14H19N5O5 [M + H]+ 338.1464, found 338.1471. (7) (2R,3R)-2′-Deoxy-N2-(2,3,4-trihydroxybut-1-yl)guanosine (15c, R,R-BDE-dGuo). 2-Fluoro-O6-TMSE-2′-deoxyinosine (14, 20 mg, 0.05 mmol), (2R,3R)-1-amino-2,3,4-butanetriol (5c, 10 mg, 0.08 mmol, 1.5 equiv), DMSO (100 µL), and DIPEA (30 µL, 0.17 mmol, 3 equiv) were combined. The mixture was stirred at 55-60 °C for 20 h. The solvent was removed by vacuum centrifugation; water was added, and the pH was adjusted to 3.0 with 10% acetic acid. The mixture was stirred for 2 h at room temperature to remove the TMSE protecting group (monitored by HPLC), followed by neutralization to pH 5-6 by 1 M KOH and lyophilization. HPLC purification, performed as described for 15a and 15b, but with a gradient of 7 to 30% B over the course of 15 min (retention time for 15c of 11 min), gave 4.5 mg (49%) of compound 15c: 1H NMR (CD3OD) δ 2.36 (m, 1H, H-2′′), 2.75 (m, 1H, H-2′), 3.43 (dd, 1H, R, J1 ) 14.0 Hz, J2 ) 7.8 Hz), 3.52-3.68 (m, 4H, R′, γ, δ, δ′), 3.74 (m, 2H, H-5′, H-5′′), 3.86 (ddd, 1H, β, J1 ) 7.6 Hz, J2 ) 3.2 Hz, J3 ) 3.0 Hz), 3.95 (m, 1H, H-4′), 4.53 (m, 1H, H-3′), 6.29 (dd, 1H, H-1′, J1 ) J2 ) 6.3 Hz), 7.96 (s, 1H, H-8); HRMS-FAB (NBA matrix) calcd for C14H22N5O7 [M + H]+ 372.1519, found 372.1537. (8) (2S,3S)-2′-Deoxy-N2-(2,3,4-trihydroxybut-1-yl)guanosine (15d, S,S-BDE-dGuo). A mixture of 2-fluoro-O6-TMSE2′-deoxyinosine (14, 10 mg, 0.03 mmol), (2S,3S)-1-amino-2,3,4butanetriol (5d, 5 mg, 0.04 mmol, 1.5 equiv), DMSO (100 µL), and DIPEA (15 µL, 0.09 mmol, 3 equiv) was stirred at 55-60 °C for 3 days. HPLC showed that the major product still retained

Chem. Res. Toxicol., Vol. 14, No. 4, 2001 383 the TMSE group. The reaction mixture was treated with 10% acetic acid as for the (R,R) isomer. The residue was purified by HPLC using the conditions described for 15c. The peak at 11 min was collected and lyophilized to give 4.5 mg (45%) of 15d: 1H NMR (CD OD) δ 2.37 (m, 1H, H-2′′), 2.74 (m, 1H, H-2′), 3.43 3 (dd, 1H, R, J1 ) 14.0 Hz, J2 ) 7.8 Hz), 3.52-3.68 (m, 4H, R′, γ, δ, δ′), 3.74 (m, 2H, H-5′, H-5′′), 3.86 (ddd, 1H, β, J1 ) 7.6 Hz, J2 ) 3.2 Hz, J3 ) 3.0 Hz), 3.95 (m, 1H, H-4′), 4.53 (m, 1H, H-3′), 6.29 (dd, 1H, H-1′, J1 ) J2 ) 6.3 Hz), 7.96 (s, 1H, H-8); HRMSFAB (NBA matrix) calcd for C14H22N5O7 [M + H]+ 372.1519, found 372.1525. Synthesis of Adducted Oligonucleotides (Scheme 4). (1) Ras 61,2-R-BDO-dAdo (17a). The 11-mer 5′-C-GGA-CXAGAA-G-3′ (X is 6-chloropurine 2′-deoxyriboside) was synthesized, deprotected with 0.1 M NaOH (75 h at room temperature), and purified as described previously (42). Oligomer 16 (50 A260 units) was mixed in a plastic tube with DIPEA (150 µL), amino alcohol (5a, 5 mg), and DMSO (500 µL). The reaction mixture was stirred at 60 °C for 48 h. The reaction was monitored by HPLC. The solvents were evaporated (vacuum centrifuge) to dryness. Water (200 µL) was added, and the reaction mixture was purified by HPLC using a YMC-ODS-AQ column (10 mm × 250 mm, flow rate of 5.0 mL/min) with a gradient of (A) 0.1 M ammonium formate at pH 6.3 and (B) acetonitrile (from 1 to 10% B over the course of 20 min, from 10 to 25% B over the course of 2 min, and from 25 to 90% B over the course of 4 min). The adducted oligonucleotide had a retention time of 19.4 min. The yield was 32 A260 units (∼64%) after HPLC purification: MS (MALDI) m/z calcd for [M - H]- 3467.7, found 3467.3. (2) Ras 61,2-S-BDO-dAdo (17b). Adducted oligonucleotide 17b was synthesized as described above for the R-BDO isomer except that amino alcohol 5b was used. The yield was 35 A260 units (∼70%) after HPLC purification: MS (MALDI) m/z calcd for [M - H]- 3467.7, found 3467.0. (3) Ras 61,2-R,R-BDE-dAdo (17c). The 6-chloropurinecontaining oligomer (16, 50 A260 units) was mixed in a plastic tube with DIPEA (25 µL), amino alcohol (5c, 5 mg), and DMSO (200 µL). The reaction mixture was stirred at 60 °C for 6 h. The reaction was monitored by HPLC. The solvents were evaporated (vacuum centrifugation) to dryness. The residue was dissolved in H2O (200 µL) and purified initially by HPLC as described above for the monoepoxide adduct; the product had a retention time of 18.5 min. The yield was 38 A260 units (∼76%). A second HPLC purification of the 3,4-diol 1,2-epoxide-adducted oligonucleotide was performed by ion-exchange chromatography on a HEMA-IEC Bio 1000 Q column (4.6 mm × 150 mm, flow rate of 1.0 mL/min) using a gradient of (A) 20 mM potassium phosphate (pH 6.4) and acetonitrile (8:2) and (B) 20 mM potassium phosphate (pH 6.4) containing 1 M KCl and acetonitrile (8:2), from 25 to 45% B over the course of 20 min. The adducted oligonucleotide had a retention time of 12.4 min: MS (MALDI) m/z calcd for [M - H]- 3501.7, found 3501.2. (4) Ras 61,2-S,S-BDE-dAdo (17d). This oligonucleotide was synthesized using aminotriol 5d and purified as described above for the (R,R) isomer. The HPLC retention time for this oligomer was essentially the same as for the (R,R) isomer. The yield was 33 A260 units (∼66%): MS (MALDI) m/z calcd for [M - H]3501.7, found 3501.5. (5) Ras 12,2-R-BDO-dGuo (19a). The 11-mer 5′-G-GCAGXT-GGT-G-3′ (18, X is 2-fluoro-O6-TMSE-dI) was synthesized as described previously (43). The oligodeoxynucleotide was cleaved from the beads and deprotected by treatment with 0.1 M NaOH (0.75 mL of NaOH/µmol) at room temperature for 12 h. The reaction was quenched by cautious neutralization with 0.1 M HOAc to pH 8-9. The crude product was purified by HPLC (YMC-ODS-AQ column, 10 mm × 250 mm, solvent A, 0.1 M ammonium formate; solvent B, CH3CN; gradient, from 5 to 11% B over the course of 5 min, from 11 to 20% B over the course of 15 min). The product fractions were collected, combined, and lyophilized. Ammonium formate was removed by passage through a Sephadex G-25 column. The yield of 18 was 25 A260 units/µmol (∼23%): MS (ES) measured 3556.5, based

384

Chem. Res. Toxicol., Vol. 14, No. 4, 2001

on 710.3 for [M - 5H]5-, 591.7 for [M - 6H]6-, and 507.1 for [M - 7H]7- (calcd 3554.8). 2-Fluoroinosine-modified oligomer 18 (45 A260 units, 0.41 µmol) was mixed in a plastic vial with DIPEA (150 µL, 0.87 mmol), (2R)-2-amino-3-buten-1-ol (5a, 10 mg, 0.11 mmol), and DMSO (150 µL). The mixture was stirred at 60 °C for 18 h, diluted with H2O, and purified by HPLC (column and solvents as for 18; gradient from 8 to 18% B over the course of 20 min, followed by 18 to 70% B over the course of 2 min) to give 28 A260 units (57%) of the TMSE-protected adducted oligomer. Deprotection with 0.1 M HOAc was performed (room temperature for 2 h), and the product was purified by HPLC with a gradient from 5 to 10% B over the course of 18 min at a flow rate of 5 mL/min (retention time of 19a, 13 min). Rechromatography on an analytical HPLC column (YMC-ODS-AQ, 4.6 mm × 250 mm; gradient from 5 to 25% B over the course of 25 min at a flow rate of 1.5 mL/min) gave a single rather broad peak at 9.7 min; despite the broadness, the product appeared to be homogeneous by CGE and MALDI MS. Other adducts in the N-ras 12 sequence which we have synthesized have had poor chromatographic properties, perhaps because of the high guanine content. Additional purification was performed by PAGE (20% T, 5%C) with 67% recovery. The final product was desalted by passage through a Sephadex G-25 column: MS (MALDI) m/z calcd for [M - H]- 3521.7, found 3522.6. Ras 12,2-S-BDO-dGuo (19b). 2-Fluoroinosine-modified oligomer 18 (50 A260 units, 0.46 µmol) was mixed in a plastic vial with DIPEA (150 µL, 0.86 mmol), (2S)-2-amino-3-buten-1-ol (5b, 5 mg, 0.06 mmol), and DMSO (200 µL). After being stirred at 60 °C for 18 h, the reaction mixture was diluted with water and purified by HPLC [YMC-ODS-AQ column (10 mm × 250 mm); solvent A, 0.1 M ammonium formate; solvent B, CH3CN; gradient from 8 to 17% B over the course of 20 min, followed by 17 to 70% B over the course of 2 min]. The product fractions were lyophilized followed by dissolution in 0.1 M HOAc (room temperature for 2 h) to remove the protecting group (TMSE) at the O6 position. The oligonucleotide was repurified by HPLC using a gradient from 7 to 10% B over the course of 18 min and desalted on a Sephadex G-25 column to give 14 A260 units (28%) of oligomer 19b. Rechromatography on an analytical column as described for 19a gave a single sharp peak at 9.6 min: MS (ES) measured 3524.4, based on 703.9 for [M - 5H]5- and 586.4 for [M - 6H]6- (calculated 3521.7). Ras 12,2-R,R-BDE-dGuo (19c). Oligomer 18 (48 A260 units, 0.44 µmol) was mixed in a plastic vial with DIPEA (50 µL, 0.29 mmol), (2R,3R)-1-amino-2,3,4-butanetriol (5c, 5 mg, 0.04 mmol), and DMSO (200 µL). The reaction mixture was stirred at 60 °C for 15 h, diluted with H2O, and purified by HPLC (gradient from 7 to 17% B over the course of 20 min, followed by 17 to 70% B over the course of 2 min). The product fractions were lyophilized to give 16 A260 units (34%) of TMSE-protected 19c. Deprotection (0.1 M HOAc, 2 h, room temperature), HPLC purification (gradient from 6 to 11% B over the course of 18 min) and desalting (Sephadex G-25) yielded 7 A260 units of 19c. This adducted oligonucleotide also gave a broad HPLC peak; it eluted slightly more quickly (retention time of 8.7 min) than the monoepoxide-adducted oligonucleotides under the analytical conditions described for 19a and 19b: MS (MALDI) m/z calcd for [M - H]- 3555.6, found 3555.9. Ras 12,2-S,S-BDE-dGuo (19d). Oligomer 18 (50 A260 units, 0.46 µmol) was mixed in a plastic vial with DIPEA (150 µL, 0.87 mmol), (2S,3S)-1-amino-2,3,4-butanetriol (5d, 6.3 mg, 0.05 mmol), and DMSO (250 µL). The reaction mixture was stirred at 60 °C for 18 h, diluted with H2O, and purified by HPLC with a gradient from 7 to 17% B over the course of 20 min, followed by 17 to 70% B over the course of 2 min. The product fractions were lyophilized and deprotected with 0.1 M HOAc (room temperature for 2 h). Additional HPLC purification (gradient from 6 to 11% B over the course of 18 min) and desalting (Sephadex G-25) gave 12 A260 units (24%) of oligomer 19d. Upon rechromatography under the analytical conditions described above, this isomer gave a slightly tailing but sharper peak

Nechev et al. Scheme 3

(retention time of 8.9 min) than the (R,R) isomer: MS (MALDI) m/z calcd for [M - H]- 3555.6, found 3554.9.

Results and Discussion For synthesis of the adducted oligonucleotides, a postoligomerization methodology was employed in which a halopurine nucleoside is introduced during oligonucleotide synthesis. The resulting halopurine-containing oligonucleotide is removed from the beads, deprotected, and purified before reaction with the amino alcohol equivalent of the epoxide under investigation (42-44). In our previous syntheses of adducted oligonucleotides using this strategy, we employed racemic amino alcohols in the case of the polycyclic aromatic hydrocarbons (PAHs) and commercially available chiral amino alcohols for the synthesis of R adducts of styrene oxide. Thus far, oligonucleotides containing diastereomeric PAH adducts have been separable by HPLC. However, we felt it was unlikely that we could achieve resolution at the oligonucleotide stage for the butadiene epoxide adducts we proposed to synthesize. Because none of the required amino alcohols were commercially available in chiral form, (2R)- and (2S)-2-amino-3-buten-1-ols and (2R,3R)and (2S,3S)-1-amino-2,3,4-butanetriols were synthesized. The chiral buten-1-ols, 5a and 5b, were prepared by the route developed by Ohfune and Kurokawa (41). The commercially available starting material (N-t-BOC-L- or D-methionine) was esterified with diazomethane, and the resulting methyl ester was reduced to the alcohol with LiAlH4. Oxidation of the sulfide to the sulfoxide with sodium periodate followed by thermal elimination of the sulfoxide produced the double bond. Removal of the t-BOC protection with acid and purification of the product by ion-exchange chromatography yielded the desired aminobuten-1-ol in ∼40% overall yield from the commercially available starting material. The chiral aminotriols, 5c and 5d, were synthesized by procedures developed in our laboratory (Scheme 3). The (2R,3R) stereoisomer (5c) was prepared from commercially available 2,3-O-benzylidene-D-tartrate (6). Careful treatment of the diester with 1 equiv of ammonia yielded monoamide 7 in 65-70% yield. Reduction of the amide ester with LiAlH4 gave protected amino alcohol 8 which was deprotected with dilute acid to give the desired aminotriol in

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Scheme 4

48% yield (Scheme 3A). (2S,3S)-1-Amino-2,3,4-butanetriol (5d) was prepared from commercially available methyl 3,4-O-isopropylidene L-threonate (9) by reaction with ammonia in methanol to form amide 10, LiAlH4 reduction of the amide to amine 11, and deprotection with 1 M HCl to give (2S,3S)-1-amino-2,3,4-butanetriol in ∼77% overall yield (Scheme 3B). Unfortunately, the D isomer of 9 is not commercially available, and thus, the latter synthetic route could not be used for the synthesis of 5c. Synthesis of Modified Nucleosides. Relatively little characterization of BDO- and BDE-modified 2′-deoxyribosides has been done with the exception of N6 BDO-dAdo adducts which were characterized by Koivisto (10). Most of the work on butadiene adducts has involved either ribosides or depurinated species. We have now synthesized the BDO adducts, (R)- and (S)-N6-(1-hydroxy-3buten-2-yl)-dAdo and N2-(1-hydroxy-3-buten-2-yl)-dGuo, and the BDE adducts, (2R,3R)- and (2S,3S)-N6-(2,3,4trihydroxybut-1-yl)-dAdo and (2R,3R)- and (2S,3S)-N2(2,3,4-trihydroxybut-1-yl)-dGuo, in a regio- and stereospecific fashion. These compounds were needed as standards for characterization of adducted oligonucleotides by enzymatic hydrolysis and should be useful for identification of compounds formed by in vitro or in vivo exposure of DNA to the butadiene epoxides. The 2′-deoxyadenosine standards 13a-d were readily prepared in 80-90% yield by reaction of 6-chloropurine 2′-deoxyriboside with amino alcohols 5a-d in DMSO or DMF containing DIPEA (Scheme 4). The synthesis of the adducted dGuo nucleosides 15a-d from 2-fluoro-O6-TMSE-2′-deoxyinosine was carried out in a similar fashion except that an additional step, removal of the O6-TMSE group, was required (Scheme 4). The TMSE group is much more labile in the product than in the starting 2-fluoro

Figure 1. Circular dichroism spectra of BDO- and BDEadducted nucleosides. Spectra were recorded in methanol at 25 °C: (A) R-BDO-dAdo (13a) and S-BDO-dAdo (13b), (B) R,RBDE-dAdo (13c) and S,S-BDE-dAdo (13d), (C) R-BDO-dGuo (15a) and S-BDO-dGuo (15b), and (D) R,R-BDE-dGuo (15c) and S,S-BDE-dGuo (15d).

compound, but to ensure complete removal, the reaction mixture was treated with dilute acetic acid at room temperature for 2 h. The structures of the adducted nucleosides were confirmed by 1H NMR and mass spectroscopy. In addition, CD spectra were obtained on the nucleosides (Figure 1). The spectra of the (R)- and (S)BDO N6 adducts of deoxyadenosine were qualitatively similar to those reported by Koivisto (10), who prepared the adducted nucleosides by reaction with optically active BDO; only the adducts at C2 of BDO exhibited CD spectra. The (R) isomer exhibited a positive band in the 270-280 nm region and a negative one around 225 nm, whereas the (S) isomer exhibited the opposite behavior in these regions. CD spectra of the BDE adducts have not previously been reported; the spectra differed only subtly from each other and from that of unmodified dAdo. The CD spectra of the R-BDO-dGuo adduct had a positive band in the 270-280 nm region and a negative one around 250 nm, and the (S) isomer exhibited the opposite pattern. These observations are similar to data reported by Neagu et al. (3) for N7 guanine adducts of optically active BDO. The data on the BDO N2 adducts are in accord with spectra reported for analogous adducts of styrene oxide (45, 46) with the (R) isomer exhibiting a positive band at the longer wavelength. The CD spectra of the BDE adducts of dGuo, unlike those of dAdo, clearly showed bands with the opposite sign in the 250 nm region and around 275-285 nm, with the (R,R) isomer having the negative band at the longer wavelength and the positive one at the shorter. The CD spectra of the BDE adducts would not be expected to be very strong, inasmuch as the adduct moiety is a weak chromophore and

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Figure 2. HPLC chromatograms depicting the time course of a typical adduction reaction: 6-chloropurine-containing oligonucleotide 16 (starting material) with 2-amino-3-buten-1-ol (5a) to give adducted oligonucleotide 17a. Reaction and HPLC conditions as described in the Experimental Section.

the site of attachment of the adduct is not at a chiral site. Synthesis of BDO- and BDE-Adducted Oligonucleotides. The target oligonucleotides for these studies were 11-mers containing either codon 61 (CA*A) or codon 12 (GG*T) of the N-ras protooncogene. We have employed these sequences previously for preparation of PAH- and styrene oxide-adducted oligonucleotides which were used subsequently by others for structural and mutagenesis studies. For the synthesis of the butadiene epoxide-adducted oligonucleotides, we used a refinement of our original postoligomerization strategy in which the halopurine-containing oligonucleotides were removed from the matrix, deprotected, and purified before reaction with the amino alcohols. This strategy is especially useful for the syntheses of alkyl-substituted oligonucleotides because the adducted oligomers do not separate as well from unadducted oligonucleotides as do PAH-adducted oligomers. The halooligonucleotides react in high yield with the relatively unhindered alkyl amino alcohols, and if the halooligonucleotide is purified before reaction, the final product is very easily purified. For the adenine adducts, the 6-chloropurine nucleoside was incorporated into the oligonucleotide at the 61,2 position (Scheme 4); the yield of the purified chloropurine-containing oligonucleotide after HPLC purification was about 45 A260 units per 1 µmol cassette. The reactions with amino alcohols 5a-d proceeded very smoothly in DMSO containing DIPEA at 60 °C for 6-48 h; yields of 70-75% were obtained after reverse-phase HPLC purification. The course of a typical reaction (oligonucleotide 16 with amino alcohol 5a to give adducted oligonucleotide 17a) is shown in Figure 2. The BDE-adducted products were further purified by ion-exchange chromatography. For the preparation of BDO- and BDE-adducted ras 12,2 oligonucleotides (Scheme 4), the halopurine-containing 11-mer 18 was synthesized using 2-fluoro-O6-TMSE2′-deoxyinosine phosphoramidite followed by treatment

Nechev et al.

Figure 3. HPLC profiles of enzymatic hydrolysates of representative adducted oligonucleotides (A) 17b (5′-CGGA-CXAGAAG-3′, where X is 13b), (B) 19a (5′-GGCA-GXT-GGTG-3′, where X is 15a), (C) 17c (5′-CGGA-CXA-GAAG-3′, where X is 13c), and (D) 19c (5′-GGCA-GXT-GGTG-3′, where X is 15c). In each case, an HPLC trace of an authentic sample of the corresponding nucleoside adduct is superimposed: (A) S-BDOdAdo, (B) R-BDO-dGuo, (C) R,R-BDE-dAdo, and (D) R,R-BDEdGuo.

with 0.1 M NaOH at room temperature for 12 h (with monitoring by HPLC) to cleave the oligomer from the beads and remove the protecting groups (except the TMSE group). After HPLC purification, approximately 25 A260 units per 1 µmol cassette of 18 was obtained. Oligomer 18 was reacted with amino alcohols 5a-d in DMSO containing DIPEA at 60 °C for 15-18 h with monitoring by HPLC. During the reaction, some of the O6-TMSE protecting group was lost from the product oligonucleotide, but to ensure complete deprotection, the pH of the crude product mixture was adjusted to pH 3 and the mixture allowed to stand at room temperature for 2 h. Reverse-phase HPLC purification was satisfactory for all of the adducts except 12,2-(R)-BDO (19a) which gave broad peaks even though it appeared to be quite homogeneous by CGE and MALDI. The oligonucleotide was purified by PAGE (67% recovery). All of the adducted oligonucleotides were characterized by CGE, enzymatic digestion, and mass spectroscopy. Typical enzyme digests are shown in Figure 3. The thermal stability of duplexes formed by annealing the adducted oligonucleotides to their complements was examined, and the results are shown in Table 1. Two observations can be made. The first is that adducts on N6 of dAdo are more destabilizing than on N2 of dGuo, perhaps because AT base pairs are stabilized by only two hydrogen bonds rather than three. The second observation is that the BDO adducts are more destabilizing than the BDE adducts. The BDO adducts, being attached at a secondary carbon, are sterically more hindered and less flexible than the BDE adducts which are bound at a primary carbon. Interestingly, the decrease in the Tm values for the N6 BDO-adducted oligonucleotides seen in this study is very similar to that observed for N6-R

BD Epoxide Adducts at Adenine N6 and Guanine N2 Table 1. Thermal Melting of Unmodified and Epoxidized Butadiene N6- and N2-Modified Oligonucleotide Duplexesa sequence

complement

5′-CGGACXAGAAG-3′ X is A (unmodified) X is N6-R-BDO-dAdo X is N6-S-BDO-dAdo X is N6-R,R-BDE-dAdo X is N6-S,S-BDE-dAdo 5′-GGCAGXTGGTG-3′ X is G (unmodified) X is N2-R-BDO-dGuo X is N2-S-BDO-dGuo X is N2-R,R-BDE-dGuo X is N2-S,S-BDE-dGuo

5′-CTTCTTGTCCG-3′ 5′-CTTCTTGTCCG-3′ 5′-CTTCTTGTCCG-3′ 5′-CTTCTTGTCCG-3′ 5′-CTTCTTGTCCG-3′ 5′-CTTCTTGTCCG-3′ 5′-CACCACCTGCC-3′ 5′-CACCACCTGCC-3′ 5′-CACCACCTGCC-3′ 5′-CACCACCTGCC-3′ 5′-CACCACCTGCC-3′ 5′-CACCACCTGCC-3′

Tm (°C)a 57 49 42 52 49 65 62 62 65 65

a Measurements were obtained at 260 nm in phosphate buffer [10 mM Na2HPO4/NaHPO4 (pH 7.0) containing 1.0 M NaCl and 50 µM EDTA] at duplex concentrations of ∼4.0 µM.

adducts of styrene oxide in the same sequence (47, 48). The Tm of the styrene oxide (R) adduct was 9 °C lower than that of the unadducted duplex, and the Tm of the (S) adduct was 16 °C lower. Similarly, for the N2-dGuo adducts, both the 12,2 R-styrene oxide (R) 11-mer and the (S) isomer had Tm values which were 3-4 °C lower than that of the unmodified duplex (49). This is understandable if a butadiene monoepoxide adduct (at C2 of the epoxide) is viewed as a truncated version of an R-styrene oxide adduct; i.e., each species has a hydroxymethyl group and a rigid CdC moiety attached to the carbon bound to the DNA.

In this paper, we have described extension of the postoligomerization strategy previously developed in our laboratory for styrene oxide and PAH diol epoxide adducts to the synthesis of a number of nucleosides and oligonucleotides containing single regio- and stereoisomers of BDO and BDE. The mutagenicity of these oligonucleotides in E. coli has been examined (50, 51). Although none of the species proved to be significantly mutagenic in E. coli, the site (N2 or N6) and stereochemistry of the adduct clearly influenced the mutational spectrum and replication by polymerases; the adenine adducts were readily bypassed in vitro, whereas the guanine adducts were almost totally blocking. Interestingly, the dAdo (R,R)-BDE adduct showed only A f G mutations, whereas the (S,S) isomer gave solely A f C mutations. In contrast, the mutations induced by the guanine N2 adducts were distributed approximately equally among G f A, G f T, and G f C substitutions. These syntheses and the subsequent biological studies represent the most comprehensive investigation thus far reported into the effect of regio- and stereochemistry of adducts of small aliphatic epoxides on their biological properties. However, in the case of the butadiene epoxides, there are still many adducts to be examined, including the C1 adducts of BDO and the adducts of the meso- (RS/SR) form of BDO2 or BDE. Relatively little attention has been paid to the possible biological role of meso-BDO2. However, it has recently been shown to be formed from BDO in amounts comparable to (()-BDO2

Chem. Res. Toxicol., Vol. 14, No. 4, 2001 387

by cDNA-expressed human cytochrome P450 2E1 and by human, rat, and mouse liver microsomes (52). Oe et al. (53) also reported recently the detection of significant amounts [∼50-100% of the (()-BDO2-derived species] of the N7-(2,3,4-trihydroxybutyl)guanine adduct derived from meso-BDO2 in the livers of mice and rats exposed to butadiene. The unstable adducts also have to be considered viable candidates for genotoxicity, especially the N1 adenine adducts which may be quite long-lived in dsDNA (13).

Acknowledgment. Generous support of this project by U.S. Public Health Service Grants ES00267 and ES07781 is gratefully acknowledged. Supporting Information Available: The 400 MHz 1H NMR spectra of compounds 5b, 5c, 7, 13a-d, and 15a-d, HPLC chromatogram showing relative retention times of all adducted nucleosides (13a-d and 15a-d), HPLC chromatograms of purified oligonucleotides 17a-d, HPLC profiles of enzyme digests of 17a-d, electropherograms of oligonucleotides 19ad, and HPLC profiles of enzyme digests of 19a-d. This material is available free of charge via the Internet at http://pubs.acs.org.

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