A Novel Synthesis of Malondialdehyde Adducts of Deoxyguanosine

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Chem. Res. Toxicol. 2004, 17, 144-149

A Novel Synthesis of Malondialdehyde Adducts of Deoxyguanosine, Deoxyadenosine, and Deoxycytidine Hao Wang,†,‡ Lawrence J. Marnett,†,‡,§,| Thomas M. Harris,†,‡,| and Carmelo J. Rizzo*,†,‡,| Department of Chemistry and Biochemistry, Center in Molecular Toxicology, and Vanderbilt Institute of Chemical Biology, VU Station B 351822, Nashville, Tennessee 37235-1822 Received August 16, 2003

Malondialdehyde (MDA) is a mutagenic product of lipid peroxidation and prostaglandin biosynthesis. MDA reacts with DNA bases to produce adducts of deoxyguanosine (M1G), deoxyadenosine (M1A), and deoxycytidine (M1C). A novel synthesis of these MDA nucleoside adducts has been developed, which significantly improves their availability. For the deoxyguanosine adduct, M1G, an amine equivalent to MDA, 4-amino-3-(phenylselenyl)butane-1,2diol, was reacted with 2-fluoro-O6-(2-(trimethylsilyl)ethyl)-2′-deoxyinosine via a nucleophilic aromatic substitution reaction followed by acid hydrolysis of the O6-protecting group to give an N2-modified deoxyguanosine intermediate. Periodate oxidation of this intermediate under slightly acidic conditions gave M1G in good overall yield via cleavage of the vicinal diol unit and concomitant oxidation of the phenylselenide group to the corresponding selenoxide and syn β-elimination. M1A and M1C were synthesized by the same strategy starting from 6-chloropurine 2′-deoxyriboside and 1-(2-deoxy-β-D-erythro-pentofuranosyl)-4-(1H-1,2,4-triazol1-yl)-2-(1H)pyrimidinone, respectively. An advantage of this approach is that similar chemistry has been shown to be directly applicable to the synthesis of site specifically adducted oligonucleotides containing activated nucleobases such as those used in this study. This strategy may offer an improved synthesis to oligonucleotides containing M1G and a feasible approach to M1A and M1C containing oligonucleotides.

Introduction MDA (1; Figure 1)1 is an endogenous mutagen that is generated as a byproduct of prostaglandin biosynthesis and through lipid peroxidation (1, 2). Base propenals such as 2 are reactive equivalents to MDA and arise from oxidative damage to DNA (3, 4). Such compounds are on the order of 100 times more reactive toward DNA than MDA itself. MDA and base propenals have been shown to be mutagenic in bacteria, and MDA is mutagenic in mouse and human kidney cells and carcinogenic in rats (5-7). The major product from the reaction of MDA or its equivalents with DNA is the pyrimido[1,2-a]purin-10(3H)one nucleoside (M1G, 3) derived from deoxyguanosine (8). Also formed are the N6- and N4-(3-oxopropenyl)modified deoxyadenosine (M1A, 4) and deoxycytidine (M1C, 5) nucleosides, respectively (Figure 1) (9, 10). Background levels of M1G have been detected in human livers, and M1A has been detected from the treatment of calf thymus DNA with 3-benzoylacrolein, a reactive equivalent of MDA (1, 11-13). It has been proposed that * To whom correspondence should be addressed. Tel: 615-322-6100. Fax: 615-343-1234. E-mail: [email protected]. † Department of Chemistry, Vanderbilt University. ‡ Center in Molecular Toxicology, Vanderbilt University. § Department of Biochemistry. | Vanderbilt Institute of Chemical Biology. 1 Abbreviations: MDA, malondialdehyde; M G, 3-(2-deoxy-β-D1 erythro-pentofuranosyl)pyrimido[1, 2-a]purin-10(3H)-one; M1A, N6-(34 oxo-1-propenyl)-2′-deoxyadenosine; M1C, N -(3-oxo-1-propenyl)-2′deoxycytidine; LDA, lithium diisopropylamide; DME, 1,2-dimethoxyethane; AcOH, acetic acid.

Figure 1.

MDA forms DNA-DNA cross-links, which represent one of the most serious forms of DNA damage (6, 14). Convenient methods for the site specific synthesis of MDA-containing oligonucleotides are required to better understand the biological consequences of MDA-mediated DNA damage and to relate the biology to structural perturbations of the DNA. The lability of all MDA-adducted nucleosides to alkaline conditions makes their preparation challenging. While these modified nucleosides have been prepared, only M1G has been site specifically incorporated into oligonucleotides, which was accomplished via the adducted phosphoramidite method (15, 16). The synthesis of the M1G phosphoramidite featured a stereoselective, enzymatic transglycosylation of the MDA-adducted guanine base and 2′-deoxycytidine as the 2-deoxyribosyl donor; however, the preparation of the adducted base proceeds in low yield largely due to the poor reaction of MDA with guanine and subsequent purification difficul-

10.1021/tx034174g CCC: $27.50 © 2004 American Chemical Society Published on Web 01/10/2004

Nucleoside Adducts of Malondialdehyde

ties making this route inconvenient for the preparation of M1G-containing oligonucleotides (16, 17). Because the standard DNA deprotection protocols require strongly basic conditions, the greater alkaline sensitivity of M1A and M1C makes the adducted phosphoramidite approach to oligonucleotides containing these lesions unfeasible using standard protecting groups. We report here a new synthesis of the MDA-adducted nucleosides M1G (3), M1A (4), and M1C (5). Of note, the adduction reactions were accomplished through the use of “activated” nucleoside bases and an amine analogue of MDA via a nucleophilic aromatic substitution reaction. In closely related systems, we have demonstrated that this chemistry can be directly applied to the site specific synthesis of modified oligonucleotides under mild, neutral conditions. This should allow for convenient access to structurally defined oligonucleotides containing these important lesions needed for biological and structural studies.

Experimental Procedures All commercially obtained chemicals were used as received, except THF, which was freshly distilled from a sodium/benzophenone ketyl. Anhydrous DME, DMSO, and diisopropylamine were obtained in Aldrich Sure-seal bottles and used as received. Proton NMR data were recorded at 300 MHz. Highresolution FAB mass spectra were obtained from the University of Notre Dame Mass Spectrometry Center using nitrobenzyl alcohol as the matrix. TLC was performed using Silica Gel 60 F254 precoated, 250 µm plates, and visualization was accomplished by a hand-held UV lamp or by staining the plate with anisaldehyde or ninhydrin stain followed by heating. Adducted nucleosides were purified on a Beckman HPLC system using a YMC ODS-AQ C-18 reversed phase column (4.6 mm × 25 mm, S-5 120 Å) using acetonitrile/water as the elutent and monitored with a diode array detector at 254 nm for M1G and 320 nm for M1A and M1C. The following solvent gradient was used for HPLC purification and analysis: 0-15 min linear gradient from 1 to 10% acetonitrile, then a 5 min linear gradient to 20% acetonitrile, then a 10 min linear gradient to 100% acetonitrile followed by a 5 min linear gradient to the initial conditions. 2-(Phenylselenyl)acetonitrile (16) (18). To a solution of diphenyl diselenide (1.00 g, 3.2 mmol) in absolute ethanol (20 mL) cooled in an ice bath was added solid NaBH4 (0.26 g, 6.9 mmol) in a single portion. A solution of chloroacetonitrile (15, 0.49 g, 6.5 mmol) in absolute ethanol (10 mL) was added dropwise. The mixture was stirred at 0° C for 1 h and then warmed to room temperature. Water (20 mL) and ether (50 mL) were added. The organic layer was separated, washed with brine, and dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. Purification by flash chromatography on silica, eluting with 5% ethyl acetate in petroleum ether, gave 16 (1.17 g, 93% yield), rf 0.43 in 20% ethyl acetate in petroleum ether. 1H NMR (CDCl3): δ 7.69 (m, 2H), 7.38 (m, 3H), 3.38 (s, 2H). 4-(tert-Butyldimethylsilyloxy)-3-hydroxy-2-(phenylselenyl)butyronitrile (18). To a stirred solution of 2-(phenylselenyl)acetonitrile (16, 235 mg, 1.2 mmol) in THF (5 mL) cooled to -78 °C under argon was added dropwise a solution of LDA (2.0 M THF, 0.65 mL, 1.3 mmol). The reaction was stirred at -78 °C for 30 min and then 2-(tert-butyldimethylsilyloxy)acetaldehyde (17, 90% purity from Aldrich, 193 mg, 1 mmol) in THF (1 mL, the THF solution of 17 was previously treated with activated 3 Å molecular sieves over 15 min) was added dropwise over 3 min. After the addition was complete, the reaction was stirred at -78 °C for 3 min and then quenched by the addition of saturated aqueous NH4Cl (10 mL). The mixture was allowed to warm to room temperature, and the organic layer was separated. The organic layer was successively washed with

Chem. Res. Toxicol., Vol. 17, No. 2, 2004 145 water and saturated brine. The combined aqueous layers were extracted twice with ether, and the combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. Purification by flash chromatography on silica gel, eluting with 5% ethyl acetate in petroleum ether, gave a mixture of 16 and 18 (310 mg total, 60% yield of the desired product was estimated by the 1H NMR of the mixture). Purification could be more easily achieved after the next step. A pure sample of 18 for characterization purposes was obtained by repeated flash chromatography rf 0.42 in 20% ethyl acetate in petroleum ether. 1H NMR (CDCl3): δ 7.71 (m, 2H), 7.39 (m, 3H), 3.85 (m, 4H), 2.82 (m, 1H, OH), 0.89 (s, 9H), 0.09 (s, 6H). 3,4-Dihydroxy-2-(phenylselenyl)butyronitrile (19). The crude 18 (207 mg, 0.56 mmol) taken directly from the procedure described above without further purification was dissolved in a stock solution of HF/pyridine in THF/pyridine solution (20 mL, ca. 1.4 N HF), which was prepared according to a literature procedure (19). The reaction mixture was stirred for 24 h at room temperature and then quenched by the addition of 1.0 M aqueous KH2PO4 (20 mL) and extracted with ethyl acetate (2 × 20 mL). The combined organic layers were washed with saturated brine (20 mL) dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. Purification by flash chromatography on silica gel, eluting with 2% methanol in chloroform, gave 19 (101 mg, 70%) rf 0.61 in 1% methanol in chloroform. 1H NMR: δ 7.71 (m, 2H), 7.39 (m, 3H), 3.87 (m, 4H), 3.33 (br, 1H, OH), 2.48-2.40 (m, 1H, OH). 4-Amino-3-(phenylselenyl)butane-1,2-diol (14). To a solution of 19 (98 mg, 0.38 mmol) in dry DME (6 mL) was added borane-methyl sulfide complex (10.0 M, 0.76 mL, 7.6 mmol), and the reaction was stirred at 40 °C under argon for 1 day. After it was cooled to 0 °C, methanol (3 mL) was added, the solution was stirred for 1 day, and then, water (2 mL) was added. The solvents were evaporated under reduced pressure, and methanol was added and evaporated (3 × 10 mL). The residue was dried under high vacuum for 1 day to give 14 (80 mg, 81% yield), which was used without further purification. 1H NMR (CD OD): δ 7.64-7.60 (m, 2H), 7.34-7.26 (m, 3H), 3 3.89-3.65 (m, 4H), 3.08-2.82 (m, 2H). FAB HRMS m/z calcd for C10H16NO2Se (M + H), 262.0346; found, 262.0363. N2-[3,4-Dihydroxy-2-(phenylselenyl)butyl]-2′-deoxyguanosine (20). A solution of 14 (4 mg, 0.015 mmol) in anhydrous DMSO (50 µL) was added to a mixture of 6 (3.7 mg, 0.01 mmol), DMSO (50 µL), and diisopropylethylamine (50 µL). The reaction mixture was stirred at 55 °C for 1 day, then cooled to room temperature, and concentrated using a centrifugal evaporator. The residue was dissolved in 5% aqueous AcOH (0.5 mL) and stirred at room temperature for 1 h. HPLC purification of the reaction mixture gave 20 as a mixture of diastereomers (2.8 mg, 55% yield). 1H NMR (CD3OD): δ 7.96, 7.95, 7.93, and 7.89 (diastereomers, s, 1H, H-8), 7.56 (m, 2H, aromatic), 7.20 (m, 3H, aromatic), 6.18 (m, 1H, H-1′), 4.51 (m, 1H, H-3′), 3.96-3.50 (m, 9H, H-4′, H-5′, H-5′′, 2H-1*, H-2*, H-3* and 2H-4*), 2.65 (m, 1H, H-2′), 2.35 (m, 1H, H-2′′). FAB HRMS m/z calcd for C20H26N5O6Se (M + H), 512.1048; found, 512.1026. 3-(2-Deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-a]purin-10-(3H)one (M1G, 3). An aqueous solution of sodium periodate (0.69 mL, 13.8 µmol, 20 mM in water) was added to a solution of 20 (2.4 mg, 4.6 µmol) in 1% aqueous AcOH (1 mL). The reaction mixture was shaken at room temperature for 3 h and then purified by HPLC to give M1G (3, 1.0 mg, 72%). 1H NMR (D2O): δ 9.27 (dd, 1H, J ) 7.2, 2.1 Hz, H-8), 8.92 (dd, 1H, J ) 4.0, 2.2 Hz, H-6), 8.28 (s, 1H, H-2), 7.25 (dd, 1H, J ) 7.2, 4.0 Hz, H-7), 6.43 (t, J ) 7.0 Hz, 1H, H-1′), 4.60 (m, 1H, H-3′), 4.02 (m, 1H, H-4′), 3.67 (m, 2H, H-5′, H-5′′), 2.77 (m, 1H, H-2′), 2.48 (m, 1H, H-2′′). FAB HRMS m/z calcd for C13H14N5O4 (M + H), 304.1046; found, 304.1026. N6-[3,4-Dihydroxy-2-(phenylselenyl)butyl]-2′-deoxyadenosine (23). A solution of 14 (4 mg, 0.015 mmol) in anhydrous DMSO (50 µL) was added to a solution of 22 (2.7 mg, 0.01 mmol), DMSO (50 µL), and diisopropylethylamine (50

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Chem. Res. Toxicol., Vol. 17, No. 2, 2004 Scheme 1

µL). The reaction mixture was stirred at 55 °C for 10 h and then cooled to room temperature. HPLC purification of the reaction mixture gave 23 as a mixture of diastereomers (3.3 mg, 66% yield). 1H NMR (CD3OD): δ 8.27, 8.26, 8.24 and 8.23 (diastereomers, 1H, H8 or H-2), 8.16 and 8.14 (diastereomers, s, 1H, H-8 or H-2), 7.54 (m, 2H, aromatic), 7.14 (m, 3H, aromatic), 6.40 (m, 1H, H-1′), 4.57 (m, 1H, H-3′), 3.90-3.40 (m, 9H, H-4′, H-5′, H-5′′, 2H-1*, H-2*, H-3* and 2H-4*), 2.80 (m, 1H, H-2′), 2.40 (m, 1H, H-2′′). FAB HRMS m/z calcd for C20H26N5O5Se (M + H), 496.1099; found, 496.1095. N6-(3-Oxo-1-propenyl)-2′-deoxyadenosine (M1A, 4). An aqueous solution of sodium periodate (20 mM, 0.43 mL, 8.6 µmol) was added to a solution of 23 (2.2 mg, 4.3 µmol) in 1% aqueous AcOH (2 mL). The reaction mixture was stirred at room temperature for 1 h and then purified by HPLC to give M1A (4, 0.8 mg, 61%). 1H NMR (D2O): δ 9.17 (d, 1H, J ) 8.7 Hz, β-vinylH), 8.60 (d, 1H, J ) 13.2 Hz, aldehyde-H), 8.40 (s, 1H, H-2 or H-8), 8.35 (s, 1H, H-8 or H-2), 6.40 (t, J ) 7.0 Hz, 1H, H-1′), 5.88 (dd, 1H, J ) 13.5, 8.7 Hz, R-vinyl-H), 4.50 (m, 1H, H-3′), 4.03 (m, 1H, H-4′), 3.65 (m, 2H, H-5′, H-5′′), 2.72 (m, 1H, H-2′), 2.42 (m, 1H, H-2′′). FAB HRMS m/z calcd for C13H16N5O4 (M + H), 306.1203; found, 306.1187. N4-[3,4-Dihydroxy-2-(phenylselenyl)butyl]deoxycytidine (25). A solution of 14 (4 mg, 0.015 mmol) in anhydrous DMSO (50 µL) was added to a solution of 24 (2.8 mg, 0.01 mmol), DMSO (50 µL), and diisopropylethylamine (50 µL). The reaction mixture was stirred at 60 °C for 1 day and then cooled to room temperature. HPLC purification of the reaction mixture gave 25 as a mixture of diastereomers (2.8 mg, 60% yield). 1H NMR (CD3OD): δ 7.85 (m, 1H, H-6), 7.58 (m, 2H, aromatic), 7.23 (m, 3H, aromatic), 6.22 (m, 1H, H-1′), 5.75 (m, 1H, H-5), 4.34 (m, 1H, H-3′), 3.95-3.40 (m, 9H, H-4′, H-5′, H-5′′, 2H-1*, H-2*, H-3* and 2H-4*), 2.31 (m, 1H, H-2′), 2.10 (m, 1H, H-2′′). FAB HRMS m/z calcd for C19H26N3O6Se (M + H), 472.0987; found, 472.0991. N4-(3-Oxo-1-propenyl)-2′-deoxycytidine (M1C, 5). An aqueous solution of sodium periodate (20 mM, 430 µL, 8.6 µmol) was added to a solution of 25 (2.0 mg, 4.3 µmol) in 1% aqueous AcOH (2 mL). The reaction mixture was stirred at room temperature for 1 h and then purified by HPLC to give M1C (5, 0.9 mg, 74%). 1H NMR (D O): δ 9.20 (d, 1H, J ) 8.6 Hz, aldehyde-H), 8.20 (d, 2 1H, J ) 13.7 Hz, β-vinyl-H), 8.07 (d, 1H, J ) 7.4 Hz, H-6), 6.18 (d, 1H, J ) 7.4 Hz, H-5), 6.13 (t, J ) 6.3 Hz, 1H, H-1′), 5.81 (dd, 1H, J ) 13.6, 8.6 Hz, R-vinyl-H), 4.31 (m, 1H, H-3′), 3.99 (m, 1H, H-4′), 3.69 (m, 2H, H-5′, H-5′′), 2.41 (m, 1H, H-2′), 2.20 (m, 1H, H-2′′). FAB HRMS m/z calcd for C12H16N3O5 (M + H), 282.1090; found, 282.1071.

Results and Discussion We and others have previously synthesized 1,N2deoxyguanosine adducts of related R,β-unsaturated aldehydes such as acrolein, crotonaldehyde, and 4-hydroxynonenal through the nucleophilic aromatic substitution reaction of O6-[2-(trimethylsilyl)ethyl]-2-fluoro-2′deoxyinosine (6) by an amine synthon of the enal (Scheme 1) (20-24). In a strategy initially developed by Johnson, a vicinal diol unit could be used as a surrogate for the

Wang et al. Scheme 2

Scheme 3

aldehyde and could be cleaved by sodium periodate after assembly of the oligonucleotide (16). A critical issue in the present case is that MDA is in a higher oxidation state than acrolein, and the synthetic approach must be adapted to account for this difference. We envisioned introducing the additional double bond of the MDA adduct through a β-elimination reaction after the adduction and periodate cleavage reactions as shown retrosynthetically in Scheme 2. Thus, the three carbon MDA synthon is represented as 14 in which the leaving group (X) must be compatible with the nucleophilic aromatic substitution conditions. Halides were judged unsatisfactory because they could undergo intra- and intermolecular self-condensation reactions. Because of this limitation, we focused on a phenylselenyl ether as the leaving group. We anticipated that the phenylselenide would be stable to the nucleophilic aromatic substitution reaction but would undergo oxidation to the corresponding selenoxide during the periodate oxidation step (25). Selenoxides are known to undergo facile unimolecular syn β-elimination at room temperature or below, particularly when R,βunsaturated carbonyls are the product (26). Selenoxide elimination is predicted to give N2-(3-oxopropenyl)deoxyguanosine (11), which is a likely intermediate in the reaction of deoxyguanosine with MDA to give M1G. Synthesis of the MDA Synthon. The MDA synthon 14 where X is a phenyselenyl group was accomplished in four steps from chloroacetonitrile according to Scheme 3. Displacement of chloroacetonitrile with the phenylselenide anion, generated in situ by the reduction of diphenyldiselenide with sodium borohydride, gave 2(phenylselenyl)acetonitrile (15) in 93% yield (18). Generation of the enolate of 16 with LDA in THF and aldol

Nucleoside Adducts of Malondialdehyde

Chem. Res. Toxicol., Vol. 17, No. 2, 2004 147

Scheme 4

Figure 2. HPLC analysis of the adduction reaction of 6 with amine 14 to give 20 (top trace) and the periodate oxidation of 20 to M1G (3, bottom trace).

condensation with commercially available protected glycolaldehyde 17 gave a mixture of diastereomeric aldol products 18. In addition to the syn- and anti-aldol products (18), we also observed a small amount of product in which the tert-butyldimethylsilyl protecting group migrated to the secondary hydroxyl group (structure not shown). The mixture was deprotected using pyridinium hydrofluoride to give nitrile 19 (19). Carbon-selenium bonds are easily reduced; thus, the choice of reducing agents for the selective reduction of the nitrile was of concern. Borane was found to smoothly reduce the nitrile to the primary amine without overreduction of the sensitive phenylselenyl group, giving the desired product in 81% yield. Synthesis of M1G. Amine 14 (X ) -SePh) was coupled with 2-fluoro-O6-(2-(trimethylsilyl)ethyl)-2′-deoxyinosine (6) under the nucleophilic aromatic substitution conditions previously worked out in our laboratory for related systems (Scheme 4). Because 14 is a mixture of syn and anti diastereomers each of which is racemic, the coupled product 20 is actually a mixture of four diastereomers (Figure 2). Because there are conformational requirements for the selenoxide syn β-elimination reaction, it is possible that the diastereomers of 20 would undergo this reaction at different rates. Although these compounds could be partially separated by HPLC, we elected to subject the mixture to the periodate oxidation reaction. We found that the optimal conditions for the concomitant periodate diol cleavage and selenide oxidation-selenoxide elimination were in the presence of a small amount of AcOH. Upon addition of sodium periodate, we observed rapid consumption of 20 to give two products, one of which was identified as M1G (3); the other was converted to M1G over 3 h. If AcOH was omitted, the conversion to M1G was slower. M1G was unambiguously identified by coelution with an authentic standard, LC/ES-MS, and its characteristic UV spectrum as observed via a diode array detector (Figure 3). The adducted nucleoside was also isolated and fully analyzed by 1H NMR spectroscopy. The identity of the initial intermediate from the periodate treatment is uncertain although it is reasonable that protic acid would promote the isomerization/cyclization of N2-(3-oxopropenyl)-2′deoxyguanosine (11) to M1G (3). The overall yield for the periodate oxidation of 20 to M1G (3) is 72%. Synthesis of M1A. In a similar fashion, amine 14 was coupled with 6-chloropurine-2′-deoxyriboside (22) to give

Figure 3. UV spectra of MDA adducted nucleosides M1G (3), M1A (4), and M1C (5).

N6-adducted adenosine 23 (Scheme 5). As for the analogous intermediate for the M1G synthesis (20), 23 was obtained as a mixture of four diastereomers (Figure 4). Treatment of this mixture with sodium periodate gave M1A (4) in 62% yield. We found that excess periodate gave unidentified byproducts; thus, the reaction was performed with stoichiometric oxidant. Although the role of trace AcOH in the periodate oxidation is less clear than in the M1G synthesis, byproducts were observed if it was omitted. Interestingly, the oxidation of 23 to M1A (4) is complete in less than 1 h, whereas the analogous reaction for the M1G synthesis required 3 h under identical conditions. M1A was characterized by its UV and 1H NMR spectra, which were identical to those previously reported (9). Synthesis of M1C. The reaction of the 4-triazolopyrimidone nucleoside (24) with amine 14 gave the N4modified 2′-deoxycytidine derivative 25 (Scheme 6) in

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Summary

Figure 4. HPLC analysis of the adduction reaction of 22 with amine 14 to give 23 (top trace) and the periodate oxidation of 23 to M1A (4, bottom trace).

Figure 5. HPLC analysis for the adduction reaction of 24 with amine 14 to give 25 (top trace) and the periodate oxidation 25 to M1C (5, bottom trace).

Scheme 5

We have developed a new strategy for the synthesis of MDA-modified nucleosides of deoxyguanosine (M1G, 3), deoxyadenosine (M1A, 4), and deoxycytidine (M1C, 5) using a nucleophilic aromatic substitution reaction of activated nucleosides 6, 22, and 24 with an amine equivalent of MDA (14). The adducted nucleosides (20, 23, and 25) are then subjected to periodate oxidation to give the desired MDA-adducted nucleosides. An advantage of this approach is that we have demonstrated that the chemistry can be directly applied to the site specific synthesis of adducted oligonucleotides in related systems (21, 22, 24, 27). The periodate oxidation step is performed under neutral or slightly acidic conditions in which the MDA adducts are stable and can be performed after the deprotection of the oligonucleotide. We have previously incorporated 6 and 22 into oligonucleotides, and oligonucleotides containing 24 have been previously reported (21, 22, 24, 28). We believe that this strategy will offer an improved synthesis of oligonucleotides containing M1G and a feasible approach to M1A and M1C containing oligonucleotides. Indeed, we have recently utilized this chemistry for the site specific synthesis of oligonucleotides containing M1G and M1A. The results of these studies as well as the reactivity and properties of the MDA-adducted oligonucleotides will be reported soon.

Acknowledgment. This work was supported by the National Institutes of Health through research Grants CA87819 (L.J.M.), ES05355 (T.M.H.), and ES11331 (C.J.R.) and center Grant ES00267. Supporting Information Available: Copies of 1H NMR spectra of 3-5, 14, 18-20, 23, and 25. This material is available free of charge via the Internet at http://pubs.acs.org.

References

Scheme 6

60% yield as a mixture of diastereomers. Periodate oxidation of 25 gave N4-(3-oxopropenyl)-2′-deoxycytidine (5, M1C) in 74% yield (Figure 5). As with the periodate oxidation of 23 to M1A (4), stoichiometric periodate in trace AcOH gave the best results. The UV (Figure 3) and 1 H NMR spectra of M1C (5) matched those previously reported (10).

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(12)

(13)

(14)

(15)

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