Stereospecific Synthesis of Oligonucleotides Containing

Irina G. Minko , Ivan D. Kozekov , Thomas M. Harris , Carmelo J. Rizzo , R. Stephen Lloyd and Michael P. Stone. Chemical Research in Toxicology 2009 2...
0 downloads 0 Views 97KB Size


Chem. Res. Toxicol. 2001, 14, 1506-1512

Stereospecific Synthesis of Oligonucleotides Containing Crotonaldehyde Adducts of Deoxyguanosine Lubomir V. Nechev, Ivan Kozekov, Constance M. Harris, and Thomas M. Harris* Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235 Received April 4, 2001

Crotonaldehyde reacts with DNA to form two diastereomeric 1,N2 cyclic adducts of deoxyguanosine. A synthesis of the two diastereomeric deoxynucleosides has been achieved by reaction of mixed diastereomers of 4-amino-1,2-pentanediol with 2-fluoro-O6-(trimethylsilylethyl)-deoxyinosine. The resulting N2-(1-methyl-3,4-dihydroxybutyl)-deoxyguanosine was treated with NaIO4, cleaving the vicinal diol to the aldehyde. Spontaneous cyclization gave the two diastereomers of the crotonaldehyde-adducted nucleoside that were readily separated by HPLC. The absolute configurations were assigned by an enantiospecific synthesis of one diastereomer from (S)-3-aminobutanoic acid. The synthetic strategy has been extended to preparation of a site-specifically adducted oligonucleotide by reaction of the mixed diastereomers of 4-amino-1,2-pentanediol with an 8-mer oligonucleotide containing 2-fluoro-O6-(trimethylsilylethyl)-deoxyinosine. The diastereomeric oligonucleotides were separated by HPLC and absolute configurations of the adducts were established by enzymatic digestion to the adducted nucleosides.

Introduction The R,β-unsaturated aldehyde crotonaldehyde is a widely dispersed product of pyrolysis and incomplete oxidation of organic materials (1). It is present in significant quantities in tobacco smoke (2), is a metabolite of N-nitrosopyrrolidone (3-5), and arises in living systems as a product of lipid peroxidation (6). Crotonaldehyde is genotoxic, mutagenic, and carcinogenic (7-11). Like other R,β-unsaturated aldehydes, it reacts with DNA to form adducts which are believed to be the basis of genotoxicity. At this juncture, only adducts of dGuo have been characterized (3, 12, 13). Adduct formation by the simpler analogue acrolein has been observed with dGuo, dAdo, and dCyd. It reacts with deoxyguanosine to form regioisomeric 1,N2-hydroxypropano adducts 1 and 2 as shown in Scheme 1 (8, 14). Adduct 1 in which the hydroxyl group is proximal to the N1 position is formed by reaction of guanine N2 with the β carbon of the aldehyde and N1 with the carbonyl group. Conversely, isomeric adduct 2 in which the hydroxyl group is distal to the N1 position is formed by attack of guanine N1 on the β carbon and attack of N2 on the carbonyl. With crotonaldehyde, only the diastereomers of proximal adduct 3 have been detected; two diastereomers are created by the stereogenic center bearing the methyl group. For both diastereomers, the hydroxyl group on the propano ring has been assigned as being predominantly trans to the methyl group (8, 15). Site-specific mutagenesis studies of the proximal deoxyguanosine adduct of acrolein have revealed it to be essentially nonmutagenic in point mutation assays although the hydroxypropano adduct blocks the WatsonCrick face of the nucleoside (16, 17). By contrast, the * To whom correspondence should be addressed. Phone: (615) 3222649. Fax: (615) 322-2649. E-mail: [email protected]

Scheme 1

unfunctionalized 1,N2-propano adduct is strongly mutagenic (18, 19). It has been proposed that the explanation for this difference is that the hydroxypropano adduct is able to undergo ring opening to form an N2,3-oxopropyl adduct (20). An NMR study by de los Santos and co-workers has confirmed that the hydroxypropano adduct reverts to the 3-oxopropyl species in double-stranded DNA (21). The lack of mutagenicity of the 3-oxopropyl adduct at the N2 position of dGuo is consistent with the recent finding that analogous adducts of butadiene monoepoxide and diolepoxide are only weakly mutagenic (22). It would be useful to examine the structures of DNA containing the diastereomers of the analogous 1,N2 dGuo crotonaldehyde adduct to see whether they also assume the de los Santos structure. The synthesis of DNA containing adducts of acrolein presented an interesting problem in that the aldehyde in equilibrium with the cyclic species is not compatible with the conditions employed for preparation of phosphoramidite reagents and assembly of oligodeoxynucleotides. Two synthetic strategies have been reported, both involving elaboration of the aldehyde after assembly of the oligodeoxynucleotide by periodate cleavage of a 1,2-diol (20, 23). This paper describes the utilization of one of these methods for enantiospecific synthesis of the crotonaldehyde adduct of deoxyguanosine and extension of the synthetic meth-

10.1021/tx0100690 CCC: $20.00 © 2001 American Chemical Society Published on Web 10/13/2001

Crotonaldehyde-Deoxyguanosine Adducts

odology to the preparation of oligodeoxynucleotides containing site-specific crotonaldehyde adducts.

Experimental Procedures General Methods. Thin-layer chromatography was performed on silica gel glass plates (Merck, Silica Gel 60 F254, layer thickness 0.25 mm). The chromatograms were visualized under UV light (254 nm) or by staining with an anisaldehyde/sulfuric acid solution, followed by heating. Column chromatography was performed using silica gel (Merck, 70-230 mesh). 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. For monitoring reactions and purification a YMC ODS-AQ column (250 × 4.6 mm, flow rate 1.5 mL/min, or 250 × 10 mm, flow rate, 5 mL/min) monitored at 260 nm was used with H2Oacetonitrile for nucleosides, and 0.1 M ammonium formateacetonitrile for oligonucleotides. Gradient A: 99% H2O for initial, 15 min linear gradient to 90% H2O, 5 min linear gradient to 80% H2O, 3 min linear gradient to 20% H2O, 2 min isocratic at 20% H2O, 3 min gradient to 99% H2O. Gradient B: 99% H2O for initial, 5 min linear gradient to 94% H2O, 15 min linear gradient to 91% H2O, 3 min linear gradient to 20% H2O, 2 min isocratic at 20% H2O, 3 min gradient to 99% H2O. Instrumentation. 1H NMR spectra were recorded at 400.13 MHz on a Bruker AM400 NMR spectrometer in D2O or DMSOd6. Low and high-resolution FAB mass spectra were obtained at the Mass Spectrometry Facility at the University of Notre Dame, Notre Dame, Indiana. Negative ion MALDI-TOF1 mass spectra of modified oligonucleotides were obtained on a Voyager Elite DE instrument (Perseptives Biosystems) using a 3-hydroxypicolinic acid (3-HPA) matrix containing ammonium hydrogen citrate (7 mg/mL) to suppress multiple sodium and potassium adducts. CD spectra were recorded in water at 25 °C on a JASCO J-700 spectropolarimeter. (S)-3-(Benzoylamino)butanoic Acid (24). Benzoyl chloride (0.50 g, 0.42 mL, 3.58 mmol) was added to a stirred solution of (S)-3-aminobutanoic acid (4S, 0.50 g, 3.58 mmol, Fluka) in 10 mL of 1 M NaOH. The mixture was cooled (ice bath) during the addition and stirred at room temperature for 24 h. TLC (ether, visualized with ninhydrin spray) showed complete consumption of the starting amino acid. The mixture was acidified (pH ∼6), extracted with ether and dried over MgSO4. The ether was evaporated and the residue was purified by a silica gel column (ether) to give 0.48 g (65%) of the product as a white solid. 1H NMR (DMSO-d6) δ 12.16 (bs, 1H, COOH), 8.28 (m, 1H, NH), 7.80 (m, 2H, o-Ph), 7.46 (m, 3H, m- and p-Ph), 4.34 (m, 1H, CH), 2.56 (dd, 1H, CH2, J1 ) 15.3 Hz, J2 ) 6.8 Hz), 2.38 (dd, 1H, J1 ) 15.3 Hz, J2 ) 4.3 Hz), 1.17 (d, 3H, CH3, J ) 6.6 Hz). HRMS (FAB+): calcd for C11H14NO3 [M + H]+, 208.0974; found, 208.0981. Methyl Ester of (S)-3-(Benzoylamino)butanoic Acid. (S)3-(Benzoylamino)butanoic acid (0.48 g, 2.32 mmol) was dissolved in 50 mL of methanol containing 4-5 drops of concentrated H2SO4. The mixture was refluxed for 1 h. Most of the methanol was evaporated, water was added and the mixture was extracted three times with ether. The combined ether layers were washed with satd. NaHCO3 solution and water and dried over MgSO4. Evaporation of the solvent gave 0.47 g (92%) of the ester, which was used in the next step without purification. 1H NMR (CDCl3) δ 7.78 (m, 2H, o-Ph), 7.46 (m, 3H, m- and p-Ph), 6.95 (bs, 1H, NH), 4.56 (m, 1H, CH), 3.72 (s, 3H, COOCH3), 2.69 (dd, 1H, CH2, J1 ) 15.9 Hz, J2 ) 5.3 Hz), 2.62 (dd, 1H, CH2, J1 ) 15.9 1 Abbreviations: DIBAL-H, diisobutylaluminum hydride; FAB MS, fast-atom bombardment mass spectrometry; MALDI TOF MS, matrixassisted laser desorption/ionization time-of-flight mass spectrometry; HRMS, high-resolution mass spectrometry; TMSE, trimethylsilylethyl. COSY, correlation spectroscopy; HMBC, heteronuclear multibond correlation spectroscopy (long-range 13C-1H scalar correlated 2D NMR experiment).

Chem. Res. Toxicol., Vol. 14, No. 11, 2001 1507 Hz, J2 ) 4.8 Hz), 1.34 (d, 3H, CH3, J ) 6.8 Hz). HRMS (FAB+): calcd for [M + H]+ C12H16NO3, 222.1130; found, 222.1122. (S)-3-(Benzoylamino)butyraldehyde (5S). Methyl (S)-3(benzoylamino)butanoate (0.47 g, 2.12 mmol) was dissolved in 20 mL of anhydrous methylene chloride. The solution was cooled to -78 °C. Diisobutylaluminum hydride (DIBAL-H) (2.12 mL of 1 M solution in hexane, 2.12 mmol) was added over 15 min; the mixture was stirred at -78 °C for 1 h. Additional DIBAL-H (2.12 mL, 2.12 mmol) was added over 15 min and the mixture was stirred for 1 h at -78 °C, followed by quenching at -78 °C with 4 mL of a saturated aqueous solution of NH4Cl. Hydrochloric acid (4%, 4 mL) was added and the mixture was warmed to room temperature. After 0.5 h, the mixture was extracted several times with CH2Cl2 and the combined organic layers were dried over MgSO4. The organic solvents were removed and the residue was purified by chromatography on a silica gel column (hexane/EtOAc, 1:1) to give 0.25 g (63%) of the substituted butyraldehyde (5S) (25). 1H NMR (CDCl3) δ 9.85 (bs, 1H, CHO), 7.75 (m, 2H, o-Ph), 7.46 (m, 3H, m- and p-Ph), 6.49 (s, 1H, NH), 4.64 (m, 1H, CH), 2.81 (m, 2H, CH2), 1.40 (d, 3H, CH3, J ) 6.9 Hz). HRMS (FAB+): calcd for C11H14NO2 [M + H]+, 192.1025; found, 192.1019. (S)-2-(Benzoylamino)-4-pentene (6S). A solution of methylidenetriphenyl-phosphorane (prepared from methyltriphenylphosphonium bromide (0.56 g, 1.57 mmol) and n-BuLi (0.560 mL of a 2.5 M solution in hexane, 1.4 mmol) in THF (20 mL) was added to a stirred solution of the above aldehyde (0.25 g, 1.13 mmol) in dry THF (5 mL) at -78 °C. The reaction was monitored by TLC by adding small aliquots to a mixture of saturated aqueous (NH4)2SO4 and EtOAc. After the starting material had been consumed, the mixture was warmed to room temperature and poured into saturated aqueous (NH4)2SO4, which was extracted with ether. The extract was dried over MgSO4, and after evaporation of the solvents, the residue was purified by chromatography on a silica gel column (hexane/ EtOAc, 2:1) to give 103 mg (42%) of (S)-2-(benzoylamino)-4pentene (6S). 1H NMR (CDCl3) δ 7.74 (m, 2H, o-Ph), 7.44 (m, 3H, m- and p-Ph), 5.95 (bs, 1H, NH), 5.84 (m, 1H, ) CH), 5.14 (m, 2H, ) CH2), 4.30 (m, 1H, CH), 2.35 (m, 2H, CH2), 1.26 (d, 3H, CH3, J ) 6.6 Hz). HRMS (FAB+): calcd for C12H16NO [M + H]+, 190.1232; found, 190.1227. 4(S)-Amino-1,2-(R and S)-pentanediol (7S). (S)-2-(Benzoylamino)-4-pentene (6S, 100 mg, 0.53 mmol) was added to a mixture of water (5 mL), acetone (2 mL), N-methylmorpholine N-oxide (71 mg, 0.6 mmol) and OsO4 (1-2 mg). The mixture was stirred overnight at room temperature and the solvents were evaporated. The residue was purified on a silica gel column (ether/methanol, 95:5) to give 93 mg (79%) of 4(S)-(benzoylamino)-1,2(R and S)-pentanediol (7S). 1H NMR (DMSO-d6) δ 8.20 (m, 1H, NH), 7.82 (m, 2H, o-Ph), 7.47 (m, 3H, m- and p-Ph), 4.49 (m, 2H, 2× OH), 4.21 (m, 1H, CHN), 3.50 (m, 1H, CHO), 3.29 (m, 2H, CH2O), 1.35-1.73 (m, 2H, CH2C), 1.16 (m, 3H, CH3). HRMS (FAB+) [M + H]+: calcd, 224.1287; found, 224.1286. The diol (90 mg, 0.4 mmol) in 6 M HCl (10 mL) was refluxed for 4 h. The solution was evaporated to dryness, water was added and the mixture was neutralized (pH 6.0). The water were evaporated again and the 4(S)-amino-1,2(R and S)-pentanediol was purified on an ion exchange column (DOWEX 50-X8). The aminodiol was eluted with 1 M NH4OH to give, after evaporation to dryness, 40 mg (83%) of amino alcohol 7S. 1H NMR (DMSOd6) δ 3.75 (m, 1H, CHOH), 3.50 (m, 1H, 1× CH2OH), 3.41 (m, 1H, CH2OH), 3.12 (m, 1H, CHNH), 1.46 (m, 2H, CH2), 1.08 (m, 3H, CH3). HRMS (FAB+) [M + H]+: calcd, 120.1025; found, 120.1002. N2-(1(S)-methyl-3(R and S),4-dihydroxybutyl)deoxyguanosine (9S). Amino alcohol 7S (9.8 mg, 0.082 mmol) was added to a mixture of O6-trimethylsilylethyl-2-fluoro-deoxyinosine (8, 20 mg, 0.054 mmol), DMSO (150 µL), and diisopropylethylamine (50 µL). The mixture was stirred at 55 °C for 7 h. The reaction was stopped and the solvents were evaporated under vacuum. The residue was dissolved in 5% acetic acid (1 mL) and stirred


Chem. Res. Toxicol., Vol. 14, No. 11, 2001

at room temperature for 1 h. HPLC purification of the reaction mixture gave 15.5 mg (78%) of N2-(1(S)-methyl-3,4-dihydroxybutyl)deoxyguanosine (9S). 1H NMR (DMSO-d6) δ 10.32 (bs, 1H, N1H), 7.83 (s, 1H, H-8), 6.30, 6.25 (2m, 1H, N2H), 6.08 (m, 1H, H-1′), 5.21 (m, 1H, 3′-OH), 4.81 (m, 1H, 5′-OH), 4.54 (m, 1H, γ-OH), 4.49 (m, 1H, δ-OH), 4.28 (m, 1H, H-3′), 4.00 (m, 1H, R-H), 3.74 (m, 1H, H-4′), 3.47 (m, 3H, γ-H, H-5′, H-5′′), 3.23 (m, 2H, δ-H), 2.40 (m, 1H, H-2′), 2.15 (m, 1H, H-2′′), 1.30-1.60 (m, 2H, β-H), 1.10 (m, 3H, CH3). HRMS (FAB+) [M+H]+: calcd. 370.1727, found 370.1732. 3-(2-Deoxy-β-D-erythro-pentofuranosyl)-8-hydroxy-6(S)methyl-5,6,7,8-tetrahydropyrimido[1,2-a]purin-10(3H)one) (3S). Sodium periodate (500 µL, 20 mM) was added to a solution of N2-(1(S)-methyl-3(R and S),4-dihydroxybutyl)deoxyguanosine (9S, 2 mg, 0.0054 mmol) in 0.05 M phosphate buffer, pH 7 (300 µL) (20). The reaction mixture was stirred at room temperature for 5-10 min. HPLC analysis showed complete disappearance of the peaks for the starting material. The reaction mixture was purified by HPLC to give 1.60 mg (88%) of the 6(S) proximal crotonaldehyde adduct (3S). 1H NMR (DMSO-d6) δ 7.83 (m, 2H, N2-H, H-2), 6.55 (bs, 1H, C8-OH), 6.12 (m, 1H, H-8), 6.03 (dd, 1H, H-1′, J1 ) J2 ) 6.8 Hz), 5.17 (d, 1H, 3′-OH, J ) 3.4 Hz), 4.84 (dd, 1H, 5′-OH, J1 ) J2 ) 5.5 Hz), 4.26 (bs, 1H, H-3′), 3.73 (m, 1H, H-4′), 3.63 (m, 1H, H-6), 3.45 (m, 2H, H-5′, H-5′′), 2.40 (m, 1H, H-2′), 2.10 (m, 1H, H-2′′), 1.95 (m, 1H, H-7), 1.38 (m, 1H, H-7), 1.15 (m, 3H, CH3). HRMS (FAB+) [M + H]+: calcd. 338.1464; found, 338.1466. Synthesis of a Mixture of 3R and 3S. (() N-Benzoyl 2-amino-4-pentene (6RS) (26) was subjected to oxidation with OsO4 followed by base hydrolysis to give a mixture of the four stereoisomers of 4-amino-1,2-pentanediol. Condensation of aminodiol 7RS with O6-trimethylsilylethyl-2-fluoro-deoxyinosine (8) gave a mixture of the four diastereomers of N2-(1-methyl-3,4dihydroxybutyl)-deoxyguanosine (9). Purification was achieved by HPLC without separation of the diastereomers. The mixture was oxidized with sodium periodate to form diastereomers 3R and 3S of the proximal crotonaldehyde adduct. The isomers were separated using gradient A (retention times of 19.76 and 20.34 min). The earlier eluting diastereomer was identical to the 3S diastereomer prepared from 4(S)-amino-1,2(R and S)-pentanediol. Accordingly, the late eluting diastereomer is compound 3(R). Synthesis of Crotonaldehyde-Modified Oligodeoxynucleotide. The preparation of O6-TMSE-2-fluorodeoxyinosine phosphoramidite and oligonucleotides containing the corresponding nucleobase is described elsewhere (27, 28). The O6TMSE-2-fluorodeoxyinosine-modified oligonucleotide was prepared on a 1 µmol scale using p-tert-butylphenoxyacetylprotected phosphoramidites (PE/Biosystems Expedite reagents). Cleavage from the solid support and deprotection were carried out by treatment with 0.1 M NaOH (∼10 h at room temperature). The neutralized crude material was purified by HPLC (YMC ODS-AQ column, 250 × 10 mm) using gradient A with 0.1 M aqueous ammonium formate and acetonitrile. The O6TMSE-2-fluoroinosine-modified oligodeoxynucleotide (10 A260 units) was mixed in a plastic test tube with diisopropylethylamine (50 µL), DMSO (100 µL), and mixed diastereomers of 4-amino-1,2-pentanediol (0.5 mg). The reaction mixture was stirred at 55 °C for 3 h. Solvents were evaporated under vacuum. The residue was dissolved in 5% acetic acid (500 µL) and stirred for 2 h at room temperature to remove the O6-TMSE group. The mixture was neutralized with 1 M NaOH and purified by HPLC to give 8.9 A260 units (∼89%) of the corresponding N2-(1-methyl3,4-dihydroxybutyl)guanine-modified oligodeoxynucleotide. The product was characterized by HPLC analysis of the enzymatic hydrolysate and MALDI-TOF MS: calcd for [M - H]- 2511.5, found 2511.3. The enzymatic hydrolysis was carried out in one step as follows: oligonucleotide (0.5 A260 units) was dissolved in 20 µL of buffer (pH 7, 0.01 M Tris‚HCl, 0.01 M MgCl2), DNase I (Promega, 5 units), alkaline phosphatase (Sigma P-4252, 1.7 units), and snake venom phosphodiesterase I (Sigma P-6877,

Nechev et al. 0.02 units) were added and the solution was incubated at 37 °C for 3 h. An aqueous solution of NaIO4 (100 µL, 20 mM) was added to a solution of N2-(1-methyl-3,4-dihydroxybutyl)guanine-modified oligodeoxynucleotide (5 A260 units) in 0.05 M phosphate buffer, pH 7 (300 µL) and the reaction mixture was stirred at room temperature for 10 min (20). The reaction resulted in two peaks in the HPLC trace representing the two isomers of the crotonaldehyde adduct. The two isomers were of approximately equal size and were separated by HPLC (gradient B, 0.1 M aqueous ammonium formate/acetonitrile); retention times of the two peaks were 18.34 and 18.99 min. The combined yield was 3.8 A260 units (∼80%). The structures were confirmed by enzymatic hydrolysis as described above and by MALDI-TOF MS: calcd. for [M - H]-, 2479.5; found for peak 1, 2479.9; found for peak 2, 2479.9. HPLC analysis of the enzyme hydrolysate (gradient A) showed that the earlier eluting peak (less lipophilic) contained the 6(S) isomer of the crotonaldehyde adduct (compound 3S).

Results The 6(S) diastereomer (3S) of the crotonaldehyde adduct was synthesized enantiospecifically from the commercially available (S)-3-aminobutanoic acid (4S) as shown in Scheme 2a. Benzoylation and esterification of 4S followed by reduction with DIBAL gave aldehyde 5S, which was converted to benzamidopentene 6S by Wittig olefination with methyltriphenylphosphonium bromide and n-butyllithium. Glycolation of the olefin with OsO4 and N-methylmorpholine N-oxide followed by hydrolytic removal of the benzoyl group gave 4(S)-amino-1,2-pentanediol 7S. The introduction of the hydroxy group at C-2 by this procedure is nonstereospecific; however, the presence of diastereomers at C-2 is of no consequence since the position is subsequently oxidized. Reaction of 7S with O6-TMSE-protected 2-fluoro deoxyinosine (8) led to (1S) N2-(1-methyl-3,4-dihydroxybutyl)-2′-deoxyguanosine (9S) (20). NMR spectra of 9S recorded in dry DMSO-d6 showed signals for four types of hydroxyl groups (two deoxyribosyl and two side chain hydroxyls) as well as signals for N2-H and N1-H, which indicated the condensation had occurred via the amino group of the aminodiol. All signals could be individually assigned via a COSY spectrum. Oxidation with NaIO4 gave aldehyde 10S which cyclized spontaneously to 3S. The analogous synthesis of the diastereomer 3R could not be performed conveniently because (R)-3-aminobutyric acid is not commercially available. As an alternative, a nonstereospecific synthesis was carried out starting from racemic 6RS (Scheme 2b) (26). Treatment with OsO4 gave a mixture of the four diastereomers of 7, which was condensed with O6-TMSE-protected 2-fluoro deoxyinosine (8) to give a mixture of the four diastereomers of 9. Preparative HPLC was used to isolate 9 without attempting to separate diastereomers. The diastereomers of the N2-(1-methyl-3,4-dihydroxybutyl)deoxyguanosine had nearly superimposable 1H NMR spectra. NMR spectra of the mixture recorded in dry DMSO-d6 showed signals for four types of hydroxyl groups (two deoxyribosyl and two side chain hydroxyls) as well as peaks for N2-H and N1-H, which indicated the condensation had occurred via the amino group of the aminodiol. All signals were individually assigned via a COSY spectrum. The mixture was subjected to oxidation of the diol with periodate to give diastereomers 3R and 3S, which were easily separated by reversed-phase HPLC. Circular dichroism spectra of 3R and 3S are shown in Figure 1;

Crotonaldehyde-Deoxyguanosine Adducts

Chem. Res. Toxicol., Vol. 14, No. 11, 2001 1509 Scheme 2

Figure 1. Circular dichroism spectra of 3S and 3R. Spectra were obtained in water at 25 °C.

the isomers exhibited opposing bands at 309 nm with the R isomer having the positive band and the S the negative. Chung and Hecht in their early work on identification of the crotonaldehyde adducts of dGuo isolated two adducts (designated as peak 4 and peak 5); when these were deglycosylated by acid treatment the resulting guanine adducts gave mirror image CD spectra with opposing bands at 280 nm (3). The spectra shown in Figure 1 are not quite mirror images because the adducts with the deoxyribose attached are diastereomeric rather than enantiomeric. Comparison of the spectra reported in this paper with the earlier spectra allows identification of the earlier eluting peak (peak 4) isolated by Chung

and Hecht as the 6(S) enantiomer and the later (peak 5) as the 6(R) isomer, the elution order on reversed-phase HPLC being the same as we have found. The NMR spectra of 3R and 3S were fully in accord with the assigned structure. The amount of uncyclized aldehyde present in the samples was below the level of detection by NMR. The one-dimensional NMR spectra were in good agreement with reported spectra for crotonaldehyde adducts (8, 15). The earlier studies had established the hydroxyl group to be trans to the methyl group in both diastereomers; no attempt was made to confirm that aspect of the structure. Long-range coupling between the methine proton (H8, δ 6.12 ppm) and the carbonyl carbon (C10, δ 157.0 ppm), observed in an HMBC two-dimensional NMR spectrum (Figure 2), indicated cyclization had occurred via N1 rather than N3 in confirmation of earlier assignments. The absolute configuration of the carbon to which the methyl group is attached in the two diastereomers had never been established; in the present investigation the synthesis from aminodiol of known configuration made it possible to establish that the less lipophilic of the two diastereomers has the S configuration at the methylated site (C-6). The configuration at C-8 in this isomer can also be assigned as S by virtue of the trans relationship between the methyl group and the hydroxy group. The synthetic method was extended to preparation of site-specifically adducted oligonucleotides (Scheme 3). The 8-mer 5′-d(AGGCXCCT), in which X represents nucleoside 8, was treated with a mixture of the four


Chem. Res. Toxicol., Vol. 14, No. 11, 2001

Nechev et al. Scheme 3

Figure 2. HMBC spectrum of compound 3S. The cross-peak at the bottom of the spectrum (indicated by the arrow) represents the correlation between H-8 and C-10 shown in the inset structure.

diastereomers of aminodiol 7. The resulting oligonucleotide containing nucleosides 9R and 9S was purified as a single fraction by reversed-phase HPLC and, after characterization by MALDI-TOF mass spectrometry and by nuclease digestion to establish the presence of nucleosides 9R and 9S, treated with NaIO4. The resulting oligonucleotides containing the 3R and 3S diastereomers of the crotonaldehyde adduct were readily separated from each other and purified by reversed-phase HPLC (Figure 3). They were characterized by MALDI TOF mass spectrometry. Enzymatic digestion yielded 3 plus the normal deoxynucleosides in the expected molar ratios. The HPLC traces for the deoxynucleoside mixtures derived from the two oligonucleotides are shown in Figure 3 along with the chromatograms of the authentic samples of 3R and 3S. The faster eluting oligonucleotide yielded deoxynucleoside 3 having the S configuration at the site of attachment of the methyl group.

Figure 3. HPLC profiles of purified adducted oligonucleotides (A) d(AGGC-3S-CCT) and (B) d(AGGC-3R-CCT). (C and D) HPLC profiles of enzymatic hydrolysates of the two oligonucleotides. In each case, the HPLC trace of an authentic sample of the corresponding nucleoside adduct is superimposed.

Discussion The present synthesis of crotonaldehyde-adducted oligodeoxynucleotides will now make it possible to investigate individually the mutagenic properties of the two diastereomers of the crotonaldehyde adduct. The results of these studies will be of much interest relative to the recently reported studies of the corresponding acrolein adduct which showed negligible mutagenic activity in point mutation assays (16, 17). The structure and conformation of the crotonaldehyde adducts in duplex DNA may be significantly different from those of the acrolein adduct. Whereas de los Santos found the acrolein adduct to undergo ring opening in duplex DNA to form the

Crotonaldehyde-Deoxyguanosine Adducts Scheme 4

Chem. Res. Toxicol., Vol. 14, No. 11, 2001 1511

cleavage of this diastereomer again yielded the 6(R) diastereomer of crotonaldehyde adduct 3. This result provides support of the hypothesis that both acetaldehyde-derived crotonaldehyde adducts arise from a common intermediate which is formed mainly by nucleophilic attack on the si face of the Schiff base (see Scheme 4). In the formation of 3 directly from crotonaldehyde, attack occurs on the β carbon of the aldehyde with a small preference for the si face to yield the 6(S) isomer.

Acknowledgment. We wish to acknowledge technical assistance by Ms. Pamela Tamura and Amanda Wilkinson and generous financial support by the National Institutes of Health (ES00267, ES05355, and ES07781).


(hydrated) N2-(3-oxopropyl) adduct (21), the methyl group on the crotonaldehyde adducts may stabilize the cyclic form of the adduct relative to the acyclic in one or both of the diastereomers and/or destabilize the acyclic structures via steric interactions of the methyl group with adjacent nucleosides in the duplex. If so, the mutagenic properties of the crotonaldehyde adducts will be more comparable to those of the unfunctionalized 1,N2-propano adduct which is highly mutagenic (18, 19). Crotonaldehyde-deoxyguanosine adducts have been detected in human and animal tissues (29-31). It has been generally believed that they arise from crotonaldehyde formed by oxidative degradation of unsaturated lipids, or from exogenous sources. However, a second route to crotonaldehyde-guanine adducts involving reactions of acetaldehyde was recently reported by Wang et al. (32). Acetaldehyde is known to undergo aldol condensation to form crotonaldehyde (33), but Wang found evidence that the adducts arising from acetaldehyde are formed by an indirect route. They observed that the two diastereomers of crotonaldehyde adduct 3 were formed from acetaldehyde in a 1:5 ratio with the more lipophilic isomer being more abundant, whereas the reaction of crotonaldehyde with calf thymus DNA yields the two diastereomers of the adduct in a 1.5:1 ratio with the less lipophilic one being more abundant. Wang has proposed the acetaldehyde reaction involves initial formation of a Schiff base at the N2 position of deoxyguanosine followed by addition of a second molecule of acetaldehyde to the imino bond. The inherent chirality of the DNA duplex is the source of the chiral selection in the second step. In support of this mechanism, the ethylidene derivative, i.e., the Schiff base, can be observed by HPLC and trapped as the N2-ethyl derivative by reduction with NaCNBH3. On the basis of order of elution on reversed-phase HPLC, the major crotonaldehyde adduct arising from the acetaldehyde reaction can be assigned as the 6(R) isomer and the major one arising directly from crotonaldehyde as the 6(S). Wang’s study also revealed that the reaction of acetaldehyde with calf thymus DNA yielded crosslinked species 11, identified as involving the N2 positions of guanines in the two strands. The sequence context of the cross-link is presently unknown. It is noteworthy that one diastereomer of 11 predominated and hydrolytic

(1) Eder, E., Budiawan, and Schuler, D. (1996) Crotonaldehyde: a carcinogenic and mutagenic air, water and food pollutant. Cent. Eur. J. Public Health 4 (Suppl.), 21-22. (2) Nath, R. G., Ocando, J. E., Guttenplan, J. B., and Chung, F. L. (1998) 1,N2-propanodeoxyguanosine adducts: potential new biomarkers of smoking-induced DNA damage in human oral tissue. Cancer Res. 58, 581-584. (3) Chung, F. L., and Hecht, S. S. (1983) Formation of cyclic 1,N2adducts by reaction of deoxyguanosine with R-acetoxy-N-nitrosopyrrolidine, 4-(carbethoxynitrosamino)butanal, or crotonaldehyde. Cancer Res. 43, 1230-1235. (4) Hecht, S. S., Young-Sciame, R., and Chung, F. L. (1992) Reaction of a-acetoxy-N-nitrosopiperidine with deoxyguanosine: oxygendependent formation of 4-oxo-2-pentenal and a 1,N2-ethenodeoxyguanosine adduct. Chem. Res. Toxicol. 5, 706-712. (5) Wang, M. Y., Chung, F. L., and Hecht, S. S. (1988) Identification of crotonaldehyde as a hepatic microsomal metabolite formed by R-hydroxylation of the carcinogen N-nitrosopyrrolidine. Chem. Res. Toxicol. 1, 28-31. (6) Chung, F. L., Chen, H. J., and Nath, R. G. (1996) Lipid peroxidation as a potential endogenous source for the formation of exocyclic DNA adducts. Carcinogenesis 17, 2105-2111. (7) Dittberner, U., Eisenbrand, G., and Zankl, H. (1995) Genotoxic effects of the R,β-unsaturated aldehydes 2-trans-butenal, 2-transhexenal and 2-trans, 6-cis-nonadienal. Mutat. Res. 335, 259265. (8) Chung, F. L., Young, R., and Hecht, S. S. (1984) Formation of cyclic 1,N2-propanodeoxyguanosine adducts in DNA upon reaction with acrolein or crotonaldehyde. Cancer Res. 44, 990-995. (9) Eder, E., Deininger, C., Neudecker, T., and Deininger, D. (1992) Mutagenicity of β-alkyl substituted acrolein congeners in the Salmonella typhimurium strain TA100 and genotoxicity testing in the SOS chromotest. Environ. Mol. Mutagen. 19, 338-345. (10) Chung, F. L., Tanaka, T., and Hecht, S. S. (1986) Induction of liver tumors in F344 rats by crotonaldehyde. Cancer Res. 46, 1285-1289. (11) Marnett, L. J., Hurd, H. K., Hollstein, M. C., Levin, D. E., Esterbauer, H., and Ames, B. N. (1985) Naturally occurring carbonyl compounds are mutagens in Salmonella tester strain TA104. Mutat. Res. 148, 25-34. (12) Wang, M., McIntee, E. J., Cheng, G., Shi, Y., Villalta, P. W., and Hecht, S. S. (2000) Identification of paraldol-deoxyguanosine adducts in DNA reacted with crotonaldehyde. Chem. Res. Toxicol. 13, 1065-1074. (13) Chung, F. L., Young, R., and Hecht, S. S. (1989) Detection of cyclic 1,N2-propanodeoxyguanosine adducts in DNA of rats treated with N-nitrosopyrrolidine and mice treated with crotonaldehyde. Carcinogenesis 10, 1291-1297. (14) Galliani, G., and Pantarotto, C. (1983) The reaction of guanosine and 2′-deoxyguanosine with acrolein. Tetrahedron Lett. 24, 44914492. (15) Eder, E., and Hoffman, C. (1992) Identification and characterization of deoxyguanosine-crotonaldehyde adducts. Formation of 7,8 cyclic adducts and 1,N2,7,8 bis-cyclic adducts. Chem. Res. Toxicol. 5, 802-808. (16) Yang, I.-Y., Hossian, M., Miller, H., Khullar, S., Johnson, F., Grollman, A., and Moriya, M. (2001) Responses to the major acrolein-derived deoxyguanosine adduct in Escherichia coli. J. Biol. Chem. 276, 9071-9076.


Chem. Res. Toxicol., Vol. 14, No. 11, 2001

(17) VanderVeen, A. A., Hashim, M. F., Nechev, L. V., Harris, T. M., Harris, C. M., and Marnett, L. J. (2001) Evaluation of the mutagenic potential of the principal DNA adduct of acrolein. J. Biol. Chem. 276, 9066-9070. (18) Burcham, P. C., and Marnett, L. J. (1994) Site-specific mutagenesis by a propanodeoxyguanosine adduct carried on an M13 genome. J. Biol. Chem. 269, 28844-28850. (19) Moriya, M., Zhang, W., Johnson, F., and Grollman, A. P. (1994) Mutagenic potency of exocyclic DNA adducts: marked differences between Escherichia coli and simian kidney cells. Proc. Natl. Acad. Sci. U.S.A. 91, 11899-118903. (20) Nechev, L. V., Harris, C. M., and Harris, T. M. (2000) Synthesis of nucleosides and oligonucleotides containing adducts of acrolein and vinyl chloride. Chem. Res. Toxicol. 13, 421-429. (21) de los Santos, C., Zaliznyak, T., and Johnson, F. (2001) NMR characterization of a DNA duplex containing the major acroleinderived deoxyguanosine adduct γ-hydroxy-1,N2-propano-2′-deoxyguanosine. J. Biol. Chem. 276, 9077-9082. (22) Carmical, J. R., Zhang, M., Nechev, L., Harris, C. M., Harris, T. M., and Lloyd, R. S. (2000) Mutagenic potential of guanine N2 adducts of butadiene mono- and diolepoxide. Chem. Res. Toxicol. 13, 18-25 and 430. (23) Khullar, S., Varaprasad, C. V., and Johnson, F. (1999) Postsynthetic generation of a major acrolein adduct of 2′-deoxyguanosine in oligomeric DNA. J. Med. Chem. 42, 947-950. (24) Estermann, H., and Seebach, D. (1988) Diastereoselective alkylation of 3-aminobutanoic acid in the 2-position. Helv. Chim. Acta 71, 1824-1839. (25) Drey, C. N. C., Ridge, R. J., and Mtetwa, E. (1980) Selective hydrogenation of 4,5-dihydro-1,3-oxazin-6-ones to carboxaldehyde derivatives; chemical differentiation between acylazetidin-2-ones and the corresponding isomeric oxazin-6-ones. J. Chem. Soc., Perkin Trans. 1 378-380.

Nechev et al. (26) Carpanelli, C., and Gaiani, G. (1982) Diels-Alder cycloadditions on N-sulfinyl-p-toluamide. I. Regioselectivity towards 1- and 2-substituted 1,3-butadienes. Gazz. Chim. Ital. 112, 187-190. (27) DeCorte, B. L., Tsarouhtsis, D., Kuchimanchi, S., Cooper, M. D., Horton, P., Harris, C. M., and Harris, T. M. (1996) Improved strategies for postoligomerization synthesis of oligodeoxynucleotides bearing structurally defined adducts at the N2 position of deoxyguanosine. Chem. Res. Toxicol. 9, 630-637. (28) Harris, T. M., and Harris, C. M. (2000) Synthesis of N2-substituted deoxyguanosine nucleosides from 2-fluoro-O6-(trimethylsilylethyl)2′-deoxyinosine. Current Protocols in Nucleic Acid Chemistry, pp 1.3.1-1.3.15, J. Wiley & Sons, New York. (29) Nath, R. G., and Chung, F.-L. (1994) Detection of exocyclic 1,N2propanodeoxyguanosine adducts as common DNA lesions in rodents and humans. Proc. Natl. Acad. Sci. U.S.A. 91, 74917495. (30) Yang, K., Fang, J.-L., Li, D., Chung, F. L., and Hemminki, K. (1999) 32P-postlabelling with high-performance liquid chromatography for analysis of abundant DNA adducts in human tissues. IARC Sci. Publ. 205-217. (31) Nath, R. G., Ocando, J. E., and Chung, F. L. (1996) Detection of 1,N2-propanodeoxyguanosine adducts as potential endogenous DNA lesions in rodent and human tissues. Cancer Res. 56, 452456. (32) Wang, M., McIntee, E. J., Cheng, G., Shi, Y., Villalta, P. W., and Hecht, S. S. (2000) Identification of DNA adducts of acetaldehyde. Chem. Res. Toxicol. 13, 1149-1157. (33) Baigrie, L. M., Lenoir, D., Seikaly, H. R., and Tidwell, T. T. (1985) Generation, alkylation, and silylation of directed enolates formed by reaction of ketenes and organolithium reagents. J. Org. Chem. 50, 2105-2109.