Synthesis of Nucleosides and Oligonucleotides Containing Adducts

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Chem. Res. Toxicol. 2000, 13, 421-429

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Synthesis of Nucleosides and Oligonucleotides Containing Adducts of Acrolein and Vinyl Chloride Lubomir V. Nechev, Constance M. Harris, and Thomas M. Harris* Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235 Received September 22, 1999

Vinyl chloride and acrolein are important industrial chemicals. Both form DNA adducts, vinyl chloride after enzymatic oxidation to chlorooxirane and acrolein by direct reaction. Reaction at the N2 position of guanine is a major pathway. The resulting 2-oxoethyl and 3-oxopropyl adducts cyclize spontaneously to hydroxyethano and hydroxypropano derivatives, respectively. The two cyclic adducts have been detected in DNA exposed to these mutagens. A new method has been developed for the synthesis of deoxyguanosine adducts of chlorooxirane and acrolein, as well as oligonucleotides containing these adducts. Reaction of O6-[(trimethylsilyl)ethyl]-2-fluoro-2′-deoxyinosine with the appropriate aminodiol followed by oxidative cleavage of the diol with NaIO4 gave the adducts in excellent yields. Reaction of oligonucleotides containing the halonucleoside with the aminodiols followed by NaIO4 efficiently created the nucleosides in the oligonucleotides. Deoxyadenosine adducts were created similarly using 6-chloropurine 9-(2′-deoxyriboside).

Introduction Bifunctional electrophiles frequently react with the nucleobases to yield annelation products. A good example is chlorooxirane (Scheme 1) which arises from vinyl chloride by enzymatic oxidation. The methylene position of the oxirane alkylates the N2 position of deoxyguanosine to give the 2-oxoethyl derivative (1); cyclization of 1 then occurs by reaction of the aldehyde with the proximal N1 atom to form 3 (1). The reaction of oxidized vinyl chloride with DNA also gives rise to cyclic etheno adducts of guanine, namely, 1,N2-etheno nucleoside 5 and the isomeric N2,3-etheno nucleoside, which have been widely studied (2-9). Interestingly, 3 does not readily undergo dehydration to adduct 5. Deoxyadenosine reacts with chlorooxirane via the N1 position to give 2-oxoethyl derivative 2 which cyclizes to 1,N6-hydroxyethano derivative 4 (10). Dehydration of 4 occurs immediately to give etheno derivative 6 in contrast to the stable hydroxyethano adduct of guanine. The reaction of cytidine is analogous to that of adenosine (10). These adducts apparently arise also from endogenous C-2 donors, although the precise identity of these species remains unknown (11). Acrolein also gives cyclic adducts (Scheme 1). Guanine forms regioisomeric 1,N2 adducts 9 and 10, presumably via N2 and N1 adducts 7 and 8, respectively (12, 13). Regioisomeric 1,N6 adducts 13 and 14 on adenine have also been reported and presumably arise via N1 and N6 oxopropyl derivatives 11 and 12 (14, 15). None of the acrolein adducts dehydrate readily. Acrolein is pervasive in the environment and a significant constituent of tobacco smoke (16). It is a metabolite of the widely used chemotherapeutic agent, cyclophosphamide; acrolein adducts of DNA have been detected in cultured cells treated with the drug (17) and in the blood of patients undergoing treatment (18). Adducts 9 and 10 have been seen to arise from acrolein formed endogenously by peroxidation of unsaturated lipids (19). The mutagenic properties of vinyl

chloride and acrolein have been studied extensively (2024). Interpretation of the results is complicated by the variety of reactions that can occur. With vinyl chloride, the situation is further complicated by the rapid rearrangement of chlorooxirane to chloroacetaldehyde. Both species react with DNA and cause mutations. However, the toxicity/mutation profiles and frequency of large deletions induced in cells treated with vinyl chloride are consistent with chlorooxirane being the main mutagenic species rather than chloroacetaldehyde (23). Extensive mutagenesis studies of cyclic etheno adducts in defined oligonucleotides have been performed (9, 25-28), but much less is known about the biological properties of the hydroxyethano (26) and hydroxypropano adducts, particularly the latter. Thus, this paper focuses on the synthesis of hydroxyethano and hydroxypropano cyclic adducts 3 and 9 of guanine and, to a lesser extent, the adducts of adenine. Site-specifically adducted DNA cannot be prepared by direct reaction with chlorooxirane or acrolein because numerous competing reactions occur. We present a simple approach for the preparation of DNA containing adducts 3, 9, and 14. The key to the process is introduction of the Ω-oxoalkyl moiety after, rather than before, assembly of the oligonucleotide. Site-specific introduction is achieved by reversal of the normal nucleophileelectrophile relationship of the nucleoside and modifying agent (29), using an amine, which is the synthetic equivalent of acrolein or chlorooxirane, in condensations with 2-fluoroinosine at the target site in the oligonucleotide. Ω-Oxoalkylamines are inherently unstable, undergoing facile intramolecular reactions to form Schiff bases. However, alkylamines containing distal 1,2-diols can be used as their synthetic equivalents. After the condensation of the amine with DNA containing a halogensubstituted nucleoside has been effected, the aldehyde function can be revealed by periodate oxidation. The

10.1021/tx990167+ CCC: $19.00 © 2000 American Chemical Society Published on Web 04/21/2000

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Chem. Res. Toxicol., Vol. 13, No. 5, 2000 Scheme 1

conditions are sufficiently mild that no other structural modifications occur in the oligonucleotides.

Experimental Procedures General Methods. 1H NMR spectra were recorded at 400.13 MHz on a Bruker AM400 NMR spectrometer in D2O or DMSOd6. DMSO was distilled under vacuum over CaH2. Other chemicals were used without further purification. Thin-layer chromatography was performed on silica gel (EM Science, Silica Gel 60F254 glass plates, layer thickness of 250 µm). 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 (EM Science, 70-230 mesh). Oligodeoxynucleotides were

Nechev et al. purified, and the reactions were followed on a Beckman HPLC system with a diode array UV detector monitoring at 260 nm. For purification of oligonucleotides, 0.1 M ammonium formate was used as solvent A and acetonitrile as solvent B with the following gradients: gradient 1, 99% A at first, 4 min linear gradient to 90% A, 21 min linear gradient to 70% A, and 4 min linear gradient to 10% A followed by 4 min linear gradient to initial conditions; gradient 2, 99% A at first, 15 min linear gradient to 90% A, 5 min linear gradient to 80% A, and 10 min linear gradient to 0% A followed by 5 min linear gradient to initial conditions; and gradient 3, 99% A at first, 20 min linear gradient to 90% A, 2 min linear gradient to 75% A, and 4 min linear gradient to 10% A followed by 7 min linear gradient to initial conditions. For purification of nucleosides, water was used as solvent A instead of aqueous ammonium formate. Mass Spectrometry. Low- and high-resolution FAB1 mass spectra were obtained at the Mass Spectrometry Facility at the University of Notre Dame (Notre Dame, IN). Mass spectra (MALDI-TOF) 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. Oligonucleotide Synthesis. Oligodeoxynucleotides were synthesized on a 1 µmol scale using a PerSeptive Expedite model 8909 Nucleic Acid Synthesis System and Expedite phosphoramidite reagents (having tert-butylphenoxyacetyl protection on exocyclic amino groups) and the standard protocol. The modified oligodeoxynucleotides were cleaved from the solid support, and the exocyclic amino groups were deprotected in a single step using 0.1 M NaOH (30). The samples were neutralized, and the oligonucleotides were purified by HPLC (YMC ODS-AQ column, 250 mm × 10 mm) using gradient 1. Enzymatic digestion with nuclease P1, snake venom phosphodiesterase (SVPD), and alkaline phosphatase (AP) were carried out under conditions described previously (31). The digests were analyzed by HPLC (YMC ODS-AQ column, 250 mm × 4.6 mm) using gradient 2. Synthesis of Nucleosides Containing Hydroxyethano and Hydroxypropano Adducts. (1) N2-(2,3-Dihydroxypropyl)deoxyguanosine (17). (()-3-Amino-1,2-propanediol (7.5 mg, 0.082 mmol, Aldrich) was added to a mixture of O6[(trimethylsilyl)ethyl]-2-fluoroinosine (30) (15, 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. To remove the (trimethylsilyl)ethyl group from the O6 position of 16, the residue was dissolved in 5% acetic acid (1 mL) and stirred at room temperature for 1 h. HPLC purification (gradient 3) of the reaction mixture gave 14 mg (76%) of N2-(2,3-dihydroxypropyl)deoxyguanosine (17) (1): 1H NMR (DMSO-d6, 65 °C) δ 7.82 (s, 1H, H-8), 6.38 (t, 1H, NH, J ) 5.2 Hz), 6.15 (dd, 1H, H-1′, J ) 6.5 Hz, J ) 7.0 Hz), 4.36 (m, 1H, H-3′), 3.83 (m, 1H, H-4′), 3.66 (m, 1H, side chain CH-O), 3.56 (m, 2H, H-5′, H-5′′), 3.47 (m, 1H, CH-N), 3.39 (m, 2H, side chain CH2-O), 3.21 (m, 1H, CHN), 2.49 (m, 1H, H-2′), 2.24 (m, 1H, H-2′′). In the spectrum of the same sample at room temperature, the exchangeable protons were visible and assigned (COSY) as follows: δ 10.5 (1H, NHamide), 5.30 (1H, 3′-OH), 5.02 (1H, CH-OH), 4.89 (1H, 5′-OH), 4.71 (1H, CH2OH); HRMS (FAB+) m/z calcd for C13H20N5O6 [M + H]+ 342.1414, found 342.1420. (2) 3-(2-Deoxy-β-D-erythro-pentofuranosyl)-3,5,6,7-tetrahydro-7-hydroxy-9H-imidazo[1,2-a]purin-9-one (Hydroxy-1,N2-ethanodeoxyguanosine, 3). Sodium periodate (500 µL, 20 mM in 0.05 M phosphate buffer, pH 7) was added to a solution of 17 (2 mg, 0.0058 mmol) in phosphate buffer (300 1 Abbreviations: FAB, fast atom bombardment mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight mass spectroscopy; HRMS, high-resolution mass spectroscopy; HMBC, heteronuclear multibond correlation (long-range 13C-1H scalar correlated two-dimensional NMR experiment); TBDMS, tert-butyldimethylsilyl; TMSE, (trimethylsilyl)ethyl; t-Boc, tert-butoxycarbonyl.

Hydroxyethano and Hydroxypropano dGuo and dAdo

Figure 1. HPLC chromatograms showing the sodium periodate-effected conversion of N2-dihydroxyalkyl-modified deoxyguanosine 17 to cyclic adduct 3 in nucleoside (A) and in oligonucleotide 5′-d(AGGC-(17)-CCT) (B). µL, pH 7). The reaction mixture was stirred at room temperature for 5-10 min. HPLC analysis showed complete disappearance of the peak for the starting material (Figure 1). The reaction mixture was purified by HPLC (gradient 2) to give 1.5 mg (84%) of hydroxy-1,N2-ethanodeoxyguanosine (3): 1H NMR (D2O) δ 7.81 (s, 1H, H-2), 6.11 (m, 2H, H-1′, H-7), 4.45 (m, 1H, H-3′), 3.96 (m, 1H, H-4′), 3.81 (m, 1H, H-6), 3.62 (m, 2H, H-5′, H-5′′), 3.45 (m, 1H, H-6), 2.60 (m, 1H, H-2′), 2.34 (m, 1H, H-2′′); HRMS (FAB+) m/z calcd for C12H16N5O5 [M + H]+ 310.1151, found 310.1144. (3) (2S)-4-Amino-1,2-butanediol. The amino alcohol was prepared from L-asparagine essentially by the procedure of Knapp et al. (32). The following modification was used to obtain the amino alcohol without t-Boc protection on the amino group. After the borane reduction, the reaction mixture was treated overnight with 4 M HCl. The solvents were evaporated. The solid residue was redissolved in water and neutralized with 10% NaOH; the sample was again evaporated to dryness. The residue was dissolved in a small amount of water and applied on an ion exchange column (Dowex 50W-X8 resin, H+ form, 5 milliequiv/g). The column was washed with 300 mL of deionized water, and the amino alcohol was eluted with 300 mL of 1 M NH4OH. This solution was concentrated under vacuum to give the pure 4-amino-1,2-butanediol as a thick, clear oil: 1H NMR (D2O) δ 1.53 (m, 1H), 1.63 (m, 1H), 2.89 (m, 2H), 3.34 (m, 2H), 3.60 (m, 1H); [R]D25 -21° (c 1.87, MeOH) [lit. (33) [R]D25 -23°]. (4) N2-(3,4-Dihydroxybutyl)deoxyguanosine (19). (2S)4-Amino-1,2-butanediol (4.3 mg, 0.041 mmol) was added to a mixture of O6-[(trimethylsilyl)ethyl]-2-fluoroinosine (15, 10 mg, 0.027 mmol), DMSO (150 µL), and diisopropylethylamine (50 µL). The mixture was stirred at 55 °C for 6 h. The mixture was cooled, and the solvents were evaporated under vacuum. The residue (18) was dissolved in 5% acetic acid (1 mL) and stirred at room temperature for 1 h. HPLC purification (gradient 2) of the reaction mixture gave 7.9 mg (82%) of N2-(3,4-dihydroxybutyl)deoxyguanosine (19): 1H NMR (DMSO-d6) δ 10.54 (s, 1H, N1-H), 7.89 (s, 1H, H-8), 6.39 (m, 1H, N2-H), 6.14 (dd, 1H, H-1′, J ) 6.4 Hz, J ) 7.5 Hz), 5.28 (m, 1H, 3′-OH), 4.87 (m, 1H, 5′OH), 4.66 (m, 1H, side chain CH-OH), 4.57 (m, 1H, side chain CH2-OH), 4.34 (m, 1H, H-3′), 3.79 (m, 1H, H-4′), 3.50 (m, 5H, H-5′, H-5′′, CH2-N, CH-O), 3.23 (m, 2H, CH2-O), 2.60 (m, 1H, H-2′), 2.19 (m, 1H, H-2′′), 1.72 (m, 1H, C-CH-C), 1.44 (m, 1H,

Chem. Res. Toxicol., Vol. 13, No. 5, 2000 423 C-CH-C); HRMS (FAB+) m/z calcd for C14H22N5O6 [M + H]+ 356.1570, found 356.1582. (5) 3-(2-Deoxy-β-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine (Hydroxy-1,N2propanodeoxyguanosine, 9). Sodium periodate (500 µL, 20 mM in phosphate buffer, pH 7) was added to a solution of 19 (2 mg, 0.0056 mmol) in phosphate buffer (300 µL, pH 7). The reaction mixture was stirred at room temperature for 5-10 min. HPLC analysis showed complete disappearance of the peak for the starting material. The reaction mixture was purified by HPLC (gradient 2) to give 1.55 mg (86%) of hydroxy-1,N2propanodeoxyguanosine (9): 1H NMR (DMSO-d6) δ 7.90 (m, 1H, NH), 7.87 (s, 1H, H-2), 6.64 (d, 1H, C8-OH, J ) 4.5 Hz), 6.23 (m, 1H, H-8), 6.07 (dd, 1H, H-1′, J ) 6.0 Hz, J ) 7.9 Hz), 5.26 (d, 1H, 3′-OH, J ) 3.9 Hz), 4.93 (t, 1H, 5′-OH, J ) 5.5 Hz), 4.32 (m, 1H, H-3′), 3.78 (m, 1H, H-4′), 3.49 (m, 2H, H-5′, H-5′′), 3.38 (m, 1H, H-6), 3.25 (m, 1H, H-6), 2.51 (m, 1H, H-2′), 2.16 (m, 1H, H-2′′), 1.97 (m, 1H, H-7), 1.68 (m, 1H, H-7); 13C NMR (DMSO-d6) δ 26.8 (C-7), 33.7 (C-6), 40.0 (hidden under DMSO peak, visible in HMBC) (C-2′), 62.1 (C-5′), 69.3 (C-8), 71.2 (C-3′), 82.7 (C-1′), 87.9 (C-4′), 115.8 (C-9a), 135.7 (C-2), 150.1 (C-3a), 151.2 (C-4a), 156.0 (C-9); HRMS (FAB+) m/z calcd for C13H18N5O5 [M + H]+ 324.1308, found 324.1322. (6) N6-(2,3-Dihydroxypropyl)deoxyadenosine (21). (()3-Amino-1,2-propanediol (10 mg, 0.11 mmol) was added to a mixture of chloronucleoside 20 (29, 34) (20 mg, 0.074 mmol), DMSO (150 µL), and diisopropylethylamine (50 µL). The mixture was stirred at 55 °C for 7 h. HPLC analysis showed complete disappearance of the starting material. The mixture was cooled, and the solvents were evaporated under vacuum. The residue was purified on a silica gel column (87:7:6 acetonitrile/water/ NH4OH mixture) to give 20.5 mg (85%) of N6-(2,3-dihydroxypropyl)deoxyadenosine (21): 1H NMR (DMSO-d6, 45 °C) δ 8.30 (s, 1H, H-8), 8.19 (s, 1H, H-2), 7.27 (bs, 1H, NH), 6.35 (dd, 1H, H-1′, J ) 6.2 Hz, J ) 7.5 Hz), 5.17 (d, 1H, 3′-OH, J ) 4.1 Hz), 5.03 (t, 1H, 5′-OH, J ) 5.0 Hz), 4.76 (1H, CH-OH), 4.48 (1H, CH2-OH), 4.41 (m, 1H, H-3), 3.88 (m, 1H, H-4′), 3.71 (m, 2H, CH2-N), 3.64-3.50 (m, 3H, H-5′, H-5′′, side chain CH-O), 3.39 (m, 2H, side chain CH2-O), 2.71 (m, 1H, H-2′), 2.27 (m, 1H, H-2′′); HRMS (FAB+) m/z calcd for C13H20N5O5 [M + H]+ 326.1464, found 326.1470. (7) 3-(2-Deoxy-β-D-erythro-pentofuranosyl)-7,8-dihydro7-hydroxy-3H-imidazo[2,1-i]purine (Hydroxy-1,N6-ethanodeoxyadenosine, 23). Sodium periodate (500 µL, 20 mM in phosphate buffer, pH 7) was added to a solution of 21 (2 mg, 0.0061 mmol) in phosphate buffer (300 µL, pH 7). The reaction mixture was stirred at room temperature for 5-10 min. HPLC analysis showed complete disappearance of the peak for the starting material; only a single product peak was observed. HPLC purification (H2O/CH3CN, gradient 2), which was only needed to separate the product from the inorganic salts in the reaction mixture, led to 1.45 mg (81%) of hydroxy-1,N6-ethanodeoxyadenosine (23): 1H NMR (D2O, pH 3.8) δ 8.55 (s, 1H, H-2), 8.38 (s, 1H, H-5), 6.45 (t, 1H, H-7, J ) 5.5 Hz), 6.36 (t, 1H, H-1′, J ) 6.5 Hz), 4.45 (m, 1H, H-3′), 4.17 (m, 1H, H-8), 3.96 (m, 1H, H-4′), 3.84 (m, 1H, H-8), 3.59 (m, 2H, H-5′, H-5′′), 2.67 (m, 1H, H-2′), 2.43 (m, 1H, H-2′′); HRMS (FAB+) m/z calcd for C12H16N5O4 [M + H]+ 294.1202, found 294.1202. (8) N6-(3,4-Dihydroxybutyl)deoxyadenosine (24). 4-Amino1,2-butanediol (6 mg, 0.057 mmol) was added to a mixture of 6-chloropurine 9-(2′-deoxyriboside) (20, 10 mg, 0.037 mmol), DMSO (150 µL), and diisopropylethylamine (50 µL). The mixture was stirred at 55 °C for 6 h, at which point the HPLC peak for the starting nucleoside had disappeared completely. HPLC purification (gradient 3) yielded 10 mg (79%) of N6-(3,4-dihydroxybutyl)deoxyadenosine (24): 1H NMR (DMSO-d6, 55 °C) δ 8.26 (s, 1H, H-8), 8.18 (s, 1H, H-2), 7.43 (bs, 1H, N6-H), 6.34 (dd, 1H, H-1′, J ) 6.3 Hz, J ) 7.4 Hz), 4.42 (m, 1H, H-3′), 3.88 (m, 1H, H-4′), 3.59 (m, 5H, H-5′, H-5′′, CH2-N, CH-O), 3.32 (m, 2H, CH2-O), 2.70 (m, 1H, H-2′), 2.28 (m, 1H, H-2′′), 1.80 (m, 1H, C-CH2-C), 1.58 (m, 1H, C-CH2-C); HRMS (FAB+) m/z calcd for C14H22N5O5 [M + H]+ 340.1621, found 340.1646.

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(9) 3-(2-Deoxy-β-D-erythro-pentofuranosyl)-3,7,8,9-tetrahydro-7-hydroxypyrimido[2,1-i]purine (Hydroxy-1,N6propanodeoxyadenosine, 14). Sodium periodate (500 µL, 20 mM in 0.05 M phosphate buffer, pH 7) was added to a solution of 24 (2 mg, 0.0058 mmol) in phosphate buffer (300 µL, pH 7). The reaction mixture was stirred at room temperature for 5-10 min; HPLC analysis showed complete disappearance of the peak for the starting material. HPLC purification (gradient 3) of the reaction mixture gave 1.46 mg (82%) of hydroxy-1,N6-propanodeoxyadenosine (14): 1H NMR (D2O, pH 3.8) δ 8.51 (s, 1H, H-2), 8.42 (s, 1H, H-5), 6.47 (t, 1H, H-1′, J ) 6.5 Hz), 6.13 (m, 1H, H-7), 4.59 (m, 1H, H-3′), 4.11 (m, 1H, H-4′), 3.75 (m, 4H, H-5′, H-5′′, 2 × H-9), 2.81 (m, 1H, H-2′), 2.58 (m, 1H, H-2′′), 2.39 (m, 1H, H-8), 2.27 (m, 1H, H-8); HRMS (FAB+) m/z calcd for C13H18N5O4 [M + H]+ 308.1359, found 308.1361. Synthesis of Oligonucleotides Containing Hydroxyethano and Hydroxypropano Adducts. The syntheses of the phosphoramidites of O6-TMSE-2-fluoroinosine (15) and 6-chloropurine deoxyriboside (20) are described elsewhere (30, 31). The oligonucleotides containing 15 and 20 were prepared using a standard DNA synthesizer cycle with phenoxyacetyl-protected phosphoramidites. Cleavage from the solid support and deprotection were carried out in one step by treating with 0.1 M NaOH (∼10 h at room temperature) (30, 35, 36). The mixture was neutralized; the product was filtered (0.45 µm) and purified by HPLC (YMC ODS-AQ column, 250 mm × 10 mm, gradient 1). Synthesis of 5′-d(AGGC-(3)-CCT). The oligodeoxynucleotide (10 A260 units) containing 15 was mixed in a plastic test tube with diisopropylethylamine (50 µL), DMSO (100 µL), and 3-amino-1,2-propanediol (0.5 mg). The reaction mixture was stirred at 55 °C for 3 h. HPLC analysis showed complete disappearance of the starting material. Solvents were evaporated under vacuum. The residue was dissolved in 5% acetic acid (500 µL) and the mixture stirred for 2 h at room temperature. The mixture was neutralized with 1 M NaOH and purified by HPLC (gradient 3) to give 7.5 A260 units (∼75%) of the corresponding N2-(2,3-dihydroxypropyl)guanine-modified oligodeoxynucleotide. The product was characterized by HPLC analysis of the enzymatic hydrolysate using the independently prepared nucleoside 17 as an authentic sample: MS (MALDI) m/z calcd for [M - H]- 2483.5, found 2483.4. A solution of NaIO4 in phosphate buffer (100 µL, 20 mM, pH 7) was added to a solution of N2-(2,3-dihydroxypropyl)guaninemodified oligodeoxynucleotide (5.0 A260 units) in phosphate buffer (300 µL, pH 7.0), and the reaction mixture was stirred at room temperature for 10 min. The reaction resulted in a single HPLC peak which was purified (gradient 2) to give 4.0 A260 units (∼80%) of the hydroxy-1,N2-ethanoguanine-modified oligodeoxynucleotide. The structure was confirmed by HPLC analysis (Phenylhexyl column, Phenomenex, Torrance, CA; gradient 2) of the enzymatic hydrolysate using the independently prepared nucleoside 3 as an authentic sample: MS (MALDI) m/z calcd for [M - H]- 2451.4, found 2451.5 (Figure 2A,B). Synthesis of 5′-d(AGGC-(9)-CCT). The oligodeoxynucleotide (10 A260 units) containing 15 was mixed in a plastic test tube with diisopropylethylamine (50 µL), DMSO (100 µL), and 4-amino-1,2-butanediol (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 the mixture stirred for 2 h at room temperature. The mixture was neutralized with 1 M NaOH and purified by HPLC to give 7.9 A260 units (∼79%) of the corresponding N2-(3,4-dihydroxybutyl)guanine-modified oligodeoxynucleotide. The material was characterized by HPLC analysis (gradient 2) of the enzymatic hydrolysate using the independently prepared nucleoside 19 as an authentic sample: MS (MALDI) m/z calcd for [M - H]2497.5, found 2497.1. A solution of NaIO4 in phosphate buffer (100 µL, 20 mM, pH 7) was added to a solution of N2-(3,4-dihydroxybutyl)guaninemodified oligodeoxynucleotide (5.0 A260 units) in phosphate

Nechev et al.

Figure 2. Characterization of oligodeoxynucleotides containing deoxyguanosine adducts 3 and 9. (A) MALDI spectrum and (B) HPLC profile of the enzymatic hydrolysate of 5′-d(AGGC-(3)CCT). (C) MALDI spectrum and (D) HPLC profile of the enzymatic hydrolysate of 5′-d(AGGC-(9)-CCT). In panels B and D, the HPLC trace of an authentic sample of the corresponding deoxyguanosine adduct is superimposed on that of the enzyme hydrolysate. buffer (300 µL, pH 7.0), and the reaction mixture was stirred at room temperature for 10 min. The reaction resulted in a single HPLC peak which was purified (gradient 1) to give 3.8 A260 units (∼80%) of the hydroxy-1,N2-propanoguanine-modified oligodeoxynucleotide. The structure was confirmed by HPLC analysis of the enzymatic hydrolysate using the independently prepared nucleoside 9 as an authentic sample: MS (MALDI) m/z calcd for [M - H]- 2465.5, found 2466.0 (Figure 2C,D). Synthesis of 5′-d(GCT-(23)-TAGC). The oligodeoxynucleotide (10 A260 units) containing 20 was mixed in a plastic test tube with diisopropylethylamine (50 µL), DMSO (100 µL), and 3-amino-1,2-propanediol (0.5 mg). The reaction mixture was stirred at 55 °C for 2 h. HPLC analysis showed complete disappearance of the starting material. Solvents were evaporated under vacuum. The residue was dissolved in water (500 µL) and purified by HPLC (gradient 3) to give 7.4 A260 units (∼74%) of the corresponding N6-(2,3-dihydroxypropyl)adeninemodified oligodeoxynucleotide. The product was characterized by HPLC analysis of the enzymatic hydrolysate using the independently prepared nucleoside 21 as an authentic sample: MS (MALDI) m/z calcd for [M - H]- 2482.5, found 2482.1. A solution of NaIO4 in phosphate buffer (100 µL, 20 mM, pH 7) was added to a solution of N6-(2,3-dihydroxypropyl)adeninemodified oligodeoxynucleotide (5.8 A260 units) in phosphate buffer (300 µL, pH 7.0), and the reaction mixture was stirred at room temperature for 10 min. The reaction resulted in a single HPLC peak which was purified (gradient 1) to give 4.5 A260 units (∼78%) of the hydroxy-1,N6-ethanoadenine-modified oligodeoxynucleotide. The structure was confirmed by enzymatic hydrolysate analysis using the independently prepared nucleoside 23 as an authentic sample: MS (MALDI) m/z calcd for [M - H]- 2450.4, found 2450.8 (Figure 3A,B). Synthesis of 5′-d(GCT-(14)-TAGC). The oligodeoxynucleotide (10 A260 units) containing 20 was mixed in a plastic test

Hydroxyethano and Hydroxypropano dGuo and dAdo

Chem. Res. Toxicol., Vol. 13, No. 5, 2000 425 Scheme 2

Figure 3. Characterization of oligodeoxynucleotides containing deoxyadenosine adducts 14 and 23. (A) MALDI spectrum and (B) HPLC profile of the enzymatic hydrolysate of 5′-d(GCT-(23)TAGC). (C) MALDI spectrum and (D) HPLC profile of the enzymatic hydrolysate of 5′-d(GCT-(14)-TAGC). In panels B and D, the HPLC trace of an authentic sample of the corresponding deoxyadenosine adduct is superimposed on that of the enzyme hydrolysate. tube with diisopropylethylamine (50 µL), DMSO (100 µL), and 4-amino-1,2-butanediol (0.5 mg). The reaction mixture was stirred at 55 °C for 2 h. Solvents were evaporated under vacuum. The residue was dissolved in water (500 µL) and purified by HPLC (gradient 3) to give 7.4 A260 units (∼74%) of the corresponding N6-(3,4-dihydroxybutyl)adenine-modified oligodeoxynucleotide. The product was characterized by HPLC analysis of the enzymatic hydrolysate using the independently prepared nucleoside 24 as an authentic sample: MS (MALDI) m/z calcd for [M - H]- 2496.5, found 2496.7. A solution of NaIO4 in phosphate buffer (100 µL, 20 mM, pH 7) was added to a solution of N6-(3,4-dihydroxybutyl)adeninemodified oligodeoxynucleotide (5 A260 units) in phosphate buffer (300 µL, pH 7.0), and the reaction mixture was stirred at room temperature for 10 min. The reaction resulted in a single HPLC peak which was purified (gradient 1) to give 4.0 A260 units (∼80%) of the hydroxy-1,N6-propanoadenine-modified oligodeoxynucleotide. The structure was confirmed by HPLC analysis of the enzymatic hydrolysate using the independently prepared nucleoside 14 as an authentic sample: MS (MALDI) m/z calcd for [M - H]- 2464.5, found 2465.1 (Figure 3C,D).

Results Chlorooxirane and Acrolein Adducts (3 and 9) of Deoxyguanosine. The reactions of amines with 2-halo derivatives of deoxyinosine and O6-protected deoxyinosine have been used in the past to prepare a variety of N2alkylated derivatives of deoxyguanosine. Examples include simple adducts such as those of styrene oxide (29) and complex ones such as bis-nucleosides tethered between the exocyclic amino groups (35). The studies

presented here were undertaken to examine the potential for application of this nonbiomimetic approach to the synthesis of the 1,N2 cyclic guanine adducts of chlorooxirane and acrolein (Scheme 2). The approach involved reaction of 2-fluoro-O6-[(trimethylsilyl)ethyl]-2′-deoxyinosine (15) with 3-amino-1,2-propanediol and 4-amino1,2-butanediol, respectively. The reactions were carried out in DMSO at 55 °C using diisopropylethylamine as an acceptor for the HF being liberated. After brief treatment with HOAc to remove the O6 protection, good yields were obtained of the respective N2-(dihydroxyalkyl)deoxyguanosine derivatives 17 and 19. Racemic aminopropanediol was used for the preparation of the ethano derivatives which led to a mixture of diastereomeric products, visible as two closely eluting peaks in the case of deoxyguanosine derivative 17. The diastereomers were carried on to the next step without separation, since the stereochemistry of the hydroxyalkyl chain would be lost in the cleavage with periodate. The most convenient synthesis of the aminobutanediol needed for the hydroxypropano derivatives proceeded from a chiral starting material with the result that the dihydroxybutano adduct 19 was a single stereoisomer. Oxidations with NaIO4, carried out at neutrality and ambient temperature, gave the corresponding N2-(Ω-oxoethyl and Ω-oxopropyl)deoxyguanosines 1 and 7 which were unstable and underwent immediate cyclization to cyclic adducts 3 and 9. The empirical formulas of dihydroxyalkyl adducts 17 and 19 were established by exact mass measurement. The 1H NMR spectrum of compound 17 showed that reaction of the aminodiol with the fluoronucleoside had occurred via the amino group rather than one of the hydroxyl groups. The 1H spectrum recorded using a DMSO-d6 solution at room temperature exhibited four hydroxyl signals, i.e., two in the deoxyribose and two in the side chain and they were individually assigned via a

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Nechev et al. Scheme 3

Figure 4. Expected connectivities in HMBC spectra of 9 and 9a.

COSY spectrum. Further evidence for the structure of 17 came from the periodate oxidation to form 3. If condensation of the aminodiol had occurred via the hydroxyl group, the amino nitrogen would have been lost from the resulting xanthosine derivative during the oxidation, leading to a product with a different empirical formula. For 19, the similarity of the 1H NMR spectrum to that of 17 provided support for the structure. In addition, it should be recognized that, had condensation with the fluoronucleoside occurred via a hydroxyl group, the resulting xanthosine would have been unable to be oxidized by periodate. It is noteworthy that the oxidation of 17 gave 3, since periodate is capable of also cleaving R-aminoaldehydes. The success of the synthesis presented here is due to the amide character of the nitrogen atom and also to the mild conditions that were used which involved only a limited excess of periodate. The oxidation products exist almost completely in the ring-closed form, i.e., 3 and 9, and are mixtures of diastereomers that equilibrate via the aldehydes. The structures are supported by exact mass measurement and by the 1H NMR spectra. The structure of the hydroxyethano adduct of guanine had previously been examined by Guengerich et al. (1) and the hydroxypropano adduct (9) of deoxyguanosine by Galliani and Pantarotto (12) and Chung et al. (13). The 1H spectrum of 9 compared favorably with the spectrum reported by Chung et al. A point of possible uncertainty in the assigned structure of 9 (and also of 3) was whether the cyclization had involved N1 or N3; the previous assignments had rested largely on correlations of UV spectra with those of known N1 derivatives. We were able to confirm the structure of 9 as involving N1 by HMBC twodimensional NMR spectroscopy; the important correlations are shown in Figure 4 where the correct linear structure (9) is compared with the incorrect angular structure (9a) (note the use of the purine numbering convention in this figure). The boldface proton can be securely assigned in the 1H spectrum as the signal at 6.23 ppm on the basis of its chemical shift. In both 9 and 9a, it will be expected to show long-range coupling to C2 of the purine nucleus. In linear structure 9, it would also show connectivity to C6 of the purine nucleus, whereas in angular structure 9a, the connectivity would be to C4. For either structure 9 or 9a, the most downfield carbon signal (156.0 ppm) can be expected to be the carbonyl carbon at C6 of the purine nucleus. In the HMBC spectrum, C8 (151.2 ppm) and C4 (150.1 ppm) are distinguished from C6 by their long-range coupling to H1′ of the deoxyribose. Thus, the HMBC experiment showing connectivity of the boldface proton to C6 proves that the ring closure occurred at the N1 of guanine. Chlorooxirane and Acrolein Adducts (23 and 14) of Deoxyadenosine. The investigations were extended to analogous deoxyadenosine adducts 23 and 14, which were prepared by reactions of the two aminodiols with

6-chloropurine deoxyribonucleoside (20) followed by NaIO4 oxidation of diols 21 and 24 to give aldehydes 22 and 12, respectively (Scheme 3). It was observed in the NMR spectra that the conversion of 22 and 12 to cyclic adducts 23 and 14 is less complete, i.e., more reversible, than had been observed with the corresponding deoxyguanosine derivatives. The equilibrium is controlled by pH. The acyclic adducts can be observed in equilibrium with the cyclic adducts under basic conditions, whereas the cyclic structures are favored in acid on account of their enhanced basicity relative to the acyclic structures. Cyclic adducts 23 and 14 show no tendency to dehydrate. Nucleoside 23 has not been observed as an adduct in DNA, although a synthesis of the base corresponding to the acyclic species has been reported by Shaw and Smallwood (37). With regard to hydroxypropano adduct 14, the 1H NMR spectrum of the present sample differed significantly from the spectrum previously reported by Sodum and Shapiro for an acrolein adduct of deoxyadenosine, to which they tentatively assigned structure 14 (15). It appears likely that the material they isolated was, in fact, isomeric adduct 13. Oligonucleotides Containing Nucleosides 3 and 9. With the success of the reactions on nucleosides, the chemistry was then applied to the synthesis of adducted oligonucleotides (Scheme 4). For the preparation of oligonucleotides containing guanine adducts, the 8-mer deoxyribonucleoside 5′-d(AGGC-(15)-CCT) was constructed by conventional solid-phase methodology. To avoid hydrolysis of the fluoronucleoside moiety during the final deprotection step, the labile phenoxyacetyl-protected phosphoramidite reagents of dAdo, dGuo, and dCyd were

Hydroxyethano and Hydroxypropano dGuo and dAdo Scheme 4

Chem. Res. Toxicol., Vol. 13, No. 5, 2000 427

oligonucleotide 5′-d(GCT-(20)-TAGC) followed by periodate oxidation. The reactions proceeded smoothly and gave the oligonucleotides containing 23 and 14 in excellent yield and purity. The structures were established by MALDI-TOF and by enzymatic hydrolysis to give the four normal deoxynucleosides plus nucleosides 23 and 14, respectively, the structures of which were confirmed by HPLC comparison with authentic samples (Figure 3).

Discussion

used so that mild deprotection conditions could be employed. Removal of the phenoxyacetyl group could be carried out with NaOH (0.1 M) at ambient temperature rather than with the usual hot, concentrated NH4OH, which had previously been shown to convert 15 into deoxyguanosine. The reactions of the fluoropurine in the 8-mer with 3-amino-1,2-propanediol and 4-amino-1,2butanediol were carried out in DMSO (3 h at 55 °C). Treatment with dilute acetic acid removed the O6 protection. The resulting oligonucleotides, 5′-d(AGGC-(17)CCT) and 5′-d(AGGC-(19)-CCT), were purified by HPLC. Their structures were established by MALDI-TOF mass spectrometry, which confirmed the composition of the oligonucleotides, and by enzymatic hydrolysis to the constituent nucleosides. The 1,2-diols were cleaved quantitatively by brief oxidation with NaIO4 (10 min, ambient temperature) to give the aldehydes which cyclized spontaneously. Figure 1 contains HPLC traces showing the course of this reaction for the N2-dihydroxypropyl nucleoside and the corresponding oligonucleotide. The molecular weights of the two oligonucleotides, obtained by MALDI-TOF mass spectrometry, indicated the oxidations had been successful. Enzymatic hydrolysis of the two oligonucleotides, followed by HPLC analysis, established that they contained dCyd, dGuo, dAdo, and dThd in the expected ratio of 3:2:1:1 plus 1 equiv of nucleosides 3 and 9, respectively, which were identified by comparison with authentic standards (Figure 2). Oligonucleotides Containing Nucleosides 23 and 14. Oligonucleotides were prepared containing adducts 21 and 24 by reaction of the aminodiols with the

The synthetic method presented here represents an excellent method for preparing two-carbon and threecarbon Ω-oxoalkyl adducts at the N2 position of deoxyguanosine; these or, more strictly speaking, the cyclization products thereof are major products of the reaction of chlorooxirane and acrolein with DNA. The method should be applicable to higher Ω-oxoalkyl analogues as well, although in those cases cyclization may not be favored. The methodology is more problematic for adenine adducts in that, with the exception of the one report mentioned above that is likely to be in error, the reaction of chlorooxirane and acrolein with deoxyadenosine has occurred by attack of N1, not N6, on the electrophile. It is worth noting that nucleosides 22 and 23, and 12 and 14, may actually be among the products formed by the reactions of chlorooxirane and acrolein with DNA, but they have not, as yet, been detected. The availability of authentic samples will facilitate the search for them among the mixture of products formed by exposure of DNA to chlorooxirane and acrolein. The N6-linked 3-oxopropyl derivative of adenine could arise either by direct alkylation of N6 or by a Dimroth rearrangement of an initially formed N1 adduct. Nucleosides 23 and 14, when incorporated into DNA, should be excellent candidates for linking reporter groups and other moieties to DNA. The reversibility of the cyclic adducts permits the aldehydes to be available for reaction with other nucleophiles, and it should be possible to couple amines with the aldehydes irreversibly by reaction in the presence of reducing agents. Routes to DNA containing guanine adducts 3 and 9 have previously been described; these routes involve introduction of the carbon skeleton for the side chain before assembly of the oligonucleotide rather than after. Guengerich and co-workers prepared DNA containing hydroxyethano adduct 3 from the preformed base in which the hydroxyl group was protected as an acetate ester (27) using an enzymatic procedure to introduce the deoxyribose moiety. The oligonucleotide was synthesized by the usual phosphoramidite procedure using labile phenoxyacetyl protection of the exocyclic amino groups so that the final deprotection could be carried out with NaOH. Guengerich et al. had previously explored the possibility of carrying out the synthesis by the strategy described herein but failed to find conditions under which periodate cleavage of diol 17 could be achieved without concurrent depurination (1). Recently, Khullar et al. reported a synthesis of DNA containing acrolein adduct 9 from a protected form of adducted nucleoside 19 (38). Their approach involved carrying out the reaction of the aminodiol with the bisTBDMS ether of the O6-p-nitrophenylethyl analogue of 15 followed by protection of the vicinal hydroxyl groups as acetate esters, and removal of the TBDMS groups from

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the deoxyribose moiety with fluoride ion before preparation of the phosphoramidite reagent. After deprotection, the aldehyde group was created by periodate oxidation. Both of these methods for preparing DNA containing guanine adducts have proven useful, but the strategy described herein offers certain advantages. First, the divergent strategy provides efficiency in the synthesis; i.e., DNA containing a variety of different cyclic adducts can be prepared from a single oligonucleotide containing the fluoroinosine synthon by using a variety of dihydroxyalkylamines. Second, the method circumvents the need for orthogonal protection during the formation of the adducted nucleoside. It should be noted that there are significant changes in hydrophobicity of the oligonucleotides on going from the halopurine to the dihydroxyalkyl purine and then to the periodate oxidation product. The resulting changes in HPLC retention times facilitate production of highly pure oligonucleotides. Thus, the method should be an attractive one for preparation of a number of related adducts such as those of crotonaldehyde and 4-hydroxy-2-nonenal. It might also be useful for preparation of DNA adducts of fecapentaenes; these adducts have been proposed to arise from conjugated enal degradation products of the enol ether (39). The hydroxyethano and hydroxypropano guanine adducts exist in an equilibrium with the N2-Ω-oxoalkyl adducts. In the nucleoside, this equilibrium lies so far toward the cyclic form that the acyclic species cannot be detected by NMR. The structure of these lesions has not been examined in duplexed DNA but may favor the acyclic species. A study of the relative mutagenicities of the 1,N2-hydroxyethano adduct and the corresponding ethano and etheno adducts revealed that, whereas the latter adducts are highly mutagenic, the hydroxyethano adduct produced only a low level of mutations (26). The structural basis of this observation is unknown, but a possible explanation is that the hydroxyethano adduct is present in the acyclic 2-oxoethyl form which permits base stacking and Watson-Crick base pairing to be maintained; on the other hand, the ethano and etheno adducts disrupt base pairing. Mutagenicity of the 1,N2propano adduct of guanine has been studied extensively (40-42), but the hydroxypropano has not yet been examined. Structural studies using high-field NMR need to be carried out on DNA duplexes containing the hydroxyethano and hydroxypropano adducts to establish whether they do indeed exist in the acyclic forms. Structural studies of duplexes containing the 1,N2-propano and malondialdehyde adducts of guanine have been reported. The former showed severe disruption of the DNA duplex (43). On the other hand, the malondialdehyde adduct which exists in the cyclic form, i.e., pyrimido[1,2a]purin-10(3H)-one, in the nucleoside and in singlestranded DNA at neutral pH, has been found to exist in duplexed DNA in the ring-opened N2-3-oxo-1-propenylguanine form with the three-carbon fragment pointing into the minor groove. This acyclic structure minimizes structural distortions and retains substantial base stacking, although Watson-Crick base pairing appears to have been disrupted (44).

Acknowledgment. We thank the National Institute for Environmental Health Sciences for generous support of this research via Grants ES00267 and ES07781.

Nechev et al. Supporting Information Available: The 400 MHz 1H NMR spectra of deoxynucleoside adducts 3, 9, 14, 17, 19, 21, 23, and 24; COSY, 13C, and HMBC spectra of 9; UV spectra of 3, 9, 14, and 23; and MALDI-TOF spectra and HPLC profiles of the enzyme digests of the oligodeoxynucleotides containing these adducts. This material is available free of charge via the Internet at http://pubs.acs.org.

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