adenine Oligodeoxynucleotide A - American Chemical Society

Chem. Res. Toxicol. 2007, 20, 641-649

641

Site Specific N6-(2-Hydroxy-3,4-epoxybut-1-yl)adenine Oligodeoxynucleotide Adducts of 1,2,3,4-Diepoxybutane: Synthesis and Stability at Physiological pH Sergey Antsypovich, Danae` Quirk-Dorr, Crystal Pitts, and Natalia Tretyakova* Department of Medicinal Chemistry and The Cancer Center, UniVersity of Minnesota, Minneapolis, Minnesota 55455 ReceiVed August 1, 2006

1,2,3,4-Diepoxybutane (DEB) is an important metabolite of 1,3-butadiene, a high volume industrial chemical classified as a human and animal carcinogen. DEB is a bifunctional alkylating agent that exhibits both mutagenic and cytotoxic activity, presumably a result of its ability to form bifunctional DNA adducts. Initial reactions of DEB with DNA produce 2-hydroxy-3,4-epoxybut-1-yl (HEB) lesions at guanine and adenine nucleobases. The epoxy group of the monoadduct is inherently reactive and can then undergo further reactions, for example, hydrolysis to the corresponding 2,3,4-trihydroxybutyl adducts and/or second alkylation to yield 2,3-butanediol cross-links. In the present work, synthetic DNA 16-mers containing structurally defined racemic N6-(2-hydroxy-3,4-epoxybut-1-yl)-2′-deoxyadenosine (N6-HEB-dA) adducts (5′-AATTATGTXACGGTAG-3′, where X ) N6-HEB-dA) were prepared by coupling 6-chloropurinecontaining oligodeoxynucleotides with 1-amino-2-hydroxy-3,4-epoxybutane. The latter was generated in situ from the corresponding Fmoc-protected amino epoxide. The N6-HEB-dA-containing DNA oligomer was isolated by reverse-phase HPLC, and the presence of N6-HEB-dA in its structure was confirmed by molecular weight determination and by HPLC-UV-ESI+-MS/MS analyses of enzymatic digests. An independently prepared N6-HEB-dA nucleoside served as an authentic standard. The fate of N6-HEB-dA within DNA at physiological conditions in the presence of various nucleophiles (e.g., cysteine, dG, and the complementary DNA strand) was investigated. Under all conditions tested, N6-HEB-dA rapidly cyclized to produce previously unidentified exocyclic dA lesions (t1/2 < 2 h at physiological conditions). Only trace amounts of hydrolyzed and cross-linked products were detected, suggesting that the rate of cyclization was much greater than the rates of other reactions at the epoxide ring. These results indicate that DEBinduced alkylation of N6-adenine in DNA is unlikely to lead to DNA-DNA cross-linking but instead can result in the formation of exocyclic dA adducts. Introduction 1,3-Butadiene (BD1) is an important industrial chemical used in the production of rubber, plastics, and other polymers (1). Aside from occupational sources, most human exposures to BD occur as a result of its high concentrations in automobile exhaust (2) and in tobacco smoke (3). BD is classified as a known human carcinogen on the basis of evidence for increased occupational cancer risk in humans and carcinogenicity in laboratory animals (4). The widespread occurrence of BD in our environment warrants investigations into the mechanisms of its biological activity. BD must be metabolically activated to its ultimate carcinogenic species by a series of epoxidation steps catalyzed by cytochrome P450 monooxygenases (Scheme 1). The first oxidation catalyzed by cytochrome P450 2E1 and 2A6 yields (R)- and (S)-3,4-epoxy-1-butene (EB) (5, 6). EB can be * Corresponding author. Tel: (612) 626-3432. Fax: (612) 626-5135. E-mail: [email protected]. 1 Abbreviations: BD, 1,3-butadiene; DEB, 1,2,3,4-diepoxybutane; DCM, dichloromethane; DIPEA, N,N-diisopropylethyl amine; DMAP, N,N-dimethylaminopyridine; DMSO, dimethylsulphoxide; DMT, 4,4′-dimethoxytrityl; dR, 2′-deoxyribose; EB, 3,4-epoxy-1-butene; EBD, 1-butene-3,4-diol; EtOAc, ethylacetate; Fmoc, 9-fluorenylmethoxycarbonyl; N6-HEB-dA, N6(2-hydroxy-3,4-epoxybut-1-yl)-2′-deoxyadenosine, HPLC-ESI-MS, high pressure liquid chromatography-electrospray ionization-mass spectrometry; mCPBA, 3-chloroperoxybenzoic acid; MeOH, methanol; TEAA, triethylammonium acetate; TMS, trimethylsilyl.

Scheme 1. Metabolism of 1,3-Butadiene to DNA-Reactive Epoxides and the Formation of Monoepoxide Adducts

hydrolyzed to 1-butene-3,4-diol or can undergo a second oxidation to yield d,l- and meso-1,2,3,4-diepoxybutane (DEB) (5, 7-9). The latter reaction can be catalyzed by P450 2E1 and 3A4. 1-Butene-3,4-diol can in turn be further oxidized to 3,4epoxy-1,2-butanediol (EBD) (5). All three epoxides can react with nucleophilic sites in DNA and proteins or undergo detoxification by epoxide hydrolysis and glutathione conjugation (10). Among BD metabolites, DEB appears to play a central role in the adverse biological effects of BD (8, 9, 11). It is much more genotoxic and mutagenic than EB and EBD (12). DEB is 50× more effective in inducing sister chromatid exchanges in human lymphocytes than EB (13) and is 2 orders of magnitude more mutagenic than EB in TK6 lymphoblasts (14). While EB

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642 Chem. Res. Toxicol., Vol. 20, No. 4, 2007

AntsypoVich et al.

Scheme 2. Synthesis of N6-(2-Hydroxy-3,4-epoxybut-1-yl)-2′-deoxyadenosine (N6-HEB-dA) and N6-HEB-dA-Containing DNA Strands

exposure results in base substitutions at GC basepairs, DEB is a potent inducer of deletions and point mutations at both GC and AT basepairs (15, 16). Efficient conversion of BD to DEB in tissues of laboratory mice has been hypothesized to bring about an increased sensitivity of this species to BD carcinogenesis (8, 17). The genotoxic effects of DEB are thought to originate from its ability to form bifunctional DNA adducts. DNA alkylation by DEB initially produces 2-hydroxy-3,4-epoxybut-1-yl (HEB) monoadducts at guanine and adenine bases. While guanine is preferentially modified at the N-7 position, adenine alkylation can take place at the N-1, N-3, N-7, and the N6 positions (1820). The N6-adenine alkylation products can result either from a direct attack of DEB at the exocyclic amino group of adenine or from a Dimroth rearrangement of the corresponding N-1 adducts (21). Because they contain an oxirane group, HEB monoadducts are inherently reactive. Previous studies have shown that they can undergo hydrolysis to the corresponding 2,3,4-trihydroxybut-1-yl monoadducts (19) or can alkylate cellular nucleophiles to yield DNA-DNA and DNA-protein cross-links (22, 23). Zhang et al. recently reported that synthetic N-1-guanine monoepoxide adducts can cyclize to form N2,N1exocyclic lesions (24-26). In contrast, no exocyclic DEBadenine products have been previously identified. In the present work, a synthetic DNA 16-mer containing a structurally defined N6-(2-hydroxy-3,4-epoxybut-1-yl)-2′-deoxyadenosine monoadduct was prepared by coupling the corresponding 6-chloropurine-containing oligodeoxyribonucleotide with 1-amino-3,4-epoxybutan-2-ol, and the stability of this lesion in the presence of various nucleophiles was investigated. Our results presented herein demonstrate that N6-(2hydroxy-3,4-epoxybut-1-yl)-2′-deoxyadenosine adducts are unstable at physiological conditions and rapidly cyclize to form fused structures. In contrast, hydrolysis to triols and cross-linking

to form bis-adducts are relatively minor pathways of epoxide decomposition.

Experimental Procedures Chemicals. 5′-O-(4,4′-Dimethoxytrityl)-3′-O-(2-cyanoethyl)-N,Ndiisopropylphosphoramidite of 6-chloropurine-2′-deoxyribose was purchased from Chemgenes Corporation (Wilmington, MA). The DNA oligonucleotide 5′-AATTATGTXACGGTAG-3′, where X ) 6-chloropurine, was synthesized by the Midland Certified Reagent Company (Midland, TX). All other chemicals and enzymes were acquired from Sigma-Aldrich Chemical Co. (Milwaukee, WI). Thin-layer chromatography was performed with silica gel F254 as the adsorbent on glass plates (Analtech, Inc., Newark, DE) or on pre-coated plastic sheets (Polygram SIL G/UV254, MachereyNagel). The chromatograms were visualized under UV (254 nm) light or by staining with ninhydrin solution, followed by heating. Column chromatography was performed using Biotage Si 25+M 1395-1 columns (40-63 µm, 60 Å, US Patent 6,294,087) on a Biotage SP1-XOA MPLC system with UV detection and a fraction collector. Instrumentation. 1H and 13C NMR spectra were acquired at 25 °C on a Varian Invova 600 MHz instrument (Varian, Inc., Palo Alto, CA). Samples were dissolved in DMSO-d6. UV spectrophotometry was performed on a Beckman DU-7400 instrument (Beckman, Fullerton, CA). Melting points were acquired on a Thomas Hoover capillary melting point apparatus (Philadelphia, PA) and are uncorrected. Accurate mass measurements were performed with a Bruker BioTOF II reflectron ESI-TOF instrument. HPLC-MS. An Agilent 1100 capillary HPLC-ion trap mass spectrometer (Agilent Technologies, Inc., Wilmington, DE) was operated in the ESI+ mode for the analyses of small molecules and in the ESI- mode for the analyses of DNA oligodeoxynucleotides. The target ion abundance value was set to 30,000, the maximum accumulation time was 300 ms, and 4 scans were taken per average. The typical fragmentation amplitude was 0.7 V, with a scan width of 1.2 amu. Nitrogen was used as a nebulizing (15 psi) and drying

Synthesis and Stability at Physiological pH

Chem. Res. Toxicol., Vol. 20, No. 4, 2007 643

Figure 1. HPLC analysis of 6-chloropurine-dR before (A) and after coupling with 1-amino-2-hydroxy-3,4-epoxybutane to produce N6-(2hydroxy-3,4-epoxybut-1-yl)-2′-deoxyadenosine (N6-HEB-dA) (B). (Inset) UV-DAD spectra of the main products.

gas (5 L/min, 200 °C). Electrospray ionization was achieved at a spray voltage of 3-3.5 kV. For analyses of reaction mixtures, samples were dissolved in a 1:1 mixture of acetonitrile and 0.1% acetic acid and infused into the MS ion source at a flow rate of 10-15 µL/min using a syringe pump. The mass spectrometer was operated in full scan mode over the range of m/z 50-350. For analyses of nucleoside adducts, a capillary Zorbax SB-C18 column (150 × 0.5 mm, 5 µm, Agilent Technologies, Inc., Wilmington, DE) was eluted at a flow rate of 15 µL/min with a gradient of 15 mM ammonium acetate (A) and 100% acetonitrile (B); the column was maintained at 25 °C. Solvent composition was kept at 0% B from 0 to 5 min and then was changed linearly from 0% B to 6% B in 25 min, further changed to 10% B in 40 min, and then to 50% B in 2 min. Product ions of m/z 338.3 [M + H]+ were monitored to detect N6-(2-hydroxy-3,4-epoxybut-1-yl)-2′deoxyadenosine (M ) 337.2 Da). For analysis of DNA oligodeoxynucleotides, a capillary Zorbax Extend-C18 column (150 × 0.5 mm, 3.5 µm, Agilent Technologies, Inc., Wilmington, DE) was maintained at 40 °C. The column was eluted at a flow rate of 12 µL/min. The solvent composition was kept at 2% B for 5 min and then changed linearly to 17% B over 45 min. HPLC-UV Analysis. HPLC was carried out with an Agilent Technologies model 1100 HPLC system equipped with a photodiode array detector (Wilmington, DE). UV absorbance was monitored at either 254 or 260 nm. Columns and solvent elution systems were as follows: System 1. A semipreparative silica Whatman Partisil 10 column (50 cm × 9.4 mm, 10 µm) was eluted isocratically with 0.5% methanol in chloroform at a flow rate of 4 mL/min with UV

Figure 2. HPLC-UV trace (A), HPLC-ESI+-MS trace (B), MS/MS spectrum (C), and MS3 spectrum (D) of synthetic N6-(2-hydroxy-3,4epoxybut-1-yl)-2′-deoxyadenosine (N6-HEB-dA).

detection (254 nm). This system was used for the separation and analysis of diasteroisomeric N-Fmoc-1-amino-2-hydroxy-3,4-epoxybutanes (compound 2 in Scheme 2). System 2. (a) A semipreparative Supelcosil LC-18-DB (25 cm × 10 mm, 5 µm) column was eluted with a linear gradient of acetonitrile (B) in water (A) at a flow rate of 3 mL/min. Solvent composition was changed from 5% to 9% B in 30 min and then further changed to 70% B in 4 min. (b) A semipreparative Zorbax Eclipse XDB-C18 (25 cm × 9.4 mm, 5 µm) column was eluted with a linear gradient of acetonitrile (B) in water (A) at a flow rate of 3 mL/min. Solvent composition was changed from 5% to 10.3% B in 35 min and then further changed to 90% B in 2 min with UV detection (254 nm). These systems were used for the analysis and purification of synthetic nucleoside standards (compound 4 in Scheme 2). System 3. A 250 mm × 4.6 mm Jupiter Proteo 90 Å C12 column (Phenomenex, Torrance, CA) was eluted with a linear gradient of acetonitrile (B) in 100 mM TEAA, pH 7.0 (A). Solvent composition was changed from 3% to 11% B in 10 min and then further changed to 11.7% B in 21 min and 50% B in 2 min. The column was eluted at a rate of 1.0 mL/min with UV detection (260 nm). This system was used for the separation of structurally modified oligodeoxynucleotides.


AntsypoVich et al.

Table 1. NMR Characterization of N6-HEB-dA (Compound 4) chemical shift (ppm) 8.34 (s, 1H) 8.21 (s, 1H) 7.57 (s, 1H) 6.34 (t, 1H) 5.29 (m, 3H) 4.40 (br s, 1H) 3.87 (d, 1H) 3.82 (m, 1H) 3.60 (d, 1H) 3.52 (m, 2H) 3.50 (d, 1H) 2.93 (m, 1H) 2.70 (m, 1H) 2.62 (br t, 1H) 2.50a (m, 1H) 2.24 (m, 1H) a

coupling constant (Hz)

Jvic ) 6.8 Jvic ) 2.1 Jvic ) 2.1 Jvic ) 3.9 Jgem ) 11.8 Jvic ) 4.0 Jvic ) 4.8 Jgem ) 11.8 Jgem ) 13.2 Jvic ) 6.6 Jvic ) 4.6 Jvic ) 4.6 Jgem ) 13.2 Jvic ) 2.7

assignment H-8 H-2 N6-H H-1′ OH groups H-3' H-4' CH B H-5'

COSY

carbon chemical shift (ppm)

3.52 (CH2 A) 2.70 (H-2′); 2.24 (H-2′)

84.0 (C-1′)

3.87 (H-4′); 2.70 (H-2′); 2.24 (H-2′) 4.40 (H-3′); 3.60 (H-5′); 3.50 (H-5′) 3.52 (CH2 A) 3.87 (H-4′); 3.50 (H-5′)

70.8 (C-3′) 87.9 (C-4′) 75.7 (CB) 61.8 (C-5′)

CH2 A H-5′ CH C H-2′

7.57 (N6-H); 3.82 (CH B) 3.87 (H-4′); 3.60 (H-5′) 2.62 (CH2 D); 2.50 (CH2 D) 6.34 (H-1′); 2.24 (H-2′)

69.8 (CA) 61.8 (C-5′) 54.0 (CC) 39.0 (C-2′)

CH2 D CH2 D H-2′

2.93 (CH C); 2.50 (CH2 D) 2.93 (CH C); 2.62 (CH2 D) 6.34 (H-1′); 2.70 (H-2′)

39.0 (C-2′)

The signal overlapped with the signal of the solvent (DMSO).

Figure 3. gHMQC NMR spectrum of synthetic N6-(2-hydroxy-3,4epoxybutan-1-yl)-dA (N6-HEB-dA).

Synthetic Procedures. (2-Hydroxybut-3-enyl)carbamic acid 9H-fluoren-9-ylmethyl Ester (1 in Scheme 2). The title compound was synthesized as described previously (27). (2-Hydroxy-3,4-epoxybutyl)carbamic acid 9H-fluoren-9-ylmethyl Ester (2 in Scheme 2). Compound 1 (289 mg, 0.932 mmol) was dissolved in dichloromethane (DCM) (20 mL) and cooled to 0 °C. Solid mCPBA (804 mg, 3.73 mmol actual) was added, and the reaction mixture was stirred for 18 h. The reaction was monitored by TLC (10%MeOH/DCM); product rf ) 0.76. Purification by silica gel chromatography eluted by hexane and hexane/ EtOAc 2:1 (v/v) yielded compound 2 as a mixture of stereoisomers that could be resolved into two components: 2a (31.2 mg, 96 µmol, 10.3%) and 2b (51.3 mg, 158 µmol, 16.9%). Compounds 2a and 2b eluted at 10.9 and 11.6 min, respectively, upon normal phase

HPLC analysis (system 1) (Figure S-1, Supporting Information). ESI+-MS: m/z 326.2 [M + H]+, MS2 m/z 326.2 f m/z 179.0 [(9methylene-9H-fluorene) + H]+, 148.0 [M + 2H-(9-methylene-9Hfluorene)]+. Compound 2a (R,S and S,R 2-Hydroxy-3,4-epoxybutyl)carbamic acid 9H-fluoren-9-ylmethyl Ester). MP ) 114-118 °C, 1H NMR (DMSO-d6) δ 2.56 (1H, m, CH2 D, J1 ) 4.6, J2 ) 2.6), 2.63 (1H, t, CH2 D), 2.84 (1H, q, CH C), 3.07 (1H, m, CH2 A), 3.14 (1H, m, CH2 A), 3.35 (1H, m, CH B), 4.17 (1H, t, 9-CH), 4.26 (2H, m, 10-CH2), 5.02 (1H, d, OH), 7.28 (1H, NH), 7.29 (2H, 2-CH and 7-CH of fluorene), 7.38 (2H, 3-CH and 6-CH of fluorene), 7.67 (2H, 1-CH and 8-CH of fluorene), 7.86 (2H, 4-CH and 5-CH of fluorene) (Figure S-2, Supporting Information). Compound 2b (R,R and S,S 2-Hydroxy-3,4-epoxybutyl)carbamic acid 9H-fluoren-9-ylmethyl Ester. MP ) 115-118 °C, 1H NMR (DMSO-d ) δ 2.48 (1H, m, CH D, partially overlapped 6 2 with a large peak of DMSO), 2.63 (1H, t, CH2 D, J1 ) 4.8, J2 ) 2.7), 2.82 (1H, q, CH C), 3.05 (1H, m, CH2 A), 3.09 (1H, m, CH2 A), 3.24 (1H, m, CH B), 4.17 (1H, t, 9-CH), 4.27 (2H, m, 10-CH ), 2 5.10 (1H, d, OH), 7.27 (1H, t, NH), 7.29 (2H, 2-CH and 7-CH of fluorene), 7.38 (2H, 3-CH and 6-CH of fluorene), 7.67 (2H, 1-CH and 8-CH of fluorene), 7.85 (2H, 4-CH and 5-CH of fluorene) (Figure S-3, Supporting Information). R,R and S,S 1-Amino-3,4-epoxybutan-2-ol (3 in Scheme 2). Compound 2b (19.5 mg, 60.0 µmol) was dissolved in a mixture of DMSO (500 µL) and DIPEA (300 µL) and incubated at 37 °C for 18 h. The resulting racemic 1-amino-3,4-epoxybutan-2-ol (compound 3) was used without further purification. 1H NMR (DMSOd6) δ 2.63 (2H, t, CH2 D), 2.81 (1H, m, CH C), 3.02 (2H, m, J ) 5.4, CH2 A), 3.20 (1H, m, J ) 5.4, CH B) (Figure S-4, Supporting Information). ESI+-MS: m/z 104.1 [M + H]+, MS2 of m/z 104.1: m/z 86.1 [M + H - H2O]+, 69.2 [M + H - H2O - NH3]+, MS3 of m/z 86.1: m/z 68.2 [M + H - 2H2O]+, 58.3 [M + H - H2O CO]+ (Figure S-5, Supporting Information). N6-(2-Hydroxy-3,4-epoxybut-1-yl)-2′-deoxyadenosine (4 in Scheme 2). Compound 2b (6.96 mg, 21.4 µmol) was dissolved in DMSO (200 µL). A solution of 6-chloropurine-2′-deoxyriboside (5) (1.45 mg, 5.36 µmol) in DIPEA (18.7 µL) was added, and the reaction mixture was incubated at 37 °C for 5 days under an Ar atmosphere. The reaction mixtures were diluted with water (600 µL), filtered, and separated by HPLC (system 2a). Under these conditions, N6-(2-hydroxy-3,4-epoxybut-1-yl)-2′-dA (compound 4 in Scheme 2) eluted as a major peak at 27.6 min (1.1 mg, 3.26 µmol, 57% yield) (Figure 1). UV (Figure 1B inset): λmax ) 268 nm. M ) 337.3 Da from ESI+-MS: m/z 338.3 [M + H]+, MS3 m/z 338.3 f 222.2 [M + 2H - dR]+ f m/z 136.0 [Ade + H]+, 148.0 [PudCH2 + H]+, 204.0 [M + 2H - dR - H2O]+ (Figure 2). Accurate mass from Q-TOF MS: Calculated for C14H20N5O5


Figure 4. Spontaneous conversion of N6-(2-hydroxy-3,4-epoxybut-1yl)-2′-dA to a cyclic product (product 1) upon storage in water for 1 h (A) and for 6 h (B).

Figure 5. HPLC-UV analysis of the DNA 16-mer, 5′-AAT TAT GTX ACG GTA G-3′ (X ) 6-chloropurine-dR), before (A) and after coupling with 1-amino-2-hydroxy-3,4-epoxybutane to produce the corresponding N6-HEB-dA containing oligomer (B). Cycle 1 and cycle 2 represent cyclic byproducts.

[M + H]+, m/z 338.1459; observed m/z 338.1463. 1H NMR (DMSO-d6), gCOSY, and gHMQC: see Table 1 and Figure 3. Spontaneous Hydrolysis of Compound 4 at Room Temperature. N6-HEB-dA (compound 4, 0.1 mg) was dissolved in 100 µL of water. Following incubation at room temperature for 6 h, the products were resolved by HPLC (system 2b) and analyzed by HPLC-ESI+ MS/MS as described above (Figure 4). Synthesis of N6-(2-Hydroxy-3,4-epoxybut-1-yl)-2′-deoxyadenosine-Containing Oligodeoxyribonucleotides. Compound 2b (651 µg, 2 µmol) was dissolved in a mixture of DMSO (70 µL) and DIPEA (10 µL) and incubated at 37 °C for 18 h to remove the N-Fmoc protective group (Scheme 2). Then the dry 6-chloropurine-containing oligomer (5′-AAT TAT GTX ACG GTA G, where X ) 6-chloropurine, 50.0 nmol) was added, followed by incubation at 37 °C for another 18 h. The reaction mixtures were diluted with water (210 µL) and filtered. HPLC purification with system 3 yielded the N6-HEB-dA-containing oligomer eluting at 19.8 min (9.5 nmol, 19%) (Figure 5). The presence of N6-HEBdA in the oligodeoxynucleotide structure was confirmed by


Figure 6. ESI--MS mass spectra (top) and deconvoluted mass spectra (bottom) of synthetic oligomers, 5′- AAT TAT GTX ACG GTA G-3′: X ) 6-chloropurine-dR (A), X ) N6-(2-hydroxy-3,4-epoxybut-1-yl)dA (B).

HPLC-ESI--MS (M ) 5030.3 Da (calcd); M ) 5030.2 Da (found), Figure 6) and by HPLC-ESI+-MS of enzymatic hydrolysates (Figure 7). Attempted Synthesis of N6-(2-Hydroxy-3,4-epoxybut-1-yl)-2′deoxyadenosine-Containing Oligodeoxyribonucleotides in the Presence of DMAP. A mixture of DMAP (217.5 µg, 1.78 µmol) and compound 2b (580.0 µg, 1.78 µmol) in 60 µL of anhydrous DMSO was added to 44.5 nmol of dry, desalted 5′-AAT TAT GTX ACG GTA G (X ) 6-chloropurine). The reaction mixture was incubated at 37 °C for 24-48 h. Enzymatic Hydrolysis of N6-(2-Hydroxy-3,4-epoxybut-1-yl)2′-deoxyadenosine-Containing Synthetic Oligodeoxynucleotide. Aliquots of synthetic oligodeoxynucleotides (1.0 nmol) were incubated with phosphodiesterase I (120 mU) and alkaline phosphatase (40 U) in 10 mM Tris-HCl/15 mM MgCl2 at pH 7.0 (10 µL total volume) at 20 °C for 5 min. The enzymatic digests were analyzed by HPLC-ESI+-MS as described above, and the resulting nucleoside adducts were identified by their UV spectra, MS fragmentation patterns, and HPLC coelution with synthetic standards. Eluting at 42.3 min, N6-(2-hydroxy-3,4-epoxybut-1-yl)-dA gave an ESI+ MS signal at m/z 338.3 [M + H]+, and its MSn fragmentation pattern was identical to that of synthetic N6-HEBdA (m/z 338.3 [M + H]+ f m/z 222.2 [M + 2H - dR]+ f m/z 136.0 [Ade + H]+, 148.0 [Ade - CH2 + H]+, 204.0 [M + 2H dR - H2O]+; Figure 7). Secondary Reactions of N6-(2-Hydroxy-3,4-epoxybut-1-yl)2′-deoxyadenosine-Containing Oligodeoxynucleotide. Freshly isolated DNA oligomer 5′-AAT TAT GTX ACG GTA G (X ) N6-HEB-dA, 1 nmol) in the HPLC eluent (∼11% of acetonitrile in 0.1 M TEAA at pH 7.0 (300 µL)) was combined with a 10-fold molar excess of the complementary strand (10 nmol) or 2′-dG (0.065 M in 300 µL of water/DMSO (1:1, v/v)) or L-cysteine (1 M solution in 300 µL of water). Following incubation for 3 days at room temperature, the reaction mixtures were concentrated under vacuum, and the oligodeoxynucleotide products were analyzed by capillary HPLC-ESI--MS as described above.

Results and Discussion Our strategy for the preparation of DNA strands containing structurally defined N6-(2-hydroxy-3,4-epoxybut-1-yl)-2′-deoxyadenosine (N6-HEB-dA) lesions involved coupling 6-chloropurine-containing DNA with synthetic 1-amino-3,4-epoxybutan2-ol (Scheme 2). This approach is conceptually similar to the postoligomerization methodology developed by the Harris group (28) and was previously employed in our laboratory for the preparation of N6-(2-hydroxy-3-buten-1-yl)-adenine containing oligomers (27). 1-Amino-3,4-epoxybutan-2-ol was generated by the epoxidation of N-Fmoc-protected 1-amino-3-buten-2-ol


Figure 7. HPLC-UV (A) and HPLC-ESI+-MS/MS/MS trace (B) of enzymatic DNA hydrolysates of a synthetic DNA 16-mer, 5′-AAT TAT GTX ACG GTA G-3′ (X ) N6-HEB-dA), and the MS3 spectrum of the peak corresponding to N6-HEB-dA (C).

(compound 1) (27), followed by deprotection (Scheme 2). To minimize the side reactions likely to occur because of the inherent reactivity of the oxirane group, the Fmoc protective group was removed in situ immediately prior to or during the coupling reaction (see below). Synthesis of 1-Amino-3,4-epoxybutan-2-ol (3 in Scheme 2). Racemic 1-amino-3-buten-2-ol (27) was converted to its NFmoc derivative (compound 1 in Scheme 2) using standard methods (29). Epoxidation of 1 in the presence of mCPBA gave rise to stereoisomers of (2-hydroxy-3,4-epoxybutyl)-carbamic acid 9H-fluoren-9-ylmethyl ester (2 in Scheme 2). Diastereomeric mixtures of N-Fmoc-1-amino-2-hydroxy-3,4-epoxybutanes (2) were resolved by normal phase chromatography to give compounds 2a and 2b (Figure S-1, Supporting Information). The 1H NMR spectra of 2a and 2b were similar (Figures S-2 and S-3, Supporting Information). Only small differences were observed between the chemical shifts for proton signals corresponding to CHB, OH, and CH2D (Figures S-2 and S-3, Supporting Information). Geminal and vicinal coupling constants for the diastereotopic methylene protons of compound 2a (CH2D) (J1 ) 4.6 and J2 ) 2.6) were slightly smaller than those of compound 2b (J1 ) 4.8 and J2 ) 2.7) (Figures S-2 and S-3, Supporting Information). Compounds 2a and 2b were identified as R,S; S,R (2-hydroxy-3,4-epoxybutyl)-carbamic acid 9Hfluoren-9-ylmethyl ester and R,R; S,S (2-hydroxy-3,4-epoxy-

AntsypoVich et al.

butyl)-carbamic acid 9H-fluoren-9-ylmethyl esters, respectively, on the basis of their elution order from a silica column (system 1) and the comparison of their NMR spectra with those of structurally analogous diepoxides, monoepoxides, and nucleoside derivatives (22, 30-32). For example, the coupling constants of the methylene protons of meso-DEB are smaller than the coupling constants of corresponding protons of the optically active DEB isomers: J1 ) 5.1 and J2 ) 1.5 for mesoDEB, and J1 ) 6.0 and J2 ) 2.4 for S,S- and R,R-DEB (22). Furthermore, carbon chemical shift for the CD atom of compound 2a (43.6 ppm) was higher than the corresponding signal for compound 2b (42.7 ppm), whereas the CC signal of 2a (52.4 ppm) was observed at a lower resonance than that for compound 2b (53.4 ppm). This is consistent with published chemical shifts for the corresponding carbons of racemic and meso-DEB and 1,2-dihydroxy-3,4-epoxybutane (22, 31, 33). On the basis of their elution order from a silica column, 2a is less polar than 2b (Figure S-1, Supporting Information), which is consistent with a greater polarity of racemic N7-(2,3,4-trihydroxybutyl)guanine as compared with that of the meso adduct (34). Because R,R; S,S (2-hydroxy-3,4-epoxybutyl)-carbamic acid 9H-fluoren9-ylmethyl ester (2b) was obtained in a greater yield, it was used for all of the following experiments. R,R;S,S 1-Amino-3,4-epoxybutan-2-ol (3 in Scheme 2) was generated in situ by the removal of the Fmoc group from 2b and used without further purification. The presence of 3 in the deprotection mixtures was confirmed by NMR and ESI+ MS analysis (Figures S-4 and S-5, Supporting Information). The cleavage of the Fmoc group was evident by the downfield shifts for H-1, H-8, and H-10 protons as well as by the disappearance of the H-9 signal (9, 27) (Figure S-4, Supporting Information). Mass spectral analysis of 3 by ESI+ MS/MS (m/z 104.1 [M + H]+ f m/z 86.1 [M + H - H2O]+, m/z 68.2 [M + H - H2O - NH3]+) was consistent with the assigned structure of 3 (Figure S-5, Supporting Information). Synthesis and Structural Characterization of N6-(2-hydroxy-3,4-epoxybut-1-yl)-2′-deoxyadenosine (Compound 4). The coupling reaction between 6-chloropurine-dR and racemic 1-amino-3,4-epoxybutan-2-ol (3) (Scheme 2) was first performed on the nucleoside level to produce N6-(2-hydroxy-3,4-epoxybut1-yl)-2′-deoxyadenosine (Scheme 2, bottom). The reaction mixtures were separated by reverse phase HPLC (system 2a) (Figure 1). We found that this coupling reaction proceeded at a pace slower than that of the analogous reaction with 1-amino3-buten-2-ol, previously performed at our laboratory (27). A significant quantity of unreacted 6-chloropurine-dR was still present following 48 h of incubation (not shown). HPLC analysis of the reaction mixture following incubation for 5 days revealed two peaks eluting at 17.5 and 27.6 min, whereas only trace amounts of the starting material were detected at 25.2 min (Figure 1B). The new peaks had distinct UV spectra (Figure 1B, inset), which were different from that of the starting material (Figure 1A, inset). HPLC fractions corresponding to the main products were collected, and their structures were examined by mass spectrometry and NMR spectroscopy. The compound eluting at 17.5 min was identified as an exocyclic DEB-dA side product on the basis of the ESI+ MS and UV data (Antsypovich, S., Quirk-Dorr, D., Pitts, C., and Tretyakova, N., unpublished work). The product eluting at 27.6 min was identified as the desired compound, N6-(2-hydroxy-3,4-epoxybut-1-yl)-2′-deoxyadenosine (N6-HEB-dA), on the basis of the following information. The molecular weight of this compound was determined as 337.3 on the basis of the ESI+-MS analysis (Figure 2B, m/z



Figure 8. HPLC-UV analysis of the reaction mixture containing the 16-mer 5′-AAT TAT GTX ACG GTA G-3′ (X ) N6-HEB-dA, peak 3) and the first-order kinetics for the spontaneous conversion of N6-HEB-dA to a cyclic product (peak 1) at physiological conditions.

338.3 [M + H]+). The accurate mass obtained from ESI+ Q-TOF experiments (338.1463 for the [M + H]+ ions) was consistent with the structure of N6-HEB-dA (calculated: 338.1459). The major MS/MS fragmentation pathway observed in the ion trap mass spectrometer was the loss of deoxyribose: m/z 338.3 f m/z 222.2 [M + 2H-dR]+ (Figure 2C), whereas further fragmentation of the m/z 222.2 ions in MS3 experiments revealed characteristic secondary fragments at m/z 136.0 [Ade + H]+, 148.0 [(AdedCH2) + H]+, and 204.0 [M + 2H - dR - H2O]+ (Figure 2D). These fragments are fully consistent with the assigned structure of N6-HEB-dA (4 in Scheme 2). Final structural assignments for N6-HEB-dA are based on the 1-D and 2-D NMR spectra (Table 1 and Figure 3). The 1H NMR spectrum of the aromatic region was characterized by single proton signals at 8.34 ppm (H-8 of Ade) and 8.21 ppm (H-2 of the adenine heterocycle). The assigned carbon and proton chemical shifts corresponding to the adenine heterocycle were comparable with those reported for N6-methyl-2′-deoxyadenosine (35, 36). Adenine substitution at the exocyclic N6-position was confirmed by 1H NMR, gCOSY, and gHMQC experiments (Table 1). The exocyclic amino group (N6H, 7.57 ppm) gave a one-proton signal characteristic for N6-alkyladenines (Table 1). The 1H NMR signals of the sugar moiety (Table 1) were analogous to the corresponding signals of 2′-deoxyribose within 2′-deoxyadenosine and N6-methyl-2′-deoxyadenosine (35). The signals of the 2-hydroxy-3,4-epoxybutane side chain of 4 were assigned on the basis of the 1H NMR, gCOSY, and 2-D proton-decoupled 1H-13C gHMQC experiments (Figure 3). The diastereotopic CH2D methylene group was observed as a multiplet at 2.50 ppm (partially obscured by the broad DMSO signal) and a broad signal at 2.62 ppm. The CHC methine proton

appeared as a multiplet at 2.93 ppm. In the proton COSY spectrum, the CH2D protons coupled with CHC methine proton and with each other (Table 1). The CHB methine proton appeared as a multiplet at 3.82 ppm. The protons of the methylene group adjacent to adenine (CH2A) were observed as a partially resolved multiplet at 3.52 ppm. The proton chemical shifts of CH2A, CHB, CHC, and CH2D were similar to the corresponding methine and methylene protons in compounds 2b and 3 (Figures S-3 and S-4, Supporting Information). As expected, COSY cross-peaks were detected between the CH2A protons and the methine proton within CHBOH. Importantly, the CH2A protons also coupled with the amine proton of N6H in the proton COSY experiment, consistent with the N6 substitution of adenine in compound 4 (Table 1). The HMBC spectra of compound 4 were also recorded; however, no correlations were observed between the aromatic carbons and the protons within the side chain. This is similar to the results for other N6-substituted deoxyadenosines (11). Taken together, our NMR data are consistent with the structure of N6-(2-hydroxy-3,4epoxybut-1-yl)-2′-deoxyadenosine (N6-HEB-dA, 4). Further studies indicated that N6-HEB-dA (4) was unstable in water and that it completely decomposed within 6 h of incubation at room temperature (Figure 4). Only trace amounts of the hydrolysis product N6-(2,3,4-trihydroxybut-1-yl)-2′-deoxyadenosine were detected (not shown). Instead, another product with the same mass as that of N6-HEB-dA but with a shorter HPLC retention time was observed (Product 1, Figure 4). Full structural characterization of this secondary product is in progress. Synthesis and Structural Characterization of N6-(2-hydroxy-3,4-epoxybut-1-yl)-2′-dA-Containing DNA Oligodeoxynucleotides. The N6-HEB-dA-containing DNA 16-mer was


Figure 9. Capillary HPLC-ESI--MS analysis of the reaction mixtures following the incubation of the N6-HEB-dA (X) containing oligonucleotide, 5′-AAT TAT GTX ACG GTA G-3′, with excess L-cysteine.

prepared from the corresponding 6-chloropurine-containing oligomer (5′-AAT TAT GTX ACG GTA G-3′) (Scheme 2, top). Our previous studies (37) revealed the preferential interstrand G-A cross-linking by DEB at 5′-GNT-3′/5′-ANC-3′ trinucleotides (N ) any base). Therefore, this sequence was designed to enable the formation of A-G DEB cross-links following the annealing of the N6-HEB-dA-containing strand to its complement. The 6-chloropurine-containing 16-mer was prepared using 6-chloropurine-2′-deoxyribose phosphoramidite according to a previously published procedure (38). Phenoxyacetyl (PAC)protected phosphoramidites were used for the assembly of the modified oligomers (38). The deprotection step was performed in the presence of 0.1 M NaOH at room temperature over 72 h. Consistent with literature (38), we observed that the exposure of 6-chloropurine-dR-containing DNA to ammonium hydroxide solution or alcoholic solution of sodium hydroxide caused the degradation of the modified nucleoside (data not shown); therefore, such harsh conditions were avoided. 6-ChloropurinedR-containing oligomers were purified by reverse-phase HPLC. To obtain the N6-HEB-dA containing 16-mer, the corresponding 6-chloropurine oligomer was incubated with 40 equiv of racemic 1-amino-3,4-epoxybutan-2-ol (3) in the presence of DIPEA in anhydrous DMSO (Scheme 2). The N-Fmoc protective group of 2b was removed in situ by incubation with DIPEA in DMSO for 18 h at room temperature. Although 1-amino3,4-epoxybutan-2-ol 3 was not isolated, its presence in reaction mixtures was confirmed by mass spectrometry and proton NMR (Figures S-4 and S-5, Supporting Information). Following deprotection, the solution of 3 in DIPEA/DMSO was added to a dry 6-chloropurine oligomer and then mixed and incubated at 37 °C for 18 h. Although some starting material still remained following 18 h of reaction, longer incubations did not improve the reaction yields; instead, they led to the formation of multiple side products, which were difficult to resolve by HPLC (results not shown).2 The reaction mixtures were diluted with water and separated by reverse-phase HPLC (Figure 5). In addition to the unreacted 6-chloropurine oligomer (tR, 21.1 min), three new oligonucleotide peaks were observed eluting at 17.6, 18.1, and 19.8 min (Figure 5). HPLC fractions containing each oligomer were collected, and structures of the products were determined by ESI--MS of the 16-mers and HPLC-ESI+-MS/MS of their 2 An oligonucleotide coupling reaction was also attempted in the presence of DMAP as a base because according to the literature, it affords a more efficient Fmoc group removal. Although the reaction proceeded somewhat faster, several additional side products were observed, reducing the overall yield of N6-HEB-dA-containing oligonucleotides. Therefore, our optimized coupling conditions employ 18 h incubation with DIPEA as a base.

AntsypoVich et al.

enzymatic digests. The first two peaks (tR 17.6 and 18.1 min) were assigned to oligomers containing cyclic DEB-dA adducts, e.g., 1,N6-(2,3-dihydroxy-1,4-butadiyl)-2′-deoxyadenosine (Antsypovich, S., Quirk-Dorr, D., Pitts, C., and Tretyakova, N., unpublished work). The third oligomer eluting at 19.8 min was identified as DNA containing N6-HEB-dA on the basis of the following evidence. The molecular weight of this oligonucleotide product was consistent with the replacement of the chlorine in 6-Cl-Pu with a molecule of 1-amino-3,4-epoxybutan-2-ol (Figure 6). ESI-MS spectral analysis of this strand yielded multiple charge states, which were deconvoluted to obtain molecular weight information, for example, m/z 1256.5 [M - 4H] 4-, 1005.1 [M - 5H] 5-, 837.4 [M - 6H] 6-, 717.7 [M - 7H] 7-, 627.7 [M - 8H] 8- (calculated M ) 5030.3 Da; measured M ) 5030.2 Da). HPLC-UV-ESI+-MS/MS analysis of the enzymatic digests of 19.8 min oligonucleotide peak (Figure 7) in parallel with an authentic standard of N6-HEB-dA (Figure 2A and B) confirmed the presence of the target nucleoside in its structure. The nucleoside composition as determined from UV peak integration (dC (1.4), dG (4.4), T (5.0), dA (4.8, sum of dA and dI), N6HEB-dA (0.6)) was found to be consistent with the theoretical ratios (dC (1.0), dG (4.0), T (5.0), dA (5.0), N6-HEB-dA (1.0)) (Figure 7). dI was formed by the deamination of dA in the presence of adenosine deaminase, a contamination present in commercial alkaline phosphatase. The amounts of N6-HEB-dA detected in enzymatic digests are lower than expected (0.6 instead of 1 equiv) because of its limited stability at the conditions of enzymatic hydrolysis (Figure 4). Stability and Further Reactions of N6-(2-hydroxy-3,4epoxybut-1-yl)-2′-dA-Containing Oligodeoxynucleotides. As was the case with the N6-HEB-dA nucleoside (Figure 4), oligodeoxynucleotide-containing N6-HEB-dA was unstable in an aqueous solution. The half-life (t1/2) of N6-HEB-dA in singlestranded DNA was calculated as 6300 ( 280 s via first-order kinetic analysis (Figure 8). Interestingly, the major decomposition product (tR, 17.6 min) was not the expected hydrolytic product, N6-2,3,4-trihydroxybutan-1-yl-adenine, but rather a cyclic DEB-dA adduct, which was also present in the coupling reaction mixtures (the 17.6 min peak in Figure 1). When oligonucleotides containing N6-HEB-dA were incubated in the presence of the complementary strand, only trace amount of the cross-linked DNA duplex was detected by HPLCESI/MS (not shown). We initially hypothesized that DNADNA cross-linking was not occurring because of the inefficient duplex formation at low concentrations of N6-HEB-dA oligomers in the reaction mixtures.3 However, only trace amounts of cross-linked products were observed when the N6-HEB-dAcontaining strand was incubated with a large molar excess of 2′-dG or L-cysteine (Figure 9). We conclude that even in the presence of other nucleophiles, N6-HEB-dA-containing oligonucleotides preferentially form exocyclic derivatives, whereas hydrolysis and cross-linking reactions are negligible. Acknowledgment. We thank Brock Matter and Melissa Goggin (University of Minnesota Cancer Center) for their assistance with the HPLC and mass spectrometry experiments and Bob Carlson (University of Minnesota Cancer Center) for his help with the preparation of the figures for this manuscript. NMR instrumentation was provided with funds from the NSF (BIR-961477), the University of Minnesota Medical School, and 3 N6-HEB-dA-containing oligomers could not be concentrated because it led to their degradation.


the Minnesota Medical Foundation. This research is supported by a grant from the NCI (R01 9CA095039). Supporting Information Available: This material is avaliable free of charge via the Internet at http://pubs.acs.org.

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Chem. Res. Toxicol., Vol. 20, No. 4, 2007 649 in exposed calf thymus DNA. Chem. Res. Toxicol. 10, 1171-1179. (20) Tretyakova, N. Y., Sangaiah, R., Yen, T. Y., and Swenberg, J. A. (1997) Synthesis, characterization, and in Vitro quantitation of N-7guanine adducts of diepoxybutane. Chem. Res. Toxicol. 10, 779-785. (21) Selzer, R. R. and Elfarra, A. A. (1996) Characterization of N1- and N6-adenosine adducts and N1-inosine adducts formed by the reaction of butadiene monoxide with adenosine: evidence for the N1-adenosine adducts as major initial products. Chem. Res. Toxicol. 9, 875-881. (22) Park, S., Anderson, C., Loeber, R., Seetharaman, M., Jones, R., and Tretyakova, N. (2005) Interstrand and intrastrand DNA-DNA crosslinking by 1,2,3,4-diepoxybutane: role of stereochemistry. J. Am. Chem. Soc. 127, 14355-14365. (23) Loeber, R., Rajesh, M., Fang, Q., Pegg, A. E., and Tretyakova, N. (2006) Cross-linking of the human DNA repair protein O6-alkylguanine DNA alkyltransferase to DNA in the presence of 1,2,3,4-diepoxybutane. Chem. Res. Toxicol. 19, 645-654. (24) Zhang, X. Y., and Elfarra, A. A. (2003) Identification and characterization of a series of nucleoside adducts formed by the reaction of 2′deoxyguanosine and 1,2,3,4-diepoxybutane under physiological conditions. Chem. Res. Toxicol. 16, 1606-1615. (25) Zhang, X. Y., and Elfarra, A. A. (2004) Characterization of the reaction products of 2′-deoxyguanosine and 1,2,3,4-diepoxybutane after acid hydrolysis: formation of novel guanine and pyrimidine adducts. Chem. Res. Toxicol. 17, 521-528. (26) Zhang, X. Y., and Elfarra, A. A. (2005) Reaction of 1,2,3,4diepoxybutane with 2′-deoxyguanosine: initial products and their stabilities and decomposition patterns under physiological conditions. Chem. Res. Toxicol. 18, 1316-1323. (27) Quirk Dorr, D., Murphy, K., and Tretyakova, N. (2006) Synthesis of DNA oligodeoxynucleotides containing structurally defined N6-(2′hydroxy-3′,4′-buten-1′-yl)-adenine adducts of 3,4-epoxy-1-butene. Chem.-Biol. Interact. Published online 6/9/2006. (28) Nechev, L. V., Zhang, M. Z., Tsarouhtsis, D., Tamura, P. J., Wilkinson, A. S., Harris, C. M., and Harris, T. M. (2001) Synthesis and characterization of nucleosides and oligonucleotides bearing adducts of butadiene epoxides on adenine N6 and guanine N2. Chem. Res. Toxicol. 14, 379-388. (29) Greene, T. W., and Wuts, P. G. M. (1999) Protection for amino group P-779. ProtectiVe Groups in Organic Synthesis. pp 506-507, WileyInterscience, New York. (30) Robbins, M. A., Devine, P. N., and Oh, T. (1999) Synthesis of chiral non-racemic diols from (S,S)-1,2,3,4-diepoxybutane: (2S,3S)-dihydroxy-1,4-diphenylbutane (2,2′-bioxirane, [S-(R*,R*)]- and 2,3-butanediol, 1,4-diphenyl-, [S-(R*,R*)]. Org. Synth. 76, 101-109. (31) Claffey, D. J. (2002) Multigram-scale stereoselective synthesis of meso1,3-butadiene bisepoxide. Synth. Commun. 32, 3041-3045. (32) Sangaiah, R., Christova-Gueorguieva, N. I., Ranasinghe, A., Tretyakova, N. Y., Gold, A., Ball, L. M., and Swenberg, J. A. (1997) Synthesis of perdeuterated analogues of the epoxide metabolites of butadiene: 1,2-epoxybut-3-ene-d6 and 1,2,3,4-diepoxybutane-d6. J. Labelled Compd. Radiopharm. 39, 731-739. (33) Nieusma, J. L., Claffey, D. J., Maniglier-Poulet, C., Imiolczyk, T., Ross, D., and Ruth, J. A. (1997) Stereochemical aspects of 1,3butadiene metabolism and toxicity in rat and mouse liver microsomes and freshly isolated rat hepatocytes. Chem. Res. Toxicol. 10, 450456. (34) Oe, T., Kambouris, S. J., Walker, V. E., Meng, Q., Recio, L., Wherli, S., Chaudhary, A. K., and Blair, I. A. (1999) Persistence of N7-(2,3,4trihydroxybutyl)guanine adducts in the livers of mice and rats exposed to 1,3-butadiene. Chem. Res. Toxicol. 12, 247-257. (35) Chang, C. J., Ashworth, D. J., Chern, L. J., Gomes, J. D., Lee, C. G., and Narayan, R. (1984) 13C NMR studies of methylnucleosides. Org. Magn. Reson. 12, 671-675. (36) Fujii, T., and Itaya, T. (1999) Systematic tables of mono- and polyN-methylated adenines: acid dissociation constants and UV and NMR spectral data. Heterocycles 51, 2255-2277. (37) Park, S., Hodge, J., Anderson, C., and Tretyakova, N. Y. (2004) Guanine-adenine cross-linking by 1,2,3,4-diepoxybutane: potential basis for biological activity. Chem. Res. Toxicol. 17, 1638-1651. (38) Kim, H., Nechev, L., Zhou, L., Tamura, P., Harris, C. M., and Harris, T. M. (1998) Synthesis and adduction of fully deprotected oligodeoxynucleotides containing 6-chloropurine. Tetrahedron Lett. 39, 6803-6806.

TX060178K

adenine Oligodeoxynucleotide A - American Chemical Society

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