Synthesis and Properties of an Acetaldehyde ... - ACS Publications

Chem. Res. Toxicol. 2005, 18, 711-721

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Synthesis and Properties of an Acetaldehyde-Derived Oligonucleotide Interstrand Cross-Link Yanbin Lao and Stephen S. Hecht* Department of Medicinal Chemistry and The Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455 Received September 28, 2004

Acetaldehyde (AA), occurring widely in the human environment, is a mutagen and carcinogen. AA can react with DNA to form AA-DNA adducts. Several types of adducts, including an interstrand cross-link 3-(2-deoxyribos-1-yl)-5,6,7,8-tetrahydro-8-(N2-deoxyguanosyl)-6-methylpyrimido[1,2-a]purine-10(3H)one (7), have been previously characterized by our laboratory. We hypothesize that cross-link 7 may be involved in determining the mutagenic and carcinogenic properties of AA. To address this question, the double-stranded oligonucleotide 13, bearing cross-link 7, was synthesized in a sequence appropriate for mutagenicity studies in human cells. Oligonucleotide 9, containing 2-fluoro-O6-(trimethylsilylethyl)deoxyinosine (dIno), was reacted with 4-amino-1,2-pentanediol, followed by treatment with NaIO4. The resulting oligonucleotide 11 containing the 1,N2-propano-deoxyguanosine (dGuo) 5 was incubated with the complementary oligonucleotide 12 to give the desired cross-link 13, which was characterized by negative-ion electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) and enzymatic hydrolysis to cross-link 7. The formation of cross-link 13 at 5′-CpG-3′ was confirmed by incubation of 11 with [15N5]12 containing a 5′-Cp[15N5]G-3′ sequence. The formation of cross-link 13 was reversible. Therefore, oligonucleotide 24 containing the irreversible analogue of cross-link 7, 1,3-bis(2′-deoxyguanos-N2-yl)butane, was synthesized for use as a control in the mutagenicity studies. Oligonucleotide 21 was reacted with 1,3-diaminobutane dihydrochloride, followed by incubation with the complementary oligonucleotide 23, to give 24. To determine the optimum distance and orientation for cross-link formation, six oligonucleotides, containing 5 at the i + 1, i + 2, and i + 3 or the i - 1, i - 2, and i - 3 positions relative to dGuo in the complementary strand, were 5′-end labeled with [γ-32P]ATP, followed by incubation of each labeled oligonulceotide with its complementary strand and then analysis by denaturing polyacrylamide gel electrophoresis. Only the oligonucleotide containing 5′-Cp5-3′ formed the cross-link with the complementary 5′-CpG-3′ sequence. The results of this study confirm the structure of an AA-derived DNA cross-link, supply characterized crosslink-containing oligonucleotides for mutagenicity studies, and provide information on the optimum distance and orientation for cross-link formation.

Introduction Acetaldehyde (1, AA) is a mutagen and carcinogen. AA causes gene mutations, sister chromatid exchanges, micronuclei and aneuploidy in cultured mammalian cells (1, 2). It induces base substitution mutations - G to A and A to T - in the HPRT1 gene of human T lymphocytes (3). When administrated to rats by inhalation, AA induces adenocarcinoma and squamous cell carcinoma of the nasal mucosa. Treatment of hamsters with AA by inhalation causes laryngeal carcinoma. AA has been associated with alcohol-related cancers in humans who * To whom correspondence should be addressed. Mailing address: The Cancer Center, University of Minnesota, MMC 806, 420 Delaware St. SE, Minneapolis, MN 55455. Telephone: (612) 626-7604. Fax: (612) 626-5135. E-mail: [email protected]. 1 Abbreviations: AA, acetaldehyde; ALDH, aldehyde dehydrogenase; ADH, alcohol dehydrogenase; DMF, N,N-dimethylformamide; DPAGE, denaturing polyacrylamide gel electrophoresis; ESI-Q-TOF-MS, electrospray ionization quadrupole time-of-flight mass spectrometry; HMBC, heteronuclear multiple bond correlation spectroscopy; HPRT, hypoxanthine-guanine phosphoribosyl transferase; MALDI-TOF-MS, matrix-assisted laser desorption ionization-time of flight-mass spectrometry; TEA, triethylamine; TEAA, triethylammonium acetate; TMSE, trimethylsilylethyl.

have an inefficient form of aldehyde dehydrogenase (ALDH) encoded by a mutant allele of the ALDH gene (ALDH2*2). AA has been evaluated by the International Agency for Research on Cancer as “possibly carcinogenic to humans” (1, 2). Human exposure to AA can be extensive. AA is ubiquitous in the environment, being present in fruits, vegetables, and auto emissions. AA is produced during sugar metabolism in vivo. A major source of human exposure to AA is alcohol drinking. AA is the major metabolite of ethanol catalyzed by alcohol dehydrogenase (ADH) (1, 2). Its concentrations can reach 119 µM in saliva due to microbiological oxidation of ethanol (4), and 2-20 µM in the blood of alcoholics (5). Cigarette smoking is another potentially important source of AA exposure. The AA concentrations in cigarette smoke (18-1400 µg/cigarette) are generally more than 1000 times higher than those of polycyclic aromatic hydrocarbons and tobacco-specific nitrosamines (6). AA reacts with DNA to form AA-DNA adducts. A number of these adducts, including an interstrand crosslink, have been characterized by our laboratory and others (7-12). The mechanism of formation of an AA-

10.1021/tx0497292 CCC: $30.25 © 2005 American Chemical Society Published on Web 03/18/2005

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Lao and Hecht

Scheme 1. Formation of an AA-Derived Interstrand Cross-Link 7a

a

dR ) 2′-deoxyribosyl.

derived interstrand cross-link is shown in Scheme 1 (12). The most reactive deoxyribonucleoside in DNA toward AA is deoxyguanosine (dGuo) (2) (10). AA reacts with the exocyclic amino group of dGuo to form an unstable Schiff base, N2-ethylidene-dGuo (3). The addition of a second molecule of AA to 3 gives rise to aldehyde intermediate 4. Cyclization of aldehyde 4 with N-1 gives 1, N2-propano-dGuo 5. Reaction of aldehyde 4 with dGuo in the opposite strand produces Schiff base 6, which then reacts with N-1 to give the dGuo-dGuo cross-link 7. Although AA had been previously shown to produce DNA cross-links as well as DNA-protein cross-links (1, 2, 13-15), cross-link 7 represents the first structural characterization of a DNA cross-link derived from AA. Cross-link 7 was also formed in the reaction of crotonaldehyde (8) with dGuo, however, a different formation mechanism was proposed (12). Interstrand cross-links, if not repaired, are likely to prevent DNA replication and transcription, leading to cytotoxic and mutagenic effects (16, 17). A number of bifunctional anticancer agents (i.e. nitrogen mustards, platinum drugs) and mutagens (i.e. diepoxybutane, malondialdehyde) share the common ability to cross-link DNA (17-19). We hypothesized that the AA-derived interstrand cross-link 7 may play a significant role in the mutagenicity and carcinogenicity of AA. Synthetic methodology has been developed by the Harris laboratory for preparation of interstrand oligonucleotide cross-links formed by acrolein and crotonaldehdye (20, 21). In this study, we employed the same approach to synthesize the double-stranded oligonucleotide 13 (Scheme 2B), which contains cross-link 7, in a sequence appropriate for ongoing mutagenicity studies. The stable cross-link 24 (Scheme 5) was also synthesized

for use as a model compound in the mutagenicity studies. The optimum distance and orientation for AA-derived cross-link formation were determined using synthetic oligonucleotides.

Experimental Procedures Chemicals. O6-TMSE-2-fluoro-deoxyinosine (dIno) and its 5′-O-(4,4′-dimethoxytrityl)-3′-O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite were purchased from Chemgenes Corporation (Wilmington, MA). The unmodified oligonucleotides and oligonucleotides containing O6-TMSE-2-fluoro-dIno were synthesized by the Midland Certified Reagent Company (Midland, TX) and used without further purification. [15N5]-N2-isobutytyl-5′O-(4,4′-dimethoxytrityl)dGuo-3′-O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite was purchased from Spectra Stable Isotopes (Columbia, MD) and the oligonucleotide containing [15N5]dGuo was synthesized on an Applied Biosystems ABI 394 DNA Synthesizer (Applied Biosystems, Foster City, CA) by the MicroChemical Facility (University of Minnesota, Minneapolis, MN). [γ-32P]ATP (6000 Ci/mmol) was purchased from Amersham Biosciences Co. (Piscataway, NJ). OptiKinase was obtained from USB Corporation (Cleveland, OH). Snake venom phosphodiesterase I (P-3243) was obtained from Sigma-Aldrich Chemical Co. (St Louis, MO). Alkaline phosphatase (567752) was purchased from Roche Molecular Biochemicals (Indianapolis, IN). All other chemicals were acquired from Sigma-Aldrich. Instrumentation. Low resolution MS were obtained on a Finnigan LCQ Deca instrument (Finnigan MAT/Thermoquest, San Jose, CA) in the positive-ion mode for small molecules or on an Agilent 1100 capillary HPLC Ion-trap MS system (Agilent Technologies, Inc. Wilmington, DE) in negative mode for singlestranded oligonucleotides. Negative-ion mode ESI-Q-TOF-MS of cross-linked oligonucleotides were performed on an ABI QSTAR instrument (Applied Biosystems) equipped with an Agilent 1100 HPLC system in the Mass Spectrometry Consortium for the Life Sciences (MSCLS) (University of Minnesota,

Acetaldehyde-Derived Oligonucleotide Cross-Link

Chem. Res. Toxicol., Vol. 18, No. 4, 2005 713

Scheme 2. Synthesis of Monoadducted and Cross-Linked Oligonucleotidesa

a

EH ) enzymatic hydrolysis.

St Paul, MN). The samples were introduced into the TurboIonSpray ionization source through a 250 mm × 3.0 mm Luna C18 (2) column (Phenomenex, Torrance, CA) with isocratic elution by 1% CH3CN in 15 mM ammonium acetate buffer (pH 6.8) for 5 min and then a gradient from 1 to 10% CH3CN over 15 min, followed by 10 to 60% CH3CN over a period of 10 min at a rate of 0.5 mL/min. NMR spectra were acquired on a Varian Inova 500 or 600 MHz instrument (Varian, Inc., Palo Alto, CA). UV

spectrometry was performed on a Beckman DU-7400 instrument (Beckman, Fullerton, CA). HPLC Analysis. HPLC was carried out with Waters Associates (Midford, MA) systems equipped with a model 991 or 996 photodiode array detector. Columns and solvent elution systems were as follows. System 1 was a 250 mm × 4.6 mm Jupiter Proteo 90 Å C12 column (Phenomenex, Torrance, CA) with isocratic elution by 3% CH3CN in 100 mM TEAA buffer (pH

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7.0) for 10 min and then a gradient from 3 to 10% CH3CN over 10 min, followed by 10 to 15% CH3CN over a period of 30 min at a rate of 1.0 mL/min with UV detection (260 nm). This system was used for monitoring the reactions of oligonucleotides. A 250 mm × 10 mm Jupiter Proteo 90 Å C12 column was used to purify the modified oligonucleotides with a flow rate of 3.0 mL/min. System 2 was a 250 mm × 4.6 mm Aqua 125Å C18 column (Phenomenex) with isocratic elution by 5% MeOH in 40 mM ammonium acetate buffer (pH 6.8) for 10 min and then a gradient from 5 to 35% MeOH over the course of 60 min at a flow rate of 0.5 mL/min with UV detection (254 nm). This system was used for analysis of the enzymatic hydrolysates of modified oligonucleotides. System 3 was a 250 mm × 4.6 mm Vydac C18 column (The Separations Group, Inc., Hesperia, CA) eluted with CH3CN and 40 mM ammonium acetate buffer (pH 6.8) using the same gradient as system 2 at a flow rate of 0.5 mL/min with UV detection (254 nm). This was used for analysis of the reaction of 17 and 18. Similarly, a 250 mm × 10 mm Vydac C18 column was eluted at a flow rate of 1.5 mL/min for purification of 19 and 20. Synthesis. 1. AA-Derived Oligonucleotide Cross-Link (13). The synthesis of AA-derived oligonucelotide cross-link 13 was based on previously published methods (21, 22). In brief, the starting oligonucleotide 9 containing O6-TMSE-2-fluorodIno (80 OD) (Scheme 2A) was mixed with 4-amino-1, 2-pentanediol (10) (10 mg, 0.08 mmol), DMSO (500 µL), and TEA (250 µL). The mixture was stirred at 75 °C for 24 h. The solvents were evaporated under vacuum, and the residue was dissolved in 5% acetic acid (4 mL) and stirred at room temperature for 2 h. After neutralization by 1M NaOH, the mixture was concentrated, and the residue was redissolved in 50 mM potassium phosphate buffer (4 mL, pH 7.0). To the mixture was added an aqueous solution of NaIO4 (2 mL, 20 mM) and it was stirred at room temperature for 30 min. HPLC purification (system 1) gave (6R,8R)- (4.0 OD) and (6S,8S)- (5.5 OD) diastereomers of oligonucleotide 11 containing 1, N2-propano-dGuo.The combined yield was 12% from 9. Each isomer was then mixed with the complementary oligonucelotide 12 (2 × OD) (Scheme 2B). The mixture was incubated at 37 °C for 1 week to give (6R,8S)- (1.72 OD, 43%) or (6S,8R)- (0.82 OD, 15%) oligonucleotide cross-link 13, respectively, after HPLC purification (system 1). The crosslinks 13 were identified by HPLC analysis of the enzymatic hydrolysates (system 2) and by negative ESI-Q-TOF-MS. MS calcd. 9108 (for pyrimidopurinone and imine) and 9126 (for carbinolamine), found 9108 and 9126 for both (6R,8S)- and (6S,8R)-13. 2. [15N5]-Labeled AA-derived Oligonucleotide CrossLink ([15N5]13). (6R,8R)-11 (2.0 OD) was mixed with [15N5]12 (4.0 OD, Scheme 2C) in 50 mM potassium phosphate buffer (200 µL, pH 7.0). The mixture was incubated at 37 °C for 1 week. HPLC purification (system 1) of the mixture gave (6R,8S)[15N5]13 (0.46 OD, 23%). The structure was characterized as above. MS calcd. 9113 and 9131, found 9113 and 9131. 3. 1,3-Bis(phthalimidyl)butane (16). To a solution of potassium phthalimide (15) (25 g, 135 mmol) in DMF (150 mL) was added 1,3-dibromobutane (14, 8 mL, 67 mmol). The mixture was heated under reflux for 6 h, and then allowed to stand at room-temperature overnight. The solvent was evaporated under vacuum, and the residue was dissolved in H2O (100 mL), and extracted with CH2Cl2 (3 × 200 mL). The combined organic layers were dried (Na2SO4), filtered, and condensed under vacuum. Purification by flash chromatography on silica gel (Aldrich, 70-230 mesh, 60 Å), with elution by hexanes/ethyl acetate (8:1, v/v), gave 16 (9.5 g, 27 mmol, 40%). mp 125-126 °C (lit 23 129-133 °C), 1H NMR (acetone-d6): δ ppm 7.75 (m, 8H, aromatic), 4.33 (m, 1H, N-CH-CH3), 3.63 (m, 2H, N-CH2-CH2), 2.50 (m, 1H, N-CH2-CHa), 2.22 (m, 1H, N-CH2-CHb), 1.44 (d, J ) 6.9 Hz, 3H, CH3), positive ESI-MS m/z 349 [M+H]+. 4. 1,3-Diaminobutane Dihydrochloride (17). Compound 16 (426 mg, 1.2 mmol) was dissolved in 6 M HCl (70 mL). The mixture was heated under reflux overnight, cooled to room

Lao and Hecht temperature, then cooled in an ice bath to precipitate phthalic acid. After filtration, the aqueous solution was extracted with ethyl acetate (3 × 100 mL), and concentrated under vacuum to give 1,3-diaminobutane dihydrochloride (17, 173 mg, 1.1 mmol, 92%). mp 166-167 °C (lit 23 169-170 °C), 1H NMR (CD3OD): δ ppm 3.30 (m, 1H, H3N+-CH-CH3), 2.93 (m, 2H, H3N+-CH2-CH2), 1.96 (m, 1H, H3N+-CH2-CHa), 1.82 (m, 1H, H3N+-CH2-CHb), 1.22 (d, J ) 6.0 Hz, 3H, CH3), positive ESI-MS m/z 89 [M- (2HCl)+H]+. 5. N2-(3-Aminobut-1-yl)dGuo (19) and 1, 3-Bis(2′-deoxyguanos-N2-yl)butane (20). Compound 17 (10 mg, 0.06 mmol) was added to a mixture of O6-TMSE-2-fluoro-dIno (18, 19 mg, 0.05 mmol), DMSO (200 µL), and TEA (100 µL). The mixture was stirred at 75 °C. After 24 h, the reaction mixture was cooled and the solvents were evaporated under vacuum. The residue was dissolved in 5% acetic acid (1 mL) and stirred at room temperature for 2 h. After neutralization (1M NH4OH), HPLC purification (system 3) gave (R/S)-19 (5 mg, 15 µmol), (S)-20 (3.7 mg, 6 µmol), and (R)-20 (4.1 mg, 7 µmol). The combined yield was 82%. (R/S)-19 1H NMR (DMSO-d6) δ ppm 8.78 (br, 1H, N1-H, tentatively assigned), 7.83 (s, 1H, H-8), 7.30 (br, 1H, N2-H, tentatively assigned), 6.60 (br, 2H, NH2, tentatively assigned), 6.14 (t, J ) 7.0 Hz, 1H, H-1′), 4.36 (m, 1H, H-3′), 3.79 (m, 1H, H-4′), 3.55 (m, 1H, H-5′a), 3.49 (m, 1H, H-5′b), 3.31 (m, 2H, HA), 3.14 (m, 1H, HC), 2.62 (m, 1H, H-2′a), 2.19 (m, 1H, H-2′b), 1.74 (m, 1H, HB-a), 1.66 (m, 1H, HB-b), 1.15 (d, J ) 7.0 Hz, 3H, CH3), UV (MeOH/pH 6.8 buffer): λmax (nm) 210.1, 253.4, 280.2 (sh), positive ESI-MS m/z 339 [M+H]+, MS/MS of m/z 339: m/z 223 [M-116+H]+ (R)- 20 1H NMR (DMSO-d6): δ ppm 10.55 (br, 1H, N1-Ha), 10.40 (br, 1H, N1-Hb), 7.88 (s, 2H, 2 × H-8), 6.55 (br, 1H, N2-Ha), 6.49 (br, 1H, N2-Hb), 6.14 (dd, J ) 6.6, 12.0 Hz, 2H, 2 × H-1′), 5.27 (br, 2H, 2 × 3′-OH), 4.86 (br, 2H, 2 × 5′-OH), 4.34 (br, 2H, 2 × H-3′), 3.98 (br, 1H, NH-CH-CH3), 3.80 (m, 2H, 2 × H-4′), 3.51-3.48 (m, 4H, 4 × H-5′), 3.34 (m, 2H, CH2-CH2-CH), 2.60 (m, 2H, 2 × H-2′a), 2.20 (m, 2H, 2 × H-2′b), 1.78 (m, 2H, CH2-CH2-CH), 1.20 (d, J ) 6.6 Hz, 3H, CH3), positive ESI-MS m/z 589 [M+H]+, 473 [M-116+H]+, 357 [M-2 × 116+H]+, MS/MS of m/z 589: m/z 473 [M-116+H]+, 357 [M-2 × 116+H]+ (S)- 20 1H NMR (DMSOd6): δ ppm 10.52 (br, 1H, N1-Ha), 10.34 (br, 1H, N1-Hb), 7.87 (s, 1H, H-8a), 7.86 (s, 1H, H-8b), 6.48 (br, 1H, N2-Ha), 6.41 (br, 1H, N2-Hb), 6.12 (dd, J ) 6.6, 13.2 Hz, 2H, 2 × H-1′), 5.26 (br, 2H, 2 × 3′-OH), 4.85 (br, 2H, 2 × 5′-OH), 4.33 (m, 2H, 2 × H-3′), 4.01 (m, 1H, NH-CH-CH3), 3.79 (m, 2H, 2 × H-4′), 3.52-3.47 (m, 4H, 4 × H-5′), 3.38 (m, 2H, CH2-CH2-CH), 2.56 (m, 2H, 2 × H-2′a), 2.20 (m, 2H, 2 × H-2′b), 1.75 (m, 2H, CH2-CH2-CH), 1.20 (d, J ) 6.6 Hz, 3H, CH3), positive ESI-MS m/z 589 [M+H]+, 473 [M-116+H]+, 357 [M-2 × 116+H]+, MS/MS of m/z 589: m/z 473 [M-116+H]+, 357 [M-2 × 116+H]+. 6. Oligonucleotide Containing (R/S)-N2-(3-Aminobut-1yl)dGuo (22). Oligonucleotide 21 containing O6-TMSE-2fluoro-dIno (8.7 OD) was mixed with 17 (20 mg, 0.12 mmol), DMSO (500 µL), and TEA (250 µL). The reaction mixture was stirred at 75 °C for 24 h, and the solvents were evaporated under vacuum. The TMSE protecting group was removed by treatment with 5% acetic acid (1 mL), with stirring at room temperature for 5 h. The mixture was neutralized (1M NH4OH), and purified by HPLC (system 1) to give oligonucleotide 22 (1.2 OD, 14%). The oligonucleotide 22 was characterized by enzymatic hydrolysis and negative ESI-MS. MS calcd. 4670, found 4671. 7. Oligonucleotide Cross-Link Containing 1,3-Bis(2′deoxyguanos-N2-yl)butane (24). Oligonucleotide 22 (16.4 OD) was mixed with a large excess of oligonucleotide 23 (100 OD) in 50 mM Na2B4O7-NaOH buffer (2 mL, pH 10). The mixture was stirred at room temperature for 1 week. HPLC purification (system 1) of the mixture gave (R)- (2.7 OD, 16.5%) and (S)(2.1 OD, 12.8%) 24. They were characterized by HPLC analysis (system 2) of the enzymatic hydrolysates and negative ESI-QTOF-MS. MS calcd. 9110, found 9110 for both (R)- and (S)-24. Enzymatic Hydrolysis. In a typical reaction, an oligonucleotide (0.05-0.1 OD) was dissolved in Tris-HCl buffer (30 µL, 10 mM Tris-HCl, 10 mM MgCl2, pH 7.0) and incubated with



Table 1. Sequences of Synthetic Oligonucleotidesa oligonucleotide

sequenceb

25 25′ 26 26′ 27 27′ 28 28′ 29 29′ 30 30′ 31

5′-CCATACTAACXATAC-3′ 3′-GGTATGATTGCTATG-5′ 5′-CCATACTACAXATAC-3′ 3′-GGTATGATGTCTATG-5′ 5′-CCATACTCAAXATAC-3′ 3′-GGTATGAGTTCTATG-5′ 5′-CCATAATAAXCATAC-3′ 3′-GGTATTATTCGTATG-5′ 5′-CCATAATAXACATAC-3′ 3′-GGTATTATCTGTATG-5′ 5′-CCATAATXAACATAC-3′ 3′-GGTATTACTTGTATG-5′ 5′-CCTACATAACGATACGTGAGTATTGCTATG-3′

a The sequences were designed by presenting the complementary strand dGuo at the i + 1 (25), i + 2 (26), and i + 3 (27) or the i - 1 (28), i - 2 (29), and i - 3 (30) positions relative to 5, denoted by X. b 25 and 28-30, X ) (6R,8R)-5; 26 and 27, X ) (6R/S,8R/S)-5.

snake venom phosphodiesterase I (8 mU) and alkaline phosphatase (10 U) at 37 °C for 90 min. The mixture was analyzed by HPLC (system 2), and the adducts were identified by their UV spectra and by HPLC coelution with synthetic standards. Reduction of 7 to 20 by NaBH4. An aliquot of (6R,8S)- or (6S,8R)-7 was dissolved in 50 mM potassium phosphate buffer (50 µL, pH 7.0) and treated with excess NaBH4. The mixture was stirred at room-temperature overnight. After neutralization by 5% acetic acid, HPLC purification (system 3) of the mixture gave (R)- or (S)-20, which was identified by UV spectra and positive ESI-MS/MS analyses. [32P]-Labeling and Gel Electrophoresis. Modified oligonucleotides 25-30 (Table 1) containing 5 were synthesized and characterized as described above, and purified by HPLC (system 1) and DPAGE. The [32P]-labeling experiment was conducted according to the manufacturer’s recommendation. Each modified oligonucleotide as well as the 30-mer standard 31 (5 pmol) was dissolved in H2O (5 µL) and mixed with 10 × reaction buffer (2.5 µL), [γ-32P]ATP (6 pmol, 3.5 µL), Optikinase (10 U, 1 µL) and H2O (13 µL). The mixture was incubated at 37 °C for 30 min. The reaction was quenched by heating at 65 °C for 10 min. Excess ATP was removed on a MicroSpinTM G-25 column (Amersham Biosciences), which was preequilibrated in 50 mM potassium phosphate buffer (pH 7.0). Each 5′-end labeled modified oligonucleotide 25-30 was mixed with its complementary oligonucleotide 25′-30′ (50 pmol) in 50 mM potassium phosphate buffer (25 µL) respectively, and the mixture was incubated at 37 °C. After 1 week, the mixture was dried by centrifugation under reduced pressure, and the residue was dissolved in formamide (4 µL) and TBE buffer (4 µL, 100 mM Tris, 100 mM boric acid, 2 mM EDTA). The mixture was then eletrophoresed on 19% denaturing polyacrylamide gel (7 M urea) in TBE buffer at constant voltage (2500 V) for 2 h. The gel was imaged on a Molecular Dynamics Storm 8400 phosphorimager using a phosphor screen (20 × 25 cm, Amersham Biosciences) and densitometry was performed using ImageQuant software.

Results Synthesis of AA-Derived Oligonucleotide CrossLink. The sequence of the targeted cross-link 13 (Scheme 2B) was designed to be appropriate for ongoing mutagenicity studies in human cells. Two strands of 15-mer oligonucleotides were linked by a four-carbon tether, derived from two molecules of AA. The linkage was formed at the N2 positions of Gua from each strand. There were four overhanging bases at the 5′-end of each strand, i.e., CCAT on the top strand and TTCC on the bottom, which were required for unidirectional insertion of the cross-link into the plasmid used in the mutagenicity studies. The remaining eleven bases were paired with

Figure 1. Chromatogram obtained upon HPLC analysis (system 1) of the incubation mixture of (6R,8R)-11 with 12 (see Scheme 2B).

each other in the two oligonucleotide strands. Based on a previous study which showed that a related acroleinderived cross-link was formed exclusively in a 5′-CpG-3′ sequence (21), the current sequence was also designed for cross-link formation at 5′-CpG-3′. Starting from O6-TMSE-2-fluoro-dIno-modified oligonucleotide 9, two diastereomers of 11 were prepared (Scheme 2A). Their structures were confirmed by negative ion ESI-MS and by enzymatic hydrolysis to (6R,8R)and (6S,8S)-5, identified by HPLC (system 2) coelution with standards (data not shown). Then, (6R,8S)- and (6S,8R)-13 were prepared by incubation of each diastereomer of 11 with its complementary strand 12 (Scheme 2B). The yield of (6S,8R)-13 (15%) was lower than that of (6R,8S)-13 (43%). Under our HPLC conditions (system 1), double-stranded cross-links 13 eluted after single strands 11 and 12 (Figure 1). Enzymatic hydrolysis of (6R,8S)- and (6S,8R)-13 gave (6R,8S)- and (6S,8R)-7, respectively (Scheme 2B). Standards 5 and 7 were fully characterized in previous studies, in which they were obtained by reacting crotonaldehyde (8) with dGuo (Scheme 3) (12). As shown in Figure 2, the early eluting isomer of 5 was identified as (6S,8S)-5 by HPLC coelution with authentic (6S,8S)-5 (22). The assignment of two diastereomers of 7 (Figure 2) was based on HPLC coelution with (6R,8S)- and (6S,8R)-7 generated from enzymatic hydrolysis of (6R,8S)- and (6S,8R)-13. Negative ESI-Q-TOF-MS of (6R,8S)- and (6S,8R)-13 gave molecular weights of 9108 and 9126, suggesting the presence of carbinolamines (Scheme 2B) (20, 21, 24). These results were consistent with previous studies (20, 21). Enzymatic hydrolysis of HPLC-purified cross-links 13 also generated peaks corresponding to 5, which suggested the reversibility of cross-link formation, as shown in Scheme 1. DPAGE analysis of HPLC-purified 13 confirmed the reversibility, showing bands corresponding to 13 as well as two single strands 11 and 12 (see Figure 19 in Supporting Information). However, the cross-link 13 was reasonably stable in buffer. Eighty-nine percent of purified cross-link 13 was intact after a month of storage at 4 °C in 15 mM ammonium acetate buffer (pH 7.0). The half-life of 13 was 20h when it was incubated at 37 °C in 10 mM Tris-HCl buffer (pH 7.4). Under the conditions used in ongoing mutagenicity studies (70 mM Tris-HCl buffer, pH 7.6, 10 mM MgCl2, 5 mM dithiothreitol, 37 °C for 1h, then 16 °C for 24h), 97% of cross-link 13 remained. Therefore, the cross-link is expected to be stable enough for mutagenicity studies. The formation of cross-link 13 at the 5′-CpG-3′ position, indicated in bold in Scheme 2C, was determined by incubation of (6R,8R)-11 with [15N5]12, which contained

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Scheme 3. Diastereomers of 5 and 7 Formed in the Reaction of Crotonaldehyde with dGuo.

[15N5]dGuo in the complementary sequence 5′-CpG-3′. Negative ESI-Q-TOF-MS analysis of (6R,8S)-[15N5]13 gave molecular weights of 9113 and 9131, 5 units more than those of unlabeled cross-link (6R,8S)-13 (9108 and 9126). Enzymatic hydrolysis of (6R,8S)-[15N5]13 gave a peak coeluting with (6R,8S)-7, as shown in Figure 3A. Positive ESI-MS/MS analysis of this peak gave m/z 592 [M+H]+, 476 [M-116+H]+ and 360 [M-(2 × 116)+H]+ (Figure 3B), 5 units more than those of unlabeled (6R,8S)-7 (Figure 3C, m/z 587 [M+H]+, 471 [M-116+H]+ and 355 [M-(2 × 116)+H]+). These results confirmed that the cross-link was formed at the 5′-CpG-3′ position between two complementary oligonucleotide strands. Synthesis of Oligonucleotide Cross-Link Containing 1,3-Bis(2′-deoxyguanos-N2-yl)butane. Similar chemistry was employed in this synthesis (25, 26). The nucleoside adducts 19 and 20 were synthesized by the reaction of O6-TMSE-2-fluoro-dIno (18) with 1,3-diaminobutane dihydrochloride (17), which was obtained by Gabriel synthesis (Scheme 4) (23). Compared to its secondary amino group, the primary amino group of 17 was highly reactive toward the fluorine atom of 18. The structures of 19 and 20 were characterized by UV, 1H NMR, and ESI-MS/MS analysis. HMBC analysis of 19 confirmed the substitution of fluorine in 18 by the primary amino group of 17, not by the secondary amino group. A correlation was observed between HA (δ 3.31

Figure 2. Chromatogram obtained upon HPLC analysis (system 2) of the products of the reaction of crotonaldehyde with dGuo (12). The assignments of diastereomers of 5 and 7 were based on HPLC coelution with authentic standards.

ppm) and C2 (δ 146 ppm) in the HMBC spectrum, but there was no correlation between HC (δ 3.14 ppm) and C2 (δ 146 ppm). These results are consistent with the greater nucleophilicity of the primary than the secondary amine. The regiospecific purity of 19 was confirmed by HPLC analysis.The (R)- and (S)-diastereomers of 19 were baseline separated from the (R)- and (S)-diastereomers of N2-(1-aminobut-3-yl)dGuo (19′), which was isolated as a minor product when the reaction of 17 with 18 was carried out in a 5:1 molar ratio (see Supporting Information). Reduction of (6R,8S)- and (6S,8R)-7 with NaBH4 gave standards (R)- and (S)-20, which were used to distinguish the (R)- and (S)- diastereomers of synthetic 20 by HPLC coelution (system 3). The mixed diastereomers of oligonucleotide 22 were prepared by the reaction of the O6-TMSE-2-fluoro-dIno-modified oligonucleotide 21 with 17 (Scheme 5). (R)- and (S)-22 were not separated under our HPLC conditions (system 1), as shown in Figure 4A. The structure of 22 was established by negative ion ESI-MS and enzymatic hydrolysis. ESI-MS analysis gave a molecular weight of 4671. HPLC analysis of an enzymatic hydrolysate of 22 gave two peaks coeluting with the diastereomers of 19 as well as four deoxyribonucleosides (Figure 4B). The purity of 22 was at least 95% based on HPLC analysis of the enzymatic hydrolysate (Figure 4B). No attempt was made to separate the diastereomers of 22. Eventually, cross-links (R)- and (S)-24 were easily separated by HPLC once they were formed from the reaction of the mixed diastereomers of 22 with O6-TMSE-2-fluoro-dIno-modified oligonucleotide 23 (Figure 4A, system 1). Each diastereomer of 24 was characterized by negative ESI-Q-TOF-MS and HPLC analysis of enzymatic hydrolysates. ESI-Q-TOF-MS analysis gave molecular weights of 9110 for both (R) and (S) (calcd. 9110). Enzymatic hydrolysis of each diastereomer of 24 gave peaks corresponding to 20 (Figure 4C and 4D). The peaks were identified by HPLC coelution with synthetic standards (R)- and (S)-20 (system 2). The yield of (R)-24 (16.5%) was similar to that of (S)-24 (12.8%). Optimum Distance and Orientation for AADerived Oligonucleotide Cross-Link Formation. Six oligonucleotides 25-30 were synthesized and characterized as above. These sequences were designed by presenting the complementary strand dGuo at the i + 1 (25), i + 2 (26), i + 3 (27) or the i - 1 (28), i - 2 (29), and i - 3 (30) positions relative to adduct 5 (denoted by X in Table 1). Except for 26 and 27, only (6R,8R) diastereo-



fied by DPAGE before 5′-end labeling with [γ-32P] ATP and Optikinase. Each labeled oligonucleotide 25-30, respectively, was incubated with the complementary strands 25′-30′ in 50 mM phosphate buffer for a week. The reaction mixtures were separated by DPAGE (Figure 5). Lane 1 was loaded with the 30-mer standard 31 (Table 1) and lane 2 with 15-mer standard 25. Lanes 3-8 were loaded with the incubation mixtures of each pair of oligonucleotides. As shown in Figure 5, the cross-link was formed only in lane 3, which was loaded with the mixture of 25 and 25′ having the sequences 5′-Cp5-3′ and 5′-CpG3′. This result indicates that 5′-CpG-3′ is the sequence with optimum distance and orientation for AA-derived cross-link formation. The yield of cross-link formation was 2.5%, calculated by dividing the amount of the crosslink by that of the cross-link plus the single stranded oligonucleotide.

Discussion

Figure 3. (A) Chromatogram obtained upon HPLC analysis (system 2) of enzymatic hydrolysate of (6R,8S)-[15N5]13 as well as standard (6R,8S)-7; structures and MS of (B) (6R,8S)[15N5]7 and (C) (6R,8S)-7. *N indicates 15N.

mers of oligonucleotides were used. The mixed diastereomers were used for 26 and 27 because the (6R,8R) and (6S,8S) diastereomers were not separated under our HPLC conditions. The optimum distance and orientation to form the interstrand cross-link was determined based on the extent of cross-link formation with these synthetic oligonulceotides. Each oligonucleotide 25-30 was puri-

In the present study, oligonucleotide interstrand crosslink 13 was site-specifically prepared in a sequence appropriate for mutagenicity studies. Enzymatic hydrolysis of oligonucleotide cross-link 13 gave the nucleoside cross-link 7, which coeluted on HPLC with the cross-link formed in the reaction of AA and DNA, confirming the structure of the AA-derived cross-link. Our previous results demonstrated that one diastereomer of cross-link 7 was predominantly formed in the AA-DNA reaction, and, upon standing for 30 days at 4 °C, gradually converted to the major diastereomer of 5 observed in the reactions of AA with DNA and dGuo (12). This diastereomer was identified as (6R,8S)-7 and the major diasteromer of 5 identified as (6R,8R)-5 by HPLC coelution with standards (system 2). The results strongly support our proposed mechanism for the formation of AA-derived 5 and 7, illustrated in Scheme 1 (12). Both (6R,8R)-5 and (6R,8S)-7 are produced from the same precursor, N2-ethylidene-dGuo (3). When the second molecule of AA approaches the Schiff base 3, the attack on the si face is favored to give the intermediate (6R)-4, which either cyclizes to form (6R,8R)-5 or reacts with an interstrand dGuo to give (6R,8S)-7 (22). The stereoselectivity of formation of 7 in the AA-DNA reaction is consistent with that of formation of oligonucleotide cross-link 13 in the synthesis. Consistent with previous results (21), the yield of (6R,8S)-13 (43%) was higher than that of (6S,8R)-13 (15%), which indicates that the cross-link 13 with R configuration at C6 has higher stability than that with S configuration in a DNA duplex context. The reason for this stereoselectivity is currently unknown. In a preliminary molecular dynamics modeling study on the structures of (6R)- and (6S)diastereomers of cross-link 13 (INSIGHT II, data not shown), we observed that the cross-link was formed in the minor groove of the B DNA helix. The methyl group with (R)- configuration points to the center of the minor groove, however, it is much closer to the phosphate backbone of DNA when bearing the (S)-configuration. Steric hindrance resulting from the interaction of the DNA backbone with the methyl group may explain the lower stability of the cross-link with (S)-configuration at C6. Further investigations including NMR studies are needed to support this hypothesis. Kozekov et al. concluded that the acrolein and crotonaldehyde-derived oligonucleotide cross-links are mix-

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Scheme 4. Synthesis of Nucleoside Adducts 19 and 20

Scheme 5. Synthesis of Oligonucleotide Cross-Links (R)- and (S)-24

tures of the carbinolamine and imine forms (21). The high melting temperature and weak signals in the MALDITOF-MS of the acrolein-derived oligonucleotide cross-link excluded the presence of a pyrimidopurinone (21). The imine was trapped by NaCNBH3 (20, 21). NMR studies detected the presence of a cabinolamine in the oligonucleotide cross-link (24), and MS analysis gave two ions corresponding to imine and carbinolamine (20, 21). Our results are supportive of the presence of imine and carbinolamine, however, the pyrimidopurinone cannot be completely excluded. We observed two m/z values in the negative-ion mode ESI-Q-TOF-MS analysis of oligonucleotide cross-link 13, corresponding to pyrimidopurinone and/or imine as well as carbinolamine. However, in contrast to weak signals for cross-links species obtained in their MALDI-TOF-MS analyses (20, 21), we observed intense signals for cross-links in ESI-Q-TOF-MS analysis. The signal for pyrimidopurinone and/or imine was predominant, and about 2-3-fold more intense than that of the carbinolamine. A plausible explanation for the different observations is that, during MALDI sample preparation, the matrix 3-hydroxypicolinic acid catalyzes the addition of H2O to the imine and dissociation of the

carbinolamine. In the ESI-Q-TOF-MS analysis, 15 mM ammonium acetate (pH 6.8) was used, which may stabilize the pyrimidopurinone and/or imine to give strong signals. Therefore we suggest that the imine may be the predominant species and be in equilibrium with the pyrimidopurinone and carbinolamine in the DNA duplex. The formation of carbinolamine or pyrimidopurinone is less favored because it would require additional adjustment of the DNA duplex conformation to accommodate the hydroxyl group or cyclization, respectively. DNA interstrand cross-links, if not repaired, are lethal DNA lesions. They can block DNA replication and transcription (16, 17). They may also induce both point and deletion mutations during cellular repair processes (27-31). Furthermore, the mechanisms by which interstrand cross-links are repaired in human cells are not clearly understood (32, 33). The availability of AA-derived oligonucleotide cross-link 13, as well as an in vivo system suitable for mutagenicity and repair studies in human cells (34-37), allows us to investigate cross-link mutagenicity and repair in human cells. The results will be helpful for understanding mechanisms of mutagenesis and carcinogenesis by AA, and repair mechanisms for



Figure 4. Chromatograms obtained upon (A) HPLC analysis (system 1) of reaction mixture of 22 with 23 (upper trace shows the retention time of (R/S)-22 in system 1); and HPLC analyses (system 2) of enzymatic hydrolysates of (B) (R/S)-22 (C) (R)-24 (D) (S)-24.

Figure 5. DPAGE analysis of incubation mixtures of six pairs of oligonucleotides (sequences see Table 1): lane 1, 30-mer standard 31; lane 2, 15-mer standard 25; lane 3, incubation mixture of 25 and 25′; lane 4, incubation mixture of 26 and 26′; lane 5, incubation mixture of 27 and 27′; lane 6, incubation mixture of 28 and 28′; lane 7, incubation mixture of 29 and 29′; lane 8, incubation mixture of 30 and 30′.

interstrand cross-links in human cells. This work is currently in progress in the laboratory of our collaborator, Dr. Masaaki Moriya, State University of New York, Stony Brook. We found that cross-link 13 is able to revert to the un-cross-linked forms. However, it was stable enough for insertion into the plasmid for the mutagenicity study (97% remaining under conditions of the experiments). We believe that the cross-link would be more stable in genomic DNA than in a short oligonucleotide duplex, because the cross-link was present in AA-treated DNA, as indicated by its detection as nucleoside cross-link 7 in enzymatic hydrolysates. To investigate the mutagenicity of reversible cross-link 13, we also synthesized the irreversible cross-link 24. The compound contains a stable branched trimethylene tether. It should give useful information pertinent to the mutagenicity of intact crosslink 13. The (R)- and (S)-diastereomers of cross-link 24

are also good model compounds for NMR studies to investigate the stereoselectivity observed in the synthesis of cross-link 13. It may not be possible to establish the structure of cross-link 13 by NMR because the sample will be a mixture of pyrimidopurinone and/or imine, carbinolamine, and un-cross-linked species. Cross-link formation by DNA cross-linking agents is often sequence specific. For example, diepoxybutane and nitrogen mustards-derived N7 G-N7 G cross-links preferentially form in the sequence 5′-GNC-3′ (38-42), and DNA cross-linking by formaldehyde is favored in the sequence 5′-ApT-3′ (43, 44). Both malondialdehyde and mitomycin C preferentially cross-link oligonucleotides containing the sequence 5′-CpG-3′ (19, 32, 45). We observed that the optimum distance for AA-derived crosslink formation was one base away from the modified G, the same as malondialdehyde and mitomycin C. This is

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reasonable because these latter three cross-links may be formed at the N2 position of G, and the tethers are 3 or 4 carbon atoms long. Therefore they could not readily span a distance greater than that between adjacent base pairs of the DNA duplex. The optimum orientation for AA-derived cross-link formation was determined to be 5′-CpG-3′, and there was no cross-link formation in a 5′-GpC-3′ sequence. The results are consistent with those of a previous study (21). Kozekov et al. demonstrated that an acrolein-derived cross-link was formed exclusively at 5′-CpG-3′. The structures of oligonucleotides containing model tether trimethylenes in both 5′-CpG-3′ and 5′-GpC-3′ sequences have been established by NMR studies and molecular dynamics (25, 26). The differences between the two structures are: 1) the two guanines and the 3-carbon-atom tether lie very nearly in a single plane in the 5′-CpG-3′ duplex, whereas there is a lack of planarity in the 5′-GpC-3′ duplex; 2) the 5′-GpC-3′ duplex is much more severely distorted than the 5′-CpG-3′ sequence. Therefore the 5′-CpG-3′ duplex is stabilized by the trimethylene cross-link, while the 5′-GpC-3′ duplex is destabilized. This model is also applicable to explain the orientation preference for the AA-derived cross-link, as observed here. Due to the reversibility, the destabilized 5′-GpC-3′ duplex would revert to the un-cross-linked form. In conclusion, we confirmed the structure of a crosslink formed in the reaction of AA and DNA, prepared characterized oligonucleotide interstrand cross-links for mutagenicity and repair studies, and determined the optimum distance and orientation for AA-derived DNA cross-link formation.

Acknowledgment. We thank Dr. Peter Villalta, Mr. Brock Matter, and Ms. Sudha Marinanikkuppam (MSCLS) for their assistance with MS experiments, and thank Dr. Shana Sturla for her help in NMR experiments. We acknowledge Dr. Masaaki Moriya, Department of Pharmacological Sciences, State University of New York at Stony Brook, for the sequence design of the targeted cross-links and DPAGE analysis of HPLCpurified cross-link 13. We thank Dr. Thomas M. Harris, Department of Chemistry, Vanderbilt University, for a generous gift of authentic standard (6S,8S)-5. We acknowledge Hansen Wong and Bob Carlson for their assistance during manuscript preparation. This study was supported by Grant ES11297 from the National Institute of Environmental Health Sciences.

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Supporting Information Available: Preparation and characterization of (R)- and (S)-19′; HPLC, UV traces, and MS spectra of two diastereomers of 19 and 19′; 1H NMR and 1H-1H COSY spectra of 19 and 19′; HMBC spectrum of 19; 1H NMR of (R)- and (S)-20; negative-ion ESI-Q-TOF-MS spectra of oligonucleotide cross-links; DPAGE analysis of purified (6R,8S)- and (6S,8R)-13. This material is available free of charge via the Internet at http://pubs.acs.org.

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