Incorporation of N 2-Deoxyguanosine Metabolic Adducts of 2

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Chem. Res. Toxicol. 2005, 18, 457-465

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Incorporation of N2-Deoxyguanosine Metabolic Adducts of 2-Aminonaphthalene and 2-Aminofluorene into Oligomeric DNA Radha R. Bonala, M. Cecilia Torres, Sivaprasad Attaluri, Charles R. Iden, and Francis Johnson* Department of Pharmacological Sciences, The State University of New York at Stony Brook, Stony Brook, New York 11794 Received August 2, 2004

In previous work we described an efficient procedure for the synthesis of the respective N2 and N6 adducts of 2′-deoxyguanosine (dG) and 2′-deoxyadenosine (dA) derived from a series of aminoaryl compounds. We now outline methods for the site-specific introduction into oligomeric DNA of the adducts dG-N2-AN (6), dG-N2-AAN (7), dG-N2-AF (8), and dG-N2-AAF (9) derived from 2-aminonaphthalene (2-AN) or 2-aminofluorene (2-AF). For the 2-AN adduct 7, containing an acetylamino group, the 5′-O-4,4′-dimethoxytrityl- (DMT-) 3′-O-phosphoramidite (14) required for automated DNA synthesis was synthesized in high yield via the sequence 10 f 11 f 14. On the other hand, introduction of the desacetyl adduct 6 into oligomeric DNA was accomplished via the N-trifluoroacetyl-DMT-phosphoramidite derivative 18. This involved a similar sequence (10 f 15 f 18) except that the order of the reactions was changed to avoid a decomposition that occurred when the silyl-protected amino derivative 11 was treated with trifluoroacetic anhydride. In the 2-AF series the 5′-O-DMT-3′-O-phosphoramidites 27a and 27b, related to 8 and 9, were prepared by similar methods. Again, however, the order of the reactions was changed to avoid the extreme insolubility associated with the N2-[3-(2-acetylaminofluoren-3yl)]dG (dG-N2-AAF, 9) adduct that we had noted previously. The incorporation into oligomeric DNA of the acetylamino compounds 7 and 9 proceeded smoothly and in high yield (95-100%). By contrast, the trifluoroacetyl analogues led in both the naphthyl and fluorenyl series to a mixture of oligomers containing the desired free amino adduct (6 or 8) accompanied by the N-acetyl adduct (7 or 9, respectively, after the deprotection step), indicating secondary acetylation by the capping agent acetic anhydride.

Introduction In a previous paper (1) we described in detail an adaptation of the Buchwald-Hartwig reaction that allowed the efficient synthesis of the N2 and N6 adducts of 2′-deoxyguanosine (dG)1 and 2′-deoxyadenosine (dA), respectively, derived from the metabolic products of selected carcinogenic arylamines. These adducts arise in DNA from a group of substances collectively known as the carcinogenic amines, the most notorious of which are 2-aminofluorene (2-AF), 2-aminonaphthalene (2-AN), 4-aminobiphenyl, and 2-aminotoluene (o-toluidine). All of these amines, and in most cases their N-acetyl derivatives, have been implicated in carcinogenesis and, as might be expected, have been shown to be significantly mutagenic both in vivo and in vitro (2-9). * To whom correspondence should be addressed: phone (631) 6328862; fax (631) 632-7394; e-mail [email protected]. 1 Abbreviations: dG, 2′-deoxyguanosine; dA, 2′-deoxyadenosine; 2-AN, 2-aminonaphthalene; 2-AAN, 2-acetylaminonaphthalene; 2-AF, 2-aminofluorene; 2-AAF, 2-acetylaminofluorene; DMT, 4,4′-dimethoxytrityl; dG-N2-AF, N2-(2-aminofluoren-3-yl)-2′-deoxyguanosine; dG-N2AAF, N2-(2-acetylaminofluoren-3-yl)-2′-deoxyguanosine; dG-N2-AN, N2(2-aminonaphth-1-yl)-2′-deoxyguanosine; dG-N2-AAN, N2-(2-acetylaminonaphth-1-yl)-2′-deoxyguanosine; dG-C8-AAF, N-acetyl-N-(2′deoxyguanosin-8-yl)-2-aminofluorene, dG-C8-AF, N-(2′-deoxyguanosin8-yl)-2-aminofluorene; N-OH-AN, N-hydroxy-2-aminonaphthalene; FAB, fast atom bombardment; HRMS, high-resolution mass spectrometry; TLC, thin-layer chromatography.

The mechanisms by which these adducts are formed are general in nature and involve primary metabolic/ oxidation of the nitrogen atom to the N-hydroxy derivative (8-10), followed by glucuronidation, O-acetylation, or O-sulfation as a consequence of secondary metabolic processes. Both the O-acetyl and O-bisulfate derivatives (1) and in some cases the hydroxylamine itself are known to undergo acid-catalyzed solvolysis to generate nitrenium ions (Scheme 1), which in the presence of DNA give rise to purine adducts. The preferred target of the nitrenium ion (2) is the C-8 position of dG, which leads largely to 8-aminoaryl or 8-acetylaminoaryl derivatives (4) of guanine residues. Some isomerization of the nitrenium ion also takes place to a proximal carbonium ion 3, which then targets the peripheral purine amino groups. In the case of dG (the principal purine target), this type of damage is characterized by general structure 5. In this paper we are principally concerned with the synthesis of the 2-AN and 2-AF N2 adducts of dG (represented in their monomeric forms as 6 and 7 and as 8 and 9, respectively, in Figure 1) and their introduction into oligomeric DNA. 2-(N-Acetyl)aminofluorene (2-AAF) is widely used as a model chemical carcinogen because it undergoes precisely the type of metabolic activation noted in Scheme 1. In this case the N2 adduct, namely, N2-(2-acetylamino-

10.1021/tx0497907 CCC: $30.25 © 2005 American Chemical Society Published on Web 02/24/2005

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Bonala et al.

Scheme 1. Principal Interactions of the Carcinogenic Amine Metabolites with Guanine Bases in DNA

fluoren-3-yl)-2′-deoxyguanosine (9, dG-N2-AAF), is a minor (5-15%) dG product (11-19). However, unlike the N-acetyl-N-(2′-deoxyguanosine-8-yl)-2-aminofluorene (dG-C8-AAF) and the N-(2′-deoxyguanosine-8-yl)-2-aminofluorene (dG-C8-AF) adducts (represented generally as 4) which are easily removed by DNA repair enzymes (14, 20, 21), the corresponding N2 adducts (5) have been found to be persistent in the tissues of animals treated with this amine (12, 13). Site-specific mutagenesis techniques have been used to explore the miscoding properties of dG-C8-AAF and dG-C8-AF adducts in simian kidney (COS-7) cells (22-24). Adducts inserted into a singlestrand shuttle vector promote principally dG f dT transversions together with a smaller number of dG f dA transitions at the lesion site (23). As part of our work in this area we had prepared a dG-N2-AAF-modified DNA oligomer by a postsynthetic method, albeit in very poor yield. This was used as a template to investigate the miscoding properties of this damage by use of the Klenow fragment of Escherichia coli DNA polymerase (25). The result revealed that dG-N2-AAF (9) is a miscoding lesion generating G f T transversions. However, the miscoding potential of this lesion has not been explored with mammalian DNA polymerases. In addition, because of the limitations of the postsynthetic method, the miscoding variations due to sequence context could not be studied. 2-AN has not been studied as extensively as 2-AF or 2-AAF. In early work Hueper et al. (26) developed an animal model in which they showed that bladder tumors could be induced in dogs that were fed either 2-AN or 4-aminobiphenyl. Further work by Case et al. (27) then implicated 2-AN as a highly probable cause of bladder cancer in industrial workers. More recently, Kadlubar

et al. (28) found that when 2-AN was fed to dogs, adducts were found in the nuclear DNA of urothelial and hepatic cells. As with 2-AF and 2-AAF, the genotoxicity is mediated by N-hydroxy metabolites formed largely by hepatic N-oxidation (10). The resulting N-hydroxyaminonaphthalene (N-HO-AN) undergoes N-glucuronidation that leads to a water-soluble product. The glucuronide enters the circulation and is then excreted in the urine (28). However, due to the acidic nature of mammalian urine (pH 5-6), this conjugate can be hydrolyzed back to N-HO-AN, which can react with DNA directly. Subsequent solvolysis leads to reactive intermediates that result in the formation of DNA adducts. N-HO-AN is mutagenic in the Ames test and was found to induce urothelial tumors (29-31). A comet assay demonstrated that 2-AN induces DNA damage in urinary bladder cells from rats or humans (32). Three DNA adducts derived from N-HO-AN, namely, N2-(2-aminonaphth-1-yl)-2′deoxyguanosine (dG-N2-AN, 6), N6-(2-aminonaphth-1-yl)2′-deoxyadenosine (dA-N6-AN), and N-(2′-deoxyguanosin8-yl)aminonaphthalene (dG-C8-AN), were identified as the major types of DNA damage, both in vivo and in vitro (10). Both N-HO-AN and N-hydroxy-2-acetylaminonaphthalene (N-HO-AAN) have been shown to be proximate bladder carcinogens (33, 34). Nevertheless, studies on the mutagenic behavior of the DNA adducts derived from these metabolites in mammalian cells have not been reported. Extension of our earlier synthetic work (1) on the N2dG adducts of 2-AN (6), 2-AAN (7), 2-AF (8), and 2-AAF (9) now has allowed us to incorporate these modified deoxynucleosides into oligomeric DNA in a site-specific manner.

Figure 1. Structure of the N2-naphthyl and -fluorenyl adducts of dG.

Synthesis of DNA Containing N2-dG Arylamines

Experimental Procedures All reagents and solvents were commercial grade and used as such unless otherwise specified. 1H NMR spectra were recorded on either a Bruker AC-250 or a Varian 300 spectrometer. Samples prepared for NMR analysis were dissolved in CDCl3, acetone-d6, or DMSO-d6. Chemical shifts are reported in parts per million (ppm) relative to TMS. Mass spectra were recorded on a Micromass Trio 2000 in fast atom bombardment (FAB) mode. High-resolution mass spectra (HRMS) were performed by the Mass Spectrometry Laboratory of the School of Chemical Sciences, University of Illinois at Urbana-Champaign. FAB-HRMS are reported for almost all compounds. Only in the cases of the phosphoramidites did it prove to be impossible to obtain either FAB-MS or FAB HRMS. Thin-layer chromatography (TLC) was performed on silica gel sheets (TiedeldeHae¨n, Sleeze, Germany) containing a fluorescent indicator. Components were visualized by UV light (λ ) 254 nm) or by spraying with a solution of phosphomolybdic acid. Flash column chromatographic separations were carried out on 60 Å (230400 mesh) silica gel (TSI Chemical Co., Cambridge, MA). All experiments dealing with moisture or air-sensitive compounds were conducted under dry nitrogen. The starting materials and reagents, unless otherwise specified, were the best grade commercially available (Aldrich, Fluka) and were used without further purification. All new products showed a single spot on TLC analysis, after purification. The reagents used to synthesize the oligomers were purchased from Applied Biosystems (Foster City, CA). All of the HPLC solvents were obtained from Fisher Scientific (Suwanee, GA), and were used as received unless otherwise specified. DNA oligomers were synthesized by standard phosphoramidite chemistry on an Applied Biosystems 394 DNA/RNA synthesizer (Foster City, CA). A Waters HPLC system (Milford, MA) consisting of a 600 E multisolvent delivery system, a U6K injecter, and a photodiode array detector was used to purify the modified oligomers. Purification was carried out on a Luna 5µ, phenylhexyl column (250 × 10 mm; Phenomenex, Torrance CA) at a 4 mL/min flow rate. A solvent gradient of 16-40% acetonitrile (30 min) in 0.1 M triethyl ammonium acetate buffer (pH 6.8) was used to collect the DMT-protected oligomers. The DMT group was removed by retreatment with 80% AcOH, and the oligomers were then rechromatographed with a gradient of 5-25% acetonitrile over 40 min. Electrospray ionization mass spectra were obtained from a Micromass Platform Instrument. N2-(2-Acetylaminonaphth-1-yl)-2′-deoxyguanosine (7). A solution of 12 (1) (0.2 g, 0.295 mmol) in pyridine (5 mL) was cooled in an ice bath and HF/Py (0.21 mL, 70% HF in pyridine from Aldrich) was added in 3 min. The reaction was stirred at room temperature overnight, then poured over crushed ice (20 g), and left for 2 h. The mixture was extracted with methylene chloride and the organic phase was washed with 10% aqueous NaHSO4 to pH 6 and then with brine, dried over MgSO4, filtered, and concentrated. The crude product was purified on a silica gel column with CH2Cl2/MeOH (95:5) as the eluant to give pure 12 (0.109 g, 90%). The physical data for compound 12 were identical with those of the reported compound (1) by 1H NMR. N2-(2-Acetylaminonaphth-1-yl)-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine (13). To a solution of 7 (0.225 g, 0.5 mmol) in pyridine (5 mL) was added DMT chloride (0.203 g, 0.6 mmol) as a solid, and the mixture was stirred at room temperature for 3 h. TLC (CH2Cl2/MeOH 97:3) of the reaction mixture showed the reaction to be about 80% complete. Methanol (5 mL) was added and the mixture was stirred for 30 min at room temperature. The solution was then concentrated under reduced pressure, aqueous saturated NaHCO3 (10 mL) was added, and the mixture was extracted with methylene chloride. The methylene chloride layer was dried over anhydrous MgSO4, filtered, and concentrated. The crude product was purified by column chromatography over silica gel with CH2Cl2/MeOH (97: 3) as the eluant. This afforded pure 13 (0.3 g, 80%). 1H NMR (CDCl3): δ 8.88 (br s, 1H), 8.20 (s, 1H), 7.99 (t, 1H), 7.82 (t, 1H), 7.66 (m, 3H), 7.42 (d, 1H), 7.38-7.04 (m, 9H), 6.77 (m, 4H),

Chem. Res. Toxicol., Vol. 18, No. 3, 2005 459 5.85 (t, 1H), 4.32 (m, 1H), 4.15 (m, 1H), 3.72 (s, 4H), 3.44 (m, 2H), 2.32 (m, 2H), 2.02 (s, 3H). FAB HRMS m/z calcd for C43H41N6O7 (M + H)+, 753.3036; found, 753.3014. N2-(2-Acetylaminonaphth-1-yl)-5′-O-(4,4′-dimethoxytrityl)3′-O-[N,N-diisopropylamino(2-cyanoethoxy)phosphinyl]2′-deoxyguanosine (14). Compound 13 (116 mg, 0.15 mmol) was coevaporated with dry toluene (3 × 10 mL). The residue was redissolved in dry methylene chloride (7 mL) and treated with triethylamine (0.200 mL) followed by N,N-diisopropylamino(2-cyanoethoxy)chlorophosphine (0.069 mL, 0.30 mmol). The reaction mixture was stirred at room temperature for 2 h under nitrogen, and then methylene chloride (25 mL) was added. The solution then was washed with aqueous saturated NaHCO3 solution, dried over Na2SO4, filtered, and concentrated under reduced pressure to yield the desired product 14 (160 mg, 100%) as an oil. 1H NMR (CDCl3): δ 8.88 (br s, 1H), 8.21 (s, 1H), 7.97 (t, 1H), 7.80 (t, 1H), 7.64 (m, 3H), 7.40 (d, 1H), 7.38-7.05 (m, 9H), 6.77 (m, 4H), 5.85 (t, 1H), 4.31 (m, 1H), 4.14 (m, 1H), 3.90 (m, 2H), 3.72 (s, 4H), 3.44 (m, 2H), 3.05 (m, 2H), 2.60 (m, 2H), 2.32 (m, 2H), 2.02 (s, 3H), 1.12 (s, 2H), 1.09 (s, 3H), 1.00 (s, 3H), 0.97 (s, 3H). 31P NMR (CDCl3): δ 150.35, 149.87. N2-(2-Nitronaphth-1-yl)-O6-benzyl-2′-deoxyguanosine (15). A solution of 10 (1) (2 g, 2.65 mmol) in pyridine (20 mL) was cooled in an ice bath, and HF/Py (2.11 mL, 70% HF in pyridine from Aldrich) was then added over 3 min. The reaction was left at stirring at room temperature overnight. The reaction mixture was poured over crushed ice (100 g) and left for 2 h. The mixture was extracted with methylene chloride and the extract was washed with 10% aqueous NaHSO4 to pH 6, followed by washing with brine, and then dried over MgSO4, filtered, and concentrated. The crude product was purified on a silica gel column with CH2Cl2/MeOH (95:5) to give pure 15 (1.26 g, 90%). 1H NMR (acetone-d6): δ 8.31 (d, 1H), 8.3 (s, 1H), 8.12 (d, 1H), 8.03 (d, 1H), 7.98 (d, 1H), 7.71 (t, 1H), 7.62 (t, 1H), 7.257.03 (m, 5H), 6.38 (t, 1H), 5.10 (s, 2H), 4.60 (m, 1H), 4.18 (m, 1H), 3.79-3.65 (m, 2H), 2.81 (m, 1H), 2.44 (m, 1H). FAB HRMS m/z calcd for C27H25N6O6 (M + H)+, 529.1835; found, 529.1825. N2-(2-Nitronaphth-1-yl)-O6-benzyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine (16). To a solution of 15 (1.056 g, 2 mmol) in pyridine (10 mL) was added DMT chloride (0.75 g, 2.2 mmol) as a solid, and the mixture was stirred at room temperature for 3 h. TLC (CH2Cl2/MeOH 97:3) of the reaction mixture at this point showed the reaction to be ∼80% complete. The reaction was quenched by addition of methanol (5 mL), and the solution was stirred for 30 min at room temperature. The reaction mixture was then concentrated on a rotary evaporator, and thereafter aqueous saturated NaHCO3 (15 mL) and methylene chloride were added. The methylene chloride layer was separated, dried over anhydrous MgSO4, filtered, and evaporated to dryness. The crude product was purified by column chromatography with CH2Cl2/MeOH (97:3) as the eluant. This led to pure 16 as a bright yellow solid (1.3 g, 80%). 1H NMR (CDCl3): δ 8.59 (br s, 1H), 8.18 (d, 1H), 8.09 (d, 1H), 7.99 (d, 1H), 7.82 (s, 1H), 7.80 (d, 1H), 7.80 (t, 1H), 7.50 (t, 1H), 7.42 (d, 1H), 7.38-7.15 (m, 13H), 6.82 (d, 4H), 6.23 (t, 1H), 5.19 (s, 2H), 4.40 (m, 1H), 4.03 (q, 1H), 3.79 (s, 6H), 3.30 (m, 2H), 2.68 (m, 1H), 2.35 (m, 1H). FAB HRMS m/z calcd for C48H43N6O8 (M + H)+, 831.3142; found, 831.3134. N 2 -(2-Trifluoroacetylaminonaphth-1-yl)-5′-O-(4,4′dimethoxytrityl)-2′-deoxyguanosine (17). To a solution of 16 (150 mg, 0.18 mmol) in ethyl acetate/methanol (1:1, 20 mL) was added a 10% palladium-on-carbon catalyst (20 mg). The flask was evacuated (50 Torr) and flushed with hydrogen three times. The reaction mixture was then shaken under hydrogen for 16 h at 50 psi. The solution was filtered through a pad of Celite to remove catalyst and then concentrated under reduced pressure. The residue was dried for 6 h in a vacuum oven and dissolved in pyridine (5 mL), and the solution was cooled to 0 °C in an ice bath and treated with trifluoroacetic anhydride (0.055 mL, 2.2 equiv). Stirring was continued for 2 h at 0 °C, and the reaction was quenched by being poured into an ice-cold mixture of methylene chloride and aqueous saturated NaHCO3

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(1:1, 50 mL). The methylene chloride layer was separated, dried over MgSO4, filtered, and concentrated. The crude product was purified by silica gel column chromatography with CH2Cl2/ MeOH (97:3) as the eluant. This led to pure 17 (116 mg, 80%). 1H NMR (CDCl ): δ 8.21 (s, 1H), 7.80 (t, 1H), 7.83 (t, 1H), 7.67 3 (m, 3H), 7.44 (d, 1H), 7.38-7.05 (m, 9H), 6.79 (m, 4H), 5.86 (t, 1H), 4.32 (m, 1H), 4.15 (m, 1H), 3.72 (s, 4H), 3.44 (m, 2H), 2.32 (m, 2H). FAB (M + H)+: 807.3. N2-(2-Trifluoroacetylaminonaphth-1-yl)-5′-O-(4,4′-dimethoxytrityl)-3′-O-[N,N-diisopropylamino(2-cyanoethoxy)phosphinyl]2′-deoxyguanosine (18). Compound 17 (100 mg, 0.12 mmol) was coevaporated with dry toluene (3 × 10 mL). The residue was redissolved in dry methylene chloride (5 mL) and treated with triethylamine (0.100 mL) and then with N,N-diisopropylamino(2-cyano-ethoxy)chlorophosphine (0.033 mL, 0.15 mmol). The reaction mixture was stirred at room temperature for 2 h under nitrogen, and then methylene chloride (20 mL) was added. The methylene chloride solution was washed with aqueous saturated NaHCO3 solution, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the desired product (126 mg, 100%) 18 as an oil. 1H NMR (CDCl3): δ 8.21 (s, 1H), 7.97 (t, 1H), 7.80 (t, 1H), 7.65 (m, 3H), 7.41 (d, 1H), 7.38-7.05 (m, 9H), 6.77 (m, 4H), 5.85 (t, 1H), 4.31 (m, 1H), 4.14 (m, 1H), 3.90 (m, 2H), 3.72 (s, 4H), 3.44 (m, 2H), 3.05 (m, 2H), 2.60 (m, 2H), 2.32 (m, 2H), 1.12 (s, 2H), 1.09 (s, 3H), 1.00 (s, 3H), 0.97 (s, 3H). 31P NMR (CDCl3): δ 150.56, 149.78. N2-(2-Nitrofluoren-3-yl)-O6-benzyl-3′,5′-bis-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine (22). An oven-dried 50 mL two-necked flask was charged with the protected purine nucleoside 21 (1.753 g. 3 mmol), cesium carbonate (1.37 g, 4.2 mmol), palladium acetate (71 mg, 0.30 mmol), BINAP (2,2′-bis(diphenylphosphino)-1,1′-binapthalene) (281 mg, 0.44 mmol), the bromonitrofluorene 20 (1.3 g, 4.5 mmol), and toluene (17 mL) under a nitrogen atmosphere. This mixture was stirred for 30 min at room temperature and then heated at 80 °C for 8 h. It was cooled to room temperature and filtered, and the solids were washed with ethyl acetate. The filtrate was evaporated to dryness and purified by silica gel column chromatography with ethyl acetate/hexane (80:20) as the eluant to give pure 22 (1.7 g, 75%). 1H NMR (CDCl3): δ 10.69 (s, 1H), 9.38 (s, 1H), 8.4 (s, 1H), 8.14 (s, 1H), 7.69 (d, 1H), 7.56 (m, 3H),7.43-7.30 (m, 5H), 6.46 (t, 1H), 5.70 (s, 2H), 4.62 (m, 1H), 4.02 (m, 1H), 3.94 (s. 2H), 3.88-3.76 (m, 2H), 2.72-2.62 (m, 1H), 2.52-2.47 (m, 1H), 0.89 (d, 18H), 0.09-0.05 (m, 12H). FAB HRMS m/z calcd for C42H55N6O6Si2 (M + H)+, 795.3721; found, 795.3714. N2-(2-Nitrofluoren-3-yl)-O6-benzyl-2′-deoxyguanosine (23). To an ice-cold solution of 22 (2.33 g, 2.95 mmol) in pyridine (25 mL) was added HF/Py (2.35 mL, 70% of HF in pyridine from Aldrich) over a period of 3 min. The mixture was stirred at room temperature overnight and then poured into ice-cold water containing NaHCO3 (5.6 g) and stirred for 2 h. The separated solids were filtered, washed with water, and dried in a vacuum oven over P2O5 to give 23 (1.6 g, 100%), which was pure enough for the subsequent reaction. 1H NMR (DMSO-d6): δ 10.28 (s, 1H), 9.01 (s, 1H), 8.37 (s, 1H), 8.33 (s, 1H), 7.87 (d, 1H), 7.647.37 (m, 7H), 6.37 (t, 1H), 5.59 (s, 2H), 5.36 (d, 1H), 4.93 (t, 1H), 4.39 (br s, 1H), 4.01 (s, 2H), 3.90 (br s, 1H), 3.53 (m, 2H), 2.68 (m, 1H), 2.37 (m, 1H). FAB HRMS m/z calcd for C30H27N6O6 (M + H)+, 567.1992; found, 567.1980. N2-(2-Nitrofluoren-3-yl)-O6-benzyl-5′-O-(4.4′-dimethoxytrityl)-2′-deoxyguanosine (24). To a solution of 23 (1.4 g, 2.5 mmol) in pyridine (15 mL) was added DMT chloride (0.932 g, 2.75 mmol) as a solid, and the mixture was stirred at room temperature for 3 h. TLC (CH2Cl2/MeOH 95:5) of the mixture showed the reaction to be ∼90% complete. The reaction was quenched by addition of methanol (5 mL), and the mixture was stirred for 30 min at room temperature and then concentrated under reduced pressure. To the residue was added aqueous NaHCO3 (25 mL), and the mixture was extracted with methylene chloride. The methylene chloride extract was dried over anhydrous MgSO4, filtered, and concentrated. The crude product was purified by silica gel column chromatography with CH2-

Bonala et al. Cl2/MeOH (95:5) as an eluant to give pure 24 (1.68, 80%). 1H NMR (CDCl3): δ 10.71 (s, 1H), 9.49 (s, 1H), 8.42 (s, 1H), 7.92 (s, 1H), 7.78 (d, 1H), 7.61-7.55 (m, 3H), 7.42-7.16 (m, 19H), 6.76 (dd, 4H), 6.50 (t, 1H), 5.69 (s, 2H), 4.63 (m, 1H), 4.13 (m, 1H), 3.95 (s, 2H), 3.75 (s, 6H), 3.45-3.31 (m, 2H), 2.83 (m, 1H), 2.61 (m, 1H). FAB HRMS m/z calcd for C51H45N6O8 (M + H)+, 869.3298; found, 869.3290. N2-(2-Acetylaminofluoren-3-yl)-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine (26a). To a solution of 24 (0.3 g, 0.35 mmol) in ethyl acetate/methanol (1:1, 20 mL) was added a 10% palladium-on-carbon (50 mg) catalyst. The flask was evacuated (50 Torr) and flushed with hydrogen three times. The mixture was shaken under hydrogen for 16 h at 50 psi with stirring, filtered through a pad of Celite to remove the catalyst, and then concentrated under reduced pressure. The residue was dried for 6 h in a vacuum oven, and then the crude product was redissolved in methylene chloride/pyridine (3:1, 20 mL), cooled in an ice bath, and treated with acetic anhydride (0.5 mL). After being stirred at room temperature for 16 h, the mixture was treated with aqueous NaHCO3. Stirring was continued for 30 min, and then the organic layer was separated and the aqueous layer was extracted once with methylene chloride (10 mL). The combined methylene chloride extracts were evaporated to dryness, the residue was dissolved in methanol (15 mL), concentrated ammonia (10 mL) was added, and stirring was continued at room temperature for 16 h. The mixture was evaporated under reduced pressure, coevaporated with toluene, and purified by column chromatography with CH2Cl2/MeOH (95:5) as the eluant to give pure 26a as a glassy solid (0.235 g, 87%). 1H NMR (DMSO-d6): δ 10.05 (br s, 1H), 9.34 (br s, 1H), 8. 18 (s, 1H), 7.74 (m, 2H), 7.63-7.38 (m, 3H), 7.38-7.05 (m, 9H), 6.68 (d, 4H), 6.12 (t, 1H), 5.70 (br s, 1H), 5.43 (br s, 1H), 5.02 (s, 1H), 4.21 (m, 3H), 3.98 (br s, 1H), 3.10 (m, 6H), 2.4 (m, 1H), 1.65 (m, 1H), 2.0 (s, 3H). FAB HRMS m/z calcd for C46H43N6O7 (M + H)+, 791.3193; found, 791.3181. N 2 -(2-Trifluoroacetylaminofluoren-3-yl)-5′-O-(4.4′dimethoxytrityl)-2′-deoxyguanosine (26b). To a solution of 24 (0.150 g, 0.17 mmol) in ethyl acetate/methanol (1:1, 20 mL) was added a 10% palladium-on-carbon (30 mg) catalyst. The flask was evacuated (50 Torr), flushed with hydrogen three times, and then shaken under hydrogen for 16 h at 50 psi. The resulting solution was filtered through a pad of Celite and concentrated under reduced pressure. The residue was dried for 16 h in a vacuum oven, dissolved in pyridine (10 mL), and cooled to 0 °C by use of an ice bath. Trifluoroacetic anhydride (0.045 mL, 0.35 mmol) was added and the mixture was kept at 0-5 °C for 1 h. At this time the starting material was absent. The mixture was quenched by pouring it into an ice-cold mixture of methylene chloride (25 mL) and aqueous NaHCO3 (0.5 g in 25 mL of water). The methylene chloride layer was separated, washed with brine, dried over MgSO4, filtered, and concentrated. The crude residue was purified by silica gel column chromatography with CH2Cl2/MeOH (95:5) as the eluant to give pure 26b as the glassy solid (113 mg, 78%). 1H NMR (CDCl3): δ 7.9 (br s, 2H), 7.81 (br s, 1H), 7.62 (br s, 1H), 7.45 (br s, 1H), 7.387.03 (m, 7H), 6.74 (d, 4H), 6.18 (t, 1H), 4.42 (m, 1H), 3.98 (m, 1H), 3.65 (s, 7H), 3.21 (m, 2H), 2.4 (m, 1H). FAB (M + H)+: 845.2. N2-(2-Acetylaminofluoren-3-yl)-5′-O-(4,4′-dimethoxytrityl)-3′-O-[N,N-diisopropylamino(2-cyanoethoxy)phosphinyl]-2′-deoxyguanosine (27a). Compound 26a (118 mg, 0.15 mmol) was coevaporated with dry toluene (3 × 10 mL), and the residue was redissolved in dry methylene chloride (5 mL) and treated with triethylamine (0.100 mL) followed by N,Ndiisopropylamino(2-cyanoethoxy)chlorophosphine (0.040 mL, 0.18 mmol). The reaction mixture was stirred at room temperature for 2 h under nitrogen, and then diluted with methylene chloride containing 1% TEA (25 mL). The methylene chloride layer was washed with aqueous saturated NaHCO3 solution, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the desired product 27b (150 mg, 100%) as an oil. 1H NMR (CDCl3): δ 8.25 (s, 1H), 8. 01 (s, 1H), 7.84 (m,

Synthesis of DNA Containing N2-dG Arylamines Table 1a mass (Da) 1 2 3 4 5 6 7 8 9 9ab

oligonucleotide

calcd

obsd

5′-d(TTX1 CTT)-3′ 5′-(CGT ACX1 CAT GC)-3′ 5′-(TCC TCC TGX1 GCT CTC)-3′ 5′-(TCC TCC TCX2 CCT CTC)-3′ 5′-(GTC AGG CX2C TGC CCC)-3′ 5′-(TTY1 TT-)3′ 5′-(TCC TCC TY1C CCT CTC)-3′ 5′-(TTY2 CTT)-3′ 5′-(TCC TCC TCY2 CCT CTC)-3′ 5′-(TCC TCC TCY2 CCT CTC)-3′

1191.4 3538.2 4692.0 4569.0 4749.0 1666.4 4614.8 1912.0 4531.9 4531.9

1193.99 ( 0.251 3537.88 ( 0.41 4692.11 ( 0.62 4569.06 ( 0.31 4749.18 ( 0.23 1667.87 ( 0.15 4614.01 ( 0.49 1913.44 ( 0.16 4532.17 ( 0.28 4532.42 ( 0.23

a X ) N2-(2-acetylaminofluoren-3-yl)dG, X ) N2-(2-amino1 2 fluoren-3-yl)dG; Y1 ) N2-(2-acetylaminonaphth-1-yl)dG; Y2 ) N2(2-aminonaphth-1-yl)dG. b No capping step.

2H), 7.66-7.41 (m, 3H), 7.38-7.05 (m, 9H), 6.76 (s, 2H), 6.75 (s, 2H). 6.26 (t, 1H), 4.60 (m, 1H), 4.18 (m, 3H), 3.98-3.25 (m, 12H), 3.00 (m, 2H), 2.72-2.5 (m, 4H), 2.0 (s, 3H), 1.29 (s, 3H), 1.27 (s, 3H), 1.26 (s, 3H), 1.24 (s, 3H). 31P NMR (CDCl3): δ 150. 20, 149.89. N2-(2-Trifluoroacetylaminofluoren-3-yl)-5′-O-(4,4′-dimethoxytrityl)-3′-O-[N,N-diisopropylamino(2-cyanoethoxy)phosphinyl]-2′-deoxyguanosine (27b). Compound 26a (95 mg, 0.11 mmol) was coevaporated with dry toluene (3 × 10 mL), dissolved in dry methylene chloride (5 mL), and treated first with triethylamine (0.100 mL) and then with N,N-diisopropylamino(2-cyanoethoxy)chlorophosphine (0.030 mL, 0.13 mmol). The reaction mixture was stirred at room temperature for 2 h under nitrogen and then diluted with methylene chloride solution containing 1% TEA (25 mL). The mixture was washed with aqueous saturated NaHCO3 solution, dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain the desired product 27b (120 mg, 100%) as an oil. 1H NMR (CDCl3): δ 8.23 (s, 1H), 7.98 (s, 1H), 7.81 (m, 2H), 7.66-7.41 (m, 3H), 7.38-7.03 (m, 9H), 6.78 (s, 2H), 6.76 (s, 2H). 6.25 (t, 1H), 4.61 (m, 1H), 4.17 (m, 3H), 3.98-3.24 (m, 12H), 2.97 (m, 2H), 2.71-2.49 (m, 4H), 1.28 (s, 3H), 1.26 (s, 3H), 1.25 (s, 3H), 1.23 (s, 3H). 31P NMR (CDCl3): δ 149.51, 149.88. Synthesis of DNA Oligomers Containing an N2-(2Aminofluorene-3-yl)-2′-deoxyguanosine (dG-N2-AF, 8) or an N2-(2-Aminonaphth-1-yl)-2′-deoxyguanosine (dG-N2AN, 6) Residue. (a) Oligomers Containing a dG-N2-AAF (9) or a dG-N2AF (8) Residue. In the synthesis of oligomers with the DMTphosphoramidite (27a) of 9, a 3:1 mixture of CH2Cl2/CH3CN was required to effect solution, whereas in the case of the trifluoroacetyl analogue 27b, pure acetonitrile was efficient. In each case, the standard 0.25 µM synthesis cycle was modified to increase the coupling time to 10 min following delivery of the phosphoramidite. The coupling efficiency of the modified nucleobase ranged from 95% to 100%. The synthesized oligomers were treated with 28% NH4OH at 55 °C overnight to liberate the control pore glass (CPG) support and to remove the protecting groups. After the initial HPLC purification, the DMT group was then removed by treatment with 80% HOAc, and the oligomers were rechromatographed with a gradient of 5-25% acetonitrile over a period of 40 min (DMT-off conditions). In the cases of the oligomers containing the 2-AAF moiety (entries 1-3, Table 1), two peaks were observed in the product region of the chromatogram. The later-eluting materials representing only ∼10% or less of the total gave mass spectra that indicated them to be lighter than the main oligomers by 15 mass units. These were not further investigated. However in the case of each of the oligomers 4 and 5 (Table 1) derived from DMT-phosphoramidite (27b), two components in almost equal concentrations were collected from the DMT-off purification. This was surprising since the ammonium hydroxide treatment was expected to have removed the more labile trifluoroacetyl group to afford the oligomer containing only the dG-N2-AF (8) adduct. Mass analysis of the collected HPLC fractions revealed that while the later-

Chem. Res. Toxicol., Vol. 18, No. 3, 2005 461 eluting material was the desired oligomer containing the nucleoside 8, the second contained the acetylated nucleoside dGN2-AAF (9). This appears to be derived from the capping agent because, as noted below in the naphthyl series, omission of the capping step gave a product containing only dG-N2-AN (6) residue. (b) Oligomers Containing a dG-N2-AAN (7) or a dG-N2AN (6) Residue. The synthesis and purification of oligomers 6-9 (Table 1) were accomplished in essentially the same manner as described above for the fluorenyl analogues. However because of solubility problems with the DMT-phosphoramidite 14, methylene chloride alone was used as the solvent. The coupling time used was the standard 45 s used in the 0.25 µM synthesis cycle. Longer coupling times led to a decrease in the coupling efficiency. In this case the DMT removal was accomplished with 80% AcOH for only 10 min. In this manner, a single product was obtained having the expected m/z value. On the other hand the N2-(2′-trifluoroacetylnaphth-1-yl)dG-DMT-phosphoramidite (18) was soluble in CH3CN and a coupling time of 10 min was used to obtain optimal efficiency (97%). Again in each case, the products contained some of the acetylated oligomer dG-N2-AAN (7), but this amounted to only ∼15% of the total. To minimize acetylation of the amino group, oligomer 9 (entry 9a in Table 1) was resynthesized but with omission of the capping step after introduction of the modified nucleotide. Acetylation of the modified oligomer thus was avoided. This aberrant acetylation is discussed further in the succeeding section.

Results and Discussion (a) Synthetic Chemistry of the Modified Nucleosides. The incorporation of any modified nucleoside into oligomeric DNA by the standard protocol (35) almost always requires the synthesis of the corresponding 5′-ODMT-3′-O-(2-cyanoethoxy)diisopropylaminophosphine. In the cases under discussion, different reaction sequences were needed for the aminoacyl nucleosides 6 and 8 in contrast to the related acetylamino compounds 7 and 9. Initially it appeared that once 7 or 9 had been introduced into DNA, simple basic hydrolysis should remove the acetyl groups, thus leading to DNA containing respectively 6 or 8. However none of the usual base deprotecting agents would effect this transformation under conditions that would leave the oligomer intact. Thus an alternative strategy was devised in which the more easily hydrolyzed trifluoroacetyl group was used in place of acetyl. This approach proved to be reasonably successful in synthesizing DNA containing 6 or 8. Nevertheless, the overall synthetic procedures that led to the successful synthesis of the four required phosphoramidites (14, 18, 27a, and 27b) varied somewhat because some of the corresponding intermediate compounds in the naphthyl and fluorenyl series showed differences in their sensitivity to specific reagents or in their solubility in normal solvents. The synthetic methods that were successful for the DMT-phosphoramidites 14 and 18 in the 2-AN series are illustrated in Scheme 2. Reduction of the previously synthesized adduct 10 (1) by catalytic hydrogenation effected both the removal of the benzyl group and conversion of the nitro to the amine 11, which was immediately treated with acetic anhydride to give 12 in high yield. Treatment of the latter compound with HF/ pyridine removed the silyl protecting groups from the 2-deoxyribose ring to give diol 7, and subsequent reaction under standard conditions of the resulting 3′,5′-diol with DMT-chloride in pyridine afforded an excellent overall yield of 13. Conversion of 13 to the desired phosphoramidite 14 was then accomplished by reaction with N,N-

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Bonala et al.

Scheme 2. Preparative Route to the N2-(2-Acylaminonaphth-1-yl)DG Precursors 14 and 18 for DNA Synthesis

diisopropylamino(2-cyanoethoxy)chlorophosphine in the presence of triethylamine in 100% yield after chromatography. It was expected that exactly the same route could be used for the synthesis of the corresponding trifluoroacetyl derivative 18. Unfortunately, when the amino compound from the initial reduction of 10 was subjected to trifluoroacetic anhydride in pyridine, a rapid decomposition occurred and none of the desired trifluoroacetyl analogue of 11 was obtained. Thus an alternate strategy was adopted in which 10 was converted to the 3′,5′-diol (15) by means of HF/pyridine. The DMT group was then introduced at this point by the standard procedure to give 16. Catalytic hydrogenation of 16 followed by treatment with trifluoroacetic anhydride led to a 3′-O-N-bis(trifluoroacetyl) derivative that lost the trifluoroacetyl group on the 3′-oxygen by hydrolysis during the workup procedure with aqueous NaHCO3

solution. This gave 17 in excellent yield. Thereafter standard methods were used to convert 17 to the desired phosphoramidite 18. The introduction of dG-N2-AAF (9) or its desacetyl derivative dG-N2-AF (8) into DNA oligomers did not prove to be as straightforward as in the cases of the corresponding naphthalene derivatives. A principal difficulty was the reported (36) extreme insolubility of 9 in normal solvents, which precluded the direct formation of the needed 5′-O-DMT derivative. This problem was eventually overcome by introducing the DMT group at an earlier stage in the synthetic sequence as outlined in Scheme 3. Thus condensation under Buchwald-Hartwig conditions of 3′,5′-O-bis(tertbutyldimethylsilyl)-6-O-benzyl-2′-deoxyguanosine 21 with 2-nitro-3-bromofluorene (20), [prepared by a known (37) two-step procedure from 19] led in excellent yield (75%)

Synthesis of DNA Containing N2-dG Arylamines

Chem. Res. Toxicol., Vol. 18, No. 3, 2005 463

Scheme 3. Preparative Route to the N2-(2-Acylaminofluoren-3-yl)DG Precursors 27a,b for DNA Synthesis

to the dG-N2-nitrofluorenyl-coupled product 22. The latter when treated with HF/pyridine then afforded the nitrodiol 23, which in contrast to 9 was reasonably soluble in most organic solvents. Reaction of 23 with DMT-Cl in methylene chloride containing pyridine led to the desired 5′-O-DMT derivative 24. Catalytic hydrogenation of 24 over a Pd catalyst followed by acylation with acetic anhydride or trifluoracetic anhydride gave respectively the bisacylated products 25a or 25b. Mild basic hydrolysis (aqueous ammonia in the case of 25a and NaHCO3 solution in the case of 25b) then selectively removed the 3′-O-acyl group, giving 26a or 26b. The latter compounds on treatment with N,N-diisopropylamino(2-cyanoethoxy)chlorophosphine in the presence of triethylamine then respectively gave the required 5′-ODMT-3′-O-phosphoramidites 27a and 27b. From the 31P NMR spectra of all these phosphoramidites, 14, 18, 27a, and 27b, it was obvious that they were contaminated with approximately 10% H-phosphonate, (iPr)2N(H)P(O)OCH2CH2CN. This latter compound fortunately does not interfere with the coupling or the purification of the

oligomers. On the other hand, this impurity appears to improve the solubility of the DMT-phosphoramidite because if the latter is purified by column chromatography it becomes very difficult to solubilize it in the solvents used in the coupling procedures. (b) Synthesis of DNA Oligomers. All four of the DMT-phosphoramidites (14, 18, 27a, and 27b) were used successfully in the synthesis of a series of oligomers (Table 1) containing respectively the residues dG-N2-AAN (7), dG-N2-AN (6), dG-N2-AAF (9), or dG-N2-AF (8). The coupling yields at the point of entry of the modified nucleosides ranged from 95% to 100%. In the cases of 14 and 27a containing an acetylamino group, the product oligomers were unique substances that were easily purified by HPLC. Electrospray mass spectrometry confirmed in each case that the residue 7 or 9 was present. However, both of the corresponding trifluoroacetyl DMTphosphoramidites 18 and 27b gave mixtures of two oligomers. Those in the naphthalene series were identified by electrospray mass spectrometry individually as containing the desired residues 6 (85%) or the related

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acetylamino residue 7 (15%). In the fluorenyl series, again by electrospray mass spectrometry individually, it was confirmed that the oligomers containing desired residues 8 and the related acetylamino residue 9 were in a 1:1 ratio. At first we thought that this might arise through hydrolysis of the trifluoroacetyl group during the oxidation step, in which an aqueous mixture of pyridine and iodine is used. However, in a recent publication Greenberg and co-workers (38) noted a similar effect during the use of “fast-deprotecting” phosphoramidites in which N-acetylated oligonucleotides were observed during solid-phase automated synthesis. This they ascribed to a secondary acetylation of the protected amino group of deoxyguanosine residues in the oligonucleotide by the capping agent (Ac2O/N-methylimidazole). This mechanism had been proposed previously by Sinha et al. (39) in connection with their development of tert-butylphenoxyacetyl as an N-protecting group. Given that the use of a capping agent is moot in the case of relatively short DNA oligomers, we felt that this step in the protocol could safely be omitted. In a similar case (40) involving the incorporation of 3,N4-ethano-dC into oligomeric DNA, where the capping agent interfered with the process, we found that omission of this step after the insertion of the modified nucleotide had no detrimental effect on the yield or quality of the oligomers. When this approach was applied to the synthesis of the 15-mer (entry 9a, Table 1), the desired oligomer containing the dG-N2-AN residue became the sole product. However, if the capping agent was omitted completely, the overall yield of the desired oligomer was lower (50-70%, depending on the sequence) and the final product was accompanied by a second oligomer that did not contain the lesion. This can be explained by the fact that without the capping step during the standard synthesis, the sites left open after addition of the modified nucleobase would couple with the next base to produce oligomer lacking the expected lesion. A longer coupling time after addition of the xenonucleoside could minimize the number of potential sites left open to couple with the next base in the sequence and thus reduce the amount of product lacking the desired modification. Each of the purified oligomers listed in Table 1 showed a single band when subjected to gel electrophoresis on polyacrylamide. In summary, the site-specific incorporation of 6, 7, 8, and 9 into oligomeric DNA now should permit (a) wideranging biological studies on both the miscoding properties and the cellular repair of these lesions in bacterial and mammalian systems and (b) structural studies (NMR) of the conformational state at the lesion sites. Studies along these lines are being pursued by other investigators in our group and will be reported in due course.

Acknowledgment. We thank Mr. Robert R. Rieger who obtained the mass spectral data. We are also especially grateful to the National Institute of Environmental Health Sciences, National Institute of Health, which provided a grant (ES04068) in support of these investigations.

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