1412
Chem. Res. Toxicol. 1997, 10, 1412-1419
Stereochemistry of the in Vitro and in Vivo Methylation of DNA by (R)- and (S)-N-[2H1,3H]Methyl-N-nitrosourea and (R)- and (S)-N-Nitroso-N-[2H1,3H]methylN-methylamine Thomas E. Spratt,* Thomas M. Zydowsky,† and Heinz G. Floss‡ Department of Chemistry, The Ohio State University, 140 West 18th Avenue, Columbus, Ohio 43210 Received June 5, 1997X
Reaction of DNA with the carcinogens N-methyl-N-nitrosourea and N-nitroso-N,N-dimethylamine produces several methylated species including the premutagenic O6-methylguanine. The mechanism of methylation is believed to be through a methanediazonium ion. We have studied the mechanism of methylation of DNA by these carcinogens by analyzing the stereochemistry of the methyl transfer. DNA was methylated in vitro by (R)- and (S)-N-[2H1,3H]methyl-Nnitrosourea and in vivo by (R)- and (S)-N-[2H1,3H]methyl-N-methyl-N-nitrosamine and (R)and (S)-N-[2H1,3H]methyl-N-nitrosourea. 7-Methylguanine, 3-methyladenine, O6-methylguanine, and the methylated phosphate backbone were isolated. The methyl groups were converted into acetic acid, and the stereochemistry was analyzed. The identity of the nucleophile did not influence the stereochemistry of the methylation reaction. It was found that the methyl group was transferred with an average of 73% inversion and 27% retention of configuration. The most likely mechanism for the retention of configuration is through multiple methylation events in which nucleophiles which initially react with the methanediazonium ion react as electrophiles with DNA.
Introduction Methylating agents have the potential of being very potent mutagens and carcinogens (1). These reagents can alkylate a multitude of macromolecules in vivo. It is believed that the methylation of DNA, especially at the O6-position of guanine and the O4-position of thymine, plays an important role in cell transformation (2-5). Alkylating agents such as N-alkyl-N-nitrosoureas and N,N-dialkyl-N-nitrosamines are believed to react via an alkane diazohydroxide intermediate which dissociates to the alkanediazonium ion (6-8). The diazonium ion can deprotonate to form the diazoalkane (6-10). Primary alkanediazonium ions are believed to decompose via an SN2 mechanism, while secondary diazonium ions have the potential to decompose via an SN1 mechanism involving ion pairs (11-12). The site on DNA which is alkylated also can play a role in the mechanism of reaction (15-20). Generally, the less reactive agents, such as iodomethane, react predominantly with the stronger nucleophiles such as the 7-position of guanine (1, 15). Highly reactive reagents, such as diazonium ions, are less selective and alkylate the less nucleophilic sites, such as the O6-position of guanine, to a greater extent (1, 15). The site of alkylation can have a large influence on the mechanism. N,N-Din-propylnitrosamine, N-n-propyl-N-nitrosourea, and N-nbutyl-N-nitrosourea react to form predominantly straightchain adducts at the 7-position of guanine and branched adducts at the O6-position (17-20). These results indicate that the more nucleophilic sites on the DNA react * Address for correspondence: Division of Pathology and Toxicology, American Health Foundation, 1 Dana Rd., Valhalla, NY 10595. † Present address: Shipley Co., 455 Forest St., Marlboro, MA 01752. ‡ Present address: Department of Chemistry, University of Washington, P.O. Box 351700, Seattle, WA 98195-1700. X Abstract published in Advance ACS Abstracts, November 15, 1997.
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with primary diazonium ions via an SN2 mechanism while the less nucleophilic sites react via an SN1 mechanism with the secondary carbonium ions produced from the decomposition and subsequent rearrangement of the primary diazonium ion. We have examined the mechanism of alkylation of DNA by alkanediazonium ions by investigating the reactions of precursors of the methanediazonium ion, the simplest alkanediazonium ion. To this end the stereochemistry of the methyl transfers from (R)- and (S)-N[2H1,3H]methyl-N-methyl-N-nitrosamine [(R)- and (S)NDMA)]1 and (R)- and (S)-N-[2H1,3H]methyl-N-nitrosourea [(R)- and (S)-MNU)] to H2O and DNA were studied.
Experimental Section Caution: N-Methyl-N-nitrosourea (MNU) and N-nitrosoN,N-dimethylamine (NDMA) are carcinogens and should be handled carefully. Syntheses of (R)- and (S)-MNU and (R)- and (S)-NDMA. (R)- and (S)-[2-2H1,3H]acetic acid were prepared by the procedure described by Kobayashi et al. (22) in 100% ee. (R)- and (S)-[2H1,3H]Methylamine Hydrochloride. A 50mL two-neck flask, equipped with a magnetic stirring bar, a bent solid addition tube, and an argon inlet valve, was charged with anhydrous (R)-[2-2H1,3H]sodium acetate (0.25 mmol, 40 mCi). Through the addition tube were added 60 mg of NaN3 (0.93 mmol) and 5.5 mL of polyphosphoric acid, which was prepared by the addition of 3 mL of orthophosphoric acid to 3 g of P2O5 at -10 °C and subsequent warming to 90 °C for 15 h under an argon atmosphere. The mixture was stirred vigorously at 45 °C for 60 h. The solution was cooled on ice, diluted with 20 mL of ice-cold H2O, and made strongly basic by the addition of solid KOH while keeping the temperature below 10 °C. The 1 Abbreviations: (R)- and (S)-N-[2H ,3H]methyl-N-nitrosourea, (R)1 and (S)-MNU; (R)- and (S)-N-nitroso-N-[2H1,3H]methyl-N-methylamine, (R)- and (S)-NDMA; 3-methyladenine, 3mA; O6-methylguanine, O6mG; 7-methylguanine, 7mG; p-toluenesulfonyl chloride, TsCl.
© 1997 American Chemical Society
Stereochemistry of DNA Methylation methylamine solution was then distilled into a receiving flask containing 10 mL of 0.1 N HCl which was cooled in an ice bath. This was carried out with a stream of nitrogen going from the distillation flask to the receiving flask and then to two bubblers filled with 0.1 N HCl and cooled at 0 °C. The distillate and traps were combined and evaporated in a rotary evaporator to afford (R)-[2H1,3H]methylamine hydrochloride (22.4 mCi) in a 56% yield. In the same manner (S)-[2H1,3H]acetic acid (0.25 mmol, 40 mCi) afforded (S)-[2H1,3H]methylamine hydrochloride (14 mCi) in 35% yield. (R)- and (S)-MNU. KNCO (60 mg, 0.63 mmol) was added to (R)-[2H1,3H]methylamine hydrochloride (0.3 mmol, 16 mCi) dissolved in 0.3 mL of H2O. H2SO4 (50 µL, 0.1 N) was added; the flask was sealed, heated at 110 °C for 12 min, and cooled in an ice bath. The solution was acidified by the addition of 0.4 mL of 10 N H2SO4. NaNO2 (42 mg, 0.60 mmol) was added in four portions over 20 min and a foamy white product appeared. The mixture was extracted with ether (8 × 2 mL), and the combined ether extracts were dried over anhydrous MgSO4 in the dark. The solution was filtered, concentrated, applied to a 0.5- × 10-cm column of Florisil, and eluted with ether. The eluent was evaporated to afford a colorless product in 80% yield (14 mCi). In the same manner (S)-[2H1,3H]methylamine hydrochloride (0.3 mmol, 12 mCi) afforded (S)-N-[2H1,3H]methyl-Nnitrosourea (8.4 mCi) in 70% yield. (R)- and (S)-N-[2H1,3H]Methyl-p-toluenesulfonamide. pToluenesulfonyl chloride (23.3 mg, 122 µmol) was added to (R)[2H1,3H]methylamine hydrochloride (7.15 mg, 106 µmol, 110 µCi) in 0.7 mL of 2.5 N NaOH. The flask was sealed with a rubber septum, heated, and shaken on a steam bath until the oily p-toluenesulfonyl chloride disappeared. The flask was cooled on ice and then heated again for 5 min on the steam bath. The reaction mixture was cooled to 0 °C, and 4 N HCl was added until a white precipitate formed. The suspension was adjusted to pH 6 with 1 N HCl and evaporated to dryness. The residue was triturated four times with CHCl3. The CHCl3 was dried over Na2SO4 and evaporated to give (R)-N-[2H1,3H]methyl-ptoluenesulfonamide (16.0 mg, 85.9 µmol, 89 µCi) in 81% yield. The S-isomer was synthesized in a similar manner (77 µmol, 79 µCi) in 90% yield. (R)- and (S)-N-[2H1,3H]Methyl-N-methyl-p-toluenesulfonamide. Methyl iodide (30 µL, 480 µmol) was added to (R)-N[2H1,3H]methyl-p-toluenesulfonamide (16.01 mg, 85.9 µmol, 89 µCi) dissolved in 1 mL of 1% (w/v) KOH in 70% aqueous ethanol. After the mixture had been stirred at room temperature for 1.5 h, 2 mL of 3 N NaOH was added and the solution was extracted with benzene. The benzene layer was washed with 3 N NaOH, dried over Na2SO4, and evaporated to dryness to produce (R)N-[2H1,3H]methyl-N-methyl-p-toluenesulfonamide (16.2 mg, 87.8 µmol, 85 µCi) in 97% yield. The S-isomer was prepared in a similar manner (65.4 µmol, 67 µCi) in 85% yield. (R)- and (S)-N-[2H1,3H]Methyl-N-methylamine. Concentrated HCl (2 mL) was added to (R)-N-[2H1,3H]methyl-N-methylp-toluenesulfonamide (16.2 mg, 87.8 µmol, 85 µCi) in a 50-mL flask which was sealed with a rubber septum and kept at 100 °C for 4 h. The mixture was diluted with 5 mL of water and washed with benzene. The benzene layer was extracted with water, and the aqueous fractions were combined and evaporated to dryness. The residue was made alkaline and distilled as previously described. The contents of the receiving flask and gas trap were combined and evaporated to dryness to give (R)N-[2H1,3H]methyl-N-methylamine hydrochloride (4.89 mg, 60 µmol, 60 µCi) in 93% yield. The S-isomer was synthesized in a similar manner (59 µmol, 62.4 µCi) in 92% yield. (R)- and (S)-NDMA. Glacial acetic acid (0.37 mL, 6.5 mmol) was added to (R)-N-[2H1,3H]methyl-N-methylamine hydrochloride (4.89 mg, 60 µmol, 60 µCi) and 4 mg of N,N-dimethylamine hydrochloride (50 µmol) dissolved in 0.5 mL of H2O. NaNO2 (0.222 g, 3.2 mmol) was added and allowed to react at room temperature for 30 min. The solution was made basic with 2.6 mL of 5 N NaOH and evaporated in an evacuated lyophilization bridge with the receiver flask cooled in liquid nitrogen. The distillate contained (R)-N-[2H1,3H]methyl-N-methylnitrosamine (7.12 mg, 96 µmol, 43 µCi). The S-isomer was synthesized in a similar manner (92.5 µmol, 49 µCi). Chromatography on Dowex
Chem. Res. Toxicol., Vol. 10, No. 12, 1997 1413 1-X8 (hydroxy form) with elution by water and then 0.1 N HCl demonstrated that the radioactive NDMA was contaminated with less than 2% N,N-dimethylamine. Methylation of DNA and Water. In Vitro Alkylation of DNA with (R)- and (S)-MNU. Calf thymus DNA (Sigma, St. Louis, MO; type I, 20 mg), dissolved in 6.0 mL of 0.10 M TrisHCl, pH 7.4, was added to a solution of (R)-MNU (1.3 mCi, 33 µmol) in 200 µL of ethanol. The solution was stirred at 37 °C for 4 h and then extensively dialyzed against H2O, pH 7.0, at 4 °C to remove noncovalently bound radioactivity. The methylation yield was 2.2% based on radioactivity. The DNA solution was lyophilized to dryness and dissolved in 5 mL of H2O, and the pH of the solution was brought to 8.0 by the addition of 1 M sodium borate. DNase I (1000 units; Sigma type II) was added, and the solution was stirred at 25 °C for 1 h. The pH of the solution was then adjusted to 1 with 1 N HCl, and the solution was stirred at 25 °C for 16 h. The pH was brought to 4 with 1 N NaOH, and 7-methylguanine (7mG), 3-methyladenine (3mA), O6-methylguanine (O6mG), and the depurinated DNA were isolated by chromatography on a Sephadex G-10 column (1.5 × 90 cm) by elution with 50 mM ammonium formate, pH 6.8, at a flow rate of 0.5 mL/min. The products were identified by comparison with authentic reference samples. A similar experiment using (S)-MNU (850 µCi) afforded methylated DNA in a 1.2% radioactive yield, which was similarly hydrolyzed and the methylated bases were isolated by chromatography. Alkylation of DNA in Deuterated Buffer. MNU (33 mg, 0.24 mmol) was added to 10 mL of 100 mM Tris, pD 7.4, in D2O (99.9 atom % D) at 37 °C and 50 mg of calf thymus DNA, which had been dissolved in D2O and lyophilized three times. After incubation for 4 h at 37 °C, the DNA was extensively dialyzed against H2O at 4 °C. The DNA was hydrolyzed as above, and 7mG and O6mG were isolated by preparative reverse-phase HPLC on previously described equipment (22). A Whatman Partisil ODS-3 4.9-mm × 50-cm column was eluted at 4.0 mL/ min with a 50-min gradient from 100% H2O to 50% methanol/ H2O. The DCI mass spectra of the guanine derivatives, dissolved in 2 M ammonium formate, were obtained on a Hewlett-Packard model 5988 mass spectrometer as described previously (22). The MS of 7mG and O6mG standards and samples from the methylation experiments are as follows. 7mG standard: DCI MS m/z (relative intensity) M + 2 (8.8), M + 1 (100). 7mG sample: DCI MS m/z (relative intensity) 167 M + 2 (16.3), 166 M + 1 (100). O6mG standard: DCI MS m/z (relative intensity) M + 2 (8.8), M + 1 (100). O6mG sample: DCI MS m/z (relative intensity) 167 M + 2 (12.5), 166 M + 1 (100). Solvolysis of (R)- and (S)-MNU. (R)- or (S)-MNU (0.25 µmol, 10 µCi) was incubated in 10 mL of 100 mM Tris-HCl buffer, pH 7.4, at 37 °C for 4 h. Methanol was isolated by addition of benzene and steam distillation. The stereochemistry of the methanol was determined as described below. Exchange of the methyl protons was examined by reaction of N-methyl-N-nitrosourea in H2O and D2O. Reactions were accomplished as above by dissolving 25 mg of N-methyl-Nnitrosourea (0.24 mmol) in 10 mL of both 100 mM Tris-HCl, pH 7.4, and 100 mM Tris-DCl, pD 7.4, at 37 °C. The deuterium incorporation into methanol was determined by first converting the methanol to the 3,5-dinitrobenzoate ester as described below: MS EI m/z (relative intensity) 226 (M+, 100), 227 (13.6). The mass spectrum of unlabeled methyl 3,5-dinitrobenzoate was also obtained: MS EI m/z (relative intensity) 226 (M+, 100), 227 (10). In Vivo Alkylation of DNA by (R)- and (S)-MNU and (R)and (S)-NDMA. In separate experiments, a total of 5.0 mCi of (R)- or (S)-MNU (125 µmol), dissolved in 0.5 mL of saline solution, was injected into the tail vein of two 250-g female Sprague-Dawley rats. The rats were sacrificed after 4 h. Prior to the injection of NDMA (72 and 24 h), the rats were induced by the administration of phenobarbital (80 mg/kg). A total of 22 mCi of (R)-NDMA (138 µmol) was injected into eight rats, and a total of 6.2 mCi of (S)-NDMA (129 µmol) was injected into four rats. After 4 h the rats were sacrificed. The livers were excised and frozen with liquid nitrogen.
1414 Chem. Res. Toxicol., Vol. 10, No. 12, 1997 DNA was isolated by tissue homogenization, phenol extraction, and hydroxyapatite chromatography (23). The DNA was depurinated by an initial DNase I digestion followed by incubation in 0.1 N HCl overnight at 25 °C. The methylated bases and depurinated DNA were then separated by chromatography with Sephadex G-10 (1.5 × 100 cm) with 25 mM sodium formate at 1 mL/min as the mobile phase. The radioactive peaks were identified by cochromatography with standards. Conversion of Methyl Groups to Acetic Acid. 3(R)[2H1,3H]Methyladenine and 7(R)-[2H1,3H]Methylguanine to (R)-[2H1,3H]Methylamine. The methylated base (0.57 µCi, 14 nmol) was dissolved in 20 mL of 5 N H2SO4, and the temperature was raised to 80 °C. After 1.5 mL of 250 mM NaNO2 (0.38 mmol) had been added dropwise over 5 min, the reaction was brought to reflux and stirred at reflux for 30 min. Subsequently, 50 mM KMnO4 was added dropwise to keep a slight excess of KMnO4 present for 3 h. Excess KMnO4 was then destroyed by the dropwise addition of 100 mM oxalic acid. The reaction mixture was cooled to 4 °C, and the pH of the solution was brought to 5 with 5 N NaOH. The solution was refluxed for 36-42 h, cooled in an ice bath, made alkaline by the addition of 2 mL of 5 N NaOH, and distilled into 0.1 N HCl as described above The distillate and the acidic solutions in the bubblers were combined and evaporated on a rotary evaporator to yield [2H1,3H]methylamine hydrochloride in 60% yield. (R)-O6-[2H1,3H]Methylguanine to (S)-[2H1,3H]Methanol. O6-[2H1,3H]Methylguanine (3 nmol) was diluted with 100 mg of unlabeled O6mG (0.61 mmol) and dissolved in 12 mL of 3 N KOH. Oxone (6.0 g, 4.8 mmol) was added in portions over 1 h to the stirred solution. The solution was heated at 40 °C for 30 h and then acidified with 6 N HCl. The acidic solution was stirred at room temperature for 12 h and then made strongly basic (pH 11.5) with 6 N KOH. After the mixture stirred for 3.5 h, benzene (40 mL) was added, and the two-phase system was refluxed for 4 h. The methanol was distilled into an icecooled flask, and the fraction distilling between 55 and 80 °C was collected. (R)-[2H1,3H]Methyl Phosphotriester Fraction to (S)[2H1,3H]Acetonitrile. The depurinated DNA (1 µCi) solution was evaporated on a rotary evaporator and dissolved in 3 mL of HMPA containing 65 mg of KCN (1 mmol) in a 10-mL recovery flask. The flask was connected via a vacuum bridge to a second 50-mL recovery flask containing 1 mL of water. The reaction flask was heated to 90 °C and the water flask cooled in liquid nitrogen. After 1 week [2H1,3H]acetonitrile was obtained in 5% radiochemical yield. (R)-[2H1,3H]Methyl Phosphotriester Fraction to (R)[2H1,3H]Methanol. Alternatively, the depurinated DNA solution (1 µCi) was lyophilized to dryness and dissolved in 0.3 mL of 72% HClO4. The solution was heated at 110 °C for 1 h, cooled to room temperature, and diluted to 2 mL with H2O. The hydrolysate was applied to a Dowex 50W-X8 column (18 × 2.5 cm) equilibrated with H2O. The column was eluted with 1 N HCl, and 6-mL fractions were collected. The early fractions which contained the methyl phosphate were combined and evaporated to dryness. After the residue was dissolved in 100 mM Tris-HCl, pH 8.0, alkaline phosphatase (2 units) was added and the mixture incubated at 37 °C for 30 min. The solution was lyophilized on an evacuated bridge, and the volatile material, which was composed of [2H1,3H]methanol and water, was treated as described below to convert the methanol to acetic acid with inversion of configuration of the methyl group in 0.2% overall radiochemical yield. (R)-N-[2H1,3H]Methylamine to (R)-N-[2H1,3H]Methyl-ptoluenesulfonamide. (R)-[2H1,3H]Methylamine hydrochloride (8.5 nmol, 0.34 µCi) was dissolved in 0.5 mL of 1 M methylamine hydrochloride solution (0.5 mmol) and cooled in an ice bath. After the addition of 190 mg of p-toluenesulfonyl chloride (1.0 mmol), the flask was sealed with a rubber septum, NaOH (0.3 mL, 5 N, 1.5 mmol) was added, and the reaction flask was heated at 80 °C for 15 min, after which it was cooled in an ice bath. After the base addition and heating were repeated, the reaction was quenched by cooling on ice and adding 10 mL of 1 N HCl. The product was extracted into CH2Cl2 and dried over
Spratt et al. MgSO4 and the solvent evaporated to give (R)-N-[2H1,3H]methylp-toluenesulfonamide (0.20 µCi) in 60% yield. (R)-N-[2H1,3H]Methyl-p-toluenesulfonamide to (R)-N[2H1,3H]Methyl-N,N-bis(p-tolylsulfonyl)amine. (R)-N-[2H1,3H]Methyl-p-toluenesulfonamide (0.29 mmol, 0.20 µCi), dissolved in 2 mL of anhydrous DMF, was added to 42 mg of NaH (50% suspension in mineral oil, 0.88 mmol) in a round-bottom flask under a nitrogen atmosphere. The solution was stirred at 60 °C for 1 h and then cooled in an ice bath. Three equivalents of p-toluenesulfonyl chloride (166 mg, 0.87 mmol) in 0.5 mL of DMF was added, and the reaction mixture was stirred for 10 h at room temperature. The reaction was quenched by the addition of 5 mL of 1 N HCl and the mixture extracted with CH2Cl2, which was dried over MgSO4 and evaporated. The product was purified by preparative TLC on silica gel with development by CHCl3 (Rf ) 0.66) to give pure product in 20% yield. (R)-N-[2H1,3H]Methyl-N,N-bis(p-tolylsulfonyl)amine to (S)-[2H1,3H]Acetonitrile. (R)-[2H1,3H]Methyl-N,N-bis(p-tolylsulfonyl)amine (58 µmol, 40 nCi), dissolved in 3 mL of anhydrous HMPA, was added to a 10-mL flask, equipped with a magnetic stir bar, containing 65 mg of KCN (1 mmol). The flask was connected via a vacuum bridge to a second 50-mL round-bottom flask containing 1 mL of H2O and a stir bar. The bridge was evacuated, the reaction flask heated with stirring to 90 °C, and the second flask cooled with liquid nitrogen. The heating was continued for 1 week and yielded (S)-[2H1,3H]acetonitrile in 15% yield. (R)-[2H1,3H]Methanol to (R)-[2H1,3H]Methyl 3,5-Dinitrobenzoate. Methanol was isolated from a 5-mL aqueous solution by the addition of 30 mL of benzene and 10 µL of methanol (250 µmol) followed by steam distillation. The distillate was collected over a temperature range of 58-70 °C into a flask cooled in an ice bath. The distillate contained 25 mL of benzene, 1.5 mL of H2O, and less than 250 µmol of methanol. 3,5Dinitrobenzoyl chloride (461 mg, 2 mmol) and N,N-dimethylaniline (253 µL, 2 mmol) were added, and the mixture was vigorously stirred for 17 h at room temperature. The benzene layer was separated and the aqueous layer extracted with benzene. The combined benzene fractions were dried with Na2SO4 and evaporated to dryness. The product was purified by preparative TLC on silica gel with development by CHCl3 (Rf ) 0.45). The product was eluted from the silica gel with acetone. The acetone was evaporated, and the residue was dissolved in benzene and washed with 0.1 N HCl to remove N,Ndimethylaniline which had coeluted with the product. The benzene solution was dried over Na2SO4, filtered, and evaporated to give the methyl ester in 60% yield. (R)-[2H1,3H]Methyl 3,5-Dinitrobenzoate to (S)-[2H1,3H]Acetonitrile. KCN (65 mg, 1 mmol) was added to (R)-[2H1,3H]methyl 3,5-dinitrobenzoate (0.25 µmol, 10 nCi) dissolved in 3 mL of anhydrous HMPA and reacted on the vacuum bridge as described above to yield (S)-[2H1,3H]acetonitrile in 48% yield. (S)-[2H1,3H]Acetonitrile to (S)-2-[2H1,3H]Acetic Acid. H2O2 (30%, 2 mL) and 75 µL of 6 N NaOH were added to (S)-[2H1,3H]acetonitrile (8.7 µmol, 6.0 nCi) dissolved in 1 mL of H2O, and the mixture was heated at 50 °C for 7 h. The solvent was evaporated to dryness to yield acetamide which was dissolved in 1 mL of H2O, cooled to 0 °C, and acidified by the dropwise addition of 1 mL of 5 N H2SO4. NaNO2 (0.5 mL, 5.8 M) was added, and the reaction mixture was stirred on ice for 1 h and then at room temperature for 5 h. The labeled acetic acid was isolated by distillation. The distillate was brought to pH 9 by the addition of 0.1 N NaOH and evaporated to dryness to afford sodium acetate in 67% yield. The chirality of the acetic acid was analyzed as described previously (24).
Results Stereochemical Analysis. The chiral acetic acid was synthesized by published procedures (21). Stereochemical analysis of acetic acid involves the enzymatic conversion of acetate into malate and then fumarate. The formation of malate from (R)- or (S)-acetic acid proceeds
Stereochemistry of DNA Methylation
Chem. Res. Toxicol., Vol. 10, No. 12, 1997 1415
Scheme 1a
Scheme 2a
a (a) Oxone; (b) HClO ; (c) alkaline phosphatase; (d) 3,54 dinitrobenzoyl chloride; (e) KCN; (f) H2O2; (g) HONO.
a (a) NaNO /heat; (b) KMnO ; (c) NaN ; (d) KNCO; (e) NaNO / 2 4 3 2 HOAc; (f) TsCl/NaOH; (g) MeI; (h) HCl; (i) TsCl, NaH; (j) KCN; (k) H2O2; (l) HONO.
with a primary kinetic isotope effect (kH/kD) of 3.8 which produces an uneven distribution of tritium between the two methylene hydrogens of L-malate. The enzymatic equilibration with fumarate is completely stereospecific: the pro-3R-hydrogen is exchanged with the solvent. Beginning with (R)-acetic acid, 21% of the tritium is removed from the substrate, while with (S)-acetic acid 79% of the radioactivity is lost. The amount of tritium retained by the substrate in the fumarase reaction is referred to as the F value of the sample. The enantiomeric excess is determined by eq 1 in which positive values refer to an excess of the R-enantiomer and negative values to the S-enantiomer. Equation 2 shows the definition of enantiomeric excess. In our hands the chirality analyses are reproducible to (2 F values which correspond to (7% ee (24). Chirality analyses for the acetic acid used to synthesize (R)- and (S)-NDMA and (R)- and (S)-MNU gave F values of 79 and 21 which correspond to 100% ee R and S.
ee ) {(F - 50)/29} × 100 (%)
(1)
ee ) {|[methyl-R] - [methyl-S]|/ ([methyl-R] + [methyl-S])} × 100 (%) (2) Synthesis of Methylating Agent. The syntheses of chiral MNU and NDMA are outlined in Scheme 1. A modified Schmidt reaction was employed to convert acetic acid into methylamine with overall retention of configuration accompanied by a small and somewhat variable degree of racemization. The chiral purity of the [2H1,3H]methylamine used to make (R)- and (S)-MNU was determined by converting the methyl group to acetic acid with inversion of configuration as illustrated in Scheme 1. Ditosylation of the amino group converted it into a leaving group which was displaced by cyanide with inversion of configuration. The resulting acetonitrile was hydrolyzed to acetic acid by a mild two-step procedure to minimize racemization. Some racemization can, however, occur during the distillation of the acetic acid (25). Analysis of the stereochemistry of the acetic acid enan-
tiomers indicated that the methylamine samples were at least 93% ee R and 86% ee S, respectively. Confirmation of the identity of chirally labeled MNU and NDMA was based on characterization of material from identical unlabeled syntheses and on the chemical properties of the products. The synthetic reactions were carried out on the same scale with unlabeled starting materials. The products of these reactions were fully characterized. Additionally, (R)- and (S)-MNU and (R)- and (S)-NDMA behaved as expected. Conversion of Methylated Products to Acetic Acid. To determine the stereochemistry of methyl transfer, the configuration and chiral purity of the methyl group in the reactant and product must be determined. However, the stereochemistry of these compounds cannot be measured directly. They must be converted into acetic acid via a pathway in which the stereochemical fate of the methyl group is known. Scheme 1 shows the procedures used to convert the nitrogen-bound methyl groups to acetic acid, while Scheme 2 shows procedures used to convert the oxygen bound methyl groups to acetic acid. The first step in the conversion of 7mG and 3mA to acetic acid is the oxidation/hydrolysis of the bases with NaNO2 and KMnO4 to form methylamine. The amino group is then converted into a leaving group by ditosylation. Reaction with KCN results in the formation of acetonitrile with inversion of configuration. This reaction is carried out in a vacuum bridge in which the HMPA solution is heated at 90 °C and the receiving flask is cooled in liquid nitrogen. This setup allows the newly synthesized acetonitrile to be distilled away from the HMPA-cyanide solution, thereby preventing additional nucleophilic attacks on the methyl group which would cause racemization. Acetonitrile is then converted into acetic acid by a twostep procedure involving oxidation to acetamide with H2O2 followed by diazotation with HONO. This procedure was followed to minimize racemization. However, the presence of a nitrile or a carboxamide, strong electron-withdrawing groups, adjacent to the chiral methyl group can make the methyl group acidic enough to undergo some racemization in basic solution, e.g., during the oxidation of acetonitrile in 0.2 N NaOH/20% H2O2 to acetamide. A small and variable amount of racemization has been found to occur during the distillation of the acetic acid (25). This partial racemization can be a potential problem. However, since the same steps are carried out in the analysis of the methylating
1416 Chem. Res. Toxicol., Vol. 10, No. 12, 1997
Spratt et al.
Table 1. Stereochemical Analysis of in Vitro and in Vivo Methylation of Rat Liver DNA by (R)- and (S)-MNU and (R)- and (S)-NDMA in vitro MNU
Table 2. Stereochemistry of Methyl Groups Involved in the in Vitro Methylation of DNA by (R)- and (S)-MNUa (R)-MNU
in vivo MNU
acetate NDMA
compound
(R)
(S)
(R)
(S)
(R)
(S)
7-methylguanine 3-methyladenine O6-methylguanine methyl phosphate methanol
76/24 78/22 74/26 74/24 78/22
78/22 76/24 76/24 66/34 69/31
76/24
70/30
71/29 69/31
62/38
74/26
68/32
71/29
a Expressed as percent inversion/percent retention of configuration. The chirality analysis is reproducible to within 7% ee.
agents and of the products, any racemization during these steps should cancel out and will not affect the important determinant, the change in configuration and chiral purity of the methyl group in going from substrate to product. The initial step in the conversion of O6mG to acetic acid involves the formation of methanol from O6mG. In this step the oxygen can come from the purine or the solvent. Isotopically labeled solvent was previously used to demonstrate that the carbon-oxygen bond is broken 7% of the time and the desired oxygen-purine bond 93% of the time (26). The oxygen is then converted into a leaving group by reaction with 3,5-dinitrobenzoyl chloride. The acid is displaced by KCN in a vacuum bridge and the resulting acetonitrile is converted into acetic acid as described above. This procedure converts O6mG into acetic acid with 93% inversion of configuration and 7% retention of configuration. This is reflected in the stereochemical purity of the methyl group of O6mG in Table 1. The in vitro methyl phosphotriester fraction was converted to acetonitrile by two different pathways (1 and 2). The methyl phosphotriester fraction obtained from (S)-MNU was directly converted into acetonitrile by heating with KCN as illustrated by pathway 1 in Scheme 2. Since this reaction was incubated at 90 °C for 1 week, the possibility existed that the methyl group reacted with other nucleophiles in the DNA before reacting with cyanide. This process would result in racemization. Therefore pathway 2 (Scheme 2) was used to convert the methyl phosphates from (R)-MNU into acetonitrile. In this procedure, DNA was hydrolyzed and methyl phosphate isolated. Methanol, obtained enzymatically, was converted into acetonitrile as described above. As shown in Table 2, the acetic acid produced from the direct cyanolysis was stereochemically less pure than that from pathway 2. The methyl phosphotriester fractions of the DNA from the in vivo methylation were subsequently analyzed by pathway 2. Stereochemistry of Methyl Transfer. Calf thymus DNA was incubated with (R)- and (S)-MNU in 100 mM Tris-HCl buffer, pH 7.4, at 37 °C. The DNA was hydrolyzed by an initial brief reaction with DNase I followed by depurination in 0.1 N HCl at 25 °C for 16 h. These conditions have been shown to minimize demethylation of O6-methylguanine. The methylated products were isolated by Sephadex G-10 chromatography in the following radiochemical yields: 7-methylguanine, 1.3%; 3-methyladenine, 0.12%; O6-methylguanine, 0.12%; and apurinic acid, 0.3%. The methyl groups were converted into acetic acid as described above, and the stereochemistry was analyzed. The results are presented in Table 2. MNU was solvolyzed in the same buffer, the methanol isolated, and the stereochemistry of the methyl group
compound MNUe 7-methylguaninef 3-methyladeninef O6-methylguanineg methyl phosphate methanolf
compound
(S)-MNU acetate
compound
Fb
configc
configd
Fb
configc
configd
25 65 66 63 64 66
93 S 52 R 55 R 45 R 48 R 55 R
93 R 52 S 55 S 48 S 48 Sf 55 S
77 34 35 36 41 39
86 R 56 S 51 S 48 S 32 S 38 S
86 S 56 R 51 R 52 R 32 Rh 38 R
a Reaction in 100 mM Tris-HCl, pH 7.4, at 37 °C. b F value of the acetic acid derived from the compound. We have observed that the F value is reproducible to within (2. c Configuration of the acetic acid sample (% ee). The configuration would have an error range of (7% ee. d Configuration of the methyl group on the compound (% ee). The configuration would have an error range of (7% ee. e Synthesized from methylamine with retention of configuration about the methyl group. Methylamine was converted to acetic acid with inversion of configuration. f Converted into acetic acid with inversion of configuration about the methyl group. g Converted into acetic acid with 93% inversion and 7% retention of configuration. h Direct displacement of phosphodiesters into acetonitrile produced some racemization.
Table 3. Stereochemistry of the Methyl Groups Involved in the in Vivo Methylation of DNA by (R)- and (S)-MNU (R)-MNU compound
(S)-MNU
acetate
compound
acetate
compound
Fa configb
configc
Fa configb
configc
MNUd
23 7-methylguaninee 65 methyl phosphatee 64
93 S 52 R 48 R
93 R 52 S 48 S
75 39 40
86 R 38 S 34 S
86 S 38 R 34 R
a F value of the acetic acid derived from the compound. The F value has an experimental error of (2. b Configuration of the acetic acid sample (% ee). The configuration would have an error range of (7 ee. c Configuration of the methyl group on the compound (% ee). The configuration would have an error range of (7% ee. d Synthesized from methylamine with retention of configuration. e Converted into acetic acid with inversion of configuration about the methyl group.
Table 4. Stereochemistry of the Methyl Groups Involved in the in Vivo Methylation of DNA by (R)- and (S)-DNA (R)-DNA compound NDMAd
(S)-NDMA
acetate
compound
acetate
compound
Fa configb
configc
Fa configb
configc
23 7-methylguaninee 62 3-methyladeninee 61 methyl phosphatee 62
93 S 41 R 38 R 41 R
93 R 41 S 38 S 41 S
75 43
86 R 24 S
86 S 24 R
a F value of the acetic acid derived from the compound. The F value has an experimental error of (2. b Configuration of the acetic acid sample (% ee). The configuration would have an error range of (7% ee. c Configuration of the methyl group on the compound (% ee). The configuration would have an error range of (7% ee. d Synthesized from methylamine with retention of configuration. e Converted into acetic acid with inversion of configuration about the methyl group.
analyzed. The stereochemistry of the methyl groups is reported in Table 2. DNA samples, isolated from the livers of rats injected with chiral NDMA and MNU, were hydrolyzed to 3mA, 7mG, and the apurinic fraction. These methylated species were converted into acetic acid, and the stereochemistry was determined. The results are presented in Tables 3 and 4. Table 1 presents a summary of the stereochemical course of the methylations. The reactions all proceed with 66-78% inversion of configuration and 22-34% retention of configuration. The reproducibility of the chirality analysis is (2 F value units which
Stereochemistry of DNA Methylation
Chem. Res. Toxicol., Vol. 10, No. 12, 1997 1417 Scheme 3
correspond to (7% ee. Therefore, within the resolution of the experiment, methyl transfer to all of the biological nucleophiles proceeded with the same stereochemistry. Formation of Diazomethane. To test whether the loss of chirality was due to reversible formation of diazomethane from the methanediazonium ion (6, 9, 10), the alkylations were carried out in deuterated buffer (99.9 atom % D). Methanol, 7mG, and O6mG were isolated. The methanol produced was converted into methyl 3,5-dinitrobenzoate, and analysis by mass spectrometry found that methanol thus produced contained 3.7% more deuterium than natural abundance. This amount of deuterium incorporation can account for only 2% of the retention of configuration. 7mG and O6mG were analyzed by mass spectrometry to determine deuterium incorporation. The deuterium incorporation, determined by comparing these samples with undeuterated controls, was 7% for 7mG and 5% for O6mG. Thus, only about 3% of the retention of configuration can come from this pathway.
Discussion Methylation of DNA and water by (R)- and (S)-MNU and (R)- and (S)-NDMA proceeded with an average of 73% inversion of configuration and 27% retention of configuration. The site of methylation did not affect the stereochemistry of methylation within the resolution of the experiment. Five possible explanations for the retention of configuration include (1) racemization during conversion of methylated species to acetic acid, (2) alkylation via an SN1 mechanism with formation of a methylcarbenium ion (Scheme 3, pathway 1), (3) formation of diazomethane during methylation of the nucleo-
philes (Scheme 3, pathway 2), (4) front-side SN2 reaction with retention of configuration (Scheme 3 pathway 3), and (4) alkylation via multiple SN2 reactions (Scheme 3, pathway 4). During the conversion of the methylated nucleoside or methanol to acetic acid, racemization of the methyl group could occur when an adjacent electron-withdrawing group makes the methyl group acidic or when the heteroatommethyl bond is broken and remade. The presence of an electron-withdrawing group gives rise to the potential for racemization when acetonitrile, acetamide, and acetic acid are manipulated. A small amount of racemization has been observed during the distillation of acetic acid (25). Racemization could also occur during the displacement of the ditosylamine or dinitrobenzoate from the methyl group by cyanide. However, these reactions are performed in the transformation of both the methylating agents and products to acetic acid. As shown in Scheme 1, the chirality of all methylamine derivatives, from MNU and NDMA to 7mG and 3mA, is determined by the same route. There is an additional step for the conversion of the methylated base to methylamine. However, the methyl-nitrogen bond was not broken, and the protons on the methyl group are not acidic, so the hydrolysis conditions should not have caused any racemization. Therefore, any racemization introduced should be reflected in both the reactant and the product and should cancel out in determining the stereochemistry of the reaction. The conversion of the oxygen-bound methyl groups to acetonitrile is different from that for the nitrogen-bound methyl groups. Therefore any losses in the stereochemical purity in this case do not necessarily cancel out. A small amount of racemization occurs when O6mG is hydrolyzed to methanol (26). This loss of stereochemistry
1418 Chem. Res. Toxicol., Vol. 10, No. 12, 1997
is taken into account in determining the stereochemistry of the methyl group on O6mG. Stereochemistry can be lost in the reaction with cyanide if the reaction does not proceed in a single SN2 reaction. The direct cyanolysis of the apurinic acid fraction of the in vitro methylated DNA (Scheme 2, pathway 1) produced acetic acid which was more racemic than that obtained via conversion of the methyl group to methanol and then to acetic acid (Scheme 2, pathway 2). During the direct cyanolysis of the apurinic fraction in HMPA, the methyl group may undergo several SN2 reactions with different phosphates along the DNA backbone acting as nucleophile and leaving group before it is intercepted by cyanide to form the volatile acetonitrile. This process may be the cause of the extra racemization observed in this reaction. Conversion of methanol to acetonitrile occurs via an SN2 attack of cyanide on the methyl group with the 3,5dinitrobenzoate as the leaving group. Racemization may occur by attack of the now free 3,5-dinitrobenzoate on unreacted methyl 3,5-dinitrobenzoate or by reaction of cyanide with acetonitrile. A reaction of cyanide with acetonitrile can be ruled out because this reaction does not occur in the similar displacement of N,N-bis(ptolylsulfonyl)-N-methylamine by cyanide that was performed in the conversion of methylamine to acetic acid. The 3,5-dinitrobenzoate is a much weaker nucleophile than cyanide and therefore will not compete in the reaction with the cyanide which is present in a large excess. The conversion of acetonitrile to acetic acid proceeds via the same steps for analysis of the oxygen-bound methyl groups and the nitrogen-bound methyl groups, and therefore any racemization will cancel out. A small amount of racemization does occur during conversion of the methylated DNA to acetic acid. For the nitrogen nucleophiles, this racemization would cancel out because the same steps are used to analyze the reactants and products. For the oxygen nucleophiles we have identified two steps in which a small amount of racemization occurs. The other reactions probably do not cause racemization. Therefore most of the racemization observed in the methylation of DNA with MNU or NDMA is not an artifact of the degradation and analyses. Racemization can be due to the formation of a methylcarbenium ion as is illustrated in pathway 1 of Scheme 3. However, the formation of a methylcarbenium ion from the methanediazonium ion would be very endothermic (44 kcal/mol) (27-30). This large activation energy would prohibit the formation of the methylcarbenium ion in physiological conditions. In addition, nitrous acid deamination of 1-butylamine and 2-butylamine proceeds with complete inversion of configuration (31). An n-butyl group would be more likely than a methyl group to form a carbenium ion. Since the n-butyldiazonium ion does not decompose via a carbenium ion, it is very unlikely that the methyldiazoniun ion does. Racemization of the methanediazonium ion can occur by deprotonation to diazomethane as is illustrated in pathway 2 of Scheme 3 (6, 9, 10). Deprotonation of the methyldiazonium ion would lead to a planar diazomethane, which can be protonated on either face, leading to racemization of the methyldiazonium ion. This possibility was tested by performing the in vitro reactions in deuterated buffer. MS analysis of the products showed 4-7% incorporation of deuterium. Thus, formation of
Spratt et al.
diazomethane can only account for approximately 3% of the retention of configuration observed. We observed less deuterium incorporation than either Smith et al. (10) or Hovinen and Fishbein (6). The difference in deuterium incorporation may be due to differences in the buffers. Smith et al. (10) reported that when MNU was solvolyzed in 1 M sodium phosphate, pD 7, approximately 50% of the methanol contained deuterium. Hovinen and Fishbein (4) reported a lower (11%) deuterium incorporation in 50 mM sodium phosphate, 1 M NaClO4, pD 6.4. General acid-catalyzed deprotonation of the methyldiazonium ion may explain the results. Retention of configuration may occur due to an SN2 reaction involving front-side attack as illustrated in pathway 3 of Scheme 3. Theoretical studies between Xand CH3X (in which X is a halide) have shown that the transition-state energy for an SN2 reaction with retention of configuration is 30 kcal/mol higher than that for an inversion mechanism (32, 33). The large differences in the activation energy would indicate that an SN2 reaction involving retention of configuration is not likely. However, the difference in energy may not be as large with a diazonium leaving group. With halides as the nucleophile and leaving group, both nucleophile and leaving group are negatively charged. Much of the energy difference between the retention and inversion transition states is due to repulsion between the negatively charged nucleophile and leaving group (32). The diazonium group is positively charged, and therefore there should not be any repulsion between the leaving group and nucleophile. However, Brosch and Kirmse (31) did not find any retention of configuration in the nitrous acid deamination of n-butylamine. If an SN2 reaction with retention of configuration can occur with diazonium ions, then we would have expected to find some racemization in the deamination of n-butylamine. Racemization may occur because of multiple SN2 displacement reactions. The in vitro reactions with MNU were carried out in 100 mM Tris-HCl, pH 7.4, at 25 °C for 4 h. Nucleophiles include the DNA, water, Tris, and chloride. At pH 7.4 the chloride concentration is calculated to be 98 mM. When methyl chloride is formed it could undergo additional reactions with DNA, water, and Tris. This double-displacement sequence would result in retention of configuration about the methyl group. In the in vivo reactions there are a multitude of nucleophiles with which the methyldiazonium ion could react to form electrophiles which could react with DNA. The multiple SN2 reactions may also occur intramolecularly in the DNA. Many of the nucleophiles on the DNA would also be good leaving groups. Methylation of DNA by methyl chloride, however, has not been demonstrated in vitro (34) or in vivo (35). Investigations into the in vitro methylation of DNA are complicated by the fact that methyl chloride is a gas and not soluble in water. Studies using the induction of O6alkylguanine-DNA alkyltransferase in Escherichia coli have indirectly implicated methylation of the phosphate backbone of the cellular DNA by methyl chloride (34). If the racemization of the methyl group occurred by multiple SN2 reactions, it is fortuitous that, within experimental error, each of the nucleophiles has the same degree of racemization. One might expect that the better nucleophiles would have more retention of configuration. The initial methylation by the methyldiazonium ion would proceed with inversion of configuration to all nucleophiles. The newly formed potential methylating species, such as methyl chloride, methyl phosphate, and
Stereochemistry of DNA Methylation
methylguanine, are poorer methylating agents than the methyldiazonium ion. The better nucleophiles would react faster in the second SN2 attack. Thus, we would expect the methyl group on 7mG to be more racemic than O6mG. In summary, DNA and water were methylated by (R)and (S)-MNU and (R)- and (S)-NDMA with an average of 73% inversion of configuration and 27% retention of configuration. The stereochemistry of methyl transfer is not dependent on the site of methylation. The most likely cause of the retention of configuration is a side reaction involving a sequential double-SN2-displacement mechanism in which an initially modified nucleophile acts as an electrophile and subsequently reacts with the DNA.
Acknowledgment. We thank Mrs. Kyungook Lee and Mrs. Hyekyung Park for numerous careful analyses of chiral acetate samples. We would like to thank Mark Kagan of the instrument facility of the American Health Foundation for the mass spectral analyses. Financial support was provided by the National Institutes of Health through research grants CA 37661 and GM 32333 (H.G.F.). Postdoctoral fellowships CA 07118 (T.M.Z.), ES 05306 (T.M.Z.), and CA 09498 (T.E.S.) are also gratefully acknowledged.
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