Chem. Res. Toxicol. 1996, 9, 593-598
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Synthesis of N2-(4-Hydroxyphenyl)-2′-deoxyguanosine 3′-Phosphate: Comparison by 32P-Postlabeling with the DNA Adduct Formed in HL-60 Cells Treated with Hydroquinone Krisztina Pongracz and William J. Bodell* Brain Tumor Research Center, Department of Neurological Surgery, School of Medicine, University of California, San Francisco, California 94143 Received June 9, 1995X
A new adduct has been isolated from the reaction of guanosine 3′-phosphate and pbenzoquinone. The structure of this adduct has been determined as N2-(4-hydroxyphenyl)guanosine 3′-phosphate. 32P-Postlabeling showed that this adduct is similar to the DNA adduct formed in HL-60 cells treated with hydroquinone. For comparison with the corresponding deoxyribonucleotide, a synthetic procedure was developed for the preparation of N2-substituted derivatives of 2′-deoxyguanosine 3′-phosphate. 2-Bromo-2′-deoxyinosine 3′-phosphate was synthesized with a combination of synthetic and enzymatic methods. Reaction of 2-bromo-2′deoxyinosine 3′-phosphate with 4-hydroxyaniline gave N2-(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate. Using 32P-postlabeling, we compared this product with the DNA adduct produced in HL-60 cells treated with hydroquinone. The results of these studies suggest that the DNA adduct formed in HL-60 cells treated with hydroquinone corresponds to N2-(4-hydroxyphenyl)2′-deoxyguanosine 3′-phosphate.
Introduction Benzene is widely used in the chemical industry as an intermediate in the production of other agents and as a fuel additive; it is also found in automobile exhaust and cigarette smoke (1). Thus, human exposure to benzene occurs from both industrial and environmental sources (1-3). Concern regarding this exposure arises from evidence that acute exposure to benzene is leukemogenic in humans (4, 5). Benzene must be metabolized to exert its toxic and leukemogenic effects (6-8). The principal metabolites of benzenesphenol, hydroquinone (HQ),1 catechol, and muconaldehyde (9-11)saccumulate in the bone marrow (12, 13), where it is proposed that they are further activated to produce the observed myelotoxic and leukemogenic effects (8, 14). The enzymatic mechanisms responsible for the activation of benzene metabolites in bone marrow have not been elucidated; however, these metabolites appear to be further activated by peroxidases (15-17), including prostaglandin H-synthase (18, 19). Our laboratory has been interested in the role of DNA adduct formation by benzene metabolites in the myelotoxic effects of benzene exposure (19). In these studies we have used HL-60 cells, a human promyelocytic cell line, to investigate the activation of p-benzoquinone (pBQ), HQ, catechol, and 1,2,4-benzenetriol to form DNA adducts (20-22). These studies have demonstrated that activation of HQ to form adducts is dependent upon * Correspondence and requests for reprints should be addressed to this author at the University of California Brain Tumor Research Center, Box 0806, San Francisco, CA 94143-0806. Tel: (415) 476-4899; Fax: (415) 476-5799; e-mail:
[email protected]. X Abstract published in Advance ACS Abstracts, March 1, 1996. 1 Abbreviations: p-BQ, p-benzoquinone; HQ, hydroquinone; LSIMS, liquid secondary ionization mass spectrometry; DCI MS, direct chemical ionization mass spectrometry; HR EI MS, high resolution electron impact mass spectrometry; ESI, electrospray ionization; tR, retention time; TSP, 3-(trimethylsilyl)-1-propanesulfonic acid sodium salt; AU, absorbance unit; PEI, poly(ethylenimine).
0893-228x/96/2709-0593$12.00/0
peroxidase activity and that treatment of cells with either HQ or p-BQ results in the production of the same DNA adduct. Recently, we have demonstrated that benzene administration results in the formation of DNA adducts in the bone marrow of male B6C3F1 mice (23). These adducts were identical to those produced in HL-60 cells or in mouse bone marrow treated in vitro with HQ or p-BQ. To characterize these DNA adducts, we have investigated the DNA adducts formed by the reaction of p-BQ with purified DNA. The DNA adducts formed were identified as 3′-hydroxy-3,N4-benzetheno-2′-deoxycytidine 3′-phosphate, 3′-hydroxy-1,N6-benzetheno-2′-deoxyadenosine 3′-phosphate, and 3′-hydroxy-1,N2-benzetheno-2′deoxyguanosine 3′-phosphate (20, 24, 25). However, these identified adducts were not the same as those produced in HL-60 cells treated with HQ or p-BQ (20). In this study, we further investigated the DNA adducts produced in HL-60 cells treated with HQ or p-BQ.
Materials and Methods 1H
NMR spectra were recorded on a QE-300 300 MHz instrument (General Electric, Fremont, CA). Liquid secondaryionization mass spectra (LSIMS) were recorded on a Kratos MS50S mass spectrometer (Manchester, U.K.) equipped with a 23kg magnet and postaccelerator detector. A Cs+ primary electron beam of 8 keV was used. Samples were run in a glycerol matrix in the negative-ion mode. High resolution direct chemical ionization (DCI) (NH4+) and electron impact (EI) mass spectra were obtained on a VG 70-SE mass spectrometer (VG Analytical, Ltd., Manchester, U.K.) at a source temperature of 180 °C (Resolution 5000). Electrospray ionization (ESI) mass spectra were recorded on a VG Biotech/Fisons Bio-Q instrument (VG Analytical, Ltd., Manchester, U.K.) equipped with an electrospray ionization source operating in the negative-ion mode. The ESI voltage and current were 5 kV and 20 mA, respectively, and the capillary temperature was 250 °C. The UV absorption spectra were recorded on a Beckman DU-7 spectrophotometer (Beckman Instruments, Inc., Fullerton, CA).
© 1996 American Chemical Society
594 Chem. Res. Toxicol., Vol. 9, No. 3, 1996 HPLC Analysis. Sample purifications were performed on a Perkin-Elmer HPLC system equipped with an ISCO gradient programmer and a 10 µm C-18 reversed-phase Econosil column eluted with a linear gradient of 0-70% methanol for 30 min in 10 mM ammonium acetate (pH 5.1), at a flow rate of 1 mL/min. UV absorption was monitored with a Perkin-Elmer LC-235 diode array detector. N2-(4-Hydroxyphenyl)guanosine 3′-Phosphate (1). Guanosine 3′-phosphate (Sigma Chemical Co., St Louis, MO; 100 mg) and p-BQ (100 mg) were dissolved in 20 mL of 200 mM HCl-KCl (pH 1) and incubated overnight at 37 °C. After adjustment of pH to 6.5 with 1 N sodium hydroxide and addition of 30 mL of water, the reaction mixture was loaded onto 5 BOND ELUT cartridges (Analytichem International, Division of Varian, Harbor City, CA), each eluted with 12 mL of 0.4 M sodium phosphate (pH 6). The eluants were desalted on SEP PAK C-18 reversed-phase cartridges (Waters Division of Millipore, Milford, MA). The products were eluted from the cartridges with 50% methanol and evaporated. This prepurified material was separated by HPLC as described above. The major product was eluted at a retention time (tR) of 23 min. UV absorption spectra: pH 6, UVmax 274 nm, UVmin 244 nm; pH 1, UVmax 275 nm, UVmin 245 nm; pH 12, UVmax 250, 272s nm, UVmin 233 nm. 1H NMR (recorded in D O with 3-(trimethylsilyl)-1-propane2 sulfonic acid sodium salt (TSP) as internal standard) 8.011 (s, 1H, C8), 7.326 (dd, 2H, C-2, C-6, J 9.0 Hz), 6.952 (dd, 2H, C-3, C-5), 5.907 (d, 1H, H-1′ J 5.9 Hz), 4.564 (m, 1H, H-2′), 4.282 (m, 1H, H-4′), 3.604 (m, 2H, H-5′) ppm. LSIMS m/z [M - H]454. Depurination Studies: N2-(4-Hydroxyphenyl)guanine (2A). One absorbance unit (AU) of 1 was heated at 90 °C in 1 mL of 0.1 N HCl for 2 h. The product was purified by HPLC and eluted at a tR of 27 min. The UV absorption spectra were as follows: pH 6, UVmax 272 nm, UVmin 242 nm; pH 1, UVmax 268 nm, UVmin 242 nm. HR EI MS m/z M+ 243.07553. N2-(4-Hydroxyphenyl)guanine (2). 2-Bromo-6-hydroxypurine (216 mg, 1 mmol) and 4-hydroxyaniline (10 mmol) were refluxed for 2 h in 5 mL of methoxyethanol-water (4:1) and cooled. The resulting crystals were filtered and dried. With HPLC, the product eluted at a tR of 27 min. 1H NMR (dimethyl sulfoxide-d6) 10.365 (s, 1H, D2O exchangeable), 9.162 (s, 1H, OH, D2O exchangeable), 8.270 (s, 2-NH, D2O exchangeable), 7.741 (s, 1H C8), 7.324 (dd, 2H, C-2, C-6 J 8.4 Hz), 6.707 (dd, 2H, C-3, C-5) ppm. DCI MS m/z [M + H]+ 244. UVmax 271 (pH 6), 267 (pH 1), 244, 270 nm (pH 12). 2-Bromo-2′-deoxyinosine (3). 2-Bromo-6-hydroxypurine (216 mg; 26, 27) and thymidine (242 mg) were dissolved in 100 mL of 20 mM potassium phosphate buffer (pH 7.4) and incubated overnight at 37 °C with 60 units of thymidine phosphorylase (EC 2.4.2.4, Sigma) and 90 units of purine nucleotide phosphorylase (EC 2.4.2.1, Sigma). After evaporation to dryness, the residue was dissolved in 50 mL of water and filtered. The solution was placed in a 250 mL round-bottom flask with 10 g of silica gel and evaporated to dryness. A silica gel column (2.5 × 25 cm) was prepared with chloroformmethanol-triethylamine (80:15:5) as the solvent, and the silica gel containing the reaction products was placed on top of the column. The same solvent system was used to elute the products. 2-Bromo-2′-deoxyinosine eluted in the late fractions of the chromatography (280 mg, 84%). The tR of this compound was 23.7 min. LSIMS m/z [M + H]+ 331, 333; 1H NMR (D2O) 8.066 (s, 1H, C8), 6.339 (t, 1H, H-1′), 4.638 (m, 1H, H-3′), 4.176 (m, 1H, H-4′), 3.828 (m, 2H, H-5′), 2,782 and 2.535 (m, 2H H-2′) ppm. UVmax 252 nm (pH 1), 254 nm (pH 6), 258 nm (pH 12). 5′-DMT-2-bromo-2′-deoxyinosine (4). 3 (280 mg) was dried with repeated evaporations from dry pyridine and dissolved in 20 mL of pyridine. Dimethoxytrityl chloride (400 mg), (dimethylamino)pyridine (10 mg), and triethylamine (500 µL) were added, and the reaction mixture was stirred at room temperature overnight. The reaction was followed by thin-layer chromatography using chloroform-methanol (9:1) as the solvent system. After the addition of 20 mL of water to the reaction mixture, the product was extracted three times with 80 mL of
Pongracz and Bodell ether. The ether extracts were dried with sodium sulfate and evaporated. The product was dissolved in a small volume of dichloromethane and slowly added to 200 mL of petroleum ether. The precipitated product was further purified by column chromatography using silica gel eluted with chloroformmethanol (9:1). The collected fractions (Rf 0.4) were evaporated and used for phosphorylation. 2-Bromo-2′-deoxyinosine 3′-Phosphate (5). 4 was dried by repeated coevaporations from dry pyridine. Ten milliliters of phosphorylating agent (310 mg of triazole, 140 µL of phosphoryl chloride, 340 µL of triethylamine, 10 mL of dioxane, 5 °C, 1 h, filtered) was added, and the solution was stirred for 1 h (28). Water (5 mL) was added, and the solution was stirred for 15 min. After the addition of 20 mL of phosphate buffer, the product was extracted three times with 50 mL of chloroform, and the collected organic phases were evaporated to dryness. Twenty milliliters of 80% acetic acid was added; the solution was kept at room temperature for 20 min and was then carefully evaporated (30 °C). The dry residue was dissolved in 100 mL of water, the pH of the solution was adjusted to 7 with diluted ammonium hydroxide, and in a sepratory funnnel it was washed twice with 50 mL of diethyl ether. The volume of the water phase was reduced to 50 mL and analyzed by HPLC. The tR of 5 was 20.5 min. The concentration of this solution was estimated by measurement at 260 nm using the relationship 25 µg/AU, and this value was used for reactions with the aminophenols. Samples for mass spectrometry were lyophilized. LSIMS m/z [M - H]- 409, 411. UVmax 257 (pH 6), 254 (pH 1), 259 nm (pH 12). N2-(4-Hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate (6). 5 (40 µmol) was reacted with 400 µmol of 4-hydroxyaniline in 2 mL of water at 37 °C for 24 h. The reaction mixture was loaded onto four previously washed SEP PAK cartridges, and the products were eluted with 50% methanol. The end product was purified by HPLC as described above. N2-(4-Hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate eluted at a tR of 23 min. 1H NMR (D2O) 8.036 (s, 1H, C8), 7.434 (dd, 2H, C-2, C-6, J 8.7 Hz), 7.014 (dd, 2H, C-3, C-5), 6.382 (t, 1H, H-1′, J 7.0 Hz), 4.776 (m, 1H, H-3′), 4.333 (m, 1H, H-4′), 3.864 (m, 2H, H-5′), 3.171, 2.778 (m, m, 2H, H-2′) ppm. ESI MS m/z [M - H]- 438. One AU of 6 was depurinated in 0.1 N HCl at 90 °C for 20 min. The product eluted at a tR of 27 min, and the UV spectra were identical with the UV spectra of 2. In Vitro Studies. HL-60 cells were cultured as previously described (20-22). The cultures were incubated at 37 °C in a humidified 5% CO2/95% air atmosphere. HQ (Aldrich Chemical Co., Milwaukee, WI) was dissolved in distilled water and added directly to the culture medium to achieve the desired final concentrations. After treatment, the HL-60 cells were centrifuged at 3000g for 10 min, washed with cold Hanks’ balanced salt solution (Ca2+ -Mg2+-free), recentrifuged at 3000g for 10 min, and stored at -70 °C until the DNA was isolated. 32P-Postlabeling. The DNA was isolated from treated cells as previously described (29). The P1 nuclease enhanced 32Ppostlabeling technique was used to analyze the DNA adducts formed in HL-60 cells (20-22). N2-(4-Hydroxyphenyl)guanosine 3′-phosphate, N2-(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate, and the HPLC fraction containing the cellular adduct were labeled directly using T4 kinase. The radioactive materials were applied to 10 × 10 cm poly(ethylenimine) (PEI)-cellulose sheets. The PEI plates were developed in the following solvent systems: D0, 0.4 M sodium phosphate buffer (pH 6.8); D1, 1.8 M lithium formate and 4.5 M urea (pH 3.5); D2, 0.36 M lithium chloride, 0.22 M Tris, and 3.8 M urea (pH 8); D3, 1.7 M sodium phosphate (pH 6). The adducts were located by autoradiography using Kodak XAR-5 film (Kodak, Rochester, NY) and a DuPont Chronex Lightning Plus intensifying screen (DuPont, Boston, MA) The areas corresponding to adducts were scraped into scintillation vials containing 5 mL of scintillation cocktail (Safety Solve, Research Products Inc., Mount Prospect, IL), and the radioactivity was determined by scintillation counting. The relative adduct levels were calculated as previously described (20-22).
DNA Adduct Formed in HL-60 Cells Treated with HQ
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Figure 1. UV spectra of N2-(4-hydroxyphenyl)guanosine 3′phosphate, recorded at pH 1, pH 6, and pH 12. Rechromatography of 32P-Postlabeled Adducts. For rechromatography experiments, the postlabeled adducts were excised from the PEI-cellulose plates, and the cellulose was extracted at room tempreature with 200 µL of 1.5 M pyridinium formate (pH 4.5), for 1 h. The solution was evaporated, and the residue was dissolved in 20 µL of water and spotted on another PEI-celluose sheet. The plates were subsequently developed in 0.5 M sodium phosphate and 3 M urea (pH 5). HPLC Isolation of Cellular DNA Adduct. HL-60 cells were treated with HQ as described above. The DNA was subsequently isolated and purified. DNA (50 µg) was digested with 80 µg of spleen phosphodiesterase and 80 µg of micrococcal nuclease in 60 µL of 20 mM sodium succinate and 8 mM CaCl2 (pH 6), for 2 h at 37 °C. The sample was diluted with water to 1 mL and heated at 100 °C for 90 s. After cooling, the sample was filtered with Millex GSO 0.22 µm filters (Millipore, Bedford, MA) before injection. The sample was separated by HLPC as described above. The eluant between 21 and 25 min was collected and lyophilized. The residue was dissolved in 10 µL of water and 32P-postlabeled.
Results From the reaction of guanosine 3′-phosphate with p-BQ at pH 1, a new product was isolated. The UV absorption spectra of this compound are shown in Figure 1. The neutral and the acidic spectra are similar to those reported for N2-(p-n-butylphenyl)-2′-deoxyguanosine (30). The molecular weight of the compound was 455 (LSIMS m/z [M - H]- 454), indicating the presence of a hydroxyphenyl group as the substituent modifying guanosine 3′phosphate. The exact position of the hydroxyl group on the phenyl ring was determined by 1H NMR (Figure 2). The aromatic protons of the phenyl ring appeared as two distorted doublets, characteristic of an AA′XX′ coupling system. These results indicated that the position of the hydroxyl group is para to the N2 amino group of guanosine. Therefore, we assigned the structure of N2-(4hydroxyphenyl)guanosine 3′-phosphate to the product. N2-(4-Hydroxyphenyl)guanosine 3′-phosphate was depurinated by acid hydrolysis, and the product was purified by HPLC. EI MS of the depurinated product determined the molecular weight as 243.07553, which corresponds to elemental composition C11H9N5O2. Two fragments with exact masses of 134.04782 (C7H6N2O) and 110.03628 (C4H4N3O) resulted from fragmentation of the sixmembered purine ring. These results are consistent with the structure of N2-(4-hydroxyphenyl)guanine for the depurinated product.
Figure 2. 1H NMR spectrum of N2-(4-hydroxyphenyl)guanosine 3′-phosphate, recorded in deuterium oxide with TSP as internal standard.
N2-(4-Hydroxyphenyl)guanine was also prepared from 2-bromo-6-hydroxypurine and 4-hydroxyaniline. 1H NMR analysis of this product confirmed the structure. Comparison of the UV spectrum and the HPLC tR of N2-(4hydroxyphenyl)guanine with those of the product derived from depurination of N2-(4-hydroxyphenyl)guanosine 3′phosphate showed them to be the same. These results further establish the structure of the p-BQ-guanosine reaction product as N2-(4-hydroxyphenyl)guanosine 3′phosphate. Previous studies have concluded that the DNA adducts formed in HL-60 cells treated with either HQ or p-BQ do not correspond to the benzetheno adducts previously identified (19, 20). Figure 3A shows the results of 32Ppostlabeling analysis of DNA isolated from HL-60 cells treated with 250 µM HQ for 16 h. Under these treatment conditions, one principal (1) and two minor (2 and 3) DNA adducts were detected; the relative adduct level was 4.4 × 10-7. N2-(4-Hydroxyphenyl)guanosine 3′phosphate was 32P-postlabeled and chromatographed in a similar fashion (Figure 3B). Comparison of these results suggested that the principal adduct formed in HL-60 cells treated with HQ was similar to N2-(4-hydroxyphenyl)guanosine 3′-phosphate. For comparison with the cellular adduct, it was necessary to synthesize the 2′-deoxyguanosine derivative. Enzymatic coupling procedures have been described to prepare both modified and unmodified deoxyribonucleotides (31, 32). We have developed a method for preparing N2-(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate; the synthetic scheme is shown in Figure 4. 2-Bromo-6-hydroxypurine was prepared using published procedures (26, 27). Enzymatic coupling of this compound to deoxyribose resulted in 2-bromo-2′-deoxyinosine; the expected molecular weight of 331 was confirmed by MS (LSIMS m/z [M + H]+ 331, 333) (data not shown). Dimethoxytritylation and phosphorylation followed by mild acid treatment gave 2-bromo-2′-deoxyinosine 3′phosphate. Reaction of 2-bromo-2′-deoxyinosine 3′phosphate with 4-hydroxyaniline produced the expected
596 Chem. Res. Toxicol., Vol. 9, No. 3, 1996
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PEI plates. As shown in Figure 6, the DNA adduct formed in HL-60 cells treated with HQ has the same chromatographic properties as N2-(4-hydroxyphenyl)2′-deoxyguanosine 3′-phosphate when analyzed by a combination of HPLC and PEI-cellulose chromatography.
Discussion
Figure 3. (A) Autoradiogram from 32P-postlabeling analysis of DNA isolated from HL-60 cells treated with 250 µM hydroquinone for 16 h. The film was exposed at -70 °C for 2 h. (B) 32P-Postlabeling analysis of N2-(4-hydroxyphenyl)guanosine 3′phosphate.
productsN2-(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate, as determined by ESI MS m/z [M - H]- 438. The UV spectra of this product were identical to the UV spectra of N2-(4-hydroxyphenyl)guanosine 3′-phosphate, and depurination of the compound produced N2-(4hydroxyphenyl)guanine (results not shown). N2-(4-Hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate was 32P-postlabeled and chromatographed on PEI-celluose sheets (Figure 5A). The chromatographic properties of the bisphospate derivative were similar to those previously observed for the ribo derivative (Figure 3B). DNA isolated from HL-60 cells treated with HQ was postlabeled, the adducts were resolved on PEI-cellulose sheets and located by autoradiography, and DNA adduct 1 was eluted from the PEI-cellulose sheets. Similar procedures were applied to 32P-postlabeled N2-(4hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate. Rechromatography experiments to compare the adducts showed that DNA adduct 1 formed in HL-60 cells treated with HQ has the same chromatographic properties as N2(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate (Figure 5B). To confirm these observations, we performed an experiment that combined HPLC separation with 32Ppostlabeling. DNA isolated from HL-60 cells treated with HQ was enzymatically digested to 3′-monophosphates. The mononucleotides were chromatographed using the same HPLC procedures used to purify N2-(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate. The eluant between 21 and 25 min after injection was collected, evaporated, 32P-postlabeled, and chromatographed on
In these studies we have investigated the reaction of p-BQ with guanylic acid at different pH levels. Guanylic acid was chosen due to its stability at acidic pHs. At pH 1, a new product was detected. Spectroscopic analysis showed that the structure of this product is N2-(4-hydroxyphenyl)guanosine 3′-phosphate. The structure of the depurinated product was confirmed by comparison with an independently prepared sample of N2(4-hydroxyphenyl)guanine. This product may form from the reaction of the N2 amino group of guanosine with one of the carbonyls of p-BQ, as has been observed in the reaction of primary amines with estrogen o-quinones (33, 34). In our previous studies of the reaction of p-BQ with DNA at neutral pH, the principal products detected by postlabeling were 3′-hydroxy-3,N4-benzetheno-2′-deoxycytidine 3′-phosphate; 3′-hydroxy-1,N6-benzetheno-2′deoxyadenosine 3′-phosphate, and 3′-hydroxy-1,N2-benzetheno-2′-deoxyguanosine 3′-phosphate (20, 24, 25). In the present investigation, in which the pH of the reaction mixture was raised above 3.5, the previously identified cyclic guanosine adduct 3′-hydroxy-1,N2-benzethenoguanosine 3′-phosphate became dominant.2 In previous investigations several modified deoxyribonucleotides and the corresponding ribonucleotides have been prepared (24).2 Both sets of adducts were 32Ppostlabeled, and the chromatographic properties on PEIcellulose sheets were analyzed. These experiments demonstrated that the bisphosphate derivative of a 2′deoxyribonucleotide or ribonucleotide with the same modified base structure chromatographed similarly on PEI-cellulose plates. We observed that 32P-postlabeled N2-(4-hydroxyphenyl)guanosine 3′-phosphate was very similar to the 32P-postlabeled adduct from HL-60 cells treated with p-BQ. Because of the general acid lability of deoxyguanylic adducts, it was necessary to develop an alternative method for preparing the corresponding N2-(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate. 2-Bromo- or 2-fluoro-2′-deoxyinosine is the most suitable starting material for the syntheses. However, all published methods for preparing these compounds involve several steps and the use of protecting groups (35-37). 2-Fluoro2′-deoxyinosine was reported in the syntheses of N2modified guanosine derivatives, but this compound had a low reactivity with 4-hydroxyaniline.2 The synthesis of 2-bromo-2′-deoxyinosine via the O6-methyl derivative has been described (38), and a stereoselective method using 2-bromo-6-hydroxypurine as the starting material has been developed (39). Recently, Chapeau and Marnett (32) described an enzymatic method for preparing deoxyribonucleoside adducts from the modified base. We developed a chemical-enzymatic strategy for the preparation of 2-bromo-2′-deoxyinosine 3′-phosphate. This compound would be a useful intermediate not only in the preparation of N2-(4-hydroxyphenyl)-2′-deoxyguanosine 2
K. Pongracz, unpublished results.
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Figure 4. Synthetic scheme for the preparation of N2-(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate. DMT ) dimethoxytrityl; TPase ) thymidine phosphorylase; PNPase ) purine nucleotide phosphorylase.
Figure 6. Autoradiogram of 32P-postlabeling analysis of HPLCenriched fractions (21-25 min) from enzymatic digestion of HL60 cells treated with hydroquinone. The films were exposed for 30 min at -70 °C.
Figure 5. (A) Autoradiogram of 32P-postlabeling of N2-(4hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate. (B) Rechromatography analysis of N2-(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate with adduct 1 formed in HL-60 cells treated with HQ: Lane a, DNA adduct 1; lane b, 1:1 mixture of DNA adduct 1 and N2-(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate; lane c, N2-(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate. The exposure times for (A) and (B) were 10 min at room temperature and overnight at -70 °C, respectively.
3′-phosphate but also in the preparation of a series of N2-modified deoxyguanosine 3′-phosphate derivatives for 32 P-postlabeling analysis. Using this procedure, we successfully prepared N2(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate. The structure of the product was confirmed by ESI MS. Using 32P-postlabeling, we compared N2-(4-hydroxyphen-
yl)-2′-deoxyguanosine 3′-phosphate with the DNA adduct formed in HL-60 cells treated with either p-BQ or HQ. In previous studies we have demonstrated that the principal adduct formed in HL-60 cells treated with either HQ or p-BQ was the same (20). The results of the present study suggested that the adduct formed in HL60 cells treated with these agents was N2-(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate. Further evidence for this conclusion was obtained by HPLC separation of deoxyribonucleotides produced by enzymatic digestion of DNA isolated from treated HL-60 cells. 32PPostlabeling of the fraction with the same retention time as N2-(4-hydroxyphenyl)-2′-deoxyguanosine 3′-phosphate led to the detection of the same adduct as with P1enhanced 32P-postlabeling. These results taken together suggest that the adduct formed in HL-60 cells treated with p-BQ or HQ corresponds to N2-(4-hydroxyphenyl)2′-deoxyguanosine 3′-phosphate. Additional proof of this conclusion will require direct structural analysis of the DNA adduct formed in these treated cells (40).
Acknowledgment. This work was supported by Grant P42ES04705 from the National Institute of Environmental Health Sciences. The authors thank Dr. G.
598 Chem. Res. Toxicol., Vol. 9, No. 3, 1996
Le´vay for treatment of the HL-60 cells, Cheryl Christensen for manuscript preparation, and Stephen Ordway for editorial assistance. Mass spectrometry was performed at the Mass Spectrometry Facility, University of California San Francisco, which is supported by Grant NIH NCRR BRTP RRO1614.
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