Substrate Specificity of Human O6

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Chem. Res. Toxicol. 1997, 10, 1234-1239

Substrate Specificity of Human O6-Methylguanine-DNA Methyltransferase for O6-Benzylguanine Derivatives in Oligodeoxynucleotides Isamu Terashima,† Hisaya Kawate,‡ Kunihiko Sakumi,‡ Mutsuo Sekiguchi,‡ and Kohfuku Kohda*,† Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabedori, Mizuho-ku, Nagoya 467, Japan, and Department of Biochemistry, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-82, Japan Received April 17, 1997X

To investigate the substrate specificity of human O6-methylguanine-DNA methyltransferase (MGMT) for O6-benzylguanine (6BG) derivatives incorporated in oligodeoxynucleotides, we prepared 25-mer lengths of sequences containing various 6BG derivatives and their related compounds and then measured the ability of these derivatives to inactivate MGMT in vitro. Oligodeoxynucleotides containing a 6BG, O6-(2-fluorobenzyl)guanine (2F-6BG), O6-(3-fluorobenzyl)guanine (3F-6BG), O6-(4-fluorobenzyl)guanine (4F-6BG), O6-benzylhypoxanthine (6BH), or O6-methylguanine (6MG) were all good substrates for MGMT, and no obvious differences were observed among them. Oligodeoxynucleotides containing N2-isobutyrylated 6BG and 6MG showed only a slightly reduced capacity for inactivating MGMT compared to N2-nonmodified forms of these derivatives. No obvious differences were observed in the corresponding double-stranded and single-stranded oligodeoxynucleotides. MGMT substrate specificity for the 6BG derivatives in the oligodeoxynucleotide was found to be quite different from that seen in our previous study [Mineura, K., et al. (1994) Int. J. Cancer 58, 706-712; (1995) Int. J. Cancer 63, 148-151. Kohda, K., et al. (1995) Biol. Pharm. Bull. 18, 424-430] and others [Moschel, R. C., et al. (1992) J. Med. Chem. 35, 4486-4491. Chae, M.-Y., et al. (1994) J. Med. Chem. 37, 342-347] using the corresponding free bases. In brief, (i) 6BG, 3F6BG, and 4F-6BG greatly inhibited human MGMT, whereas 2F-6BG, 6BH, and 6MG displayed much weaker activity; (ii) any modifications at the 2-amino group of the 6BG resulted in severe reductions in the ability to inactivate MGMT. These results obtained by the experiments using oligodeoxynucleotides and free bases suggest that human MGMT has low substrate specificity for 6BGs in oligodeoxynucleotides. Conformational changes in human MGMT which favor binding to oligodeoxynucleotides containing 6BG derivatives and the subsequent transfer of their benzyl groups may account for the difference in substrate specificity between the incorporated 6BG derivatives and their free base form.

Introduction Alkylating carcinogens such as N-alkyl-N-nitrosoureas and N-alkyl-N′-nitro-N-nitrosoguanidines (alkyl: methyl, ethyl, propyl, butyl, etc.) react with cellular DNA and produce many types of alkylated products (1). Among alkylated bases, O6-alkylguanine (6AG)1 is considered to represent the most mutagenic and carcinogenic of base modifications (lesions) (2). However, cells contain a repair enzyme, O6-methylguanine-DNA methyltransferase (MGMT), which can repair 6AG by transferring the alkyl group to the cysteine residue at the active site of the enzyme (3). This process abolishes any further enzyme activity (3). * To whom correspondence should be addressed. Tel: (81)-52-8363408. Fax: (81)-52-834-9309. E-mail: [email protected]. † Nagoya City University. ‡ Kyushu University. X Abstract published in Advance ACS Abstracts, October 15, 1997. 1 Abbreviations: 6AG, O6-alkylguanine; MGMT, O6-methylguanineDNA methyltransferase; 6MG, O6-methylguanine; 6BG, O6-benzylguanine; 2F-6BG, O6-(2-fluorobenzyl)guanine; 3F-6BG, O6-(3-fluorobenzyl)guanine; 4F-6BG, O6-(4-fluorobenzyl)guanine; 6BH, O6-benzylhypoxanthine; ib-6MG, N2-isobutyryl-O6-methylguanine; ib-6BG, N2-isobutyryl-O6-benzylguanine; MNU, N-methyl-N-nitrosourea; DBU, 1,8diazabicyclo[5.4.0]undec-7-ene; ds, double-stranded; ss, single-stranded.

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BCNU, CCNU, and ACNU are bifunctional alkylating anticancer agents whose cytotoxic activities appear after they initially chloroethylate at the O6-position of the guanine residues in cellular DNA (4). Since MGMT repairs this modification, its presence results in a reduction of the cytotoxic potential of these alkylating anticancer agents. O6-Methylguanine (6MG) can serve as a substrate for MGMT, although the substrate specificity is far lower than in DNA (5); therefore, pretreatment of cells with an excess of 6MG depletes MGMT activity, resulting in potentiation of the cytotoxicity of these agents (6). When the size of the alkyl group was increased from a methyl to either an ethyl or a butyl, the depletion of MGMT activity by 6AG was reduced (5). Dolan et al. reported that O6-benzylguanine (6BG) inactivates mammalian MGMT to a much greater extent than does 6MG, although the O6-benzyl group is bigger than the O6-methyl group (7). To generate more effective lowmolecular-weight substrates for MGMT for use as potentiators of alkylating anticancer agents, numerous 6BG derivatives and their analogues have been synthesized. As a result of studies on structure-activity relationships, a large body of knowledge has accumulated concerning structures suitable as substrates for MGMT (8-14). We reported previously that all 6BG derivatives that were © 1997 American Chemical Society

Substrate Specificity of MGMT for O6-Benzylguanines

modified at the ortho-position of the benzyl group have a much weaker capacity for inhibiting human MGMT than 6BG itself, whereas 6BG derivatives modified at the meta- or para-position have the same inhibiting activity as 6BG (14, 15). 6BG derivatives modified at the 2-amino group with a methyl or acyl group and 6BG derivatives lacking the 2-amino group both showed a remarkable reduction in their ability to inhibit MGMT (9, 11-13). MGMT repair of 6AG in DNA or oligodeoxynucleotides has been previously investigated (16-21). MGMT prefers 6AG in DNA or oligonucleotides as a substrate over their free base forms (5, 16). The rate of MGMT repair of 6AG in DNA decreases with an increase in the size of the alkyl group (16), as observed for 6AG in free base form (5). 6BG in an oligodeoxynucleotide is reported to be repaired by mammalian MGMT at the same rate as 6MG in an oligodeoxynucleotide (22). This substrate specificity is quite different from that of 6MG and 6BG in free base forms where 6MG has a much lower ability to inactivate MGMT than 6BG (7). Further investigation of 6BGs in DNA, however, has not been carried out. In this study, we prepared 25-mer oligodeoxynucleotides containing a 6BG derivative (o-, m-, and pfluorobenzyl derivatives as well as derivatives modified at the 2-amino group) and investigated their reactivity toward human MGMT. Remarkable differences in the relationship of structure to activity of 6BG derivatives in oligodeoxynucleotides compared to their free base forms are discussed.

Experimental Procedures 1H

General. NMR spectra were recorded on a JEOL EX 270, GSX 400, or ALPHA 500 spectrometer (Tokyo, Japan), and chemical shifts are reported in ppm using TMS as the internal standard. Me2SO-d6 was used as the solvent. Mass spectra were obtained with a JEOL DX-300 spectrometer (Tokyo, Japan). Column chromatography was performed using Merck silica gel 60 (70-230 mesh, 63-200 µm). HPLC analyses were carried out using a Shimadzu LC10AD apparatus equipped with a photodiode array UV detector, SPD-M6A (Kyoto, Japan). For analysis of O6-benzyl-2′-deoxyguanosine derivatives, a LiChrospher 100 RP-18(e) column (Merck; 4 × 125 mm, 5 µm) was used and was eluted with 50 mM sodium phosphate buffer (pH 7.0)-MeOH (0-15% linear gradient of MeOH over 20 min, then a 15-100% gradient over 80 min) at a flow rate of 1 mL/min. Oligodeoxynucleotides were synthesized using an Applied Biosystems automated solid-phase DNA synthesizer, model 392 (CA). Syntheses of O6-Benzyl-2′-deoxyguanosine Derivatives. O6-Benzyl-2′-deoxyguanosine was prepared from 2′-deoxyguanosine by a procedure described by Pauly et al. (23). The other O6-benzyl-2′-deoxyguanosine derivatives were synthesized using the procedure described below. O6-(2-Fluorobenzyl)-2′-deoxyguanosine. 3′,5′-O-Diacetyl2′-deoxyguanosine (250 mg, 0.71 mmol) (24) was sonicated in 5 mL of dry dioxane for 30 min under nitrogen. Triphenylphosphine (465 mg, 1.78 mmol) and 2-fluorobenzyl alcohol (191 µL, 1.78 mmol) were added to the suspension, and the mixture was heated at 100 °C with stirring. Diethyl azodicarboxylate (289 µL, 1.78 mmol) was then added dropwise, and the mixture was left standing for 15 min. The reaction mixture was concentrated by evaporation, and the product was separated by column chromatography (silica gel, 2 × 35 cm; MeOH/CHCl3, 1/99 to 10/90). The fractions containing the product were collected, and the solvent was removed by evaporation. MeOH (10 mL) and 25% NH4OH (10 mL) were added to the residue, and the mixture was allowed to stand for 90 min with stirring. After the solvent was removed by evaporation, the residue was applied to a silica gel column to obtain a brownish syrup. The yield of the product was 197 mg (74%): 1H NMR δ 2.22 (m, 1H, 2′-HR), 2.58 (m, 1H, 2′-Hβ), 3.47-3.60 (m, 2H, 5′-H), 3.83 (m, 1H, 4′-H), 4.35

Chem. Res. Toxicol., Vol. 10, No. 11, 1997 1235 (m, 1H, 3′-H), 4.98 (t, 1H, 5′-OH, J ) 5.4 Hz), 5.27 (d, 1H, 3′OH, J ) 3.9 Hz), 5.54 (s, 2H, PhCH2), 6.22 (t, 1H, 1′-H, J ) 6.8 Hz), 6.51 (br s, 2H, NH2), 7.24 (m, 2H, Ph-H), 7.43 (m, 1H, PhH), 7.62 (m, 1H, Ph-H), 8.09 (s, 1H, H-8); FAB-MS m/z 376 (M + H)+, 260 (base moiety + 2H)+. O6-(2-Fluorobenzyl)-N2-isobutyryl-2′-deoxyguanosine. 6 O -(2-Fluorobenzyl)-2′-deoxyguanosine (112 mg, 0.30 mmol) was coevaporated three times with dry pyridine (1 mL each time). Dry pyridine (2 mL) was added to the residue, and the mixture was cooled in an ice-cold water bath. Chlorotrimethylsilane (180 µL, 1.50 mmol) was next added, and the mixture was left standing for 30 min. Isobutyric anhydride (240 µL, 1.50 mmol) was then added, and the mixture was allowed to stand at room temperature for another 2 h. After the mixture was cooled again in an ice-cold water bath, cold water (400 µL) was added and the mixture was stirred for 15 min. A 25% solution of NH4OH (500 µL) was then added, and the mixture was stirred for 30 min. After the solvent was removed, the residue was applied to a silica gel column (2.5 × 20 cm; MeOH/CHCl3, 1/99) to obtain a white syrup. The yield of the product was 133 mg (100%): 1H NMR δ 1.11 [d, 6H, CH(CH ) , J ) 6.7 Hz], 2.28 (m, 1H, 3 2 2′-HR), 2.69 [m, 1H, 2′-Hβ or CH(CH3)2], 2.89 [m, 1H, 2′-Hβ or CH(CH3)2], 3.51 (m, 1H, 5′-Ha), 3.59 (m, 1H, 5′-Hb), 3.85 (m, 1H, 4′-H), 4.41 (m, 1H, 3′-H), 4.89 (hump, 1H, 5′-OH), 5.31 (hump, 1H, 3′-OH), 5.67 (s, 2H, PhCH2), 6.33 (t, 1H, 1′-H, J ) 6.7 Hz), 7.25 (m, 2H, Ph-H), 7.45 (m, 1H, Ph-H), 7.71 (m, 1H, Ph-H), 8.44 (s, 1H, H-8), 10.41 (s, 1H, 2-NH). 5′-O-(4,4′-Dimethoxytrityl)-O6-(2-fluorobenzyl)-N2-isobutyryl-2′-deoxyguanosine. O6-(2-Fluorobenzyl)-N2-isobutyryl2′-deoxyguanosine (133 mg, 0.30 mmol), 4-(dimethylamino)pyridine (2 mg, 0.015 mmol), and triethylamine (58 µL, 1.4 equiv) were dissolved in 1 mL of dry pyridine. 4,4′-Dimethoxytrityl chloride (121 mg, 0.42 mmol) was then added, and the mixture was left standing at room temperature for 3 h. To carry out the reaction, additional 4,4′-dimethoxytrityl chloride (50 mg) was added and the mixture was allowed to stand for 1 h. Cold water (2 mL) was then added to the reaction mixture, and the product was extracted three times using diethyl ether (5 mL each time). The ether layers were concentrated, and the product was purified by means of silica gel column chromatography (1.5 × 30 cm; MeOH/CHCl3, 0/100 to 5/95). Fractions of the product were collected and evaporated to obtain a brownish foam. The yield of the product was 64 mg (29%): 1H NMR δ 1.07-1.10 [m, 6H, CH(CH3)2], 2.34 (m, 1H, 2′-HR), 2.87 [m, 2H, 2′-Hβ, CH(CH3)2], 3.10 (m, 1H, 5′-Ha), 3.29 (m, 1H, 5′-Hb), 3.69 and 3.70 (each s, each 3H, OCH3), 3.96 (m, 1H, 4′-H), 4.51 (m, 1H, 3′-H), 5.31 (d, 1H, 3′-OH, J ) 4.4 Hz), 5.66 (s, 2H, PhCH2), 6.36 (t, 1H, 1′-H, J ) 6.4 Hz), 6.72 (d, 2H, 3-H and 5-H of CH3O-Ph, J ) 8.8 Hz), 6.76 (d, 2H, 3-H and 5-H of CH3O-Ph, J ) 9.3 Hz), 7.11-7.19 (m, 7H, 2-H and 6-H of two CH3O-Ph, 3-H, 4-H, and 5-H of Ph), 7.22-7.30 (m, 4H, two H of PhCH2, 2-H and 6-H of Ph), 7.45 (m, 1H, H of PhCH2), 7.72 (m, 1H, H of PhCH2), 8.31 (s, 1H, 8-H), 10.36 (s, 1H, 2-NH); FAB-MS m/z 748 (M + H)+, 330 (base moiety + 2H)+. The O6-(3- and 4-fluorobenzyl)-2′-deoxyguanosine derivatives were also obtained by the same procedure as described for the synthesis of O6-(2-fluorobenzyl)-2′-deoxyguanosine derivatives. O6-(3-Fluorobenzyl)-2′-deoxyguanosine: 1H NMR δ 2.21 (m, 1H, 2′-HR), 2.58 (m, 1H, 2′-Hβ), 3.50 (m, 1H, 5′-Ha), 3.57 (m, 1H, 5′-Hb), 3.82 (m, 1H, 4′-H), 4.35 (m, 1H, 3′-H), 4.98 (t, 1H, 5′-OH, J ) 5.5 Hz), 5.27 (d, 1H, 3′-OH, J ) 3.7 Hz), 5.51 (s, 2H, PhCH2), 6.22 (t, 1H, 1′-H, J ) 6.7 Hz), 6.50 (br s, 2H, NH2), 7.18 (ddd, 1H, 4-H of PhCH2), 7.32-7.35 (m, 2H, 2-H and 6-H of PhCH2), 7.44 (m, 1H, 5-H of PhCH2), 8.10 (s, 1H, H-8); EIMS m/z 375 M+, 259 (base moiety + H)+. O 6 -(3-Fluorobenzyl)-N 2 -isobutyryl-2′-deoxyguanosine: 1H NMR δ 1.11 [d, 6H, CH(CH3)2, J ) 6.6 Hz], 2.28 (m, 1H, 2′-HR), 2.68 [m, 1H, 2′-Hβ or CH(CH3)2], 2.88 [m, 1H, 2′Hβ or CH(CH3)2], 3.52 (m, 1H, 5′-Ha), 3.59 (m, 1H, 5′-Hb), 3.86 (m, 1H, 4′-H), 4.41 (m, 1H, 3′-H), 4.89 (hump, 1H, 5′-OH), 5.31 (hump, 1H, 3′-OH), 5.67 (d, 2H, PhCH2, J ) 1.8 Hz), 6.33 (t, 1H, 1′-H, J ) 6.7 Hz), 7.18 (ddd, 1H, 4-H of PhCH2), 7.38-7.46 (m, 3H, 2-H, 5-H, and 6-H of PhCH2), 8.41 (s, 1H, 8-H), 10.40 (s, 1H, 2-NH).

1236 Chem. Res. Toxicol., Vol. 10, No. 11, 1997 5′-O-(4,4′-Dimethoxytrityl)-O6-(3-fluorobenzyl)-N2-isobutyryl-2′-deoxyguanosine: 1H NMR δ 1.07-1.10 [m, 6H, CH(CH3)2], 2.34 (m, 1H, 2′-HR), 2.86 [m, 2H, 2′-Hβ and CH(CH3)2], 3.11 (m, 1H, 5′-Ha), 3.28 (m, 1H, 5′-Hb), 3.69 and 3.70 (each s, each 3H, OCH3), 3.96 (m, 1H, 4′-H), 4.51 (m, 1H, 3′-H), 5.31 (d, 1H, 3′-OH, J ) 4.4 Hz), 5.63 (s, 2H, PhCH2), 6.37 (t, 1H, 1′-H, J ) 6.1 Hz), 6.73 (d, 2H, 3-H and 5-H of CH3OPh, J ) 8.8 Hz), 6.77 (d, 2H, 3-H and 5-H of CH3OPh, J ) 9.3 Hz), 7.11-7.21 (m, 8H, 2-H and 6-H of two CH3OPh, 3-H, 4-H, and 5-H of Ph, 4-H of PhCH2), 7.29 (d, 2-H and 6-H of Ph, J ) 8.3 Hz), 7.397.48 (m, 3H, 2-H, 5-H and 6-H of PhCH2), 8.32 (s, 1H, 8-H), 10.37 (s, 1H, 2-NH); FAB-MS m/z 748 (M + H)+, 330 (base moiety + 2H)+. O6-(4-Fluorobenzyl)-2′-deoxyguanosine: 1H NMR δ 2.21 (m, 1H, 2′-HR), 2.58 (m, 1H, 2′-Hβ), 3.50 (m, 1H, 5′-Ha), 3.57 (m, 1H, 5′-Hb), 3.82 (m, 1H, 4′-H), 4.35 (m, 1H, 3′-H), 4.99 (t, 1H, 5′-OH, J ) 5.8 Hz), 5.23 (d, 1H, 3′-OH, J ) 3.7 Hz), 5.51 (s, 2H, PhCH2), 6.21 (t, 1H, 1′-H, J ) 6.7 Hz), 6.49 (br s, 2H, NH2), 7.22 (m, 2H, 3-H and 5-H of PhCH2), 7.56 (m, 2H, 2-H and 6-H of PhCH2), 8.08 (s, 1H, H-8); FAB-MS m/z 376 (M + H)+, 260 (base moiety + 2H)+. O 6 -(4-Fluorobenzyl)-N 2 -isobutyryl-2′-deoxyguanosine: 1H NMR δ 1.11 [d, 6H, CH(CH3)2, J ) 6.6 Hz], 2.27 (m, 1H, 2′-HR), 2.67 [m, 1H, 2′-Hβ or CH(CH3)2], 2.87 [m, 1H, 2′Hβ or CH(CH3)2], 3.52 (m, 1H, 5′-Ha), 3.58 (m, 1H, 5′-Hb), 3.86 (m, 1H, 4′-H), 4.42 (m, 1H, 3′-H), 4.89 (hump, 1H, 5′-OH), 5.31 (hump, 1H, 3′-OH), 5.60 (s, 2H, PhCH2), 6.33 (t, 1H, 1′-H, J ) 6.7 Hz), 7.22 (m, 2H, 3-H and 5-H of PhCH2), 7.64 (m, 2H, 2-H and 6-H of PhCH2), 8.42 (s, 1H, 8-H), 10.40 (s, 1H, 2-NH). 5′-O-(4,4′-Dimethoxytrityl)-O6-(4-fluorobenzyl)-N2-isobutyryl-2′-deoxyguanosine: 1H NMR δ 1.07-1.10 [m, 6H, CH(CH3)2], 2.34 (m, 1H, 2′-HR), 2.85 [m, 2H, 2′-Hβ and CH(CH3)2], 3.11 (m, 1H, 5′-Ha), 3.28 (m, 1H, 5′-Hb), 3.69 and 3.70 (each s, each 3H, OCH3), 3.96 (m, 1H, 4′-H), 4.51 (m, 1H, 3′-H), 5.31 (d, 1H, 3′-OH, J ) 6.3 Hz), 5.60 (s, 2H, PhCH2), 6.36 (t, 1H, 1′-H, J ) 4.4 Hz), 6.72 (d, 2H, 3-H and 5-H of CH3OPh, J ) 8.8 Hz), 6.77 (d, 2H, 3-H and 5-H of CH3OPh, J ) 9.3 Hz), 7.11-7.25 (m, 9H, 2-H and 6-H of two CH3OPh, 3-H, 4-H, and 5-H of Ph, 3-H and 5-H of PhCH2), 7.29 (d, 2H, 2-H and 6-H of Ph, J ) 8.7 Hz), 7.64 (m, 2H, 2-H and 6-H of PhCH2), 8.31 (s, 1H, 8-H), 10.35 (s, 1H, 2-NH); FAB-MS m/z 748 (M + H)+, 330 (base moiety + 2H)+. Syntheses of O6-Benzyl-2′-deoxyinosine Derivatives. 3′,5′-O-Diacetyl-2′-deoxyinosine. 3′,5′-O-Diacetyl-2′-deoxyinosine was prepared by the same procedure as that for 3′,5′-Odiacetyl-2′-deoxyguanosine (24). Briefly, acetic anhydride (10 mL, 106 mmol) was added to a suspension of 2′-deoxyinosine (1.0 g, 3.97 mmol), triethylamine (10 mL, 71.1 mmol), and 4-(dimethylamino)pyridine (48 mg, 0.4 mmol) in acetonitrile (50 mL), and the mixture was left standing at room temperature for 60 min with stirring. The solvent was removed by evaporation with repeated additions of water (50 mL), and evaporation (three times) yielded a solid residue. The residue was washed once with water (50 mL) and dried in vacuo to obtain a brownish solid. The yield of the product was 919 mg (69%): 1H NMR δ 2.02 and 2.09 (each s, each 3H, CH3), 2.56 (m, 1H, 2′-HR), 3.04 (m, 1H, 2′-Hβ), 4.18-4.30 (m, 3H, 4′-H and 5′-H), 5.37 (m, 1H, 3′-H), 6.34 (t, 1H, 1′-H, J ) 7.0 Hz), 8.07 and 8.30 (each s, each 1H, 2-H and 8-H), 12.42 (br s, 1H, NH). O6-Benzyl-2′-deoxyinosine. 3′,5′-O-Diacetyl-2′-deoxyinosine (750 mg, 2.23 mmol) was benzylated and deacetylated by the same procedure as reported for the synthesis of O6-benzyl-2′deoxyguanosines from 3′,5′-O-diacetyl-2′-deoxyguanosine (24). Products were separated to obtain O6-benzyl-2′-deoxyinosine (238 mg, 31% yield) and 1-benzyl-2′-deoxyinosine (489 mg, 64% yield). Both products were obtained as a brownish syrup. 1-Benzyl-2′-deoxyguanosine was identified with an authentic specimen prepared by reacting 2′-deoxyinosine with benzyl chloride (25). O6-Benzyl-2′-deoxyinosine: 1H NMR δ 2.33 (m, 1H, 2′-HR), 2.74 (m, 2H, 2′-Hβ), 3.53 (m, 1H, 5′-Ha), 3.62 (m, 2H, 5′-Hb), 3.89 (m, 1H, 4′-H), 4.43 (m, 1H, 3′-H), 5.00 (t, 1H, 5′-OH, J ) 5.8 Hz), 5.33 (d, 1H, 3′-OH, J ) 4.3 Hz), 5.64 (s, 2H, PhCH2), 6.44 (t, 1H, 1′-H, J ) 6.7 Hz), 7.36 (m, 1H, 4-H of PhCH2), 7.41 (m, 2H, 3-H of PhCH2), 7.51 (d, 2H, 2-H of PhCH2,

Terashima et al. J ) 7.2 Hz), 8.56 and 8.60 (each s, each 1H, 2-H and 8-H); FABMS m/z 343 (M + H)+, 227 (base moiety + 2H)+. O6-Benzyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyinosine. O6Benzyl-2′-deoxyinosine (100 mg, 0.29 mmol) was trityrated by the same procedure as already described, and O6-benzyl-5′-O(4,4′-dimethoxytrityl)-2′-deoxyinosine was obtained as a yellowish foam (121 mg, 65% yield): 1H NMR δ 2.37 (m, 1H, 2′-HR), 2.91 (m, 1H, 2′-Hβ), 3.18-3.22 (m, 2H, 5′-H), 3.70 and 3.71 (each s, each 3H, OCH3), 4.02 (m, 1H, 4′-H), 4.50 (m, 1H, 3′-H), 5.39 (d, 1H, 3′-OH, J ) 4.9 Hz), 5.62 (s, 2H, PhCH2), 6.46 (t, 1H, 1′-H, J ) 6.3 Hz), 6.75 (d, 2H, 3-H and 5-H of CH3OPh, J ) 8.8 Hz), 6.79 (d, 2H, 3-H and 5-H of CH3OPh, J ) 9.3 Hz), 7.137.21 (m, 7H, 2-H and 6-H of two CH3OPh, 3-H, 4-H, and 5-H of Ph), 7.30 (d, 2H, 2-H and 6-H of Ph, J ) 7.7 Hz), ca. 7.36 (m, 1H, 4-H of PhCH2), 7.43 (m, 2H, 3-H and 5-H of PhCH2), 7.51 (d, 2H, 2-H and 6-H of PhCH2, J ) 6.8 Hz), 8.47 and 8.49 (each s, each 1H, 2-H and 8-H); FAB-MS m/z 645 (M + H)+, 227 (base moiety + H)+. Preparation of Oligodeoxynucleotides. Oligodeoxynucleotides containing a modified deoxynucleoside were prepared by the phosphoramidite method using an automated solid-phase DNA synthesizer. 3′-O-Diisopropylphosphoramidites of the 2-amino- and/or 5′-OH-protected nucleosides, the precursors of DNA synthesis, were prepared by a previously reported procedure and were used for oligodeoxynucleotide syntheses without further purification (26, 27). The two oligonucleotides prepared were 5′-CCGCTAXCGGGTACCGAGCTCGAAT-3′, in which X is a 2′-deoxyguanosine or O6-benzyl-2′-deoxyguanosine derivative. Oligodeoxynucleotide syntheses were carried out using a 1-µmol column in the trityl-ON mode. Deprotection of the oligomer was carried out with concentrated NH4OH at 55 °C for 15 h. Since the N2-isobutyryl group of O6-alkyl-N2-isobutyryl-2′-deoxyguanosine residues is resistant to concentrated NH4OH treatment under these conditions (23), the oligodeoxynucleotides were further treated with 10% 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in MeOH for 1 week at room temperature. We also prepared oligodeoxynucleotides containing an O6-alkylN2-isobutyryl-2′-deoxyguanosine without DBU treatment. The oligodeoxynucleotides thus obtained were purified by electrophoresis on 15% denatured polyacrylamide gels, and their nucleoside compositions were determined by HPLC after they were hydrolyzed with nuclease P1 and then with alkaline phosphatase (27). Annealing of Oligodeoxynucleotides. Each oligodeoxynucleotide (0.05 OD260 unit) and an equimolar amount of its complement were mixed in 20 µL of a solution containing 10 mM Tris-HCl, 1 mM EDTA, and 0.1 M NaCl (pH 8.0). The solution was heated at 80 °C for 10 min and was allowed to cool slowly to room temperature. Complete annealing was confirmed by electrophoresis on 15% polyacrylamide gels. Purification of Human Methyltransferase. Purification of human methyltransferase was performed as described (27, 28) with slight modifications. Plasmid PHH24 (29) carrying human methyltransferase cDNA was introduced into the methyltransferase-deficient Escherichia coli strain, KT233 (ada-, ogt-). The cells were grown at 37 °C in LB medium containing 50 µg/mL ampicillin and 1 mM isopropyl-β-D-thiogalactopyranoside. Using the harvested cells, a crude extract was prepared by sonication. After ammonium sulfate precipitation, human methyltransferase was purified by column chromatography on DEAE-Sephacel and MonoS columns obtained from Pharmacia LKB Biotechnology Inc. Purification was monitored by measuring methyltransferase activity. The MonoS fraction was used as the human enzyme preparation (27, 28). Inhibition Assay of Methyltransferase Activity by O6Benzylguanine Derivatives. Methyltransferase activity was determined as described (27, 30, 31). One unit of methyltransferase activity represents transfer of 1 pmol of methyl group from methylated DNA, and remaining methyltransferase activity refers to activity remaining after treatment of MGMT with each oligodeoxynucleotide, as previously reported (27). Briefly, the reaction mixture (200 µL) containing 70 mM Hepes-KOH (pH 7.8), 1 mM dithiothreitol, 5 mM EDTA, various amounts of oligodeoxynucleotide, and 0.80-1.55 units of MGMT was incubated at 37 °C for 15 min. Then, 10 µL of PBS solution that

Substrate Specificity of MGMT for O6-Benzylguanines

Chem. Res. Toxicol., Vol. 10, No. 11, 1997 1237

Chart 1. Oligodeoxynucleotides Containing 6BG Derivatives and Their Related Compounds

Figure 1. Inactivation of human MGMT by treatment with ds-oligodeoxynucleotides containing a 6BG. The oligodeoxynucleotide and MGMT were incubated at 37 °C for 15 min. Then, [3H]MNU-treated DNA was added, and the mixture was incubated for another 15 min. Radioactivity of MGMT was determined as described in Experimental Procedures.

contained 3H-labeled N-methyl-N-nitrosourea (MNU)-treated calf thymus DNA (1.5 µg) was added, and the mixture was incubated at 37 °C for another 15 min. The reaction was terminated by the addition of 400 µL of 2 M perchloric acid and 200 µL of 1 mg/mL BSA. The mixture was heated at 70 °C for 60 min to hydrolyze DNA, and methyl-accepting MGMT was collected by centrifugation and washed twice with 1 M perchloric acid at 4 °C. The pellet was dissolved in 10 µL of 0.1 N NaOH and then neutralized with 10 µL of 10 N HCl. Radioactivity was determined in a liquid scintillation counter. The IC50, the concentration of test compound capable of inhibiting the repair activity of MGMT by 50%, was then obtained from the doseresponse curve (31). For the experiment with free base compounds, a test compound dissolved in Me2SO was added instead of oligodeoxynucleotide (final concentration of Me2SO was less than 0.1%). The effect of DNA on inactivation of MGMT by 6BG and 2F-6BG in free base forms was examined by adding calf thymus DNA to the reaction mixture.

Results Preparation of Oligodeoxynucleotides Containing 6BG Derivatives. The sequence of the oligodeoxynucleotide employed and the structures of 6BG derivatives and their related compounds contained in the oligodeoxynucleotide are shown in Chart 1. O6-Methyl2′-deoxyguanosine and O6-benzyl-2′-deoxyguanosine were synthesized as previously reported (23). Three O6(monofluorobenzyl)-2′-deoxyguanosine derivatives, O6-(2fluorobenzyl)-, O6-(3-fluorobenzyl)-, and O6-(4-fluorobenzyl)2′-deoxyguanosines, were synthesized using the same procedure as described for O6-benzyl-2′-deoxyguanosine. O6-Benzyl-2′-deoxyinosine was also synthesized using the same procedure. These nucleosides were protected at the 2-amino and/or 5′-OH groups. 3′-Phosphoramidites of these compounds were synthesized and subjected to oligodeoxynucleotide synthesis using an automated solidphase DNA synthesizer (26, 27). For deprotection, DBU treatment was employed after the standard treatment with concentrated NH4OH since the N2-isobutyryl groups of O6-alkyl-N2-isobutyryl-2′-deoxyguanosine are resistant to treatment with concentrated NH4OH (23). We also prepared oligodeoxynucleotides that contain an N2-isobutyryl-O6-methylguanine or an O6-benzyl-N2-isobutyrylguanine using the standard procedure without DBU treatment. The oligodeoxynucleotides thus obtained were

purified by means of electrophoresis. A portion of each oligodeoxynucleotide was enzymatically digested into nucleosides, and the nucleoside composition was confirmed by HPLC using an authentic specimen (data not shown) (27). Inactivation of Human MGMT by 6BG Derivatives in Oligodeoxynucleotides. The ability of the oligodeoxynucleotides to inhibit human MGMT was determined by measuring the activity remaining after MGMT was preincubated with each oligodeoxynucleotide. As an example, the dose-response curve of the remaining MGMT activity after MGMT was treated with doublestranded (ds) oligodeoxynucleotides containing a 6BG is shown in Figure 1. The IC50 value, the concentration of test compound at which the repair activity of MGMT is inhibited by 50%, was obtained using the dose-response curve, and results are summarized in Table 1. dsOligodeoxynucleotides containing 6MG, 6BG, 2F-6BG, 3F-6BG, 4F-6BG, or 6BH showed almost the same inhibition of MGMT activity with IC50’s of 1.4-3.0 nM. Isobutyrylation at the 2-amino group of 6MG and 6BG reduced inhibition by these derivatives to about one-third and one-seventh, respectively. With single-stranded (ss) oligodeoxynucleotides, inhibition similar to that of dsoligodeoxynucleotides was observed. Using this assay system, the ability of 6BG and 2F-6BG in free base forms to inhibit MGMT was also examined. IC50’s of 6BG and 2F-6BG were 2.7 and 150 µM, respectively, and these values were about 1000 and 100 000 times those of the corresponding derivatives in oligodeoxynucleotides. As a reference, IC50 values of 6AG derivatives obtained by other assay systems are also shown in Table 1. Among the 6BG derivatives and analogues examined, 6BG, 3F6BG, and 4F-6BG showed almost the same ability to inactivate human MGMT (Table 1) whereas 6MG, 2FBG, 6BH, and N2-acetyl-O6-benzylguanine had activities at least 50 times weaker than 6BG (11). Effect of DNA on Inactivation of MGMT by 6BG and 2F-6BG in Free Base Forms. Goodtzova et al. reported that transfer of the alkyl group of free base 6BG by human MGMT was potentiated by DNA (32). Figure 2A shows the enhanced inhibition of MGMT activity by free base 6BG in the presence of 10 µg of calf thymus DNA. However, for F-6BG, this addition of DNA did not result in any enhancement of inhibition (Figure 2B).

Discussion In this study, we used 25-mer oligodeoxynucleotides and non-self-complementary compounds. It has been

1238 Chem. Res. Toxicol., Vol. 10, No. 11, 1997

Terashima et al.

Table 1. Inactivation of Human MGMT by O6-Benzylguanine Derivatives in Oligodeoxynucleotides or in Their Free Base Formsa IC50 in oligodeoxynucleotide (nM) compd

ds

ss

guanine 6MG ib-6MG 6BG ib-6BG 2F-6BG 3F-6BG 4F-6BG 6BH

>50 2.9 ( 0.3 10.0 ( 5.7 2.1 ( 1.4 14.0 ( 7.2 1.4 ( 0.3 3.0 ( 0.6 1.9 ( 0.9 2.6 ( 1.0

3.3 ( 0.8 6.0 ( 1.2 1.6 ( 0.6 14.4 ( 1.4 5.1 ( 1.5 4.5 ( 0.7 3.8 ( 0.7 3.7 ( 1.7

in free base form (µM) this study

literatureb

literaturec 350

2.7 ( 0.2 150 ( 18

0.9 >10 1.3 0.8

0.2 (24)d 0.2 85

a Oligodeoxynucleotides and human MGMT were incubated at 37 °C for 15 min, [3H]MNU-treated DNA was added, and the mixture was incubated for another 15 min. The radioactivity of MGMT was then determined. The IC50 value, the concentration of the test compound at which the repair activity of MGMT is inhibited by 50%, was obtained from the dose-response curve. Data represent mean ( SD (n > 4). b HeLa S3 cell-free extract was incubated with a 6AG derivative for 2 h (11). c HT29 cell-free extract was incubated for 30 min (9). d IC 2 6 50 of N -acetyl-O -benzylguanine using the same conditions as stated in footnote c (12).

Figure 2. Effect of DNA on inactivation of MGMT by 6BG and 2F-6BG in free base forms. (A) 6BG was incubated with MGMT in the presence (4) or absence (b) of 10 µg of DNA. (B) 2F-6BG was incubated with MGMT in the presence (4) or absence (b) of 10 µg of DNA. After the reaction, remaining MGMT activity was determined as described in Figure 1.

reported that MGMT has a preference for 6MG in oligodeoxynucleotides of more than 8-mer (19) or 15-mer (20); therefore, the 25-mer length employed in this experiment was thought to be sufficient for investigating the substrate specificity of MGMT. 6BG in ds-oligodeoxynucleotides inhibited human MGMT as well as did 6MG in ds-oligodeoxynucleotide, as previously reported (22). 2F-6BG, 3F-6BG, 4F-6BG, and 6BH in ss- and ds-oligodeoxynucleotides were also found to have the same ability to inactivate MGMT as 6MG in oligodeoxynucleotides (Table 1). These results are quite different from those obtained with 6BG derivatives in free base forms. In brief, (i) 6MG exhibits a much lower capacity for inactivating human MGMT than does 6BG (7); (ii) modification at the ortho-position of the benzyl group of 6BG resulted in a severe reduction in the ability of this derivative to inactivate human MGMT, whereas modifications at the meta- or ortho-position did

not (14, 15); (iii) 6BH has a much weaker activity than 6BG (9). With regard to this last point, similar evidence revealed that Ada protein repairs O6-methylhypoxanthine (6MH) in ds-oligodeoxynucleotide faster than did 6MG in oligodeoxynucleotides whereas 6MG inactivates MGMT more strongly than does 6MH (21, 33). In oligodeoxynucleotides, the activity of N2-isobutyryl-O6methylguanine (ib-6MG) and O6-benzyl-N2-isobutyrylguanine (ib-6BG) was slightly weaker than that of 6BG (Table 1). These results are also quite different from those obtained with 6BG in free base form where any modifications at the 2-amino group resulted in remarkable reductions in the ability to inactivate MGMT (11, 12). Thus, human MGMT exhibits a relatively low substrate specificity for 6BG derivatives incorporated in oligodeoxynucleotides compared with the free base forms of these derivatives. It was reported that addition of DNA activates the repair of 6BG by recombinant human MGMT (32). MGMT inhibition as a result of this activity was also increased with the addition of 10 µg of calf thymus DNA (Figure 2A). On the other hand, the MGMT-inhibitory activity of 2F-6BG was not enhanced by this addition, as shown in Figure 2B. When 50 µg of DNA was employed, however, MGMT inhibition of 2F-6BG was observed at a 100 µM dose (data not shown). These results indicate that the ortho-position of the benzyl group plays an important role in 6BG derivatives, allowing them to interact with MGMT, and that F substitution at the ortho-position of the benzyl group hinders complex formation with MGMT. For 2F-6BG, addition of 10 µg of DNA may not be sufficient to induce a conformational change in MGMT, allowing it to interact with 2F-6BG; however, addition of an excess of DNA (50 µg) may result in enough of a conformational change to permit the interaction. Unlike the free base form, 2F-6BG in oligodeoxynucleotides may effectively interact with MGMT and inhibit MGMT activity as does 6BG in oligodeoxynucleotides. Further study is required to elucidate the mechanism involved. According to a previous report, the binding affinities of MGMT to ds-DNA and ss-DNA are similar and an identical conformational change may occur (32). The results of the present work, showing that 6BG derivatives of ss- and ds-oligodeoxynucleotides have the same ability to inactivate MGMT, are in agreement with the above report. The crystal structure of the 19 kDa C-terminal domain of the Ada protein was previously determined, and it was

Substrate Specificity of MGMT for O6-Benzylguanines

shown that the cysteine residue of the alkyl acceptor site is buried (34, 35) and that MGMT must undergo a conformational change when carrying out repair of the O6-alkylguanine lesion in DNA. The results obtained by fluorescence analysis and circular dichroism also support this idea of a need for a conformational change in MGMT when binding to DNA or oligodeoxynucleotides (19, 36). In conclusion, MGMT undergoes a conformational change around its active site when binding to oligodeoxynucleotides, and the altered structure of the protein confers a lower substrate specificity for O6-alkylguanines.

Acknowledgment. We would like to thank Professor Y. Kawazoe of Nagoya City University for his helpful advice and encouragement. We also thank Mr. Y. Minoura and Dr. K. Tanabe for the syntheses of oligodeoxynucleotides. One of the authors (I.T.) gratefully acknowledges the postgraduate fellowship from the Japan Society for the Promotion of Science.

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