Deoxyguanosine−C8 Adducts with Heterocyclic Amines - American

May 10, 2006 - Takeji Takamura-Enya,*,† Satoko Ishikawa,‡ Masataka Mochizuki,‡ and Keiji Wakabayashi†. Cancer PreVention Basic Research Projec...
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Chem. Res. Toxicol. 2006, 19, 770-778

Chemical Synthesis of 2′-Deoxyguanosine-C8 Adducts with Heterocyclic Amines: An Application to Synthesis of Oligonucleotides Site-Specifically Adducted with 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine Takeji Takamura-Enya,*,† Satoko Ishikawa,‡ Masataka Mochizuki,‡ and Keiji Wakabayashi† Cancer PreVention Basic Research Project, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan, and Kyoritsu UniVersity of Pharmacy, 5-30 Shibakoen 1-chome, Minato-ku, Tokyo 105-8512, Japan ReceiVed October 23, 2005

Synthesis of 2′-deoxyguanosine-C8 adducts (dG-C8 adducts) with mutagenic/carcinogenic heterocyclic amines (HCAs) was achieved via the Buchwald-Hartwig arylamination reaction. By using tris(dibenzylideneacetone)dipalladium (Pd2dba3) and 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (xantphos) with a cesium carbonate (Cs2CO3) base at a reaction temperature of 100∼120 °C, we obtained derivatives of dG-C8 adducts with 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole (Glu-P-1), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP) in 69%∼97% yield from the cross-coupling of an 8-bromodeoxyguanosine derivative. In the case of PhIP, it was found that dimethyl sulfoxide (DMSO) was the critical solvent for the arylamination reaction. Subsequent deprotection of the resulting dG-C8 adduct derivatives yielded authentic samples of dG-C8 adducts with HCAs. The dG-C8-PhIP adduct was further converted into a suitably protected phosphoramidite derivative for automated DNA synthesis. Synthesis of oligonucleotides wherein PhIP adducted on each G within a triple G sequence in codon 869 (TCC GGG AAC) of rat Apc genes was performed with a modification in the coupling time and deprotection procedures. Introduction Heterocyclic amines (HCAs)1 are widely known food-derived mutagens and/or carcinogens, 10 of which have proven to be carcinogenic in animals (1). From biochemical studies, it is now understood that the initial event in carcinogenesis induced by HCAs is metabolic activation and subsequent covalent bond formation with DNA (1, 2). Exocyclic amino groups of HCAs are generally oxidized to hydroxyamino groups by the predominant action of cytochrome P450 1A1 or 1A2, or both (1-4). Esterification of the resulting hydroxylamines with acetyltransferases or sulfotransferases yields N-acetoxy or N-sulfonyloxy

derivatives, which undergo facile cleavage of N-O bonds; this results in the formation of nitrenium ions which are sufficiently reactive to attack cellular DNA and form DNA adducts. Generally, 2′-deoxyguanosine (dG) residues in DNA are the major targets of the nitrenium ions, thus, forming N-(2′deoxyguanosin-8-yl)-HCA (dG-C8-HCA) adducts as the major products (1, 5-8); in this case, the covalent bond is formed between the C8 position of dG and the N atom of the exocyclic amino groups of HCAs. The N2 position of dG also tends to react with carbenium ions, resonance-stabilized from parent aryl nitrenium ions, to form (2′-deoxyguanosin-N2-yl)HCA (dG-N2-HCA) adducts; however, the amount of this type of adduct formed varies among HCAs (1, 5).

* To whom correspondence should be addressed. E-mail, tenya@ gan2.res.ncc.go.jp; tel, +81-3-3547-5201; fax, +81-3-3543-9305. Present address: e-mail, [email protected]; tel, +81-46-291-3072; fax, +81-46-242-8760. † Cancer Prevention Basic Research Project, National Cancer Centre Research Institute. ‡ Kyoritsu University of Pharmacy. 1 Abbreviations: BINAP, 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl; Bn, benzyl; BOC, t-butoxycarbonyl; tBuOK, potassium t-butoxide; DMF, N,N-dimethylformamide; DMF-DMA, N,N-dimethylformamide dimethyl acetal; dG, 2′-deoxyguanosine; DMSO, dimethyl sulfoxide; DMTr, 4,4′dimethoxytrityl; DMTrCl, dimethoxytrityl chloride; Glu-P-1, 2-amino-6methyldipyrido[1,2-a:3′,2′-d]imidazole; HCAs, heterocyclic amines; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; Trp-P-1, 3-amino-1,4-dimethyl5H-pyrido[4,3-b]indole; LiHMDS, lithium hexamethyldisilazide; MALDITOF, matrix-assisted laser desorption ionization time-of-flight; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline; NBA, 3-nitrobenzoic acid; ODS, octadecylsilyl; PAC, phenoxyacetyl; PAH, polyaromatic hydrocarbon; Pd2dba3, tris(dibenzylideneacetone)dipalladium; PhIP, 2-amino-1-methyl6-phenylimidazo[4,5-b]pyridine; STABASE, 1,1,4,4-tetramethyldisilylazacyclopentane; TBDMS, tert-butyldimethylsilyl; xantphos, 9,9-dimethyl-4,5bis(diphenylphosphino)xanthene; dd, double doublet; dt, double triplet; dq, double quartet; ddd, double double doublet; tt, triple triplet.

Thus far, authentic specimens of these DNA adducts have been obtained in many laboratories by performing biological analogous reactions with N-acetoxyarylamines and nucleosides (1, 5-8). When this method was used, some DNA adducts with HCAs were structurally determined, and oligonucleotides, sitespecifically adducted with HCAs, such as PhIP, were prepared and subjected to MS and NMR analysis in order to understand the higher-order structures of the adducted oligonucleotides (911). In addition, in vivo mutational analysis has been performed on such site-specifically modified oligonucleotides with PhIP (12). These biological mimetic reactions are good tools for determining the chemical properties of the resulting nitrenium ions and their reactivity toward nucleobases. Moreover, the reactions are also useful in understanding the formation of unusual types of DNA adducts (13-16). However, structural determination and oligonucleotide incorporation studies of HCA-DNA adducts involve tedious methodology with several

10.1021/tx050296s CCC: $33.50 © 2006 American Chemical Society Published on Web 05/10/2006

Synthesis of dG-C8 Adducts of HCAs

Figure 1. Various protective groups for arylamination. From top to bottom: STABASE, isobutyryl, DMTr. Bn, benzyl; dR′, silyl-protected deoxynribose.

repetitions of HPLC separation-fractionation because of difficulties in handling unstable hydroxylamines or N-acetoxyamines derived from HCAs and the low yields of adducts formed via nitrenium ion intermediates. Moreover, in the case of the preparation of site-specific adducted oligonucleotides, the number of the most reactive sites (i.e., dG) is strictly limited to one or two (12). To overcome these disadvantages, an efficient and practical method for obtaining HCA-DNA adducts is required. Independent chemical synthesis of the general types of DNA adducts has been recently introduced by several groups and applied to generate site-specifically modified oligonucleotides (17-28). To achieve high yields of DNA adducts, every synthesis method employs the Buchwald-Hartwig arylamination reaction, which is an efficient coupling method for amines and halogenated arenes using Pd as a catalyst (29, 30). When this method was used with 2,2′-bis(diphenylphosphino)-1,1′binaphthyl (BINAP) as a phosphine ligand, synthesis of dGN2 adducts of arylamines was initially reported by De Riccardis et al. (17). The synthesis of cross-linked nucleosides generated from nitrous acid treatment of DNA has been demonstrated using a similar methodology (18). In the case of dG-C8 adducts, Wang and Rizzo reported that an 8-bromodeoxyguanosine derivative was coupled with IQ-type HCAs on Pdcatalyzed arylamination, where lithium hexamethyldisilazide (LiHMDS) was used as a base and a 1,1,4,4-tetramethyldisilylazacyclopentane (STABASE) group was applied for the N2 protection of dG (19). More recently, Johnson’s group successfully prepared a dG-C8 adduct with PhIP and incorporated it into oligonucleotides, thereby revealing the tolerance of isobutyryl or STABASE protection of the N2 position to the conditions of Pd(0), 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (xantphos), and LiHMDS in toluene (see Figure 1) (20). For successful preparation of a wide variety of dG-C8 adducts, it is important to select appropriate protective groups at the N2 position of dG. Wang and Rizzo also showed the efficiency of bis(t-butoxycarbonyl) (BOC) protection for this purpose with biphenyl-based phosphine ligands (19). This protective group may allow the synthesis of many types of DNA adducts; however, it might require severe conditions, such as 50% trifluoroacetic acid, to achieve deprotection. More recently,

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Rizzo’s group presented a method in which N2 protection was not required and extended its adaptability to various mutagens. Further, they applied the method for synthesis of oligonucleotides site-specifically adducted with IQ (24). An isobutyryl group, which is commonly used for the protection of the N2 position of dG in solid-phase oligonucleotide synthesis, could also be employed; however, the coupling reaction required a long time (2 or 3 days), and in some cases, only moderate yields of the coupling compounds were obtained (20, 22). Practical protection of the N2 position of dG can be ensured using a 4,4′-dimethoxytrityl (DMTr) group, as shown by Gillet and Scharer in the synthesis of several dG-C8 adducts from an 8-bromo-dG derivative (Figure 1) (21, 22). In addition, our earlier studies showed the compatibility of DMTr protection for synthesis of dG-C8 adducts from an 8-amino-dG derivative coupled with bromoarenes; this was developed as an inverse strategy for the method outlined above (23). We have also shown that high yields are obtained with the proposed approach through the use of xantphos as a critical phosphine ligand (23, 24). Among earlier reports on dG-C8 adduct synthesis, in the case of HCAs, adducts of IQ-type compounds and PhIP were successfully prepared. In this report, we further explored efficient and universal methods for preparing dG-C8 adducts from N2-dimethoxytrityl-protected 8-bromo-dG derivatives with several types of HCAs, such as, IQ, Glu-P-1, Trp-P-1, MeIQx, and PhIP (Figure 2). We also described here successful preparation of oligonucleotides with dG-C8-PhIP in the rat Apc gene by solid-phase oligonucleotide chemistry. Among these HCAs-derived DNA adducts, only PhIP was reported to induce unique -1 frameshift mutation in Apc genes when subjected to rats. Oligonucleotids modifed with PhIP will be a valuable tool to understand the relationship between structure alteration and mutation induced by PhIP.

Experimental Procedures General. All solvents were of organic synthesis grade and used without further purification. HPLC was performed using a Shimadzu LC10Avp system equipped with a Shimadzu SPD-M10Avp photodiode array detector. All the HPLC eluents applied were of HPLC grade. NMR measurements were conducted using a JEOL JNMA400 spectrometer, and the J values are given in Hz. EI-MS, FABMS, and high-resolution mass spectrometry (HRMS) were performed on a JEOL JMSR-700 mass spectrometer. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)-MS analysis was carried out on an Applied Biosystems Voyager System 2073 and electron spray ionization-mass spectrometry on a Waters ZQ 2000 mass spectrometer. Melting temperature analysis was performed with an Agilent 8453 UV-visible spectrophotometer equipped with an 8909A Perilier temperature controller (Agilent Technologies, Inc. (Palo Alto, CA)). Coupling Procedure. Nucleoside derivative 1 (0.1 mmol), arylamine (0.1 mmol), Pd2dba3 (0.01 mmol), xantphos (0.03 mmol), and Cs2CO3 (0.2 mmol) were dissolved in toluene and stirred at 100 °C for 4∼8 h. After the starting material disappeared on TLC, the solvent was evaporated and chromatographed to afford the desired compound 2. N2-(O6-Benzyl-3′,5′-di-t-butyldimethylsilyl-N2-4,4′-dimethoxytrityl-2′-deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo[4,5f]quinoxaline (2c). 1H NMR (CDCl3) δ: 8.60 (s, 1H), 7.78 (d, J ) 8.8, 1H), 7.50 (d, J ) 8.8, 1H), 7.35-7.20 (m, 14H), 6.77 (dt, J ) 9.7, 2.6, 4H), 6.72 (t, J ) 7.3, 1H), 5.97 (s, 1H), 4.78 (s, 2H), 4.70 (br, 1H), 3.97-3.92 (m, 1H), 3.78-3.75 (m, 8H), 3.75-3.73 (m, 1H), 3.73 (s, 3H), 2.73 (s, 3H), 0.94 (s, 9H), 0.86 (s, 9H), 0.14 (s, 6H), 0.02 (s, 3H), 0.00 (s, 3H). 13C NMR (CDCl3): 157.8, 155.9, 154.7, 152.9, 152.8, 152.6, 152.2, 146.2, 143.6, 138.5, 137.3, 136.9, 130.9, 130.6, 130.0, 128.8, 128.1, 127.8, 127.4, 127.0, 126.3, 122.3, 112.7, 111.4, 108.1, 86.9, 82.6, 73.3, 70.1, 67.4, 63.4, 55.1, 28.6,

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Figure 2. Structure of the selected heterocyclic amines.

25.9, 25.8, 22.3, -4.55, -5.16, -5.28. FAB-HRMS (3-nitrobenzoic acid (NBA)) m/z: calcd for C61H75N10O6Si2 (M + H), 1099.54095; found 1099.5269. N3-(O6-Benzyl-3′,5′-di-t-butyldimethylsilyl-N2-4,4′-dimethoxytrityl-2′-deoxyguanosin-8-yl)-3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (2d). 1H NMR (CDCl3) δ: 7.53 (d, J ) 7.9, 1H), 7.41 (d, J ) 7.6, 3H), 7.32-7.17 (m, 14H), 7.36-7.17 (m, 17H), 6.79 (d, J ) 8.7, 4H), 6.58 (br, 1H), 5.97 (br, 1H), 4.71 (br, 3H), 4.05 (br, 1H), 3.97 (s, 1H), 3.82-3.77 (m, 1H), 3.77 (br, 1H), 3.75 (s, 6H), 3.72 (br, 1H), 2.54-2.10 (m, 6H), 0.96 (s, 9H), 0.76 (s, 9H), 0.15 (s, 3H), 0.14 (s, 3H), -0.05 (s, 3H), -0.09 (s, 3H). 13C NMR (CDCl3): 157.9, 155.3, 153.1, 146.5, 141.5, 138.8, 137.5, 130.1, 128.9, 127.9, 127.5, 127.2, 127.1, 126.3, 125.6, 122.1, 120.1, 119.6, 112.7, 111.9, 110.2, 86.7, 82.9, 73.2, 70.2, 66.8, 64.1, 55.1, 25.9, 25.8, 22.7, 18.3, 18.1, 14.2, -4.41, -4.56, -5.33, -5.38. FAB-HRMS (NBA) m/z: calcd for C63H77N8O6Si2 (M + H), 1097.55051; found 1097.9054. N2-(O6-Benzyl-3′,5′-di-t-butyldimethylsilyl-N2-4,4′-dimethoxytrityl-2′-deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (2e). 1H NMR (CDCl3) δ: 8.84 (d, J ) 3.7, 1H), 8.58 (d, J ) 8.3, 1H), 7.91 (d, J ) 8.8, 1H), 7.62 (d, J ) 8.8, 1H), 7.41-7.20 (m, 16H), 6.78 (d, J ) 8.5, 4H), 6.71 (t, J ) 6.7, 1H), 6.03 (s, 1H), 4.86 (s, 2H), 4.73 (br, 1H), 3.97 (s, 1H), 3.82-3.77 (m, 8H), 3.57-3.42 (m, 1H), 2.06 (s, 3H), 0.96 (s, 9H), 0.88 (s, 9H), 0.16 (s, 6H), 0.04 (s, 3H), 0.02 (s, 3H). 13C NMR (CDCl3): 157.9, 156.2, 154.0, 153.6, 152.6, 151.4, 147.8, 146.1, 144.9, 138.3, 136.9, 130.2, 130.3, 129.5, 128.8, 128.4, 128.2, 127.6, 127.5, 126.4, 122.5, 120.4, 118.1, 112.8, 112.2, 104.8, 87.0, 82.5, 73.3, 70.2, 67.2, 63.4, 55.1, 35.4, 31.6, 29.7, 28.6, 25.9, 25.8, 22.7, 18.5, 18.1, 14.2, 14.1, -4.50, -5.14, -5.25. FAB-HRMS (NBA) m/z: calcd for C61H74N9O6Si2 (M + H), 1084.5301; found 1084.4768. N2-(O6-Benzyl-3′,5′-di-t-butyldimethylsilyl-N2-4,4′-dimethoxytrityl-2′-deoxyguanosin-8-yl)-2-amino-6-methyldipyrido[1,2-a: 3′,2′-d]imidazole (2f). 1H NMR (CDCl3) δ: 8.49-8.45 (m, 1H), 8.21-8.19 (m, 1H), 7.37-7.12 (m, 17H), 6.81-6.72 (m, 4H), 6.10 (s, 1H), 5.04 (br, 2H), 4.72 (br, 2H), 4.53 (br, 1H), 4.06 (dd, J ) 7.2, 4.2, 1H), 3.89-3.82 (m, 2H), 3.78 (s, 6H), 2.66 (s, 3H), 1.010.81 (m, 18H), 0.33-0.03 (m, 12H). 13C NMR (CDCl3): 158.4, 157.8, 157.4, 156.1, 152.7, 151.6, 151.5, 147.7, 147.4, 146.1, 145.3, 139.8, 139.3, 138.4, 138.3, 138.1, 136.8, 136.6, 132.2, 130.0, 129.8, 129.0, 128.9, 128.1, 127.7, 127.6, 127.5, 127.3, 126.3, 121.7, 120.6, 113.0, 112.8, 111.5, 110.3, 99.9, 87.7, 84.9, 82.3, 81.4, 73.3, 72.7, 70.2, 67.5, 63.5, 62.9, 55.1, 38.9, 35.5, 31.6, 25.9, 22.6, 18.3, 18.1, 17.1, 14.2, -4.65, -5.19, -5.29, -5.35. FAB-HRMS (NBA) m/z: calcd for C61H74N9O6Si2 (M + H), 1084.5301; found 1084.5085.

N2-(O6-Benzyl-3′,5′-di-t-butyldimethylsilyl-N2-4,4′-dimethoxytrityl-2′-deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (2g). 1H NMR (CDCl3) δ: 8.35 (d, J ) 2.0, 1H), 7.60-7.55 (m, 2H), 7.47-7.41 (m, 3H), 7.36-7.17 (m, 17H), 6.78-6.72 (m, 4H), 6.68 (t, J ) 7.1, 1H), 5.98 (s, 1H), 4.78 (s, 2H), 4.70 (br, 1H), 3.96-3.92 (m, 1H), 3.78-3.75 (m, 8H), 3.723.69 (m, 1H), 3.65 (s, 3H), 0.93 (s, 9H), 0.86 (s, 9H), 0.13 (s, 6H), 0.01 (s, 3H), 0.00 (s, 3H). 13C NMR (CDCl3): 157.9, 156.1, 155.1, 152.3, 151.7, 147.4, 146.1, 140.3, 138.6, 138.3, 136.4, 130.6, 130.0, 128.8, 128.3, 128.1, 127.6, 127.5, 127.2, 127.0, 126.4, 125.9, 112.7, 112.2, 87.0, 82.6, 70.1, 67.5, 63.4, 55.1, 28.6, 25.9, 25.8, 18.3, 18.1, 14.2, -4.55, -5.18, -5.29. FAB-HRMS (NBA) m/z: calcd for C63H76N9O6Si2 (M + H), 1110.54571; found 1010.1091. Detritylation Procedure. To a solution of 2 in methanol/CH2Cl2 was added 3% trichloroacetic acid in CH2Cl2. After confirmation of disappearance of the initial spot, the reaction mixture was then added to a buffer solution of ammonium formate (pH 6.8). The organic layer was extracted with brine, dried over Na2SO4, and evaporated. The residues were chromatographed on silica gel to give 3 in 70%∼80% yields. N2-(O6-Benzyl-3,5′-di-t-butyldimethylsilyl-2′-deoxyguanosin8-yl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (3c). 1H NMR (CDCl3) δ: 8.63 (s, 1H), 7.83 (d, J ) 8.8, 1H), 7.59 (d, J ) 6.8, 2H), 7.54 (d, J ) 8.8, 1H), 7.41-7.28 (m, 3H), 6.77 (t, J ) 7.3, 1H), 5.60 (s, 2H), 4.78 (s, 2H), 4.81-4.76 (m, 1H), 4.65 (s, 2H), 3.99-3.90 (m, 2H), 3.79-3.73 (m, 4H), 3.59-3.52 (m, 1H), 2.73 (s, 3H), 2.12 (dq, J ) 13.0, 3.3, 1H), 0.95 (s, 9H), 0.88 (s, 9H), 0.15 (s, 6H), 0.04 (s, 3H), 0.03 (s, 3H). 13C NMR (CDCl3): 156.9, 156.3, 153.2, 152.6, 147.3, 137.4, 136.8, 130.9, 130.5, 130.0, 128.2, 128.0, 127.7, 126.3, 122.6, 111.4, 109.4, 87.0, 82.7, 73.2, 68.1, 63.4, 35.8, 28.6, 25.9, 22.5, 18.4, 18.1, -4.50, -5.12, -5.25. FAB-HRMS (NBA) m/z: calcd for C40H57N10O4Si2 (M + H), 797.4102; found 797.4137. N3-(O6-Benzyl-3′,5′-di-t-butyldimethylsilyl-2′-deoxyguanosin8-yl)-3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (3d). 1H NMR (CDCl3) δ: 7.62-6.60 (m, 10H), 5.50-5.25 (m, 2H), 4.85-4.45 (m, 2H), 4.25-3.90 (m, 2H), 3.50-3.25 (m, 1H), 2.75-2.20 (m, 9H), 0.98 (s, 9H), 0.84 (s, 9H), 0.18 (s, 3H), 0.16 (s, 3H), 0.06 (br, 6H). 13C NMR (CDCl3): 153.3, 146.7, 141.8, 137.6, 134.8, 139.2, 128.3, 127.6, 125.6, 120.5, 119.9, 113.3, 112.1, 110.3, 87.1, 82.5, 73.2, 70.2, 67.7, 64.1, 41.2, 26.1, 18.6, 18.3, -4.15, -4.34, -5.02. FAB-HRMS (NBA) m/z: calcd for C42H58N8O4Si2 (M + H), 795.4198; found 795.4136. N2-(O6-Benzyl-3′,5′-di-t-butyldimethylsilyl-2′-deoxyguanosin8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (3e). 1H NMR (CDCl3) δ: 8.84 (dd, J ) 4.1, 1.5, 1H), 8.58 (d, J ) 8.3, 1H), 7.91

Synthesis of dG-C8 Adducts of HCAs (d, J ) 8.8, 1H), 7.60 (d, J ) 8.8, 1H), 7.54 (d, J ) 7.1, 2H), 7.45-7.20 (m, 5H), 6.74 (t, J ) 7.3, 1H), 5.58 (s, 2H), 4.81-4.75 (m, 1H), 4.67 (br, 2H), 3.99-3.95 (m, 1H), 3.92 (dd, J ) 16.6, 6.3, 1H), 3.78 (s, 3H), 3.76 (dt, J ) 10.1, 4.5, 1H), 3.58-3.50 (m, 1H), 2.13 (dq, J ) 13.0, 3.3, 1H), 3.57-3.42 (m, 1H), 2.06 (s, 3H), 0.95 (s, 9H), 0.88 (s, 9H), 0.15 (s, 6H), 0.04 (s, 3H), 0.03 (s, 3H). 13C NMR(CDCl3): 157.3, 155.2, 153.7, 152.6, 151.8, 147.8, 144.6, 136.8, 129.7, 128.8, 128.3, 127.9, 127.8, 122.5, 120.5, 117.8, 112.3, 106.1, 87.0, 82.5, 73.1, 67.2, 63.4, 35.7, 29.7, 28.6, 26.0, 25.9, 18.4, 18.1, 14.2, -4.40, -4.50, -5.15, -5.25. FAB-HRMS (NBA) m/z: calcd for C40H56N9O4Si2 (M + H), 782.3993; found 782.3959. N2-(O6-Benzyl-3′,5′-di-t-butyldimethylsilyl-2′-deoxyguanosin8-yl)-2-amino-6-methyldipyrido[1,2-a:3′,2′-d]imidazole (3f). 1H NMR (CDCl3) δ: 8.42 (br, 1H), 8.27-8.15 (m, 2H), 7.50 (d, J ) 7.1, 2H), 7.37-7.12 (m, 17H), 6.72 (br, 2H), 6.30 (s, 1H), 5.56 (br, 2H), 4.72 (br, 2H), 4.64 (br, 1H), 4.05 (br, 1H), 3.91 (d, J ) 5.1, 2H), 3.02 (s, 1H), 2.65 (s, 3H), 2.30 (s, 1H), 0.90 (s, 9H), 0.80 (s, 9H), 0.12 (s, 6H), 0.04 (s, 3H), -0.02 (s, 3H). 13C NMR (CDCl3): 158.6, 157.4, 153.3, 147.7, 147.3, 145.5, 139.8, 136.8, 132.2, 129.8, 128.2, 127.7, 127.5, 121.7, 120.6, 112.4, 111.7, 110.4, 87.4, 84.6, 72.0, 67.9, 62.9, 55.1, 38.8, 31.9, 29.7, 25.9, 25.8, 18.4, 18.1, 17.1, 14.2, -4.51, -4.60, -5.28, -5.31. FAB-HRMS (NBA) m/z: calcd for C40H56N9O4Si2 (M + H), 782.3993; found 782.4252. N2-(O6-Benzyl-3′,5′-di-t-butyldimethylsilyl-2′-deoxyguanosin8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (3g). 1H NMR (CDCl3) δ: 8.34 (d, J ) 1.4, 1H), 7.57 (d, J ) 7.4, 1H), 7.41 (d, J ) 7.3, 1H), 7.47-7.28 (m, 9H), 7.36-7.17 (m, 17H), 6.73 (t, J ) 7.2, 1H), 5.46 (s, 1H), 4.78 (s, 2H), 4.79-4.76 (m, 1H), 4.63 (s, 2H), 3.98-3.92 (m, 1H), 3.92 (dd, J ) 16.7, 6.2, 1H), 3.76-3.71 (m, 1H), 3.63 (s, 3H), 3.57-3.50 (m, 1H), 2.11 (ddd, J ) 12.9, 6.7, 3.1, 1H), 0.95 (s, 9H), 0.88 (s, 9H), 0.15 (s, 6H), 0.04 (s, 3H), 0.03 (s, 3H). 13C NMR (CDCl3): 157.1, 156.6, 153.1, 152.8, 152.1, 146.5, 140.2, 138.4, 136.5, 130.8, 128.8, 128.4, 128.3, 127.9, 127.3, 127.0, 125.8, 112.5, 109.1, 87.0, 82.6, 73.1, 67.7, 63.5, 60.3, 35.7, 28.0, 25.9, 25.8, 18.4, 18.1, 14.2, -4.55, -5.20, -5.29. FAB-HRMS (NBA) m/z: calcd for C42H57N9O4Si2 (M + H), 808.4150; found 808.4149. Deprotection Procedure for O6-Benzyl and Silyl Protective Groups of 3. The benzyl group at the O6 position of 3 was removed by catalytic hydrogenation with Pd black/H2. Typically, 3 (0.1 mmol) was dissolved in THF. Pd black (20∼30 mg) was added into the solution and stirred under a balloon filled with H2 gas until the initial spot on TLC disappeared. After the reaction was completed, the catalyst was removed by filtration and washed with THF. The filtrate was further treated with 300 µL of a triethylamine‚ trihydrogen fluoride (TEA‚3HF) complex. After overnight reaction, the solvent was evaporated to dryness and the resulting residue was subjected to octadecylsilyl (ODS) column chromatography to yield 5 almost quantitatively. N2-(2′-Deoxyguanosin-8-yl)-2-amino-3,8-dimethylimidazo[4,5f]quinoxaline (5c) (28). 1H NMR (DMSO-d6) δ: 10.76 (s, 1H), 8.79 (s, 1H), 7.93 (d, J ) 8.8, 1H), 7.85 (d, J ) 8.8, 1H), 6.54 (t, J ) 7.4, 1H), 6.30 (s, 2H), 4.52-4.45 (m,1H), 3.81 (dd, J ) 7.9, 5.0 1H), 3.73 (s, 3H), 3.69 (dd, J ) 11.5, 4.9, 1H), 3.54 (dd, J ) 11.6, 5.0, 1H), 3.35-3.35 (m, 1H), 2.77 (s, 3H), 2.73 (s, 3H), 2.05 (ddd, J ) 13.0, 6.8, 2.6, 1H). 13C NMR (DMSO-d6): 154.3, 153.5, 152.4, 151.4, 150.1, 149.0, 144.2, 136.7, 130.7, 129.1, 124.1, 122.4, 112.4, 109.8, 87.2, 82.0, 71.3, 62.3, 36.2, 28.5, 22.1. FAB-HRMS (NBA) m/z: calcd for C21H23N10O4 (M + H), 479.1903; found 479.1930. N3-(2′-Deoxyguanosin-8-yl)-3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (5d). 1H NMR (DMSO-d6) δ: 10.69 (s, 1H), 7.98 (br, 1H), 7.46 (br, 1H), 7.40 (br, 1H), 7.22 (br, 1H), 6.33 (br, 3H), 5.11 (br, 1H), 4.36 (br, 1H), 3.77 (br, 1H), 3.63 (dd, J ) 11.6, 3.8, 1H), 3.55-3.47 (m, 1H), 3.07-3.09 (m, 1H), 2.28 (s, 3H), 2.051.95 (m, 1H). 13C NMR (DMSO-d6): 157.9, 153.1, 141.5, 138.8, 130.1, 127.8, 127.5, 127.2, 127.1, 126.3, 125.6, 122.0, 120.0, 119.6, 112.7, 86.7, 82.9, 73.2, 70.2, 66.8, 31.6. FAB-HRMS (NBA) m/z: calcd for C23H25N8O4 (M + H), 477.1998; found 477.2076.

Chem. Res. Toxicol., Vol. 19, No. 6, 2006 773 N2-(2′-Deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (5e) (19, 31). 1H NMR (DMSO-d6) δ: 10.98 (s, 1H), 8.79 (d, J ) 2.7, 1H), 8.69 (d, J ) 7.8, 1H), 7.86 (d, J ) 9.0, 1H), 7.75 (d, J ) 9.0, 1H), 7.55 (dd, J ) 8.4, 4.2, 1H), 6.48 (s, 2H), 6.46 (t, J ) 7.6, 1H), 4.46 (t, J ) 2.9, 1H), 3.78 (d, J ) 2.7, 1H), 3.70 (s, 3H), 3.65 (dd, J ) 11.7, 5.1, 1H), 3.50 (q, J ) 5.8, 1H), 2.05-2.01 (m, 1H). 13C NMR (DMSO-d6): 153.4, 153.2, 152.5, 148.8, 148.4, 147.5, 143.7, 129.6, 128.2, 121.4, 120.7, 117.3, 113.3, 103.7, 87.3, 81.9, 71.2, 62.2, 35.8, 28.6. FAB-HRMS (NBA) m/z: calcd for C21H22N9O4 (M + H), 464.1795; found 464.1870. N2-(2′-Deoxyguanosin-8-yl)-2-amino-6-methyldipyrido[1,2-a: 3′,2′-d]imidazole (5f) (Partially Identified in Ref 32). 1H NMR (DMSO-d6) δ: 10.59 (br, 1H), 9.68 (br, 1H), 8.63 (br, 1H), 8.15 (br, 1H), 8.09 (br, 1H), 7.26 (br, 1H), 6.85 (br, 1H), 6.36 (s, 2H), 6.26 (s, 1H), 5.55 (br, 1H), 5.20 (br, 1H), 4.39 (br, 1H), 3.87 (br, 1H), 3.72 (d, J ) 16.8, 2H), 2.75 (s, 1H), 2.49 (s, 3H), 2.03 (s, 1H). 13C NMR (DMSO-d6): 155.6, 152.7, 149.7, 148.8, 146.6, 141.8, 139.4, 130.9, 129.1, 127.3, 126.8, 122.4, 112.6, 110.6, 87.1, 83.2, 71.5, 61.9, 55.1, 16.5. FAB-HRMS (NBA) m/z: calcd for C21H22N9O4 (M + H), 464.1795; found 464.1885. N2-(2′-Deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (5g) (33, 34). 1H NMR (DMSO-d6) δ: 8.28 (d, J ) 1.8, 1H), 7.88 (d, J ) 1.8, 1H), 7.63 (d, J ) 7.4, 2H), 7.37 (d, J ) 7.6, 1H), 7.35 (d, J ) 7.6, 1H), 7.25 (t, J ) 7.3, 1H), 6.38 (t, J ) 7.3, 1H), 6.25 (s, 2H), 4.36 (t, J ) 2.9, 1H), 3.67 (dd, J ) 7.9, 5.1, 1H), 3.55 (dd, J ) 12.8, 6.1, 1H), 3.53 (s, 3H), 3.40 (dd, J ) 11.4, 5.7, 1H), 3.18-3.12 (m, 1H), 1.93 (ddd, J ) 12.9, 6.9, 2.5, 1H). 13C NMR (DMSO-d6): 154.1, 152.7, 152.6, 149.6, 148.8, 139.4, 137.9, 129.8, 128.9, 126.7, 125.8, 113.4, 108.6, 87.3, 82.0, 71.2, 62.3, 28.1. FAB-HRMS (NBA) m/z: calcd for C23H24N9O4 (M + H), 490.1951; found 490.1743. N2-(N2-Dimethylaminomethylene-3′,5′-di-t-butyldimethylsilyl2′-deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (6g). Compound 5g (200 mg) was dissolved in a solvent of N,N-dimethylformamide dimethyl acetal (DMF-DMA, 10 mL) with N,N-dimethylformamide (DMF, 2 mL) and stirred at room temperature. As the reaction proceeded, the solid materials were separated from the solution. The precipitate was collected and washed with methanol (72%). 1H NMR (DMSO-d6) δ: 9.25 (br, 1H), 8.50 (s, 1H), 8.33 (s, 1H), 7.56 (d, J ) 7.3, 2H), 7.47-7.40 (m, 3H), 6.73 (t, J ) 7.2, 1H), 4.67 (s, 1H), 3.94 (br, 1H), 3.823.77 (m, 2H), 3.61 (s, 3H), 3.57-3.47 (m, 1H), 3.15 (s, 3H), 3.10 (s, 1H), 2.15-2.07 (m, 1H), 0.93 (s, 9H), 0.86 (s, 9H), 0.13 (s, 6H), 0.02 (s, 3H), 0.00 (s, 3H). 13C NMR (DMSO-d6): 157.0, 156.1, 154.6, 151.5, 148.3, 144.8, 140.1, 138.5, 131.0, 128.9, 127.3, 127.0, 125.6, 112.3, 86.9, 82.6, 73.3, 67.9, 63.7, 41.3, 36.8, 35.1, 27.9, 25.9, 25.8, 18.4, 18.0, -4.42, -4.49, -5.06, -5.26. FAB-HRMS (NBA) m/z: calcd for C38H57N10O4Si2 (M + H), 773.4102; found 773.4642. N2-(N2-Dimethylaminomethylene-2′-deoxyguanosin-8-yl)-2amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (7g). To a stirred suspension of 6g in THF was added triethylamine trihydrogenfluoride (800 µL). A clear solution was obtained, and it was stirred for 24 h. The resulting precipitate was then recovered by filtration and washed with methanol to afford pure 7g (85%). 1H NMR (DMSOd6) δ: 8.37 (s, 1H), 8.30 (d, J ) 1.7, 1H), 7.90 (d, J ) 1.5, 1H), 7.64 (d, J ) 7.3, 2H), 7.37 (t, J ) 7.6, 2H), 7.26 (t, J ) 7.3, 1H), 6.49 (t, J ) 7.3, 1H), 4.41 (s, 1H), 3.70 (q, J ) 4.1, 1H), 3.57 (d, J ) 13.3, 6.5, 1H), 3.54 (s, 3H), 3.43 (dd, J ) 10.8, 5.4, 1H), 3.02 (s, 3H), 2.91 (s, 3H), 2.85-2.77 (m, 1H), 2.02-1.92 (m, 1H). 13C NMR (DMSO-d6): 157.6, 156.1, 155.2, 152.1, 150.3, 147.5, 145.2, 139.4, 137.8, 130.1, 128.8, 127.2, 126.7, 125.6, 113.4, 112.7, 87.2, 82.6, 71.5, 62.2, 45.7, 36.8, 34.5, 28.1. FAB-HRMS (NBA) m/z: calcd for C26H29N10O4 (M + H), 545.2373; found 545.2404. N-[N2-Dimethylaminomethylene-5′-(4,4′-dimethoxytrityl)-2′deoxyguanosin-8-yl]-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (8g). To a suspended solution of 7g (80 mg) in lutidine/ DMF (1:1) were added triethylamine (3 equiv) and 4,4dimethoxytrityl chloride (DMTrCl, 3 equiv). After the addition, the reaction mixture was allowed to warm at 80 °C and stirred for 2 h. Additional DMTrCl (3 equiv) was then added, and the mixture was

774 Chem. Res. Toxicol., Vol. 19, No. 6, 2006 further stirred at 80 °C for 2 h. The solvent was evaporated under reduced pressure, and the residue was purified by column chromatography on silica gel to yield 8g (80%).1H NMR (DMSO-d6) δ: 11.53 (s, 1H), 8.49-8.36 (m, 2H), 8.04 (d, J ) 8.4, 1H), 7.807.72 (m, 2H), 7.52-7.01 (m, 13H), 6.83 (d, J ) 8.9, 2H), 6.72 (d, J ) 8.9, 1H), 6.65 (d, J ) 8.8, 1H), 6.60 (t, J ) 7.7, 1H), 4.654.5 (m, 1H), 3.95-3.80 (m, 1H), 3.70-3.0 (m, 19H), 2.25-2.05 (m, 1H). 13C NMR (DMSO-d6): 157.5, 157.4, 154.4, 148.2, 147.2, 144.6, 139.9, 135.3, 135.2, 129.8, 129.3, 129.1, 128.7, 128.6, 127.3, 127.2, 127.1, 126.6, 126.1, 112.5, 87.1, 85.0, 79.7, 71.0, 62.1, 59.6, 54.9, 54.8, 54.7, 34.5, 28.2, 28.0, 20.7, 14.1. FAB-HRMS (NBA) m/z: calcd for C47H47N10O6 (M + H), 847.3680; found 847.3593. N-{3′-[(N,N-Diisopropylamino)-2-cyanoethoxyphosphinyl]N2-dimethylaminomethylene-5′-(4,4′-dimethoxytrityl)-2′-deoxyguanosin-8-yl}-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (9g). A premixture solution of N,N-diisopropylammonium tetrazolide (1.2 equiv) and N,N,N′,N′-2-cyanoethyl tetraisopropyl phosphorodiamidite (1.2 equiv) was added to a solution of 6g (80 mg) in methylene chloride. The reaction mixture was stirred for 2 h, and the solvent was removed. The residue was purified by column chromatography on neutral silica gel and eluted with chloroform containing 2% triethylamine to yield 9g (73%). 1H NMR (DMSOd6) δ: 8.35-8.26 (m, 2H), 7.51 (d, J ) 7.3, 2H), 7.39 (t, J ) 7.6, 2H), 7.32-6.95 (m, 12H), 6.70-6.55 (m, 5H), 5.05-4.87 (m, 1H), 4.20-4.02 (m, 1H), 3.81-3.10 (m, 19H), 2.98 (s, 3H), 2.91 (s, 3H), 2.61 (tt, J ) 12.6, 5.9, 1H), 2.54 (t, J ) 6.2, 1H), 2.42-2.20 (m, 1H), 1.32-1.01 (m, 12H). 13C NMR (DMSO-d6): 158.2, 157.1, 156.0, 154.7, 151.3, 148.7, 148.6, 144.8, 144.7, 144.6, 140.0, 138.4, 135.8, 131.0, 130.9, 130.0, 129.9, 129.8, 128.8, 128.1, 128.0, 127.5, 127.4, 127.3, 127.0, 126.5, 126.4, 125.4, 117.5, 116.7, 115.1, 115.0, 112.7, 112.1, 86.0, 85.9, 82.2, 81.8, 70.4, 64.2, 68.7, 58.1, 55.1, 45.8, 45.2, 43.3, 41.1, 36.4, 35.0, 34.4, 29.6, 27.9, 24.7, 23.3, 22.9, 20.1, 14.8. 31P NMR (DMSO-d6): 149.4, 149.1. FAB-HRMS (NBA) m/z: calcd for C56H63N12O7 (M + H), 1047.4658; found 1047.4556. Preparation of Oligonucleotides 10g, 11g, and 12g Containing 5g. Oligonucleotides 5′-d(TCCGG-5g-AAC) 10g, d(TCCG-5gGAAC) 11g, and d(TCC-5g-GGAAC) 12g were prepared on a 1 µmol scale using general isobutyryl- or benzoyl-protected cyanoethyl phosphoramidites and modified phosphoramidite 5g. The manufacturer’s standard synthesis protocol was followed, except for a prolonged coupling reaction time of 15 min. Deprotection of the oligonucleotides was achieved by treatment with concentrated ammonia containing 0.25 M mercaptoethanol at 50 °C for 17 h. Purification was performed with a Sep-PAK C18 column followed by HPLC (column, Kratos Unison US-C18 3.0 mm × 150 mm; column temperature, 50 °C; eluent, a linear gradient of 3%-40% acetonitrile at a flow rate of 0.5 mL/min for 30 min) to yield 10g (16.5% yield), 11g (11.2% yield), and 12g (15.8% yield) (matrixassisted laser desorption ionization time-of-flight (MALDI-TOF)MS analysis calculated for 10g, 11g, and 12g: [M - 1]-: m/z 2943.59; found 10g, 2943.49; 11g, 2944.53; 12g, 2943.57). The modified oligonucleotides (5 µg) were dissolved in 50 µL of Tris HCl buffer (10 mM, pH 7.8) containing NaCl (20 mM), MgCl2 (2 mM), and dithiothreitol (0.2 mM). The DNA was digested to deoxynucleosides by the addition of phosphosdiesterase I (1.1 units) and bacterial alkaline phosphatase (1.5 units) at 37 °C for 6 h. Enzymatic digestion yielded 5g accompanied with the four normal nucleosides in the correct stoichiometric ratio (HPLC conditions: column, Cosmosil AR-II ODS column 4.6 mm × 250 mm; eluent, 50 mM ammonium acetate pH4.5 to acetonitrile gradient of 2%10% over 18 min, 10%-100% over 15 min, and then isocratic over 10 min at a flow rate 1 mL/min). Tm Measurement of Normal and Modified Oligonulcotides. For melting temperature analysis, all oligonucleotide (TCCGGGAAC, 10g, 11g, 12g) solutions were prepared in a phosphate buffer containing 1 M NaCl, 10 mM Na2HPO4, and 1 mM Na2EDTA at pH 7.0. Melting curves of DNA duplexes were obtained for the solutions containing a 1:1 strand ratio with an increase in temperature from 20 to 90 °C at a rate of 0.2 °C/min.

Takamura-Enya et al. Scheme 1. Cross-Coupling Reaction of 8-Bromo-2′-deoxyguanosine Derivative 1 and Aminoarenes

Table 1. Arylamination Reaction of 8-Bromonucleoside Derivative 1 with Selected Arylamines to Yield Compound 2a (Scheme 1) compound

Ar-NH2

yield (%)b

2a 2b 2c 2d

2-aminofluorene 1-aminopyrene MeIQx Trp-P-1

52 55 0 0

a Reaction conditions: Pd dba (10 mol %), xantphos (30 mol %), tBuOK 2 3 (0.1 mmol), 1 (0.1 mmol), ArNH2(0.1 mmol). b Isolation yields.

Table 2. Reaction Conditions to Yield 2c from 8-Bromodeoxyguanosine Derivative 1 with MeIQx (Scheme 1) entry

Pd

ligand

base

temp. (°C)

yield (%)

1 2 3 4 5

Pd2dba3 Pd2dba3 Pd2dba3 Pd(OAc)2 Pd2dba3

xantphos xantphos BINAP xantphos xantphos

tBuOK tBuOK tBuOK tBuOK Cs2CO3

100 80 100 100 100

0 trace 0 0 97

Results Synthesis of dG-C8-HCA Adducts. We have already reported an efficient method for performing coupling reactions between several brominated polyaromatic hydrocarbons (PAHs) and an 8-aminonucleoside derivative under the conditions of xantphos, Pd2dba3 (10 mol %), and tBuOK at 80∼100 °C (23). These conditions were initially applied for the preparation of dG-C8-HCA adducts from an 8-bromo-dG derivative 1. In this method, the phosphine ligandsBINAPsused in Gillet and Scharers’ method, is simply replaced with xantphos (Scheme 1) (21). As shown in Table 1, the coupling reactions of 2-aminofluorene and 1-aminopyrene proceeded smoothly to yield the desired coupling compounds dG-C8-aminofluorene derivative 2a and dG-C8-aminopyrene derivative 2b in 52% and 55% yields, respectively. The yields were lower than those in an 8-amino-nucleoside approach where 2a and 2b were obtained in 75% and 87% yields, respectively (23). In the case of MeIQx and Trp-P-1, numerous reaction products were formed and none of the coupling products could be ascertained from the TLC analyses. Neither the change in the reaction temperature from 100 to 80 °C nor a change of the catalytic systems (Pd(OAc)2 or BINAP) mediated the couplings (Table 2, entries 1-4). In contrast, it was found that the change in the base from tBuOK to a weaker base Cs2CO3 was extremely effective in yielding dG-C8-MeIQx derivative 2c; however, the reaction time extended to 6 h (Table 2, entry 5). Other structurally different HCAs, such as Trp-P-1, IQ, and Glu-P-1, were also found to afford the corresponding cross-coupling dG-C8 adducts 2d-f in good yields when Cs2CO3 was used as a base (Table 3). The coupling yield of IQ with 8-bromoguanine derivatives 1 is comparable to those achieved by Rizzo’s group (19). However, in the case of PhIP, no coupling compound was obtained under the applied conditions (Table 3, compound 2g).

Synthesis of dG-C8 Adducts of HCAs

Chem. Res. Toxicol., Vol. 19, No. 6, 2006 775

Table 3. Arylamination Reaction to Yield Compound 2 from 8-Bromo-2′-deoxyguanosine Derivative 1 with Selected HCAsa compound

ArNH2

yield (%)b

2d 2e 2f 2g

Trp-P-1 IQ Glu-P-1 PhIP

69 77 92 trace

a Reaction conditions: Pd dba (10 mol %), xantphos (30 mol %), Cs CO 2 3 2 3 (0.1 mmol), 1 (0.1 mmol), ArNH2(0.1 mmol) in toluene; reaction temperature, 100 °C. b Isolation yields.

Table 4. Effect of Solvents of the Arylamination Reaction to Yield 2ga entry

solvent

temp (°C)

yield (%)

1 2 3 4 5 6

toluene xylene dioxane pyridine DMF DMSO

100 120 100 100 120 120

trace trace trace trace ∼ 30 77

a Reaction conditions: Pd dba (10mol %), xantphos (30 mol %), Cs CO 2 3 2 3 (0.1 mmol), 1 (0.1 mmol), PhIP (0.1 mmol).

Scheme 2. Deprotection Procedure to Yield 5

Because of the low solubility of toluene in the applied solvent, the solid white PhIP material was constantly observed in the reaction vessel during the course of the reaction. This was overcome by changing the solvent (Table 4). The yields of a derivative of dG-C8-PhIP adduct 2g increased significantly when a polar solvent such as DMF or DMSO was used at a higher reaction temperature of 120 °C. Among the solvents tested in this study, DMSO was found to be the most effective, affording dG-C8-PhIP derivative 2g in 77% yield (Table 4, entry 6), which was higher than the yields of the coupling reaction using an N2-isobutylryl-8-bromo-dG derivative as a starting material. The deprotection of HCA-derived dG-C8 adduct derivatives 2c-g was performed in a general manner (Scheme 2). Glycosidic bonds of dG-C8 adducts 2c-g were found to be relatively resistant to acid treatment since they were distinct from those of PAH-derived dG-C8 adducts (23). Therefore, the deprotection of a 4-dimethoxytrityl group at the N2 position was performed with 3% trichloroacetic acid in CH2Cl2 at room temperature without yielding any depurination products. The detritylation products 3 were generally obtained in around 70%∼80% yields. The quantitative removal of a benzyl group

and the subsequent desilylation reaction were performed with Pd black/H2 followed by treatment with a TEA‚3HF complex in THF. After chromatography, using ODS as the stationary phase, the desired dG-C8 authentic samples 5c-g were obtained almost quantitatively. Synthesis of Oligonucleotides Site-Specifically Adducted with PhIP. The successful synthesis of dG-C8 adduct standards also led us to attempt the synthesis of oligonucleotides containing a dG-C8-PhIP adduct 5g by a general solid-phase oligonucleotide synthetic approach (Scheme 3). For oligonucleotide synthesis with dG-C8-PhIP 5g, a 3′-phosphoramidite derivative of suitably protected dG-C8-PhIP adduct was required. However, in the large-scale preparation of a dG-C8PhIP adduct, the low solubility of dG-C8-PhIP 5g in an organic solvent would make it difficult to prepare the desired phosphoramidite with a high yield. Therefore, for convenience in handling, we used silyl-protected dG-C8-PhIP 4g as the starting material. Johnson’s group initially prepared the phosphoramidite derivative of dG-C8-PhIP 5g with isobutyryl protection of the N2 position, which had been preattached prior to the arylamination reaction (20). Although N2-isobutyryl protection of dG-C8-PhIP 5g was reported to be efficient for the preparation of the desired amidite, isobutyrylation of the coupling compound 5g was found to be tedious, and only a small amount of the desired products could be recovered in our hands. Therefore, we selected an amidine protection of N2 because it was already found some 8-amino-dG derivatives can be efficiently converted to amidine-protected derivatives (25). The treatment of DMF-DMA with this silyl-protected dG-C8PhIP 4g with a small amount of DMF immediately afforded the desired N2-amidine-protected dG-C8-PhIP 6g, which was separated out from the reaction as a precipitate. The silylprotective groups of 6g were cleaved by treatment with TEA‚ 3HF in THF to afford the desired product 7g, which was again precipitated from the solution during the reaction with a purity that was sufficient for the subsequent tritylation reaction. A general 5′-OH tritylation procedure of DMTrCl/pyridine was not successful; this was probably due to the extremely low solubility of 7g in pyridine. Elevated temperature and/or addition of 4-(dimethylamino)pyridine afforded none of the trytilation products. However, we found that the treatment of DMTrCl in a 50% solution of lutidine/DMF at 80 °C efficiently dimethoxytritylated the 5′-OH position along with a small amount of the concomitant 3′-O-,5′-O-bis-dimethoxytritylation product. The phosphitylation of 3′-OH of the resulting 8g was performed with 2-cyanoethyl-N,N,N′,N′-tetraisopropyl phosphorodiamidite and N,N-diisopropylammonium tetrazolide in CH2Cl2 to afford the desired phosphoramidite of the dG-C8-PhIP adduct derivative 9g in 73% yield. Phosphoramidite 9g was dissolved in acetonitrile and applied to automatic solid-phase DNA synthesis using standard acyl (isobutyryl or benzoyl)-protected phosphoramidites. A sequence of oligonucleotides was selected for the rat Apc gene, which is the target gene for -1 frameshift mutation when F344 rats were treated with PhIP (35). Oligonucleotides with a PhIP adduction on each guanine within a triple G sequence in codon 869 (TCC GGG AAC)s10 g, 11g, and 12gswere considered for synthesis. The solid-phase oligonucleotide synthesis protocol was slightly modified by the general preparation method. As mentioned previously, DNA adducts derived from HCAs were tolerant to 3% trichloroacetic acid, and thus, the detritylation procedure was retained as the general method. The coupling time was prolonged from 35 s to 15 min. After the completion of the oligonucleotide synthesis, the solid support and the base-labile

776 Chem. Res. Toxicol., Vol. 19, No. 6, 2006

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Scheme 3. Synthetic Method of Oligonucleotides Containing 5g

protective groups were removed by 28% ammonia at 50 °C in the presence of 0.25 M mercaptoethanol in order to avoid oxidative degradation of the dG-C8 adduct in alkaline conditions (36). The resulting ‘trityl-on’ oligonucleotide was then purified using an ODS column cartridge. The recovered tritylon oligonucleotide was then treated with 80% acetic acid for the final detritylation, and the resulting tritanol was extracted with ethyl acetate. After neutralization by the addition of triethylamine to the aqueous phase, the solvent was evaporated

Figure 3. HPLC profile of the reaction mixture of the synthesis of 11g after cartridge column purification. The arrow shows the peak of 11g.

to dryness. The HPLC analyses of the residues showed the presence of the desired oligonucleotides with dG-C8-PhIP along with the failure of the oligonucleotide without PhIP modification in either of the cases of the modified oligonucleotides 10g, 11g, or 12g (Figure 3, see Supporting Information for PAGE analysis of purified oligonucleotides 10g, 11g, or 12g). The UV spectra of these oligonucleotides showed the presence of an absorption peak within 350∼450 nm, which was derived from the PhIP moiety (Figure 4). MALDI-TOF-MS analyses of purified oligonucleotides 10g, 11g, and 12g showed that their molecular ion peaks agreed closely with the theoretical value. The coupling yields of the modified amidite 9g were estimated to be below 30%∼50%, as ascertained from the UV absorbance of the cleaved DMTr protective group during oligonucleotide synthesis, thereby resulting in a moderate final yield in the oligonucleotides. The HPLC analyses of enzymatic digestion of oligonucleotides 10g, 11g, and 12g also showed the presence of the dG-C8-PhIP 5g peak along with the peaks of normal nucleotides (Figure 5, see Supporting Information for LC-ESI-MS analysis of the enzymatic digestion product of oligonucleotide 11g). The UV spectrum of the digested peak at a retention time of 30 min was also confirmed to be coincident with those of the authentic dG-C8-PhIP 5g samples.

Discussion

Figure 4. UV spectrum of 11g.

In the present study, we developed the general procedure to obtain dG-C8 adduct standard samples derived from several HCAs. In this methodology, we used xantphos as a phosphine ligand with Cs2CO3 as a base. DMSO was also found to be an alternative solvent for the cross-coupling reaction between PhIP and 8-bromo-2′-deoxyguanosine derivative 1. Other phosphine ligands such as BINAP or biphenyl-type ligands have not been

Synthesis of dG-C8 Adducts of HCAs

Chem. Res. Toxicol., Vol. 19, No. 6, 2006 777

Figure 5. HPLC profile of 11g after enzymatic digestion. The arrows show the peak of 5g.

Figure 6. Synthesized phosphoramidite derivatives of dG-C8-PhIP 5g.

tested thus far; however, based on the published literature, it might be possible for them to lead to successful coupling to some extent. In a work by the Rizzo group, a STABASE protective group at the N2 position of silylated deoxyguanosine was utilized to afford dG-C8 adducts from IQ-type HCAs. Subsequently, the group discovered that N2 protection was not necessary for the coupling reactions of an 8-bromodeoxyguanosine derivative and arylamines (19, 25). In both cases, they used a stronger LiHMDS base, which might restrict the use of a wide variety of HCA substrates, since the use of tBuOK in the present study failed to yield the desired coupling compound. The synthesis of oligonucleotides containing a site-specific adduct with PhIP was successful in a moderate yield. Johnson’s group initially prepared phosphoramidite derivatives of dGC8-PhIP with isobutyryl protection of the N2 position, which had been preattached to dG prior to the arylamination reaction (20). Here, we presented an efficient preparation method for amidine protection of the N2 position of dG, which could easily be introduced and cleaved under mild conditions. The same protection strategy was reported by Rizzo’s group for the synthesis of phosphoramidites of MeIQx. All of these phosphoramidites were stable enough for oligonucleotide synthesis, leading to the successful preparation of modified oligonucleotides with wide varieties of sequence contexts. Although phosphoramidites of other types of HCAs might be prepared with similar protective groups for oligonucleotide synthesis, advanced synthetic methods should be explored for each dGC8-HCAs adducts. We found our tritylation methodology of DMTrCl in lutidine/DMF was effective only for PhIP adduct 7g but not for N2-amidine-protected dG-C8-Glu-P-1. It was found that the latter compound was not able to be tritylated by the general method of DMTrCl in pyridine with/without 4-(dimethylamino)pyridine. In our proposed strategy, O6 and N8 protection was not necessary; however, the low solubility in the solvent for oligonucleotide synthesis may decrease the coupling yield of the phosphoramidite derived from dG-C8-PhIP. To overcome the low solubility of dG-C8-PhIP in the commonly used organic solvents, we further synthesized two dG-C8-PhIP amidites 13 and 14, attached with an O6-benzyl protective group that was expected to be removed after oligonucleotide synthesis (Figure 6, see Supporting Information for the synthesis of amidites, 13 and 14 in Figure 6). Two phosphoramidites bearing O6-benzyl protection were prepared, and the N8 position of both compounds was differently protected by a BOC or a benzyl protective group. A BOC group at the N8 position of dG-C8PhIP was found to be extremely labile due to a biguanide moiety and was easily cleaved after treatment with triethylamine in methanol for 2 days. It was possible to convert each phosphoramidite thus obtained into the desired oligonucleotides, although the conventional procedures (Pd black/ammonium formate, Pd/C

Table 5. Tm Values of Modified Oligonucleotides 10g, 11g, and 12ga entry

oligonucleotidesb

Tm (°C)

1 2 3 4

TCC GGG AAT TCC G*GG AAT10g TCC GG*GAAT11g TCC GGG*AAT12g

42.8 27.2 29.4 27.1

aT m values were measured with 1:1 ratio with the complement oligonucleotide AGGCCCTTA. b G* ) dG-C8-PhIP 5g.

with H2, and Pd/C with cyclohexadiene) for further deprotection of the O-benzyl groups failed. It is noteworthy that, in each case with N8 protection by a BOC or benzyl group, the protection of N2 with a phenoxyacetyl (PAC) group and dimethoxytritylation at 5′-OH occurred easily. The latter was performed using a general tritylation methodsdimethoxytrityl tetrafluoroborate (DMTrBF4) with lithium carbonate (Li2CO3) in lutidine at room temperature. This was a milder condition as compared to that without N8 protection (37). In the case of a dG-C8-IQ adduct without N8 protection, PAC protection reportedly failed (25). Codons 635, 869, and 1413 in the nontranscribed Apc genes are hot spots for -1 frameshift mutation induced by PhIP in rat colon tumors (35). These three codons have common sequences of 5′-GGGA-3′ (35). It is estimated that the basic mechanism for -1 frameshift involves the looping out of dGC8-PhIP from a DNA strand due to preferential syn conformation with respect to the glycosidic bond (10, 12). The Tm values of the three oligonucleotides 10g, 11g, and 12g were measured and found to be around 27 °C; this was drastically different from the temperature of 48 °C for the original nonmodified duplex (Table 5). This result indicates that the higher-order structures in a duplex DNA containing 5g have changed from the original structure. We are currently undertaking studies to elucidate the relationships between the alteration in the DNA structures modified by the mutagen, sequences, and mutations. Acknowledgment. This study was supported by a Grantin-Aid for Cancer Research from the Ministry of Health, Labor, and Welfare, Japan. Supporting Information Available: LC-ESI-MS analysis of enzymatic hydrolysates of11g, PAGE analysis of 10g, 11g, and 12g, UV melting curves of oligonucleotides 10g and TCCGGGAAT, synthesis methods for amidites 13 and 14, and 1H NMR charts of 2c-g and 5c-g. This material is available free of charge via the Internet at http://pubs.acs.org.

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