O6-Allyl Protected Deoxyguanosine Adducts of ... - ACS Publications

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Chem. Res. Toxicol. 2001, 14, 708-719

O6-Allyl Protected Deoxyguanosine Adducts of Polycyclic Aromatic Hydrocarbons as Building Blocks for the Synthesis of Oligonucleotides Heiko Kroth,† Haruhiko Yagi,† Jane M. Sayer,† Subodh Kumar,‡ and Donald M. Jerina*,† Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, The National Institutes of Health, Bethesda, Maryland 20892, and Environmental Toxicology and Chemistry, Great Lakes Center, State University of New York College at Buffalo, 1300 Elmwood Avenue, Buffalo, New York 14222 Received December 27, 2000

We describe a synthetic strategy for the preparation of oligonucleotides using N2-alkylated and O6-allyl protected deoxyguanosine phosphoramidite building blocks derived from cis- and trans-opened (()-7β,8R-dihydroxy-9R,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and (()7β,8R-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and from trans-opened (()3R,4β-dihydroxy-1R,2R-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene. The appropriately blocked phosphoramidite building blocks were obtained as mixtures of the cis- and trans-opened diol epoxide adducts upon initial reaction of the diol epoxides with O6-allyl-3′,5′-di-O-(tertbutyldimethylsilyl)-2′-deoxyguanosine. Key to the present approach is the removal of the O6allyl protecting group utilizing a palladium catalyst prior to release of the constructed oligonucleotide with ammonia from the solid support. The methodology described enables a very convenient access to oligonucleotides containing cis- and trans-N2-deoxyguanosine adducts of polycyclic aromatic hydrocarbons in different sequence contexts.

Introduction Polycyclic aromatic hydrocarbons (PAH)1 are ubiquitous environmental pollutants which are formed on incomplete combustion of fossil fuel and organic matter and are carcinogenic in rodent bioassays (1). Benzo[a]pyrene (B[a]P), one of the most widely studied PAH, and benzo[c]phenanthrene (B[c]Ph) are metabolized to vicinal bay- or fjord-region diol epoxides (DE) which form covalent adducts with DNA, resulting in their carcinogenic activity (2, 3). These DE are metabolically formed as pairs of diastereomers in which the benzylic hydroxyl group is either cis (DE-1) or trans (DE-2) to the epoxide oxygen. Covalent binding of these DE to the exocyclic amino groups of the purine bases, deoxyadenosine (dAdo) and deoxyguanosine (dGuo), in DNA occurs by either cisor trans-opening of the oxirane ring and gives rise to stable adducts thought to be responsible for cell transformation (4, 5). The most tumorigenic of the four stereoisomeric B[a]P DE (6), the (+)-(R,S,S,R)-DE-2, forms predominately trans-N2-dGuo adducts on reaction with DNA and induces mutations at GC base pairs (2, 7). In contrast, all four stereoisomers of B[c]Ph DE have * To whom correspondence should be addressed. Phone: (301) 4964560. Fax: (301) 402-0002. E-mail: [email protected]. † National Institute of Diabetes and Digestive and Kidney Diseases. ‡ State University of New York College at Buffalo. 1 Abbreviations: PAH, polycyclic aromatic hydrocarbon; B[a]P, benzo[a]pyrene; B[c]Ph, benzo[c]phenanthrene; DE, diol epoxide; DE1, diol epoxide in which the epoxide oxygen and the benzylic hydroxyl group are cis; DE-2, diol epoxide in which the epoxide oxygen and the benzylic hydroxyl group are trans; CPG, controlled-pore glass; DMA, N,N-dimethylacetamide; DMTr, 4,4′-dimethoxytrityl; DMAP, 2,6-(dimethylamino)pyridine; HRMS, high-resolution mass spectrometry; allyl, propen-1-yl; TEA, triethylamine; DCI, 4,5-dicyanoimidazole.

been found to be highly mutagenic and tumorigenic in bacterial and mammalian systems (2, 8, 9) and form a large proportion of N6-dAdo adducts (10). To determine the relationship between the biological consequences of these adducts and their structural differences, it is of utmost importance to synthesize modified oligonucleotides containing stereochemically defined adducts in different sequence contexts. Direct synthetic strategies (11) which consist of alkylation of DNA with enantiopure DE were used for the synthesis of short model oligonucleotides containing dGuo adducts of B[a]P DE-2 for study of their structural (12-14) and biological properties (15-17) and dAdo adducts of B[c]Ph DE-2 for structural investigations (18). Modification of bases in the oligonucleotide by this approach is random and therefore only one dGuo or dAdo residue in the sequence is allowable in order to prevent complex reaction mixtures. A recent report established that it is possible to separate individual dGuo modified oligonucleotides obtained on direct reaction of a DE with an oligonucleotide containing a -GGG- sequence. However, the direct synthetic approach required the use of enantiopure DE (19). A total synthetic approach, though more labor intensive, avoids many of the problems associated with the direct alkylation route in that racemic DE can be used for the synthesis of appropriately blocked phosphoramidites for use in semiautomated DNA synthesis (20). Although mixtures of two diastereomeric oligonucleotides are produced, such mixtures have generally proven to be relatively easy to separate. The total synthetic approach has been extensively used in the preparation of oligonucleotides containing dAdo adducts (21-23) for structural (24-26) and biological (27-31) investigations.

10.1021/tx0002637 CCC: $20.00 © 2001 American Chemical Society Published on Web 05/12/2001

Synthesis of DNA Containing Diol Epoxide Adducts at dGuo

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Figure 1. Synthesis of O6-allyl protected and deprotected N2-dGuo adducts derived from cis- and trans-opening of B[c]Ph DE-2; (i) DMA (90-100 °C); (ii) HPLC; (iii) Ac2O; (iv) HPLC.

However, only a few attempts have been made to prepare dGuo adducts of sterically crowded PAH such as B[a]P or B[c]Ph by total synthetic methods (32, 33) because these methods have been plagued by low yields (32) on coupling of the hydrocarbon and nucleoside derivatives. Recently, our laboratory has developed a very convenient method for the synthesis of cis- and trans-opened N2dGuo adducts of B[a]P DE in which the O6-position of the dGuo is blocked by the allyl protecting group (34). In the present report, we describe the synthesis of O6allyl protected N2-dGuo adducts which are formed by opening of B[c]Ph DE-2 (Figure 1) in order to demonstrate the general applicability of the direct epoxide opening method (34) for the synthesis of N2-dGuo adducts of a highly hindered PAH DE. We further demonstrate the smooth conversion of the fully protected N2-dGuo adducts of B[a]P DE-1, B[a]P DE-2, and B[c]Ph DE-2 to their O6-allyl protected N2-dGuo phosphoramidites (Figure 2) and their incorporation into various oligonucleotides (Tables 1, 2, and 3). Selective and complete cleavage of the O6-allyl protecting group (Figure 3) from the modified oligonucleotides prior to ammonolysis from the CPG solid support was achieved by employing a palladium catalyst.

Experimental Procedures Caution: Benzo[a]pyrene 7,8-dihydrodiol, benzo[c]phenanthrene 3,4-dihydrodiol, and diol epoxides DE-1 and DE-2 are carcinogenic and mutagenic and must be handled carefully in accordance with NIH guidelines (35). General Methods. 1H NMR spectra were measured on a Varian Gemini-2000 at 300 MHz in acetone-d6. Chemical shifts (δ) are reported in ppm and coupling constants (J) are in hertz. For adducts and related compounds, singly primed numbers are used for the protons of the deoxyribose moiety (1′-5′) and the purine protons are doubly primed (8′′). For the vinyl protons of the allyl protecting group, Hv designates the vinyl hydrogen adjacent to the methylene and Hc as well as Ht are the terminal vinyl protons cis and trans to Hv. 31P NMR spectra were recorded on the same spectrometer with CD3CN as solvent and 85% H3PO4 as external standard. High-resolution mass spectroscopy (HRMS) was performed using a JEOL SX 102a mass spectrom-

eter. All chemicals used, except for 4,5-dicyanoimidazole (DCI, Glen Research, Sterling, VA), were purchased from Aldrich Chemical Co. (Milwaukee, WI), were of highest quality, and were used without further purification. Methylene chloride, pyridine and acetonitrile were dried prior to use over 4 Å molecular sieves. 4,4′-Dimethoxytrityl chloride was thoroughly dried in vacuo before use. O6-Allyl-3′,5′-di-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine 1 was prepared from 2′-deoxyguanosine in 40% overall yield (36). The diol epoxides of B[a]P and B[c]Ph were synthesized from their dihydrodiols by published methods (37, 38). The diastereomeric mixtures of the N2-dGuo adducts N2-[10-(7,8,9-triacetoxy-7,8,9,10-tetrahydrobenzo[a]pyrenyl)]-O6-allyl-3′,5′-di-O-(tert-butyldimethlsilyl)-2′-deoxyguanosine 9 derived from cis-opening of (()-B[a]P DE-1 and N2[10-(7,8,9-triacetoxy-7,8,9,10-tetrahy-drobenzo[a]pyrenyl)]-O6allyl-3′,5′-di-O-(tert-butyldimethlsilyl)-2′-deoxyguanosine 10 derived from trans-opening of (()-B[a]P DE-1 as well as N2[10-(7,8,9-triacetoxy-7,8,9,10-tetrahydrobenzo[a]pyrenyl)]-O6-allyl-3′,5′-di-O-(tert-butyldimethlsilyl)-2′-deoxyguanosine 11 derived from cis-opening of (()-B[a]P DE-2 and N2-[10-(7,8,9triacetoxy-7,8,9,10-tetrahydrobenzo[a]pyrenyl)]-O6-allyl-3′,5′-diO-(tert-butyldimethlsilyl)-2′-deoxyguanosine 12 derived from trans-opening of (()-B[a]P DE-2 were synthesized as previously reported (34; see Scheme 1 therein for structures). Column chromatography was conducted on silica gel 60 (220-440 mesh) from Fluka (Buchs, Switzerland). The Higgins DNA HPLC column used for purification of oligonucleotides is available from Thomson Instrument Co. (Clear Brook, VA). Diastereomeric N2-[1-(2,3,4-Trihydroxy-1,2,3,4-tetrahydrobenzo[c]phenanthryl)]-O6-allyl-3′,5′-di-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine Adducts Derived from cis (3)- and trans (4)-Opening of (()-B[c]Ph DE-2 (2). A mixture of 1 (2.16 g, 4.04 mmol) and 2 (288 mg, 1.01 mmol) in dry DMA (2.5 mL) was heated at 90-100 °C for 5 h under nitrogen. The resulting clear yellow solution was cooled, and the DMA was removed under reduced pressure. The resulting yellow oil was purified by chromatography on silica gel first eluted with EtOAc-hexane (3:1) followed by 5-10% MeOH in CH2Cl2 to yield 1.4 g (54% recovery) of unreacted 1 followed by 350 mg (43%) of the crude mixture of the two cis- (3) and two transN2-dGuo (4) adducts. This adduct mixture was separated by HPLC using a 5 µm, 10 × 250 mm Axxiom silica gel column eluted at 5 mL/min with EtOAc-hexane (3:1). The two transN2-dGuo diastereomers (mixture 4) had TR(early) ) 8.1 min and TR(late) ) 8.3 min while the two cis-N2-dGuo diastereomers

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Figure 2. Synthesis of the diastereomeric mixtures of the O6-allyl-N2-dGuo phosphoramidite building blocks derived from cis- (25) and trans-opening of B[c]Ph DE-2 (26), from cis- (27) and trans-opening of B[a]P DE-1 (28), and from cis- (29) and trans-opening of B[a]P DE-2 (30); (i) HF; (ii) DMTr-Cl; (iii) 2-cyanoethyl tetraisopropylphosphorodiamidite. (mixture 3) eluted as a single peak with TR ) 11.6 min; total trans-dGuo diastereomers, 154 mg (19%), and total cis-dGuo diastereomers, 55 mg (7%). N2-[1-(2,3,4-Triacetoxy-1,2,3,4-tetrahydrobenzo[c]phenanthryl)]-O6-allyl-3′,5′-di-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine Adducts (6a) and (6b) Derived from trans Opening of (()-B[c]Ph DE-2. A mixture of 4 (120 mg, 148 µmol), acetic anhydride (1.5 mL), and DMAP (20 mg) were combined in pyridine (5 mL) and stirred overnight at room temperature. After evaporation of the solvents under reduced pressure, the remaining residue was purified by chromatography on silica gel using CH2Cl2-MeOH (98:2) to yield 134.2 mg (97%) of the mixture of acetylated diastereomers. For analytical purposes, 25 mg of the mixture of trans-N2-dGuo diastereomers were separated by HPLC using a 5 µm, 10 × 250 mm Axxiom silica gel column eluted at 5 mL/min with EtOAc-hexane (1: 3). The trans-N2-dGuo adducts had TR(early) ) 18.0 min and TR(late) ) 19.6 min (early diastereomer 6a, 10.8 mg; late diastereomer 6b, 8.7 mg). N2-[1S-(2R,3S,4R-Triacetoxy-1,2,3,4-tetrahydrobenzo[c]phenanthryl)]-O6-allyl-3′,5′-di-O-(tert-butyldimethylsilyl)2′-deoxyguanosine Adducts (6a). 1H NMR δ 8.77 (d, 1 H12, J ) 8.8), 8.17-8.08 (m, 1H8′′, 1H6), 8.00 (dd, 1H9, J ) 8.8, 1.5), 7.90-7.84 (m, 1H7,1H8), 7.59 (d, 1H5, J ) 7.8), 7.58-7.54 (m, 1H10), 7.32-7.27 (m, 1H11), 6.44 (d, 1H4, J ) 8.3), 6.39-6.26 (m, 1H1, 1H2, 1H1′), 6.20-6.14 (m, 1Hv(allyl)), 6.03 (dd, 1H3, J ) 8.3, 2.4), 5.43 (d, 1Ht(allyl), J ) 16.6), 5.21 (d, 1Hc(allyl), J ) 10.3), 5.09-5.05 (m, 2H, CH2(allyl)), 4.67 (br s, 1H3′), 3.94 (br s, 1H4′), 3.84 (br s, 2H5′,5′), 2.41 (m, 1H2′), 2.20-1.89 (3s, 9H, OAc), 1.55 (m, 1H2′), 0.87 (tert-butyl methyls), 0.11-0.04 (Si bonded methyls); LRMS (FAB+) m/z 940 ([M + H]+); HRMS calcd for C49H65O10N5Si2Cs 1072.3324, found 1072.3459 N2-[1R-(2S,3R,4S-Triacetoxy-1,2,3,4-tetrahydrobenzo[c]phenanthryl)]-O6-allyl-3′,5′-di-O-(tert-butyldimethylsilyl)-

2′-deoxyguanosine Adducts (6b). 1H NMR δ 8.70 (d, 1 H12, J ) 8.8), 8.11-8.07 (m, 1H8′′, 1H6), 8.00 (d, 1H9, J ) 8.8), 7.897.84 (m, 1H7, 1H8), 7.59-7.53 (m, 1H5, 1H10), 7.24-7.19 (m, 1H11), 6.48 (d, 1H4, J ) 8.8), 6.38-6.22 (m, 1H1, 1H2, 1H1′), 6.156.10 (m, 1Hv(allyl)), 6.02 (dd, 1H3, J ) 8.8, 2.7), 5.41 (d, 1Ht(allyl), J ) 17.6), 5.20 (d, 1Hc(allyl), J ) 10.7), 4.98-4.92 (m, 2H, CH2(allyl)), 4.60 (br s, 1H3′), 3.89-3.67 (m, 1H4′, 2H5′,5′), 2.322.28 (m, 1H2′), 2.20-1.96 (3s, 9H, OAc), 0.89-0.69 (tert-butyl methyls), 0.12-0.37 (Si bonded methyls); LRMS (FAB+) m/z 940 ([M + H]+); HRMS calcd for C49H65O10N5Si2Cs 1072.3324, found 1072.3475. N2-[1-(2,3,4-Triacetoxy-1,2,3,4-tetrahydrobenzo[c]phenanthryl)]-O6-allyl-3′,5′-di-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine Adducts (5a) and (5b) Derived from cis Opening of (()-B[c]Ph DE-2. Acetylation of 46 mg (57 mmol) of 3 as described provided 52.3 mg (98%) of the mixture of acetylated diastereomers. For analytical purposes, 16 mg of the mixture of cis-N2-dGuo diastereomers was separated by HPLC using a 5 µm, 10 × 250 mm Axxiom silica gel column eluted at 5 mL/ min with EtOAc-hexane (1:3). The cis-N2-dGuo adducts had TR(early) ) 14.4 min and TR(late) ) 16.2 min (early diastereomer 5a, 6.2 mg; late diastereomer 5b, 5.6 mg). N2-[1S-(2S,3R,4S-Triacetoxy-1,2,3,4-tetrahydrobenzo[c]phenanthryl)]-O6-allyl-3′,5′-di-O-(tert-butyldimethylsilyl)2′-deoxyguanosine Adducts (5a). 1H NMR δ 8.70-8.68 (m, 1 H12), 8.08-7.98 (m, 1H8′′, 1H6), 7.87-7.77 (m, 1H7, 1H8, 1H9), 7.51 (d, 1H5, J ) 8.8), 7.38 (p-t, 1H10, J ) 7.8), 7.09-7.04 (m, 1H1, 1H11), 7.04 (m 1H1), 6.91 (d, 1H4, J ) 7.8), 6.50-5.78 (m, 1H2, 1H1′, 1Hv(allyl)), 5.69 (dd, 1H3, J ) 7.8), 5.39-5.28 (m, 1Ht(allyl), 1Hc(allyl)), 4.78-4.55 (m, 2H, CH2(allyl), 1H3′), 4.06-3.68 (m, 1H4′, 2H5′,5′), 2.68-2.60 (m, 1H2′), 2.16-1.86 (3s, 9H, OAc), 0.95-0.87 (tert-butyl methyls), 0.19-0.04 (Si bonded methyls); LRMS (FAB+) m/z 940 ([M + H]+); HRMS calcd for C49H65O10N5Si2Cs 1072.3324, found 1072.3458.


Figure 3. Selective cleavage of the O6-allyl protecting group and complete deblocking is illustrated for oligonucleotides containing N2-dGuo adducts derived from cis- and trans-opening of B[c]Ph DE-2 and B[a]P DE-2; (i) Pd(PPh3)4; (ii) NH4OH. The rings shown with heavy lines are common to both hydrocarbons. N2-[1R-(2R,3S,4R-Triacetoxy-1,2,3,4-tetrahydrobenzo[c]phenanthryl)]-O6-allyl-3′,5′-di-O-(tert-butyldimethylsilyl)2′-deoxyguanosine Adducts (5b). 1H NMR δ 8.60-8.55 (m, 1 H12), 8.08-7.97 (m, 1H8′′, 1H6), 7.85-7.78 (m, 1H7, 1H8, 1H9), 7.51 (d, 1H5, J ) 8.8), 7.38 (p-t, 1H10, J ) 6.8), 7.20-6.88 (m, 1H1, 1H11), 6.74 (d, 1H4, J ) 8.0), 6.49-5.61 (m, 1H2, 1H1′, 1Hv(allyl), 1H3), 5.38-4.54 (m, 1Ht(allyl), 1Hc(allyl), 2H, CH2(allyl), 1H3′), 4.20-3.57 (m, 1H4′, 2H5′,5′), 2.60-2.56 (m, 1H2′), 2.42-2.38 (m, 1H2′), 2.17-1.89 (3s, 9H, OAc), 0.96-0.79 (tert-butyl methyls), 0.12-0.11 (Si bonded methyls); LRMS (FAB+) m/z 940 ([M + H]+); HRMS calcd for C49H65O10N5Si2Cs 1072.3324, found 1072.3458. N2-[1S-(2R,3S,4R-Triacetoxy-1,2,3,4-tetrahydrobenzo[c]phenanthryl)]-3′,5′-di-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine Adducts (8a) and N2-[1R-(2S,3R,4S-triacetoxy1,2,3,4-tetrahydrobenzo-[c]phenanthryl)]-3′,5′-di-O-(tertbutyldimethylsilyl)-2′-deoxyguanosine Adducts (8b). A solution of either 7 mg (7.5 µmol) of 6a or 7 mg (7.5 µmol) of 6b dissolved in 5 mL of CH2Cl2, 62 µL (700 µmol) of morpholine, and 0.9 mg (0.75 µmol) of Pd(PPh3)4 was stirred for 30 min to produce a light yellow solution. The reaction mixture was diluted with 50 mL EtOAc and extracted with 25 mL brine (pH 2.0), 25 mL of saturated NaHCO3 and 25 mL of water. The organic layer was separated, dried with MgSO4 and evaporated under reduced pressure. The resulting material was purified by HPLC using a 5 µm, 10 × 250 mm Axxiom silica gel column eluted at 5 mL/min with EtOAc-hexane (9:1) to yield 8a (3.1 mg, 46%, TR ) 9.6 min) or 8b (6.2 mg, 92%, TR ) 15.3 min). 8a: 1H NMR δ 8.71 (d, 1 H12, J ) 8.8), 8.12 (d, 1H6, J ) 8.8), 8.02 (dd, 1H9, J ) 8.8, 1.9), 7.96 (br s, 1H8′′,), 7.88 (AB system, 1H7, 1H8, J ) 7.8), 7.61-7.59 (m, 1H5, 1H10), 7.46-7.43 (m,

Chem. Res. Toxicol., Vol. 14, No. 6, 2001 711 1H11), 7.07 (br s, 1HNH), 6.50-6.47 (m, 1H4, 1H1), 6.33-6.25 (m, 1H2, 1H1′), 5.89-5.86 (m, 1H3), 4.70 (br s, 1H3′), 3.96-3.90 (m, 1H4′, 2H5′,5′), 2.63-2.61 (m, 2H2′,2′), 2.18-1.91 (3s, 9H, OAc), 0.95-0.89 (tert-butyl methyls), 0.13 (Si bonded methyls); LRMS (FAB+) m/z 900 ([M + H]+); HRMS calcd for C46H61O10N5Si2Cs 1032.3011, found 1032.2980 8b: 1H NMR δ 8.63 (d, 1 H12, J ) 8.8), 8.13 (d, 1H6, J ) 8.8), 8.02 (d, 1H9, J ) 6.6), 7.91-7.86 (m, 1H8′′, 1H7, 1H8), 7.61-7.58 (m, 1H5, 1H10), 7.40-7.37 (m, 1H11), 7.20 (br s, 1HNH), 6.49 (d, 1H4, J ) 8.2), 6.31-6.19 (m, 1H1, 1H2, 1H1′), 5.90 (d, 1H3, J ) 8.2), 4.60 (br s, 1H3′), 3.90-3.89 (m, 1H4′), 3.77-3.69 (m, 2H5′,5′), 3.19-3.16 (m, 1H2′), 2.32-2.28 (m, 1H2′), 2.17-1.96 (3s, 9H, OAc), 0.88-0.68 (tert-butyl methyls), 0.12-(-)0.38 (Si bonded methyls); LRMS (FAB+) m/z 900 ([M + H]+); HRMS calcd for C46H61O10N5Si2Cs 1032.3011, found 1032.3051 N2-[1S-(2S,3S,4R-Triacetoxy-1,2,3,4-tetrahydrobenzo[c]phenanthryl)]-3′,5′-di-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine Adducts (7a) and N2-[1R-(2R,3R,4S-triacetoxy1,2,3,4-tetrahydrobenzo-[c]phenanthryl)]-3′,5′-di-O-(tertbutyldimethylsilyl)-2′-deoxyguanosine Adducts (7b). Either 4 mg (4.3 µmol) of 5a or 4 mg (4.3 µmol) of 5b was dissolved in 3 mL of CH2Cl2. Then 38 µL (430 µmol) of morpholine and 0.5 mg (0.43 µmol) of Pd(PPh3)4 were added, and the mixture was stirred for 30 min to produce a light yellow solution. Identical work up and HPLC purification yielded 7a (2.9 mg, 76%, TR ) 12.5 min) or 7b (3.4 mg, 89%, TR ) 18.5 min). 7a: 1H NMR δ 8.59 (d, 1 H12, J ) 8.8), 8.00 (d, 1H6, J ) 8.2), 7.90-7.75 (m, 1H8′′, 1H7, 1H8, 1H9), 7.53 (d, 1H5), 7.42 (p-t, 1H10, J ) 7.7), 7.17 (p t, 1H11), 7.03 (d, 1H1, J ) 3.3), 6.67 (d, 1H4, J ) 8.2), 6.56 (p t, 1H1′, J ) 6.6), 6.21 (s, 1H2), 5.72 (dd, 1H3, J ) 8.2, 1.9), 4.79-4.77 (m, 1H3′), 4.09-4.04 (m, 1H4′), 3.95-3.93 (m, 2H5′,5′), 2.67-2.63 (m, 1H2′), 2.16-1.87 (3s, 9H, OAc), 0.96-0.95 (tert-butyl methyls), 0.20-0.14 (Si bonded methyls); LRMS (FAB+) m/z 900 ([M + H]+); HRMS calcd for C46H61O10N5Si2Cs 1032.3011, found 1032.3008 7b: 1H NMR δ 8.46 (d, 1 H12, J ) 8.8), 8.00 (d, 1H6, J ) 8.2), 7.99-7.76 (m, 1H8′′, 1H7, 1H8, 1H9), 7.51 (d, 1H5), 7.44 (p-t, 1H10, J ) 7.4), 7.11 (p-t, 1H11), 6.86 (d, 1H1, J ) 3.8), 6.72 (d, 1H4, J ) 8.8), 6.43 (dd, 1H1′, J ) 8.2, 5.5), 6.20 (s, 1H2), 5.61 (dd, 1H3, J ) 8.8, 1.9), 4.95-4.93 (m, 1H3′), 4.17-3.92 (m, 1H4′, 2H5′,5′), 3.42-3.33 (m, 1H2′), 2.45-2.40 (m, 1H2′), 2.17-1.89 (3s, 9H, OAc), 0.96-0.82 (tert-butyl methyls), 0.26-0.07 (Si bonded methyls); LRMS (FAB+) m/z 900 ([M + H]+); HRMS calcd for C46H61O10N5Si2Cs 1032.3011, found 1032.3008 Synthesis of O6-Allyl-N2-dGuo Phosphoramidite Building Blocks. (1) Desilylation of the Sugar Hydroxyl Groups. The diastereomeric mixtures of the O6-allyl protected cis- and trans-opened N2-dGuo adducts 5 (111 mg) and 6 (190 mg) derived from B[c]Ph DE-2, 9 (103 mg) and 10 (145 mg) derived from cis- and trans-opening of B[a]P DE-1 as well as 11 (136 mg) and 12 (99 mg) derived from cis- and trans-opening of B[a]P DE-2 were dissolved in 3 mL of a 4:1 mixture of acetonitrile/ pyridine in a polyethylene vial, and a 16-fold excess of a 20% HF/pyridine (39) solution was added. The reaction mixture was stirred overnight, diluted with 150 mL of ethyl acetate and extracted with 50 mL of saturated NaHCO3, 50 mL of saturated NaCl and 50 mL of water. The organic layer was separated, dried with MgSO4 and the solvents removed. The resulting yellow oil was further purified by column chromatography on silica gel using CH2Cl2-MeOH (95:5) as solvent. Recovered starting material was treated under the same conditions until all starting material was converted to the desired products. The combined yields were 58 mg (70%) for 13, 109 mg (75%) for 14, 89 mg (86%) for 15, 64 mg (81%) for 16, 64 mg (81%) for 17, and 81 mg (74%) for 18, respectively. FAB HRMS calcd for C37H37O10N5 712.2619 (B[c]Ph-derivatives), found 712.2632 (13), found 712.2626 (14) and FAB HRMS calcd for C39H37O10N5Cs 868.1595 (B[a]P-derivatives), found 868.1617 (15), found 868.1636 (16), found 868.1592 (17), and found 868.1582 (18). Synthesis of 5′-O-Dimethoxytrityl Derivatives. The desilylated triacetates 13, 14, 15, 16, 17, and 18 were transformed into their 5′-O-DMTr-derivatives by treating with a 2.25-fold

712


excess of DMTr-Cl and a 1.75-fold excess of DMAP in 3 mL of dry pyridine (40). The reaction mixtures were stirred for 6 h and then concentrated. The resulting solid material was purified by column chromatography on silica gel using EtOAc with 0.2% TEA as mobile phase. Yields were 81 mg (97%) for 19, 148 mg (96%) for 20, 69 mg (79%) for 21, 106 mg (92%) for 22, 85 mg (68%) for 23, and 78 mg (83%) for 24, respectively. FAB HRMS calcd for C58H55O12N5Cs 1146.2902 (B[c]Ph-derivatives), found 1146.2881 (19) 1146.2854 (20) and FAB HRMS calcd for C60H55O12N5Cs 1170.2902 (B[a]P-derivatives), found 1170.2925 (21), found 1170.2896 (22) found 1170.2878 (23) and found 1170.2880 (24). 3′-(2-Cyanoethyl-N,N′-diisopropyl)phosphoramidites. The DMTr-derivatives 19, 20, 21, 22, 23, and 24 were combined with a 10-fold excess of N,N-diisopropylammonium tetrazolide (41) and dried in vacuo for 4 h. The solids were dissolved in 20 mL of dry CH2Cl2 followed by a 10-fold excess of 2-cyanoethyl tetraisopropylphosphorodiamidite in 2 mL of CH2Cl2. The clear solution was stirred for 3-4 h and the solvent removed in vacuo. The remaining yellow oil was loaded onto a silica gel column under an argon atmosphere and eluted with EtOAc-n-pentane (40:60) containing 0.1% TEA until all of the excess reagent had eluted. The objective phosphoramidite diastereomers were eluted with EtOAc-n-pentane (60:40) containing 0.2% TEA. Fractions containing the products were pooled and evaporated. Then 1 mL of n-pentane was added to the colorless residue, and the mixture was sonicated for about 1 min to provide the products as white solids. After filtration and drying in vacuo, the phosphoramidites 25 (78 mg, 80%), 26 (153 mg, 86%) 27 (67 mg, 81%), 28 (111 mg, 87%), 29 (82 mg, 81%), and 30 (76 mg, 90%) were stored at -20 °C. 31P NMR (CD CN) B[c]Ph-derivatives δ 149.3 and 149.0 (25), 3 δ 149.1, 148.9 and 148.7 (26); B[a]P-derivatives δ 149.2 and 149.0 (27), δ 149.9, 149.1 and 148.9 (28), δ 150.3, 149.2 and 148.8 (29), δ 149.1 and 148.8 (30). Oligonucleotide Syntheses. The two modified oligonucleotides 5′-(AAA AAG ACT TGG* AAA AAT TTT T)-3′ with a modified deoxyguanosine residue (G*) corresponding to the adduct formed by trans opening of the epoxide ring of (+)- or (-)-B[a]P DE-2 by the 2-amino group of dGuo were prepared from the diastereomeric mixture 30 (trans-N2-dGuo B[a]P DE-2 phosphoramidite). Syntheses were carried out on a 2 µmol scale using an Applied Biosystems model 392 synthesizer to generate the sequence 3′ to the modified nucleoside, with a manual step for coupling of the adducted phosphoramidite as previously described (21, 42). A typical 2 µmol synthesis utilized 25 mg of commercially available (Glen Research, Sterling, VA) dThdsubstituted controlled pore glass [500 Å; high load (80 µmol/g)] with addition of 8 µmol of the appropriate dGuo-adducted phosphoramidites 30 and 50 µL of 0.5 M 4,5-dicyanoimidazole (DCI) in acetonitrile in the manual coupling step. Coupling was allowed to proceed for 20 h at room temperature, at the end of which time the synthesis was completed on the synthesizer. The efficiency of the manual coupling step ranged from 10 to 20% (determined by a spectrophotometric (495 nm) assay of the DMTr cation released upon detritylation of the coupling product). Because of the prolonged coupling time for the manual step which could lead to a loss of the 5′-DMTr group of successfully incorporated adducts, endcapping after the manual step was omitted. After removal of the last 5′-DMTr group on the synthesizer, the O6-allyl protecting group on the modified base (G*) was removed while the oligonucleotide was still bound to the CPG-material. The allyl group was quantitatively removed by treating the support bound oligonucleotides sequentially with 3 portions of a palladium catalyst solution [35 mg Pd(PPh3)4 and 700 µL of morpholine in 5 mL of CH2Cl2)] (43, 44) over a period of 1 h. After washing the CPG support with CH2Cl2 to remove traces of the catalyst, the oligonucleotide was cleaved from the solid support and deprotected by ammonolysis (20 h, 58 °C). The six oligonucleotides 5′-(TGG* AAA AAT TTT T)-3′, 5′-(T TGG* AAA AAT TTT T)-3′ and 5′-(CT TGG* AAA AAT TTT

Kroth et al. Table 1. HPLC Retention Times and Absolute Configurations of Oligonucleotides Containing N2-Deoxyguanosine Adducts Corresponding to trans Opening of B[a]P DE-2a parent diol epoxide

N2-dGuo adduct

retention time (min)

configuration at C-10d

5′-AAA AAG ACT TGG* AAA AAT TTT T-3′b (7S,8R,9R,10S)-DE-2 trans 19.0 R (7R,8S,9S,10R)-DE-2 trans 19.8 S 5′-TGG* AAA AAT TTT T-3′b (7S,8R,9R,10S)-DE-2 trans 20.7 (7R,8S,9S,10R)-DE-2 trans 22.9

R S

5′-T TGG* AAA AAT TTT T-3′b (7S,8R,9R,10S)-DE-2 trans 21.1 (7R,8S,9S,10R)-DE-2 trans 22.7

R S

5′-CT TGG* AAA AAT TTT T-3′c (7S,8R,9R,10S)-DE-2 trans 23.0 (7R,8S,9S,10R)-DE-2 trans 25.5

R S

a The modified base is indicated as G*. b On a Hamilton PRP-1 column (10 × 250 mm, 7 µm) eluted at 3 mL/min with a gradient from 0 to 17.5% acetonitrile in 0.1 M (NH4)2CO3, pH 7.5, over 20 min, followed by a ramp to 50% acetonitrile over the next 8 min. c On a Zorbax Eclipse XDB-C18 column (4.6 × 250 mm, 5 µm) eluted at 1.5 mL/min with a gradient from 5 to 11% acetonitrile in 0.1 M (NH4)2CO3, pH 7.5, over 20 min, followed by maintaining 11% acetonitrile for the next 10 min. d Assignments based on the CD spectra of the oligonucleotides or of the individual N2-dGuo adducts after enzymatic digestion (4, 5).

T)-3′ were also prepared using the diastereomeric mixture 30. The four oligonucleotides 5′-TTC G*AA TCC TTC CCC C-3′, as well as the four oligonucleotides 5′-GGG G*TT CCC GAG CGG C-3′, containing a run of four N2-dGuo residues, were prepared from the diastereomeric mixtures 27 (cis-N2-dGuo B[a]P DE-1 phosphoramidite) and 29 (cis-N2-dGuo B[a]P DE-2 phosphoramidite). The R/S pair of modified oligonucleotides 5′-(AAA AAG ACT TGG* AAA AAT TTT T)-3′ as well as the three pairs of oligonucleotides 5′-(GG* AAA AAT TTT T)-3′, 5′-(TGG* AAA AAT TTT T)-3′ and 5′-(T TGG* AAA AAT TTT T)-3′ were prepared using the diastereomeric mixture 26 (trans-N2-dGuo B[c]Ph DE-2 phosphoramidite). The modified oligonucleotides containing the trans-N2-dGuo B[c]Ph DE-2 adducts were purified by reverse-phase HPLC on either a Hamilton PRP-1 column (10 × 250 mm, 7 µm), a Zorbax Eclipse XDB C-18 column (4.6 × 250 mm, 5 µm), a Phenomenex Luna ODS column (4.6 × 250 mm, 5 µm), a Higgins DNA column (4.6 × 100 mm, 5 µm) or combinations of these columns. Retention times for the individual N2-dGuo adducted oligonucleotides as well as the chromatographic conditions for separation of the R/S-pairs of N2dGuo adducted oligonucleotides are presented in Tables 1, 2, and 3. Isolated yields ranged from 2.8 to 20 A260 units of each oligonucleotide. Assignment of absolute configuration for the diastereomeric oligonucleotides containing N2-dGuo adducts derived from B[a]P DE was based on the CD spectra of the oligonucleotides which showed positive, long wavelength bands for 10R- and negative bands for 10S-adducted oligonucleotides (29) (Figures 4 and 5). For oligonucleotides which showed only extremely weak long wavelength bands and for the oligonucleotides containing N2-dGuo adducts derived from B[c]Ph DE-2 (Figure 6), the assignment of their absolute configuration was based on the CD spectra of the individual cis- and trans-dGuo adducts (4, 5, 45) obtained after enzymatic digestion to the nucleoside level (5, 46).

Results and Discussion Direct opening of the highly hindered fjord-region B[c]Ph DE-2 2 with the O6-allyl protected N2-dGuo building block 1 in DMA at 90-100 °C (Figure 1) occurs but results in lower yields (43%) than that for the opening reaction of the less hindered B[a]P DE-2 (65%) (34). We

Synthesis of DNA Containing Diol Epoxide Adducts at dGuo Table 2. HPLC Retention Times and Absolute Configurations of Oligonucleotides Containing N2-Deoxyguanosine Adducts Corresponding to trans Opening of B[c]Ph DE-2a parent epoxide epoxide

N2-dGuo adduct



5′-AAA AAG ACT TGG* AAA AAT TTT T-3′b (1S,2R,3R,4S)-DE-2 trans 19.7 R (1R,2S,3S,4R)-DE-2 trans 20.9 S 5′-GG* AAA AAT TTT T-3′c (1S,2R,3R,4S)-DE-2 trans 17.5 (1R,2S,3S,4R)-DE-2 trans 19.1

R S

5′-TGG* AAA AAT TTT T-3′c (1S,2R,3R,4S)-DE-2 trans 16.8 (1R,2S,3S,4R)-DE-2 trans 18.8

R S

5′-T TGG* AAA AAT TTT T-3′c (1S,2R,3R,4S)-DE-2 trans 17.5 (1R,2S,3S,4R)-DE-2 trans 19.4

R S

a The modified base is indicated as G*. b On a Higgins DNA column (4.6 × 100 mm, 5 µm) at 40 °C eluted at 1.5 mL/min with a gradient from 5 to 11% acetonitrile in 0.1 M (NH4)2CO3, pH 7.5, over 20 min, followed by maintaining 11% acetonitrile for the next 5 min. c On the same column and under the same conditions except with a gradient from 5 to 12.5% acetonitrile in 0.1 M (NH4)2CO3, pH 7.5, over 20 min, followed by maintaining 12.5% acetonitrile for the next 5 min. d Assignments based on CD spectra of the oligonucleotides or of the individual N2-dGuo adducts after enzymatic digestion (45).

Table 3. HPLC Retention Times and Absolute Configurations of the Oligonucleotides Containing N2-Deoxyguanosine Adducts Corresponding to cis Opening of B[a]P DE-1 and DE-2a parent diol epoxide

N2-dGuo adduct



5′-TTC G*AA TCC TTC CCC C-3′ (7R,8S,9S,10R)-DE-2 cis 13.2b (7S,8R,9R,10S)-DE-2 cis 16.0b (7S,8R,9S,10R)-DE-1 cis 19.3c (7R,8S,9R,10S)-DE-1 cis 20.8c

R S R S

5′-GGG G*TT CCC GAG CGG C-3′c (7R,8S,9S,10R)-DE-2 cis 9.5 (7S,8R,9R,10S)-DE-2 cis 12.0 (7S,8R,9S,10R)-DE-1 cis 13.3 (7R,8S,9R,10S)-DE-1 cis 14.4

R S R S

a The modified base is indicated as G*. b On a Phenomenex Luna C-18 column (4.6 × 250 mm, 5 µm) at 60 °C eluted at 1.5 mL/min with a gradient from 5 to 11% acetonitrile in 0.1 M (NH4)2CO3, pH 7.5, over 20 min. c On a Zorbax Eclipse XDB C-18 column (4.6 × 250 mm, 5 µm) at 50 °C eluted at 1.5 mL/min with a gradient from 5 to 11% acetonitrile in 0.1 M (NH4)2CO3, pH 7.5, over 20 min. d Assignments based on CD spectra of the oligonucleotides or of the individual N2-dGuo adducts after enzymatic digestion (4, 5).

speculate that the sterically more crowded fjord-region of B[c]Ph DE-2 leads to more severe steric interactions with the N2-dGuo building block 1. In contrast to the opening of B[a]P DE-2 where the cis/trans-ratio was (60: 40), direct opening of B[c]Ph DE-2 resulted in a cis/trans ratio of (25:75). This suggested that opening of the B[c]Ph DE-2 2 by the dGuo building block 1 occurred primarily through an SN2 pathway. We assume that formation of a carbocation from B[c]Ph DE-2, due to its lower carbocation stabilizing energy [∆Edeloc ) 0.600β for B[c]Ph versus ∆Edeloc ) 0.794β for B[a]P (47)], is energetically less favored than formation of the corresponding carbocations from B[a]P DE-1 and DE-2. This might explain the prolonged reaction time required for reaction of B[c]Ph DE-2, 5 h compared to just 2 h for B[a]P DE-1 and B[a]P DE-2 (34). Because of the increased steric


hindrance in the fjord-region of B[c]Ph and the greater difficulty of carbocation formation compared to B[a]P, the direct opening reaction of B[c]Ph DE-2 with the N2-dGuo building block 1 represents a “worst case” for this reaction. It is expected that DE of other PAH with more easily formed carbocations and/or less steric hindrance will result in better adduct coupling yields. Separation of the mixture of cis- and trans-opened N2dGuo DE-2 adducts from the reaction of 1 and B[c]Ph DE-2 2 was performed in two steps. First the crude mixture of the 4 diastereomers was separated by HPLC (EtOAc in hexane on silica) into a mixture of cis-opened (3) and trans-opened adducts (4). After acetylation of 3 and 4, the individual cis-5a,b and trans-6a,b dGuo DE-2 diastereomers were separated by HPLC (EtOAc in hexane on silica). The stereochemistry of the cis-5a,b and trans-6a,b N2-dGuo adducts, as well as the cis-7a,b and trans-8a,b adducts with the allyl group removed (see below), was not apparent from the coupling constants of their saturated benzo-ring signals (Table 4). These were large for J3,4 and small for J2,3, and are typical for both cis- and trans-opened adducts of B[c]Ph DE-2 (45). However, we had previously observed that the chemical shifts for H1 are significantly farther downfield in the cis opened relative to the trans opened DE-2 tetraol tetraacetate (38, 45), and thus the assignment of relative stereochemistry for the acetylated dGuo adducts (cf. 45) is based primarily on the chemical shift of this proton (Table 4). Measurement of the NMR spectra of the O-allyl substituted derivatives cis-5a,b in chloroform and decoupling experiments in acetone at 500 MHz significantly improved the signals for the tetrahydro benzo-ring methine protons and permitted their unequivocal identification. As in our previous report of the O6-allyl-protected B[a]P DE N2-dGuo adducts (34), the CD spectra (Figure 7) of the present O6-allyl protected B[c]Ph DE-2 dGuo adducts did not permit direct assignment of their absolute configuration. The CD spectra of the O6-allyl cis-N2-dGuo adducts 5a and 5b and the trans-N2-dGuo adducts 6a and 6b were different from those of the O6-unprotected N2-dGuo adducts (45) and from each other, and exhibited no clearly diagnostic bands. Therefore, it was necessary to remove the O6-allyl protecting group from the N2-dGuo adducts 5a, 5b, 6a, and 6b, using a palladium catalyst as described (34). The resultant O6-deblocked N2-dGuo adducts 7a, 7b, 8a, and 8b, as their disilyl triacetates, had CD spectra (Figure 7) that were identical in shape with those reported (45) for the fully unprotected nucleoside adducts of known absolute configuration obtained from the optically active diol epoxides. Thus, unambiguous assignment of configuration to these disilyl triacetates and their O6-allyl precursors was possible. For example, the positive CD band at 258 nm for 8a, which was obtained on O6-deprotection of 6a, establishes that these two related compounds have (1S)-configuration and were formed from (-)-(1R,2S,3S,4R)-B[c]Ph DE-2 by inversion at C-1. The O6-allyl protected N2-dGuo adducts 9, 10, 11, and 12 corresponding to cis- and trans-opening of B[a]P DE-1 and B[a]P DE-2 (34) and the newly synthesized N2-dGuo adducts 5 and 6 derived from cis- and trans-opening of B[c]Ph DE-2 were used as their diastereomeric mixtures as starting material for the synthesis of their corresponding phosphoramidites (Figure 2). Desilylation of the dGuo

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Figure 4. Circular dichroism spectra of the synthesized oligonucleotides containing trans-opened N2-dGuo adducts derived from B[a]P DE-2 in 0.02 M phosphate buffer, pH 7, with ionic strength adjusted to 0.1 M with NaCl. All spectra are normalized to 1.0 absorbance unit at 260 nm. The pyrene bands at 300-350 nm in the oligonucleotides are positive for those containing 10R adducts (solid lines) and negative for those containing 10S adducts (broken lines).

adducts was achieved with 20% HF/pyridine (39) rather than tetra-butylammonium fluoride (TBAF) to prevent loss of the acetate protecting groups. Presence of the bulky hydrocarbon moiety somehow hampered cleavage of the tert-butyldimethylsilyl (TBDMS) sugar protecting groups. Even with a 16-fold excess of 20% HF/pyridine, the cleavage reaction was not complete after 24 h (∼50% remaining starting material by TLC). Adding more HF/ pyridine or increasing the initial concentration only resulted in the formation of depurination products. Therefore, the complete conversion of the starting material 5, 6, 9, 10, 11, and 12 into their desilylation products 13, 14, 15, 16, 17, and 18 was achieved by repeating the desilylation reaction two to three times with recovered starting material. This strategy avoided depurination and enabled the isolation of 13, 14, 15, 16, 17, and 18 in 7486% overall yield. Selective protection of the 5′-OH group in the deoxyribose moiety was achieved by using DMTr-Cl in pyridine (40). Initial experiments with a 1.5-fold excess of DMTrCl showed that after 24 h only 10% of the starting material was converted to the desired product. However, increasing the DMTr-Cl concentration to a 2.25-fold excess and addition of DMAP (1.75-fold) resulted in formation of the desired DMTr protected N2-dGuo adducts 19, 20, 21, 22, 23, and 24 in 68-96% yield within 6 h. Presence of the allyl protecting group at the O6 position of dGuo enabled synthesis of the phosphoramidite building blocks 25, 26, 27, 28, 29, and 30 from their corresponding DMTr protected precursors by using 2-cyanoethyl tetraisopropylphosphorodiamidite (41) instead of

2-cyanoethyl diisopropylchlorophosphoramidite (48). By using the less reactive tetraisopropyl-phosphordiamidite rather than the harsher monochloro-reagent which leads to the formation of more side products and results in lower yields of the desired dGuo phosphoramidites, the desired phosphoramidites 25, 26, 27, 28, 29, and 30 were produced in 81-90% yields. Furthermore, the presence of the O6-allyl protecting group resolved the problem of limited solubility of the O6-unprotected N2-dGuo phosphoramidites in acetonitrile in the manual coupling step during oligonucleotide synthesis. There are two possible ways for cleavage of the O6allyl protecting group after incorporation of the phosphoramidite into oligonucleotides. The cleavage reaction can be done while the oligonucleotide is bound to the solid support or after the ammonia deblocking step that releases the oligonucleotide into solution. Cleavage of the allyl group in solution has the advantage that progress of the cleavage reaction can be monitored by HPLC whereas the same reaction done on the solid support bound oligonucleotide reduces the risk of possible contamination of the final product with the catalyst. In our initial experiments, we used concentrated ammonia for 3 days at 70 °C. This methodology has been employed for the cleavage of allyl protecting groups at phosphotriester internucleotide linkages of DNA (49). However, these conditions failed to cleave the allyl group from the O6-position, and the starting oligonucleotide was recovered unchanged. Further attempts to cleave the O6-allyl protecting group from the oligonucleotides in solution using a water soluble palladium-catalyst (50, 51) also failed. In addition, starting material could not be recov-



Figure 5. Circular dichroism spectra of the synthesized oligonucleotides containing cis-opened N2-dGuo adducts derived from B[a]P DE-1 and B[a]P DE-2. Conditions as in Figure 4. The pyrene bands at 300-350 nm [positive for 10R adducts (solid lines) and negative for 10S adducts (broken lines)] were only detectable in one pair of oligonucleotides containing cis DE-1 adducts in a CG*A sequence context. Therefore, the assignment of configuration for other oligonucleotides was based on enzymatic hydrolysis to the nucleoside derivative (4, 5).

ered due to the formation of some form of complex between the catalyst and the oligonucleotide. Selective and complete cleavage of the O6-allyl protecting group from the oligonucleotides was finally achieved by treating the support bound oligonucleotide with tetrakis-triphenylphosphine-(0)-palladium (Figure 3) (43, 44). Whereas cleavage of the O6-allyl protecting group from the dGuo adducts 5, 6, 9, 10, 11, and 12 was accomplished with 10 mol % of catalyst in CH2Cl2 within 30 min (34), it was necessary to increase the amount of catalyst 30-fold and double the reaction time (1 h) to ensure the complete removal of the O6-allyl protecting group from the support bound oligonucleotides. Complete removal was verified by HPLC comparison of the retention times for the oligonucleotide products 5′-(AAA AAG ACT TG*G AAA AAT TTT T)-3′ containing the N2-dGuo adducts derived from trans opening of B[a]P DE-2 at (G*) before and after treatment with the catalyst solution. The R/S pair of oligonucleotides containing the allyl group were poorly resolved on HPLC and had longer retention times [20.9 min (early) and 21.4 min (late); data not shown] compared to the fully deblocked oligonucleotides [(18.3 min (early) and 19.8 min (late)] which were separated into their diastereomers (52). To confirm that the allyl group had been completely removed, the oligonucleotides 5′-(AAA AAG ACT TG*G AAA AAT TTT T)3′ were digested to the nucleoside level with snake venom phosphodiesterase (5). CD spectra (data not shown) of the adducts obtained from the digested oligonucleotides matched the CD spectra reported in the literature (4, 5), whereas the N2-B[a]P-dGuo adducts still bearing the O6-

allyl protecting group had markedly different CD spectra (34). Oligonucleotide Syntheses. We have made use of this new methodology to synthesize several oligonucleotides of biochemical interest. Syntheses and purifications of the oligonucleotides reported in this study generally followed procedures previously developed in this laboratory (21, 42). Two points are worthy of special mention. (1) In earlier syntheses of relatively short oligonucleotides from B[a]P DE-adducted phosphoramidites, we routinely used a CPG support with a pore size of 170 Å since this permitted a very high loading of the bound oligonucleotide (∼100 µmol/g). Upon attempting to extend this methodology in the present work to the synthesis of oligonucleotides longer than 16-mers, we found that this pore size was unsatisfactory for the synthesis of oligonucleotides consisting of more than 18 bases. A significant falloff (∼50% for each coupling step beyond the 18th base) was observed with this small poresize CPG. Use of a CPG support with a pore size of 500 Å (now commercially available with ∼80 µmol/g loading) avoided this problem. (2) We observed an empirical correlation between chromatographic elution order and the absolute configuration at the point of attachment of the diol epoxide moiety to the dGuo base. Thus, for all the oligonucleotides reported in this study, those containing a hydrocarbon adduct with R configuration at the point of attachment to the dGuo base eluted earlier on reverse phase HPLC than did their S diastereomers. The 22-mer oligonucleotides described above, 5′-(AAA AAG ACT TG*G AAA AAT TTT T)-3′, containing the

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Figure 6. Circular dichroism spectra of the synthesized oligonucleotides containing trans-opened N2-dGuo adducts derived from B[c]Ph DE-2. Conditions as in Figure 4. The three shorter oligonucleotides (12-14-mers) showed strong CD bands around 258 nm [(positive for 1S (broken lines) and negative for 1R (solid lines)] which were diagnostic for the absolute configuration of the corresponding N2-dGuo adducts. For the 22-mer these bands were no longer diagnostic and the assignment of the absolute configuration was based on enzymatic hydrolysis to the nucleoside derivative (45). Table 4. Comparison of the BcPh DE-2 Benzo-Ring 1H NMR Data (300 MHz, acetone-d6) for the O6-Allyl Protected cisand trans-Opened N2-dGuo Adducts and the O6-Allyl Deprotected cis- and trans-Opened N2-dGuo Adducts as Their Disilyl Triacetates compd (1S)b

H1

5a cis-dGuo-OAll-DE-2 5b (1R)b

7.04

6a (1S)b trans-dGuo-OAll-DE-2 6b (1R)b

6.39

7a (1S) cis-dGuo-DE-2 7b (1R)

7.03

8a (1S) trans-dGuo-DE-2 8b (1R)

6.50-6.47

7.08-6.84

6.38

6.86

6.31-6.19

H2 J1,2 ) NAa J1,2 ) 4.0 J1,2 ) 2.6 J1,2 ) 2.6 J1,2 ) 3.3 J1,2 ) 3.8 J1,2 ) NA J1,2 ) NA

H3

6.10

J2,3 ) 2.0

6.18

J2,3 ) 2.2

6.27

J2,3 ) 2.4

6.3

J2,3 ) 2.7

6.21

J2,3 ) 1.9

6.20

J2,3 ) 1.9

6.33-6.25 6.31-6.19

J2,3 ) NA J2,3 ) NA

5.69 5.61 6.03 6.02 5.72 5.61 5.89-5.86 5.90

H4 J3,4 ) 7.8 J3,4 ) 8.0 J3,4 ) 8.3 J3,4 ) 8.8 J3,4 ) 8.2 J3,4 ) 8.8 J3,4 ) NA J3,4 ) 8.2

6.91 6.74 6.44 6.48 6.67 6.72 6.50-6.47 6.49

a NA ) not assigned due to broad or overlapping peaks. b Assignments and coupling constants were confirmed by decoupling at 500 MHz.

adducts derived from trans opening of the (+)- and (-)enantiomers of B[a]P DE-2 at (G*) whose characterization was previously reported (52), are substrates for topoisomerase I. Along with the oligonucleotides containing adducts derived from cis-opening (52) of B[a]P DE-2 at the same position, these oligonucleotides were used to study the effects of minor groove bound (trans-opened) or intercalated (cis-opened) N2-dGuo adducts of B[a]P DE-2 on cleavage and religation of DNA by this enzyme (52). In addition, the oligonucleotides containing trans-

opened N2-dGuo adducts of B[a]P DE-2 at the other centrally located (G*) in this same sequence, 5′-(AAA AAG ACT TGG* AAA AAT TTT T)-3′, were synthesized (this study; Table 1) as further mechanistic probes for topoisomerase I, and the set of six marker oligonucleotides (13-15-mers) 5′-(CT TGG* AAA AAT TTT T)-3′, 5′(T TGG* AAA AAT TTT T)-3′, and 5′-(TGG* AAA AAT TTT T)-3′ were prepared as standards in order to establish the site of cleavage of this 22-mer by the enzyme. For these syntheses, the phosphoramidite mix-



Figure 7. Circular dichroism spectra (normalized to 1.0 absorbance unit at λmax, methanol) of the O6-allyl protected cis- and transopened N2-dGuo adducts 5a, 5b, 6a, and 6b and of the O6-allyl deprotected cis- and trans-opened N2-dGuo adducts 7a, 7b, 8a, and 8b. The strong CD bands at 261 nm for the O6-allyl deprotected cis-opended N2-dGuo adducts 7a and 7b and at 258 nm for the O6-allyl deprotected trans-opened N2-dGuo adducts 8a and 8b are diagnostic for their absolute configuration [positive for 1S (solid lines) and negative for 1R (broken lines) (45)] for these B[c]Ph DE adducts.

ture 30 (trans-N2-dGuo B[a]P DE-2 phosphoramidite) was used. Yet another set of topoisomerase substrates and markers, again with the same sequence but incorporating a trans-opened DE-2 adduct of B[c]Ph (Table 2), was prepared to explore both the generality of the synthetic method and the effect of varying the hydrocarbon moiety on topoisomerase I. For these oligonucleotides, the starting material was the phosphoramidite mixture 26 (trans-N2-dGuo B[c]Ph DE-2 phosphoramidite). Biochemical studies of these oligonucleotides with topoisomerase I are in progress. We had previously published an investigation of the effect of adduct stereochemistry and sequence context on the mutational consequences of trans-opened N2-dGuo adducts derived from B[a]P DE-1 and -2 upon site-specific incorporation into M13mp7L2 bacteriophage and replication of the phage in E. coli (29). The oligonucleotides used for ligation into the phage were the 16-mers, 5′-TTC G*AA TCC TTC CCC C-3′and 5′-GGG G*TT CCC GAG CGG C-3′, both of which correspond to sequences from the supF gene. To complete this systematic study, we utilized the phosphoramidite mixtures 27 (cis-N2-dGuo B[a]P DE-1 phosphoramidite) and 29 (cis-N2-dGuo B[a]P DE-2 phosphoramidite) to synthesize these same two sequences containing the corresponding cis opened B[a]P dGuo adducts (Table 3). With the synthesis of these oligonucleotides, comparison of the mutagenicity for all the cis- and trans-opened N2-dGuo adducts from B[a]P DE-1 and B[a]P DE-2 in these two different sequence contexts has now been possible (53).

In summary O6-allyl protected N2-dGuo phosphoramidite building blocks 25, 26, 27, 28, 29, and 30 derived from B[c]Ph DE-2, B[a]P DE-1 and B[a]P DE-2 (Figure 2), together with the selective and complete removal of the allyl protecting group from oligonucleotides still bound to the solid support (Figure 3) were effectively exploited in the synthesis of different oligonucleotides containing cis- and trans-N2-dGuo adducts. Synthesis of oligonucleotides containing the elusive cis-N2-dGuo adducts derived from B[a]P DE-1 in a run of four dGuo residues (GGGG*) established the potential of our approach. With the synthesis of the O6-allyl protected N2dGuo phosphoramidites from B[c]Ph DE-2 and their subsequent incorporation into oligonucleotides, we have demonstrated that this methodology can be used for the synthesis of oligonucleotides containing N2-dGuo adducts for any given hydrocarbon.

References (1) IARC. (1983) International Agency for Research on Cancer Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Polynuclear Aromatic Compounds, Part 1, Chemical, Environmental and Experimental Data, Vol. 32, IARC, Lyon, France. (2) Jerina, D. M., Chadha, A., Cheh, A. M., Schurdak, M. E., Wood, A. W., and Sayer, J. M. (1991) Covalent bonding of bay-region diol epoxides to nucleic acids. In Biological Reactive Intermediates IV. Molecular and Cellular Effects and Their Impact on Human Health (Witmer, C. M., Snyder, R., Jollow, D. J., Kalf, G. F., Kocsis, J. J., and Sipes, I. G., Eds.) pp 533-553, Plenum Press, New York. (3) Dipple, A. (1994) Reactions of polycyclic aromatic hydrocarbons with DNA. In DNA Adducts: Identification and Biological

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