Structure Elucidation of the Adducts Formed by ... - ACS Publications

University of Nebraska Medical Center, 986805 Nebraska Medical Center,. Omaha, Nebraska 68198-6805, Department of Chemistry, Washington University,...
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Chem. Res. Toxicol. 1999, 12, 758-767

Structure Elucidation of the Adducts Formed by Fjord-Region Dibenzo[a,l]pyrene 11,12-Dihydrodiol 13,14-Epoxides and Deoxyadenosine Kai-Ming Li,† Mathai George,‡ Michael L. Gross,‡ Albrecht Seidel,§ Andreas Luch,§,| Eleanor G. Rogan,† and Ercole L. Cavalieri*,† Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, Nebraska 68198-6805, Department of Chemistry, Washington University, One Brookings Drive, St. Louis, Missouri 63130-4899, and Institute of Toxicology, University of Mainz, Obere Zahlbacher Strasse 67, 55131 Mainz, Germany Received August 18, 1998

Model adducts to be used in the identification of biologically formed adducts were synthesized by reaction of fjord-region dibenzo[a,l]pyrene 11,12-dihydrodiol 13,14-epoxides (DB[a,l]PDE) and deoxyadenosine (dA). The (()-anti-DB[a,l]PDE was reacted with dA in dimethylformamide at 100 °C for 30 min to give four DB[a,l]PDE-14-N6dA adducts: (-)-anti-trans (26%), (+)anti-trans (26%), (-)-anti-cis (17%), and (+)-anti-cis (17%). The (()-syn-DB[a,l]PDE was reacted with dA under the same conditions to yield four DB[a,l]PDE-14-N6dA adducts and one N7Ade adduct: (+)-syn-cis (19%), (+)-syn-trans (13%), (-)-syn-cis (19%), (-)-syn-trans (13%), and (()syn-DB[a,l]PDE-14-N7Ade (22%). The structures of the eight stereoisomers of DB[a,l]PDE14-N6dA were unequivocally assigned by reacting optically pure (-)-anti-DB[a,l]PDE and (+)syn-DB[a,l]PDE with dA and by a combination of NMR, circular dichroism, and fast atom bombardment mass spectrometry. Reactions at 100 °C yielded mainly the trans-opened adducts at the benzylic C-14 position for both (()-anti-DB[a,l]PDE and (-)-anti-DB[a,l]PDE, whereas (()-syn-DB[a,l]PDE and (+)-syn-DB[a,l]PDE afforded mainly cis-opened adducts. At room temperature, however, only trans-opened adducts were obtained from (()-anti-DB[a,l]PDE and only cis-opened adducts from (()-syn-DB[a,l]PDE. Steric hindrance created by the fjord region may be an important factor for the stereoselectivity observed at room temperature.

Introduction Dibenzo[a,l]pyrene1 (DB[a,l]P)2 is the most potent carcinogen among the polycyclic aromatic hydrocarbons (PAHs) (1-4). Metabolic activation of DB[a,l]P leading to tumor initiation occurs by two main pathways: oneelectron oxidation to yield the DB[a,l]P radical cation and monooxygenation to produce fjord-region DB[a,l]P 11,12-dihydrodiol 13,14-epoxides (DB[a,l]PDE) (5). Identification of DNA adducts formed in cell cultures revealed that both syn- and anti-DB[a,l]PDE are produced (6) and that the activation occurs with the same stereoselectivity characteristics (7) that are found in other fjord- and bay-region PAHs, including benzo[c]phenanthrene and benzo[a]pyrene (8). DB[a,l]P is stereoselectively converted to (+)-syn- and (-)-anti-DB[a,l]PDE with 11S,12R,13S,14R- and 11R,12S,13S,14R-configurations, respectively. DB[a,l]P 11,12-dihydrodiol, the metabolic precursor of the ultimate reactive fjord-region DB[a,l]PDE, is a very potent carcinogen, although less active than DB[a,l]P in dose-response studies in mouse skin * To whom correspondence should be addressed. † University of Nebraska Medical Center. ‡ Washington University. § University of Mainz. | Present address: Institute of Toxicology and Environmental Hygiene, Technical University of Munich, Lazarettstrasse 62, 80636 Munich, Germany. 1 IUPAC systematic name of dibenzo[def,p]chrysene.

(2, 3, 9). The racemic syn- and anti-DB[a,l]PDE are tumor initiators in mouse skin, although much weaker than DB[a,l]P and its 11,12-dihydrodiol (9). These diol epoxides are carcinogenic in newborn mice (10) and rat mammary gland (11). These data suggest that the DB[a,l]PDE intermediates play a significant role in the carcinogenic activity of DB[a,l]P. To identify DB[a,l]P-DNA adducts formed in biological systems, it is necessary to synthesize prospective model adducts that will serve as reference standards. On the basis of the activation mechanisms of DB[a,l]P in biological systems, two kinds of standards are needed: oneelectron oxidation adducts formed by DB[a,l]P radical cation and the adducts formed by reaction of DB[a,l]PDE. The synthesis of some DB[a,l]P adducts formed by oneelectron oxidation was reported previously (12); others are reported in an accompanying article (13). In this article, the synthesis and structure elucidation of adducts formed by reaction of DB[a,l]PDE with deoxyadenosine (dA) are described. 2 Abbreviations: CAD, collisionally activated decomposition; CD, circular dichroism; COSY, two-dimensional chemical shift correlation spectroscopy; dA, deoxyadenosine; DB[a,l]P, dibenzo[a,l]pyrene; DB[a,l]PDE, dibenzo[a,l]pyrene 11,12-dihydrodiol 13,14-epoxide(s); DMF, dimethylformamide; FAB MS, fast atom bombardment mass spectrometry; PAH, polycyclic aromatic hydrocarbon; PDA, photodiode array.

10.1021/tx980197x CCC: $18.00 © 1999 American Chemical Society Published on Web 08/10/1999

DB[a,l]P Dihydrodiol Epoxide-dA Adducts

Experimental Procedures Caution: DB[a,l]PDEs are hazardous chemicals and were handled according to NIH guidelines (14). General Procedures. UV absorbance spectra were recorded by using a Waters 990 photodiode array (PDA) detector during elution from HPLC with the CH3CN/H2O gradient as described below. Proton and homonuclear two-dimensional chemical shift correlation spectroscopy (COSY) NMR spectra were recorded in Me2SO-d6 at 25 °C on a Varian Unity 500 spectrometer (499.843 MHz). The chemical shifts are reported relative to that of Me2SO at 2.50 ppm. Circular dichroism (CD) spectra were recorded on a Jasco J-710 spectropolarimeter (Japan Spectroscopic Co. Ltd., Tokyo, Japan) in Me2SO at 22 °C. Collisonally activated decomposition (CAD) spectra were recorded on a VG ZAB-T (Manchester, U.K.), a four-sector tandem mass spectrometer of BEBE design (15). All the CAD mass spectra were acquired by using the array detector with an array angle of 15° with respect to the central ion beam. Samples were dissolved in 20 µL of Me2SO, and a 1 µL aliquot was placed on the probe along with 1 µL of matrix, a mixture of glycerol and Me2SO (1:1) containing 1% heptafluorobutyric acid. The collision cell was floated at 4 kV. The MS1 system was operated at a resolving power of 1000. The MS2 resolving power (on the array) was approximately 1500 (full width at half-height definition). Ten to 20 spectra comprising eleven 1 s exposures were signal-averaged for each spectrum. Data acquisition and data workup were controlled by using a VAX 3100 workstation equipped with Opus software. HPLC was conducted on a Waters 600E solvent delivery system equipped with a Waters 700 WISP autoinjector. Effluents were monitored for UV absorbance (254 nm) with a Waters 990 PDA detector, and the data were collected on an APC-IV Powermate computer. Analytical runs were conducted by using a YMC ODS-AQ 5 µm, 120 Å column (6.0 mm × 250 mm) (YMC, Wilmington, NC). The column was eluted for 5 min with 20% CH3CN in H2O, followed by a 75 min linear gradient to 100% CH3CN at a flow rate of 1.0 mL/min. A second solvent system, used for the final purification of adducts, was an isocratic eluent consisting of 70% CH3OH in H2O. Preparative HPLC was conducted by using a YMC ODS-AQ 5 µm, 120 Å column (20 mm × 250 mm) at a flow rate of 6.0 mL/min. The CH3CN/H2O gradient and CH3OH/H2O isocratic eluent were used for adduct purification. Molecular Modeling and Theoretical Calculations. Molecular modeling, energy minimization, and dihedral angle calculations for DB[a,l]PDE-dA adducts were conducted by utilizing methods of molecular mechanics, and energy minimization was performed by using the Cambridgesoft Chem3D program (version 3.2 from Cambridgesoft Corp., Cambridge, MA). The Chem3D force field was generated by using the MM2 method, with default parameters. The calculations, which take into account van der Waals forces, electrostatic contributions, dipole interactions, and torsion, were evaluated for the purpose of obtaining energy-minimized three-dimensional structures of the adducts. The starting structures of the DB[a,l]PDE-dA adducts were refined until the restricted movement of atoms was less than 0.01 kcal/mol. Dihedral angles were calculated from the minimized structures. Theoretical coupling constants were obtained by using the Karplus equation that establishes a relationship between vicinal proton-proton coupling constants and the related dihedral angles (16). Chemicals. (()-anti-DB[a,l]PDE and (()-syn-DB[a,l]PDE (9) were obtained from Chemsyn Science Laboratories (Lenexa, KS). They were approximately 99% pure as determined by HPLC and were used as received. (+)-syn-DB[a,l]PDE and (-)-antiDB[a,l]PDE were synthesized as previously described (17, 18) and were used as received. The dA was purchased from Aldrich (Milwaukee, WI) and was desiccated over P2O5 under vacuum at 110 °C for 48 h prior to being used. Commercially available dimethylformamide (DMF, Aldrich) was purified by refluxing

Chem. Res. Toxicol., Vol. 12, No. 9, 1999 759 over CaH2, followed by distillation under vacuum; the distillate was stored over 4 Å molecular sieves under argon. Chemical Synthesis of DB[a,l]PDE Adducts. DB[a,l]PDE (racemic or optically pure, 5 mg, 0.0142 mmol) was dissolved in 1 mL of dry DMF at room temperature under argon. The dA (35 mg, 0.142 mmol) was added to the above solution, and the reaction was carried out for either 30 min at 100 °C or 8 h at room temperature. After the reaction was complete, DMF was removed under vacuum, the residue was dissolved in a Me2SO/ CH3OH mixture (1:1), and an aliquot was analyzed by HPLC with the CH3CN/H2O gradient. The product yields were calculated from the peak areas, monitored at 254 nm because all of the adducts have similar molecular extinction coefficients. Purification of all adducts was conducted by preparative HPLC with the CH3CN/H2O gradient, followed by a second separation using the CH3OH/H2O isocratic eluent. The purity of all adducts after preparative HPLC separation was independently checked by analytical HPLC in the two solvent systems. (1) (11S,12R,13S)-Trihydroxy-(14R)-N6dA-11,12,13,14tetrahydro-DB[a,l]P [(+)-anti-trans]: UV λmax 228, 244, 258, 267, 280, 296, 329, 342 nm; 1H NMR δ 2.10-2.15 (m, 2 H, 2′bH2), 3.44-3.53 (m, 2 H, 5′a,b-H2), 3.66 (m, 1 H, 4′b-H), 3.73 (m, 1 H, 4′a-H), 4.04 (m, 1 H, 12b-H), 4.15 (m, 1 H, 12a-H), 4.26 (m, 1 H, 3′a,b-H), 4.81 (dd, 1 H, 13a-H, J13,14 ) 5.0 Hz, J13,13OH ) 8.5 Hz), 5.10 (m, 1 H, 5′a,b-OH, can be exchanged with D2O), 5.175.26 [m, 4 H, 13b-H, 3′a,b-OH, 12a,b-OH, 11a,b-H, two protons can be exchanged with D2O, J11,12 ) 8.0 Hz (D2O/Me2SO), J12,13 ) 6.0 Hz (D2O/Me2SO)], 5.31 (bs, 1 H, 13a-OH, can be exchanged with D2O), 5.40 (bs, 1 H, 13b-OH, can be exchanged with D2O), 5.68 (t, 1 H, 1′b-H, J1′,2′ ) 6.5 Hz), 5.83 (d, 1 H, 11a,b-OH, can be exchanged with D2O), 6.12 (t, 1 H, 1′a-H, J1′,2′ ) 6.5 Hz), 6.20 [s, 1 H, 2a-H(Ade)], 6.42 (bs, 1 H, 14a-H), 6.83 (bs, 1 H, 14b-H), 7.44 [s, 1 H, 2b-H(Ade)], 7.67-7.79 (m, 2 H, 2b-H, 3b-H), 7.857.87 (m, 3 H, 6b-H, 2a-H, 3a-H), 7.93-8.03 (m, 3 H, 6a-H, 8a,b-H, 9a,b-H), 8.14 (d, 1 H, 7b-H, J6,7 ) 7.5 Hz), 8.20 (d, 1 H, 7a-H, J6,7 ) 7.5 Hz), 8.30-8.39 [m, 2 H, 10-H, 8a,b-H(Ade)], 8.46 (m, 1 H, 1a,b-H), 8.53 [bs, 1 H, 6-NbH(Ade), can be exchanged with D2O], 8.92-9.03 (m, 2H, 5a,b-H, 4a,b-H), 9.98 [bs, 1 H, 6-NaH(Ade), can be exchanged with D2O]; FAB MS [M + H]+ for C36H30N5O6 calcd 604.2196, found 604.2185. (2) (11R,12S,13R)-Trihydroxy-(14S)-N6dA-11,12,13,14tetrahydro-DB[a,l]P [(-)-anti-trans]: UV λmax 228, 245, 257, 268, 282, 296, 329, 342 nm; 1H NMR δ 2.20 (m, 1 H, 2′-H), 2.63 (m, 1 H, 2′-H), 3.46-3.64 (m, 2 H, 5′-H2), 3.86 (m, 1 H, 4′-H), 4.07 (t, 1 H, 12-H, J12,13 ) 8.0 Hz), 4.18 (bs, 1 H, 12-OH, can be exchanged with D2O), 4.36 (m, 1 H, 3′-H), 4.94 (d, 1 H, 13-H, J13,14 ) 3.5 Hz), 5.13 (t, 1 H, 11-H, J11,12 ) 7.0 Hz), 5.19 (bs, 1 H, 5′-OH, can be exchanged with D2O), 5.29 (bs, 2 H, 3′OH, 13-OH, can be exchanged with D2O), 5.76 (m, 1 H, 11-OH, can be exchanged with D2O), 6.23 (t, 1 H, 1′-H, J1′,2′ ) 6.5 Hz), 6.53 [s, 1 H, 2-H(Ade)], 6.56 (d, 1 H, 14-H, JNH,14 ) 10.0 Hz), 7.00 (m, 1 H, 2-H), 7.38 (t, 1 H, 3-H, J2,3 ) 7.0 Hz, J3,4 ) 7.5 Hz), 8.02 (t, 1 H, 6-H, J5,6 ) J6,7 ) 7.5 Hz), 8.08-8.13 (m, 2 H, 8-H, 9-H), 8.24 (d, 1 H, 7-H, J6,7 ) 7.5 Hz), 8.37 [s, 1 H, 8-H(Ade)], 8.40 (s, 1 H, 10-H), 8.50 (d, 1 H, 1-H, J1,2 ) 8.0 Hz), 8.70 (d, 1 H, 4-H, J3,4 ) 7.5 Hz), 8.92 (d, 1 H, 5-H, J5,6 ) 7.5 Hz), 10.06 [bs, 1 H, 6-NH(Ade), can be exchanged with D2O]; FAB MS [M + H]+ for C34H30N5O6 calcd 604.2196, found 604.2175. (3) (11S,12R,13S)-Trihydroxy-(14S)-N6dA-11,12,13,14tetrahydro-DB[a,l]P [(+)-anti-cis]: UV λmax 217, 228, 243, 267, 283, 294, 327, 340 nm; 1H NMR δ 2.30 (m, 1 H, 2′-H), 2.79 (m, 1 H, 2′-H), 3.49-3.64 (m, 2 H, 5′-H2), 3.88 (m, 1 H, 4′-H), 4.36-4.49 [m, 3 H, 12-H, 13-H, 3′-H, J12,13 ) 3.0 Hz (D2O/Me2SO)], 4.91 [bs, 2 H, 11-H, 13-OH, can be exchanged with D2O, J11,12 ) 7.5 Hz (D2O/Me2SO)], 5.10-5.20 (m, 2 H, 5′-OH, 12OH, can be exchanged with D2O), 5.31 (m, 1 H, 3′-OH, can be exchanged with D2O), 6.25 (s, 1 H, 11-OH, can be exchanged with D2O), 6.40 (t, 1 H, 1′-H, J1′,2′ ) 6.5 Hz), 6.63 (d, 1 H, 14-H, JNH,14 ) 10.0 Hz), 7.33 (dd, 1 H, 2-H, J1,2 ) 8.0 Hz, J2,3 ) 7.0 Hz), 7.74 (dd, 1 H, 3-H, J2,3 ) 7.0 Hz, J3,4 ) 8.0 Hz), 8.09 (m, 1 H, 6-H), 8.19 (s, 2 H, 8-H, 9-H), 8.30 (d, 1 H, 7-H, J6,7 ) 7.5 Hz),

760 Chem. Res. Toxicol., Vol. 12, No. 9, 1999 8.39 (s, 1 H, 10-H), 8.40 [s, 1 H, 2-H(Ade)], 8.49 (d, 1 H, 1-H, J1,2 ) 8.0 Hz), 8.52 [s, 1 H, 8-H(Ade)], 8.64 [d, 1 H, 6-NH(Ade), can be exchanged with D2O], 8.97 (d, 1 H, 4-H, J3,4 ) 8.0 Hz), 9.05 (d, 1 H, 5-H, J5,6 ) 7.5 Hz); FAB MS [M + H]+ for C34H30N5O6 calcd 604.2196, found 604.2170. (4) (11R,12S,13R)-Trihydroxy-(14R)-N6dA-11,12,13,14tetrahydro-DB[a,l]P [(-)-anti-cis]: UV λmax 217, 228, 243, 267, 283, 294, 327, 340 nm; 1H NMR δ 2.31 (m, 1 H, 2′-H), 2.79 (m, 1 H, 2′-H), 3.51-3.68 (m, 2 H, 5′-H2), 3.92 (m, 1 H, 4′-H), 4.31 (dd, 1 H, 12-H, J12,13 ) 4.0 Hz, J11,12 ) 6.0 Hz), 4.39 (d, 1 H, 13-H, J12,13 ) 4.0 Hz), 4.44 (bs, 1 H, 3′-H), 4.85 (bs, 1 H, 11-H, J11,12 ) 6.0 Hz, J11,11OH ) 7.0 Hz), 4.92-4.96 (m, 2 H, 12OH, 13-OH, can be exchanged with D2O), 5.17 (m, 1 H, 5′-OH, can be exchanged with D2O), 5.34 (m, 1 H, 3′-OH, can be exchanged with D2O), 5.80 (d, 1 H, 11-OH, can be exchanged with D2O, J11,11OH ) 7.0 Hz), 6.42 (t, 1 H, 1′-H, J1′,2′ ) 7.0 Hz), 6.50 (d, 1 H, 14-H, JNH,14 ) 10.0 Hz), 7.24 (dd, 1 H, 2-H, J1,2 ) 8.5 Hz, J2,3 ) 6.5 Hz), 7.67 (dd, 1 H, 3-H, J2,3 ) 6.5 Hz, J3,4 ) 8.0 Hz), 8.06 (dd, 1 H, 6-H, J5,6 ) 7.5 Hz, J6,7 ) 8.0 Hz), 8.138.19 (m, 2 H, 8-H, 9-H), 8.26-8.29 [m, 2 H, 7-H, 6-NH(Ade), can be exchanged with D2O], 8.39 [s, 1 H, 2-H(Ade)], 8.45 (s, 1 H, 10-H), 8.50 [s, 1 H, 8-H(Ade)], 8.67 (d, 1 H, 1-H, J1,2 ) 8.5 Hz), 8.96 (d, 1 H, 4-H, J3,4 ) 8.0 Hz), 9.02 (d, 1 H, 5-H, J5,6 ) 7.5 Hz); FAB MS [M + H]+ for C34H30N5O6 calcd 604.2196, found 604.2185. (5) (11S,12R,13R)-Trihydroxy-(14S)-N6dA-11,12,13,14tetrahydro-DB[a,l]P [(+)-syn-trans]: UV λmax 242, 274, 285, 293, 327, 341 nm; 1H NMR δ 2.30 (m, 1 H, 2′-H), 2.76 (m, 1-H, 2′-H), 3.51-3.63 (m, 2 H, 5′-H2), 3.88 (m, 1 H, 4′-H), 3.99 (dd, 1 H, 13a-H, J12a,13a ) 6.5 Hz), 4.30 [bs, 1 H, 13b-H, J12,13 ) 9.0 Hz (D2O/Me2SO)], 4.43 (m, 1 H, 3′-H), 4.70 (s, 1 H, 13-OH, can be exchanged with D2O), 4.94 (d, 1 H, 11a-H, J11a,12a ) 6.0 Hz), 5.16 (s, 1 H, 12-OH, can be exchanged with D2O), 5.35-5.46 (bd, 2 H, 3′-OH, 5′-OH, can be exchanged with D2O), 5.47 (d, 1 H, 11bH, J11b,12b ) 7.5 Hz), 6.16 [bs, 1 H, 14-H, J13,14 ) 8.0 Hz (D2O/ Me2SO)], 6.30 (t, 1 H, 1′a-H, J1,2 ) 6.5 Hz), 6.41 (t, 1 H, 1′b-H, J1′,2′ ) 6.5 Hz), 6.71 (bs, 1 H, 11-OH, can be exchanged with D2O), 7.20 (bs, 1 H, 2-H), 7.62 (dd, 1 H, 3-H, J2,3 ) 7.5 Hz, J3,4 ) 8.0 Hz), 8.00 (dd, 1 H, 6-H, J5,6 ) 8.0 Hz, J6,7 ) 7.5 Hz), 8.11 (d, 1 H, 9-H, J8,9 ) 15.0 Hz), 8.17 (s, 1 H, 8-H, J8,9 ) 15.0 Hz), 8.24 (d, 1 H, 7-H, J6,7 ) 7.5 Hz), 8.29 [s, 1 H, 8-H(Ade)], 8.37 [s, 1 H, 2-H(Ade)], 8.40 (s, 1 H, 1-H), 8.50 (bs, 1 H, 10-H), 8.56 [d, 1 H, 6-NH(Ade), can be exchanged with D2O, JNH,14 ) 10 Hz], 8.90 (d, 1 H, 4-H, J3,4 ) 8.0 Hz), 8.97 (d, 1 H, 5-H, J5,6 ) 8.0 Hz); FAB MS [M + H]+ for C34H30N5O6 calcd 604.2196, found 604.2185. (6) (11R,12S,13S)-Trihydroxy-(14R)-N6dA-11,12,13,14tetrahydro-DB[a,l]P [(-)-syn-trans]: UV λmax 242, 274, 285, 293, 327, 341 nm; 1H NMR δ 2.36 (m, 1 H, 2′-H), 2.85 (m, 1 H, 2′-H), 3.24 (m, 1 H, 12-H), 3.55-3.69 (m, 2 H, 5′-H2), 3.92 (m, 1 H, 4′-H), 4.03 (t, 1 H, 13-H, J12,13 ) 7.5 Hz, J13,13OH ) 6.5 Hz), 4.46 (bs, 1 H, 3′-H), 4.73 (bs, 1 H, 13-OH, can be exchanged with D2O), 5.19 (bs, 1 H, 5′-OH, can be exchanged with D2O), 5.35 (bs, 1 H, 12-OH, can be exchanged with D2O), 5.39 (bs, 1 H, 3′-OH, can be exchanged with D2O), 5.52 (bs, 1 H, 11-H), 5.89 (bs, 1 H, 11-OH, can be exchanged with D2O), 6.20 (m, 1 H, 14-H), 6.44 (t, 1 H, 1′-H, J1′,2′ ) 6.5 Hz), 7.23 (bs, 1 H, 2-H), 7.66 (dd, 1 H, 3-H, J2,3 ) 7.5 Hz, J3,4 ) 8.5 Hz), 7.91 [s, 1 H, 8-H(Ade)], 8.05 (dd, 1 H, 6-H, J5,6 ) 8.0 Hz, J6,7 ) 7.0 Hz), 8.148.22 (m, 2 H, 8-H, 9-H), 8.28 (d, 1 H, 7-H, J6,7 ) 7.0 Hz), 8.43 (bs, 1 H, 1-H), 8.48 [s, 1 H, 2-H(Ade)], 8.54 (s, 1 H, 10-H), 8.59 [d, 1 H, 6-NH(Ade), can be exchanged with D2O, JNH,14 ) 6.5 Hz], 8.94 (d, 1 H, 4-H, J3,4 ) 8.5 Hz), 9.01 (d, 1 H, 5-H, J5,6 ) 8.0 Hz); FAB MS [M + H]+ for C34H30N5O6 calcd 604.2196, found 604.2185. (7) (11S,12R,13R)-Trihydroxy-(14R)-N6dA-11,12,13,14tetrahydro-DB[a,l]P [(+)-syn-cis]: UV λmax 241, 274, 285, 294, 327, 342 nm; 1H NMR δ 2.32 (m, 1 H, 2′-H), 2.84 (m, 1 H, 2′-H), 3.14 (m, 1 H, 13-H), 3.55-3.70 (m, 2 H, 5′-H2), 3.93 (m, 1 H, 4′-H), 4.11 (t, 1 H, 12-H, J12,13 ) 7.5 Hz), 4.47 (bs, 1 H, 3′-H), 4.96 (bs, 1 H, 11-H), 5.06 (d, 1 H, 13-OH), 5.25 (d, 1 H, 12-OH, can be exchanged with D2O), 5.30 (d, 1 H, 3′-OH, can be

Li et al. exchanged with D2O), 5.37 (m, 1 H, 5′-OH, can be exchanged with D2O), 6.42 (t, 1 H, 1′-H, J1′,2′ ) 6.5 Hz), 6.66 (dd, 1 H, 14H, J13,14 ) 3.0 Hz, JNH,14 ) 10 Hz), 6.92 (s, 1 H, 11-OH), 7.39 (dd, 1 H, 2-H, J1,2 ) 8.0 Hz, J2,3 ) 7.0 Hz), 7.77 (dd, 1 H, 3-H, J2,3 ) 7.0 Hz, J3,4 ) 8.5 Hz), 8.10 (dd, 1 H, 6-H, J5,6 ) 8.0 Hz, J6,7 ) 7.5 Hz), 8.18-8.20 (m, 2 H, 8-H, 9-H), 8.30-8.32 [m, 2 H, 7-H, 2-H(Ade)], 8.38 (s, 1 H, 10-H), 8.46 [s, 1 H, 8-H(Ade)], 8.60 (d, 1H, 1-H, J1,2 ) 8.0 Hz), 8.78 [bd, 1 H, 6-NH(Ade), can be exchanged with D2O], 8.98 (d, 1 H, 4-H, J3,4 ) 8.5 Hz), 9.06 (d, 1 H, 5-H, J5,6 ) 8.0 Hz); FAB MS [M + H]+ for C34H30N5O6 calcd 604.2196, found 604.2197. (8) (11R,12S,13S)-Trihydroxy-(14S)-N6dA-11,12,13,14tetrahydro-DB[a,l]P [(-)-syn-cis]: UV λmax 241, 274, 285, 294, 327, 342 nm; 1H NMR δ 2.34 (m, 1 H, 2′-H), 2.85 (m, 1 H, 2′-H), 3.14 (m, 1 H, 13-H), 3.53-3.68 (m, 2 H, 5′-H2), 3.92 (m, 1 H, 4′-H), 4.11 (dd, 1 H, 12-H, J11,12 ) 6.5 Hz, J12,12OH ) 4.5 Hz), 4.46 (bs, 1 H, 3′-H), 4.96 (d, 1 H, 11-H, J11,11OH ) 3.5 Hz), 5.05 (bd, 1 H, 13-OH, can be exchanged with D2O), 5.24-5.34 (m, 3 H, 3′-OH, 5′-OH, 12-OH, three protons can be exchanged with D2O), 6.42 (t, 1 H, 1′-H, J1′,2′ ) 7.0 Hz), 6.65 (dd, 1 H, 14-H, JNH,14 ) 10.5 Hz, J13,14 ) 3.5 Hz), 6.91 (s, 1 H, 11-OH, can be exchanged with D2O), 7.38 (dd, 1 H, 2-H, J1,2 ) 8.0 Hz, J2,3 ) 7.5 Hz), 7.76 (dd, 1 H, 3-H, J2,3 ) 7.5 Hz, J3,4 ) 8.5 Hz), 8.07 (t, 2 H, 6-H, J5,6 ) 8.5 Hz, J6,7 ) 7.5 Hz), 8.18-8.22 (m, 2 H, 8-H, 9-H), 8.30-8.33 [m, 2 H, 7-H, 2-H(Ade)], 8.38 (s, 1 H, 10-H), 8.45 [s, 1 H, 8-H(Ade)], 8.60 (d, 1 H, 1-H, J1,2 ) 8.0 Hz), 8.77 [d, 1 H, 6-NH(Ade), can be exchanged with D2O, JNH,14 ) 10.5 Hz], 8.98 (d, 1 H, 4-H, J3,4 ) 8.5 Hz), 9.06 (d, 1 H, 5-H, J5,6 ) 8.5 Hz); FAB MS [M + H]+ for C34H30N5O6 calcd 604.2196, found 604.2175. (9) (()-syn-DB[a,l]PDE-14-N7Ade: UV λmax 240, 271, 283, 294, 326, 340 nm; 1H NMR δ 4.06 (dd, 1 H, 13-H, J12,13 ) 2.0 Hz, J13,14 ) 2.5 Hz), 4.11 (dd, 1 H, 12-H, J11,12 ) 9.0 Hz, J12,13 ) 2.0 Hz), 5.40 (d, 1 H, 11-H, J11,12 ) 9.0 Hz), 5.43 (d, 1 H, 14-H, J13,14 ) 2.5 Hz), 7.69 (dd, 1 H, 2-H, J1,2 ) 8.0 Hz, J2,3 ) 7.0 Hz), 7.78 (dd, 1 H, 3-H, J2,3 ) 7.0 Hz, J3,4 ) 7.5 Hz), 7.92 [s, 1 H, 2-H(Ade)], 8.01 (dd, 1 H, 6-H, J5,6 ) 8.5 Hz, J6,7 ) 7.0 Hz), 8.13 (dd, 2 H, 8-H, 9-H, J8,9 ) 9.0 Hz), 8.25 (d, 1 H, 7-H, J6,7 ) 7.0 Hz), 8.40 [s, 1 H, 8-H(Ade)], 8.42 (s, 1 H, 10-H), 8.93 (d, 1 H, 4-H, J3,4 ) 7.5 Hz), 8.98 (d, 1 H, 5-H, J5,6 ) 8.5 Hz), 9.01 (d, 1 H, 1-H, J1,2 ) 8.0 Hz); FAB MS [M + H]+ 487 and fragments of m/z 459, 413, 391, 353, 307, and 115.

Results and Discussion Isolation of Adducts. After the reaction of (()-antiDB[a,l]PDE or (()-syn-DB[a,l]PDE with dA, the pair of cis-opened dA adducts can be easily separated from the trans-opened dA adducts by using a CH3CN/H2O gradient (Figures 1 and 3). Under isocratic HPLC conditions (CH3OH/H2O), the two cis-opened isomers and the two transopened adducts can be separated from each other (Figures 1 and 3). For (-)-anti-DB[a,l]PDE and (+)-synDB[a,l]PDE, the cis-opened and trans-opened reaction products formed with dA can be separated with the CH3CN/H2O gradient (Figure 2). When (()-anti-DB[a,l]PDE reacted with dA in DMF at 100 °C for 30 min, four adducts were produced (Figure 1 and Table 1): (+)-anti-trans (26%), (-)-anti-trans (26%), (+)-anti-cis (17%), and (-)-anti-cis (17%). Two of these adducts are identical to those obtained from (-)anti-DB[a,l]PDE (Figure 2A), and the other two are the stereoisomers derived from the (+)-anti-DB[a,l]PDE enantiomer. In these reactions, both cis and trans opening of the oxirane ring by the exocyclic amino group of dA occur to produce a pair of epimers differing only in the stereochemistry at C-14 of the PAH moiety. The N6dA adducts were isolated from both the racemic and the optically pure anti-DB[a,l]PDE in various yields (Table 1). When (()-anti-DB[a,l]PDE was reacted with dA at

DB[a,l]P Dihydrodiol Epoxide-dA Adducts

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Figure 1. HPLC profile of the products of the reaction between (()-anti-DB[a,l]PDE and dA at 100 °C (top) and at room temperature (bottom).

room temperature for 8 h, only the two trans-opened products, (+)-anti-trans and (-)-anti-trans, were obtained and isolated by HPLC under isocratic conditions (Figure 1). Reaction of (()-syn-DB[a,l]PDE with dA at 100 °C for 30 min afforded five adducts that were isolated by successive gradient and isocratic HPLC runs (Figure 3 and Table 1): (+)-syn-trans (13%), (-)-syn-trans (13%), (+)-syn-cis (19%), (-)-syn-cis (19%), and the depurination product (()-syn-DB[a,l]PDE-14-N7Ade (22%). Reaction of (+)-syn-DB[a,l]PDE with dA under the same conditions gave (+)-syn-trans (14%) and (+)-syn-cis (56%) (Figure 2B and Table 1). Reaction of (()-syn-DB[a,l]PDE with dA at room temperature for 8 h afforded predominantly the two cis-opened products, (+)-syn-cis and (-)-syn-cis (Figure 3), and a small amount of (()-syn-DB[a,l]PDE14-N7Ade (not shown in Figure 3). In the reaction of (+)syn-DB[a,l]PDE with dA at 100 °C, (+)-syn-DB[a,l]PDE14-N7Ade was not formed (Figure 2B). Structure Elucidation of Adducts. Structure elucidation of DB[a,l]PDE-dA adducts was achieved via the synthesis of these adducts using the two optically pure diol epoxides, (-)-anti-DB[a,l]PDE and (+)-syn-DB[a,l]PDE, and by a combination of UV, NMR, fast atom bombardment tandem mass spectrometry (FAB MS/MS), and CD data. Mass Spectrometry of N6dA Adducts. The elemental compositions of all the dA adducts were established by mass measurements of the [M + H]+ ions that were produced by fast atom bombardment. The average error between the calculated and measured exact mass was 2.4 ppm (the maximum error was 4.3 ppm). The fragmentation of the various isomers was followed by tandem mass spectrometry (high-energy collisional activation of

Figure 2. HPLC profile of the products of the reaction between (-)-anti-DB[a,l]PDE and dA at 100 °C (A) and (+)-syn-DB[a,l]PDE and dA at 100 °C (B).

the [M + H]+) (Figure 4). The m/z 488 precursor fragments by loss of H2O and of Ade to give product ions of m/z 470 and 353. Successive losses of H2O and CO from the m/z 353 ion yield products of m/z 335, 317, and 307, respectively, which are the expected products for the polyhydroxylated PAH. The product ions of m/z 252, 136, and 117 are protonated dA, protonated Ade, and protonated deoxyribose, respectively. The adducts fragment to release the deoxyribose and Ade moieties, as is common for PAH diol epoxide-dA adducts (19). However, the relative abundance of the specific ions observed in the DB[a,l]PDE-dA adducts does not allow one to distinguish among different stereoisomers. (11R,12S,13R)-Trihydroxy-(14S)-N6dA-11,12,13,14tetrahydro-DB[a,l]P [(-)-anti-trans]. This compound is a major product from the reaction of (()-anti-DB[a,l]PDE or (-)-anti-DB[a,l]PDE with dA at 100 °C (26 and 60% yields, respectively; Scheme 1 and Table 1), and was found as one of the two products from the reaction of (()anti-DB[a,l]PDE with dA at room temperature, as indicated by HPLC (Figure 1). The NMR spectrum of this adduct shows one proton resonance as a broad singlet at 10.06 ppm, which can be exchanged with D2O (Figure 5B). This signal was assigned to the NH of dA, because it is the only possible proton expected to resonate at this downfield region, suggesting that this adduct was formed at the NH2 group of dA. The 14-H signal of this adduct appears at 6.56

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Figure 4. CAD spectra of (A) the [M + H]+ ion (m/z 604) of (-)-anti-trans-DB[a,l]PDE-14-N6dA and (B) the m/z 488 fragment ion, presumably the protonated base adduct formed from the [M + H]+ of (-)-anti-trans-DB[a,l]PDE-14-N6dA.

Figure 3. HPLC profile of the products of the reaction between (()-syn-DB[a,l]PDE and dA at 100 °C (top) and at room temperature (bottom). Table 1. Adducts Obtained by Reaction of (-)-anti-, (()-anti-, (+)-syn-, or (()-syn-DB[a,l]PDE with dA at 100 °C for 30 min

adduct (+)-anti-trans (-)-anti-trans (+)-anti-cis (-)-anti-cis (+)-syn-trans (-)-syn-trans (+)-syn-cis (-)-syn-cis (()-syn-DB[a,l]PDE14-N7Ade

yield from (()-anti- yield from (-)-antior or (()-syn-DB[a,l]PDE (+)-syn-DB[a,l]PDE (%) (%) 26 26 17 17 13 13 19 19 22

60 24 14 56

ppm, considerably downfield compared to the chemical shifts of the other methine protons of the tetrahydro ring. Furthermore, the NMR spectrum of this compound shows the signals of all the protons of deoxyribose. On the basis of the NMR data, this compound was assigned as the N6dA adduct attached at the C-14 position of the (-)-antiDB[a,l]PDE moiety. All the other protons were assigned by COSY and D2O exchange NMR spectra (Figure 5B). The coupling constant J13,14 of 3.5 or 5.0 Hz [(+)-antitrans] (Table 2) provides strong evidence that 13-H and 14-H are trans to each other. In fact, the cis-opened adducts have such a small J13,14 (e0.5 Hz) that the value cannot be determined. Validation of the assignment of the structures is provided by comparison of the experimental coupling constants J13,14 with the corresponding calculated ones (see below). Because the (-)-anti-DB[a,l]PDE has 11R,12S,13S,14R-configuration and trans opening of the oxirane ring at the C-14 position occurs under inversion, the absolute stereochemistry of the carbon center attached to the dA moiety must be 14S. Thus, this compound is the (-)-anti-trans stereoisomer.

(11R,12S,13R)-Trihydroxy-(14R)-N6dA-11,12,13,14tetrahydro-DB[a,l]P [(-)-anti-cis]. This compound is the minor dA adduct from the reaction of (()-anti-DB[a,l]PDE or (-)-anti-DB[a,l]PDE with dA (Scheme 1 and Table 1). Therefore, this adduct was assigned as the (-)anti-cis stereoisomer. The NMR data support this assignment (Figure 6B). In fact, the J13,14 is small and cannot be determined (e0.5 Hz, Table 2). This corroborates the fact that 13-H and 14-H must be cis to each other, and thus, the absolute stereochemistry of the adduct must be (11R,12S,13R)-trihydroxy-(14R)-N6dA11,12,13,14-tetrahydro-DB[a,l]P [(-)-anti-cis]. The NMR spectrum of this compound (Figure 6B) indicates that a minor conformer (