Synthesis and structure determination of the adducts formed by

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Chem. Res. Toricol. 1993, 6, 554-560

554

Synthesis and Structure Determination of the Adducts Formed by Electrochemical Oxidation of the Potent Carcinogen Dibenzo[ s,l]pyrene in the Presence of Nucleosides N. V. S. RamaKrishna,? N. S. Padmavathi,? Ercole L. Cavalieri,*p+ Eleanor G. Rogan,t Ronald L. Cerny,J and Michael L. Gross3 Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, Nebraska 68198-6805, and Midwest Center for Mass Spectrometry, Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0362 Received January 2, 1993

Because dibenzo[a,l]pyrene (DBP) is the most potent known carcinogenic aromatic hydrocarbon, reference adducts formed by reaction of deoxyribonucleosides with electrophilic intermediates of DBP are essential for identifying the structures of adducts formed in biological systems, Electrochemical oxidation of DBP in the presence of nucleosides leads to adducts from DBP*+.When 6.8 equivof charge are consumed, three adducts are formed with dG: 7-(DBP10-y1)Gua (89%), 8-(DBP-lO-yl)dG(2%),and8-(DBP-lO-yl)Gua (2%). With loequivofcharge, however, only two adducts are formed: 7-(DBP-lO-yl)Gua (89%)and 8-(DBP-lO-yl)Gua (4%). Anodic oxidation of 8-(DBP-lO-yl)dG yields 8-(DBP-lO-yl)Gua. Anodic oxidation of DBP in the presence of G produces 7-(DBP-lO-yl)Gua (27%)and84DBP-lO-yl)G (9%1. Anodicoxidation of DBP in the presence of dA affords two adducts, N6-(DBP-10-yl)dA (28%) and 7-(DBP-10y1)Ade (12%), whereas anodic oxidation in the presence of A produces only N6-(DBP-10-yl)A (24%), The structures of the adducts were elucidated by using UV, NMR, and MS.Formation of these adducts demonstrates that DBP*+reacts a t C-10 with nucleophiles. The most reactive nucleophilic groups for the Gua moiety are the N-7 and C-8,whereas for the Ade moiety they are N-7 and the 6-amino group.

Introduction Dibenzo[a,llpyrenel (DBP12is the most potent known carcinogen not only among the various dibenzo[a]pyrenes (1)but also among all polycyclic aromatic hydrocarbons (PAH) (2, 3). Metabolic activation of PAH leading to tumor initiation occurs by two main pathways: oneelectron oxidation to yield reactive intermediate radical cations (4,5) and monooxygenationto produce bay-region diolepoxides (5-7). Metabolism of DBP by cytochromeP-450 produces three major metabolites (8). One of them is DBP-11,12dihydrodiol, the precursor to the ultimate reactive bayregion diolepoxide. The tumor-initiating activity of this metabolite is similar to that of the parent compound in dose-response studies in mouse skin (2),although at very low doses the activity of the dihydrodiol is less than that of DBP (3). These preliminary data suggest that the diolepoxide intermediate may play a role in the carcinogenic activity of DBP. * To whom correspondence should be addressed. + University of Nebraska Medical Center.

University of Nebraska-Lincoln. 1 IUPAC systematic name: dibenzo[def,plchrysene. * Abbreviations: BP, benzo[alpyrene; CAD, collisionally activated decomposition;dA, deoxyadenosine;DBP, dibenzo[a,llpyrene;DBP-10CMG, a(dibenzo[a,Zlpyren-l0-yl)deoxyguanoeine;DBP-lO-C8G,&(dibenzo[a,Z]pyren-10-y1)guanosine; DBP-10-CSGua,8-(dibenzo[a,llpyren-l0y1)guanine;DBP-10-N7A, 7-(dibenzo[a,lIpyren-lO-yl)adenosine;DBP10-N7Ade, 7-(dibenzo[a,lIpyren-lO-yl)adenine; DBP-lO-NBdA, Ne(dibenzo[a,l]pyren-10-y1)deoxyadenosine; DBP-lO-N7Gua, 7:(dibenzo[a,l]pyren-lO-yl)guanine; dC, deoxycytidine;2D COSY, twodunensionalchemical shift correlationspectroscopy;dG,deoxyguanosine; DMF, dimethylformamide;dT, thymidine; FAB MS/MS, fast atom bombardment tandem mass spectrometry; PAH, polycyclic aromatic hydrocarbon(s);PDA, photodiode array. 8

The DBP*+ generated by Mn(0Ac)a reacts regiospecifically at C-10 (9). This result is similar to that of the benzo[alpyrene (BPI radical cation, which reacts at C-6 with acetate ion (10) and with nucleosides (11, 12). Furthermore, the predominant DNA adducts formed by BP in vitro (13) and in vivo (14) are derived from BP*+. To identify DBP adducts formed in biological systems, it is necessary to synthesize prospective model adducts that will serve as reference standards. Thus, synthesis of two classes of adducts is needed: those arising from DBP diolepoxide and the DBP'+. In this paper, we report the synthesis of radical cation adducts obtained by anodic oxidation of DBP in the presence of nucleosides. Determination of the structure of the resulting adducts has been accomplished by using NMR and fast atom bombardment tandem mass spectrometry (FAB MS/MS). The mass spectra reported here are among the first obtained with a prototype four-sector tandem mass spectrometer.

Experimental Section General Procedures. (A) UV. UV absorbancespectra were recorded with a Waters 990 photodiode array (PDA) detector during elution from HPLC with CHaOH/H20 or CH&N/H20 gradients, as described below. (B) NMR. lH-NMR spectra were recorded in dimethyl sulfoxide (MezSO)-deat 30 "C as previously reported (11). (C) FAB MS/MS. Collisionally activated decomposition (CAD)spectra were obtained by using a VG ZAB-T, a four-sector tandem mass spectrometer of BEBE design. MS1 is a standard high-resolution ZAB of reverse geometry. MS2, also capable of high resolution, is a prototype Mattuch-Herzog type design, incorporating a standard magnet and an inhomogeneous planar

oa93-22ax19312~~6-0554$04.~100 1993 American Chemical Society

Chem. Res. Toxicol., Vol. 6, No. 4, 1993 555

One-Electron Oxidation of Dibenzo[a,llpyrene Table I. Adducts of DBP Formed with Various Nucleosides electron equiv of nucleoside" charge consumed adduct (yield, % ) DBP-lO-N7Gua (89) dG 10.0 DBP-lO-C8Gua (4) dG 6.8 DBP-lO-N7Gua (89) DBP-10-CSdG (2) DBP-lO-C8Gua (2) G 3.8 DBP-lO-N7Gua (27) DBP-10-CSG (9) dA 3.0 DBP-10-NdA (28) DBP-lO-N7Ade (12) A 2.8 DBP-10-IPA (24) a dC and dT afforded no detectable products. electrostatic analyzer. This design allows the use of a PDA for simultaneous detection of ions over a variable mass range and a single-point detector for scanning experiments. For experiments reported here, sample quantities were sufficiently large so that the single-point detector was adequate. Samples were dissolved in 25 pL of MezSO, and a 1-pL aliquot was placed on the probe along with 1pL of matrix, a 1:lmixture of 3-nitrobenzyl alcohol and glycerol. A Cs+ion gun operated at 30 keV was used to desorb the ions from the probe. The instrument accelerating voltage was 8 kV. CAD spectra were obtained after precursor ion activation in the third field-free region (between MS1 and MS2) by collisions with He, which had been added to the collision cell to attenuate the ion beam by 50%. MS1 was operated at a resolution of 1000; MS2 resolution was set to 1200 (full width at half-height definition). Ten to fifteen 25-s scans were signal-averaged for each spectrum. Data acquisition and data workup were controlled by using a VAX 3100 workstation equipped with OPUS software. (D)HPLC. 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 usingaYMCODS-AQ5-pm,120-Acolumn(6.0X 250") (YMC, Overland Park, KS). After the column was eluted for 5 min with 30% CH&NinHzO,a70-mincurvilineargradient(CV7)to 100%

Scheme

CH&N was run at 1.0 mL/min. A second solvent system, which was used for purity check and final purification of adducts, consisted of elution for 5 min with 30% CHSOHin HzO followed by a 75-min linear gradient (CV6) to 100% CHaOH, run at 1.0 mL/min. Preparative HPLC was conducted using a YMC ODSAQ 5-pm, 120-A column (20 X 250 mm) at a flow rate of 6.0 mL/min. Both CH&N/H20 and CHaOH/H20 gradients were used for adduct purification. Materials. DBP was obtained from the National Cancer Institute Chemical Carcinogen Repository (Bethesda, MD). It was more than 99% pure (mp 161-162 "C) and was used as received. The deoxyribonucleosidesdeoxyguanosine(dG), deoxyadenosine (dA),deoxycytidine (dC), and thymidine (dT) and the ribonucleosides Guo and Ado were purchased from either Aldrich (Milwaukee,WI) or Sigma ChemicalCo. (St. Louis, MO) and were desiccated over P205 under vacuum at 110 "C for 48 h prior to use, Commercially available dimethylformamide (DMF) (Aldrich) was purified by heating to reflux over CaHz, followed by vacuum distillation just prior to use, and was stored over 4-A molecular sieves under argon. KC104 (Aldrich) was used as obtained. Caution: DBP is a hazardous chemical and was handled according t o NIH guidelines (15). Electrochemical Synthesis of Adducts. Electrochemical syntheses were conducted with a previously described apparatus (EG&G Princeton Applied Research, Princeton, NJ) (11). The oxidation potential used for the synthesis of DBP adducts was 1.10 V, slightly less than ita anodic peak potential of 1.14 V, measured by cyclic voltammetry (Model CV27, Bioanalytical Systems, Lafayette, IN) in DMF. All of the individual deoxyribonucleosideshave anodic peak potentials 21.36 V. Thus, during adduct synthesis at 1.10 V, none of the nucleosideswere oxidized. Glassware, syringes, needles, electrochemical cell, platinum working, and reference electrodes were dried at 150 OC prior to use. The electrochemical cell and working electrode were assembled while hot and allowed to cool under argon. Coupling between DBP and the nucleophilic groups of nucleosides was accomplished by selective anodic oxidation of DBP in the presence of the nucleoside. Typical experimental conditions were as previously reported for the oxidation of other PAH (16). After the reaction was complete, DMF was removed under vacuum, the adducts were extracted four times from the solid (KC10,) by using a solvent mixture of ethanol/chloroform/ acetone (2:1:1), and the resulting extract was filtered through a

Electrochemical Oxidation of DBP in the Presence of (A) d G or

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ANODIC OXIDATION

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566 Chem. Res. Toxicol., Vol. 6, No. 4, 1993

Whatman fluted filter paper. The combined solvent mixture was evaporated under vacuum, and the residue was dissolved in 3 mL of MezSO, filtered through a 0.45-pm filter, and analyzed by HPLC with the CH&N/H20 gradient. Purification of the adducts waa conducted by preparative HPLC in CH&N/H20, followed by the CHsOH/H20 gradient. The purity of all of the adducts after preparative HPLC separations was independently checked by analytical HPLC in the two solvent systems, CHsCN/ H20 and CHsOH/H20. The adducts were isolated from the reaction of DBP with the nucleosides in various yields (Table I). DBP: UV, A, (nm) 238, 270, 302,316, 372,390; 1H NMR, 6 7.78-7.89 (m, 4 H, 2-H, 3-H, 12-H, 13-H), 8.01 (d, 1 H, 8-H, J = 9.3 Hz), 8.07-8.15 (m, 2 H, 6-H, 9-H), 8.24 (d, 1 H, 7-H, J = 7.5 Hz), 8.42 (dd, 1H, 11-HI, 8.75 (9, 1 H, lO-H), 9.04-9.13 (m, 3 H, 1-H, 4-H, 5-H), 9.10-9.20 (dd, 1 H, 14-H). 8-(DBP-lO-yl)dG(DBP-10-C8dG):UV, A, (nm) 238,271, 306, 320, 383, 403. 7-(DBP-lO-yl)Gua(DBP-lO-N7Gua): UV, A, (nm) 239, 271,306,320,383,403; ‘H NMR, 6 6.32 (be, 2 H, 2-NHdGuaI, exchanged with D2O). 7.37 (d, 1H, 9-H, J = 9.4 Hz), 7.60 (d, 1 H, 11-H, J = 8.4 Hz), 7.76-7.97 (m, 4 H, 2-H, 3-H, 12-H, 13-H), 8.05 (d, 1H, 8-H, J = 9.4 Hz), 8.16 (dd, 1H, 6-H, JH = J&7 = 7.8 Hz), 8.22 (8, 1 H, 8-H[Gua]), 8.28 (d, 1 H, 7 - H , J = 7.8 Hz), 9.09 (d, 1H, 1-H,J = 7.9 Hz), 9.10-9.28 (m, 3 H, 4-H, 5-H, 14-H); MS, (M H)+ CaHlaNaO calcd 452.1511, obsd 452.1504. 8-(DBP-10-y1)Gua(DBP-10-C8Gua): UV, A,, (nm) 240, 271,306,320,384,403; lH NMR, 6 6.60 (bs, 2 H, 2-NHz[Gua], exchanged with DzO), 7.73-7.94 (m, 5 H, 3-H, 2-H, 13-H, 12-H, 8-H), 8.01 (d, 1H, 9-H, J = 9.3 Hz), 8.10-8.17 (m, 2 H, 6-H, 7-H), 8.26 (d, 1H, 11-H, J = 7.9 Hz), 9.06 (d, 1 H, 1-H, J = 8.6 Hz), 9.11-9.23 (m, 3 H, 4-H, 5-H, 14-H); MS, (M + H)+ CSHI$J5O calcd 452.1511, obsd 452.1491. 7-(DBP-lO-yl)Ade(DBP-lO-N7Ade): UV, A, (nm) 240, 272,306, 320, 384, 404: lH NMR, 6 5.72 (bs, 2 H, 6-NHzfAde1, exchanged with DzO), 7.30 (d, 1 H, 9-H, J = 9.1 Hz), 7.47 (d, 1 H, 11-H, J = 8.7 Hz), 7.77-7.98 (m, 4 H, 12-H, 13-H, 3-H, 2-H), 8.12 (d, 1H, 8-H, J = 9.1 Hz), 8.19 (dd, 1 H, 6-H, 5 6 7 = 7.6 Hz,

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TIME, MINUTES J~=7.8Hz),8.32(d,lH,7-H,J=7.6Hz),8.39(s,lH,2-H[Adel), 8.69 (8, 1 H, 8-H[Ade]), 9.11 (d, 1 H, 1-H, J = 7.7 Hz), 9.19 (d, Figure 1. HPLC (CH&N/H20 gradient) profiles of the products

obtained by electrochemical oxidation of DBP in the presence 1H, 4-H, J = 8.0 Hz), 9.23-9.32 (m, 2 H, 5-H, 14-HI; MS, (M + of dG. (A) 6.8 electron equiv of charge consumed; (B) 10.0 electron H)+ CaHlaNs calcd 436.1562, obsd 436.1553. equiv of charge consumed. Na-(DBP-10-y1)dA(DBP-10-MdA): UV, A, (nm) 240,270, 308,320,384,402; 1H NMR, 6 2.30 (bm, 1H, 2’-H), 2.79 (bm, 1 yielded two primary adducts, DBP-lO-C8dG and DBPH,2’-H), 3.59 (m, 2 H, 5’-H2),3.90 (bs, 1H,4’-H),4.43 (bs, 1H, 3’-H), 5.27 (bm, 2 H, 3’-OH, 5’-OH, exchanged with DzO), 6.40 10-N7Gua, and one secondary adduct, DBP-10-C8Gua (t, 1H, 1’-HI, 6.85 (8, 1 H, 2-H[Adel), 7.67-7.87 (m,4 H, 2-H, (Scheme IA), when the reaction consumed 6.8 electron 3-H, 12-H, 13-H), 7.94 (d, 1H, 8-H, J = 9.4 Hz), 8.06-8.14 (m, equiv of charge (Table I). Upon consumption of 10electron 3 H, 6-H, 9-H, 8-H[Ade]), 8.21 (d, 1H, 7-H, J = 7.4 Hz), 8.40 (d, equiv of charge, the reaction afforded two adducts, DBP1H, 11-H, J = 7.6 Hz), 9.01-9.24 (m, 4 H, 1-H, 4-H, 5-H, 14-H), 10-C8Guaand DBP-lO-N7Gua.In this case, DBP-lO-C8dG 10.50 (bs, 1H, 6-NH[Ade], exchanged with Dz0); MS, (M + H)+ was electrochemically converted to DBP-lO-C8Gua. A CMHBNaOs cdcd 552.2036, obsd 552.2031. similar conversion was seen previously in the electro8-(DBP-lO-yl)G(DBP-10-CBG):UV, A, (nm) 242,272,307, chemical oxidation of BP in the presence of dG (12).The 320,385,403; 1H NMR, 6 3.55 (m, 2 H, 5’-H2),4.00 (bs, 1H, 4’-H), structure assignments will be discussed below. 4.30 (bs, 1H, 3’-H),4.65 (bs, OH, exchanged with D20), 4.94 (bs, The analytical HPLC profiles of the products differed 1 H, 2’-H), 5.44 (m, 1 H, l’-H), 6.73 (bs, 2 H, 2-NHdGua1, exchanged with D20), 7.75-7.95 (m, 5 H, 2-H, 3-H, 8-H, 12-H, under these two reaction conditions (Figure 1, panels A 13-H), 8.02 (d, 1 H, 9-H), 8.10-8.20 (m, 2 H, 6-H, 7-H), 8.29 (d, and B). In changing from 6.8 (Figure 1A) to 10 (Figure 1 H, 11-H), 9.04-9.30 (m, 4 H, 1-H, 4-H, 5-H, 14-H); MS, (M + 1B) electron equiv, the first product (eluting at 56 min) H)+ C ~ H ~ N cdcd 5 0 ~584.1934, obsd 584.1950. disappeared, having been converted to the second product Na-(DBP-lO-yl)A(DBP-10-MA): UV, A, (nm), 241,270, (eluting at 58min). The products obtained with 10electron 307,320,384,403; lH NMR, 6 3.55 (m, 2 H, 5’-H2), 4.00 (bs, 1H, equiv of charge (Figure 1B) were identified as DBP-104’-H), 4.30 (bs, 1 H, 3’-H), 4.65 (bs, OH, exchanged with DzO), C8Gua and DBP-lO-N7Gua (see below). Isolation of the 4.94 (bs, 1H, 2’-H), 5.97 (bs, 1H, l’-H), 6.81 ( 8 , 1H, 2-H[Adel), product eluting at 56 min in Figure 1A was unsuccessful 7.67-7.87 (m, 4 H, 2-H, 3-H, 12-H, 13-H), 7.93 (d, 1H, 8-H, J = because it was converted to DBP-10-C8Guabefore it could 9.1Hz),8.03-8.11(m,3H,6-H,9-H,8-H[Adel),8.18~d,1H,7-H, J = 7.4 Hz),8.36 (d, 1H, 11-H, J = 8.1 Hz), 8.99-9.20 (m, 4 H, be isolated and the NMR recorded. These experimental 1-H, 4-H, 5-H, 14-H),10.43 (bs, 1H, 6-NH[Adel, exchanged with observations suggest that the product eluting at 56 min D2O); MS, (M H)+C~HzsNa04calcd 568.1985, obsd 568.1990. (Figure 1A) is DBP-lO-C8dG. Loss of deoxyribose from

+

Results and Discussion Synthesis of Adducts by Electrochemical Oxidation. Anodic oxidation of DBP in the presence of dG

DBP-10-C8dG by hydrolysis is even easier than for the corresponding adduct obtained withBP (12). Thus, DBP10-CSdGis converted under electrochemical oxidation or hydrolysis to DBP-lO-C8Gua.

Chem. Res. Toxicol., Vol. 6, No.4,1993 557

One-Electron Oxidation of Dibenzo[a,llpyrene

Scheme 11. Electrochemical Oxidation of DBP in the Presence of (A) Guo or (B)Ado A 0

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Upon consumption of 3.8 equiv of charge, oxidation of DBP in the presence of the ribonucleosideG afforded DBP10-C8G and DBP-lO-N7Gua (Scheme IIA and Table 1). When more equiv of charge were consumed, no additional products were formed and DBP-lO-C8G was not oxidized to DBP-10-C8Gua. This result parallels that of the corresponding adduct obtained by electrochemical oxidation of BP in the presence of Guo (12). Furthermore, the yield of DBP-lO-N7Gua was much lower than that obtained from DP[a,llP + dG (Table I). This result is also similar to that obtained with BP (12). No appreciable amounts of adducts were formed by anodic oxidation of DBP in the presence of dA at a molar ratio of l:lO, respectively. A t a molar ratio of DBP:dA of 1:20 and with 3 electron equiv of charge consumed, the reaction produced DBP-10-WdA and DBP-lO-N7Ade (Scheme IB and Table I). Upon consumption of 2.75 electron equiv of charge, DBP in the presence of the ribonucleoside A at a molar ratio of DBP:A of 1:20 produced one adduct, DBP-10-N6A (Scheme IIB and Table I). No products were detected after anodic oxidation of DBP in the presence of dC or d T at molar ratios of DBP to nucleoside of 1:lO or 1:20. S t r u c t u r e Elucidation of Adducts. Evidence for the structure of these adducts was obtained by a combination of UV, NMR, and FAB MS/MS. Above 300nm, UV spectra of the adducts showed absorbance maxima red-shifted by 3-11 nm, which is characteristic of substitution at C-10 of DBP, as in the case of C-6 of BP (11,12, 17). (A) DBP-lO-N7Gua. The NMR spectrum (Figure 2B) shows the absence of the signal for the singlet proton at (2-10 (8.75 ppm) of the DBP moiety, indicating that DBP is substituted a t that position. This observation is further substantiated by the shielding of the protons 9-H and 11-H due to the peri effect of Gua substituted a t C-10. The sharp singlet at 8.22 ppm is assigned to the C-8proton of Gua, indicating that Gua is not substituted at (2-8.The signal at 6.32 ppm, which disappears upon exchange with D20,is for the two protons of the NH2 of Gua and

demonstrates that no substitution occurred a t the amino group. This molecule does not contain the deoxyribose moiety, as seen from the absence of proton resonances in the aliphatic region. This is consistent with substitution of dG at N-7, which destabilizes the glycosidic bond (11, 12,16). The remaining aromatic protons are assigned by comparing their chemical shifts with those of the parent compound DBP (Figure 2A) and by using two-dimensional chemical shift correlation spectroscopy (2D COSY). FAB MS of DBP-lO-N7Gua produces an (M + H)+of mlz 452. The determined value for the exact mass was A 6H/W

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Figure 2. NMR spectra of (A) DBP; (B) DBP-lO-N7Gua.

568 Chem. Res. Toxicol., Vol. 6, No. 4, 1993

RamaKrishna et al.

Figure 3. Portion of the CAD mass spectra of (M + H)+ions of mlz 452 from (A) DBP-lO-N7Gua;(B) DBP-10-CSGua. (C)Portion of the CAD mass spectrum of DBP-lO-C8G.

within 1.5 ppm of the theoretical value for CzsHleNaO,as reported in the experimental section. Upon collisional activation (CA), the ions of mlz 452 decompose to form major fragment ions of mlz 435 (M + H - NH3)+,mlz 327 (C24H13NC)*+,mlz 314 (CZ~HIZN)*+, mlz 302 (C24H14)*+, mlz 300 (C24H12)'+, mlz 289 (C23H13)+, and m/z 287 (C23Hll)+(Figure 3A). The larger relative abundance of the ions of mlz 302 compared to that of the ions of mlz 327 is characteristic of ring nitrogen (N-7) rather than carbon substitution at C-8 of the Gua moiety. This is consistent with previous results for adducts of BP and Gua (11, 12). Formation of (C24H&N)*+ from the N-7substituted Gua is less favored than the formation of (C24H&N)*+ from the C-8-substituted Gua (Figure 3B), owing to the lower relative stability of the former species. The ions of mlz 287 and 289 likely result from excision of C-10 from DBP by the modified base followed by ring closure to form a five-membered ring. (B) DBP-10-C8Gua. The NMR spectrum of DBP10-C8Gua (Figure 4A) shows the absence of two sharp singlets, one at 8.75 ppm corresponding to C-10 of DBP (Figure 2A) and the other at 8.22 ppm corresponding to the C-8 of Gua. This indicates that DBP is bound a t C-10 to the C-8 of Gua. This adduct does not contain the deoxyribose moiety, as the corresponding resonances are absent in the aliphatic region of the NMR spectrum (not shown). The broad singlet a t 6.60 ppm integrates to two protons and is assigned as the 2-NH2 of Gua. This is supported by disappearance of this resonance after exchange with DzO (not shown). Assignment of the other aromatic protons is made by comparison of their chemical shifts with those of the parent compound DBP (Figure 2A) and by 2D COSY. It is noteworthy to point out that, for the C-8 adduct, the upfield shift of 9-H and 11-H is not observed LS it is in the case of the corresponding N-7 adduct (Figure 2B). The FAB mass spectrum of DBP-10-C8Gua shows the (M + H)+ species of mlz 452. The CAD spectrum (Figure

3B) contains the same series of ions observed in the CAD spectrum of the (M + H)+ ions of the N-7-substituted adduct (Figure 3A). The relative abundance of the ions, however, is markedly different. The most abundant fragment ion is of mlz 327 (C24H&N)*+ (Figure 3B). The ions of mlz 300 (C24HlZ)" and 302 (C24H14)" are of lower abundance. This is indicative of substitution at a ring carbon rather than a nitrogen and, when coupled with the

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Figure 4. NMR spectra of (A) DBP-10-CSGua; (B) DBP-10N7Ade.

Chem. Res. Toxicol., Vol. 6, No. 4, 1993 559

One-Electron Oxidation of Dibenzo[a,l]pyrene

results obtained for BP adducts (11, 12), points to the generality of distinguishing substitution at N-7 and C-8 of a nucleobase. (C) DBP-lO-N7Ade. The NMR spectrum of this adduct (Figure 4B) contains the two sharp singlets at 8.39 and 8.69 ppm of the C-2 and C-8 protons, respectively, of Ade, indicating that these positions are not substituted. The broad singlet a t 5.72 ppm, which disappears after D2O exchange, corresponds to the resonance of the two protons of the 6-NH2 of Ade, demonstrating that no substitution occurred at the amino group. The absence of proton resonances in the aliphatic region indicates that this molecule does not contain the deoxyribose moiety. This is consistent with substitution of Ade at N-7, which destabilizes the glycosidic bond, resulting in elimination of the sugar moiety. The absence of the characteristic singlet for the proton at C-10 of DBP at 8.75 ppm suggests that the covalent bond is between the N-7 of Ade and (2-10 of DBP. This conclusion is further substantiated by the shielding effect of the peri protons 9-H and l l - H due to substitution of N-7 of Ade at C-10 of the DBP moiety, as was also observed for DBP-lO-N7Gua (Figure 2B). The remaining aromatic proton resonances are assigned by using 2D COSY and by comparing their chemical shifts with those of the parent compound DBP (Figure 2A). It would be expected that the CAD spectrum of the N-7-substituted Ade (not shown) would resemble that of the N-7-substituted Gua (Figure 3A). Their overall appearance is similar; for example, there is a larger relative abundance of the (C24H14)" ions as compared to the (CaH13NC)*+ions of mlz 327, which indicates substitution at nitrogen. With Ade, however, the (C24H12)*+ions of mlz 300 are more abundant than the (C24H14)'+ ions of mlz 302, in contrast to the relative abundances in the CAD spectrum of the N-7 Gua adduct (Figure 3A). In addition, there is a pronounced series of higher mass ions of mlz 419, 407, 392, 380, 365, 353, and 338 that are due to fragmentations of the Ade six-membered ring. These ions are formed by losses of NH3, CHzNH, HNCHNH2, CHzNCHNH, NHCHNCHNH2, CHzNCHNCHNH, and HNCHNCHNCHNHz, respectively. Both sets of observations are consistent with those observed for the BP-6N7Ade adduct (12). (D) DBP-10-N6dA. The NMR spectrum of DBP-10NBdA (Figure 5A) is consistent with the assigned structure. The absence of a sharp singlet at 8.75 ppm indicates that substitution is at the (2-10 position of the DBP moiety. The protons of DBP are assigned by comparing their chemical shifts with those of the parent compound DBP (Figure 2A) and by using 2D COSY. The protons 1'-H, 2'-H2, 3'-H, 4'-H, and 5'-H2 in the aliphatic region are assigned by 2D COSY. The broad singlet a t 5.27 ppm integrates to two protons and is tentatively assigned as due to the 5'-OH and 3'-OH. This signal disappears with D2O exchange (not shown), substantiating assignment of these protons. The sharp singlet at 8.06 ppm is assigned as C-8 of Ade. The other sharp singlet a t 6.85 ppm is tentatively assigned as the C-2 proton of the dA moiety; this proton undergoes a 1.4 ppm upfield shift compared to the corresponding proton of dA (not shown). From our earlier experience, this proton often undergoes an upfield shift, depending on how the dA is oriented with respect to the aromatic ring current. An upfield shift of 1.0 ppm was observed in the corresponding signal of BP diolepoxide-10-N6dAisomers (12). The absence of the broad singlet at 7.40 ppm, corresponding to the NH2 of dA, and

b &

iI 9

n 8

-

:

I " " " ' " I ' " " " ' I " ' ' ~ ' ' "

7

PPM(,)

Figure 5. NMR spectra of (A) DBP-10-NBdA; (B) DBP-10N'A; (C) DBP-lO-C8G.

the appearance of a DzO-exchangeableproton at 10.5ppm suggest that dA is substituted at the N6 position. FAB of DBP-10-N6dA produces both an (M H)+ species of mlz 552 and ions of mlz 436, formed by hydrogen transfer from the sugar to the base and elimination of C5Ha03. This latter ion is formally the (M + H)+ species of the modified base and is the most abundant fragment observed in the CAD spectrum of mlz 552 (not shown). Higher mass ions of mlz 464 (M - 88)+and 462 (M - 90)+ result from ring cleavages of the sugar moiety and are characteristic of a deoxyribose (18). The lower mass portion of the spectrum contains the same types of ions observed in the CAD spectrum of DBP-lO-N7Ade (not shown). The CAD spectrum of source-produced ions of mlz 436 is nearly identical to this portion of the CAD spectrum of mlz 552. The most abundant lower mass fragment ions are of mlz 302 and 300, (C24H14)" and (C24H12)*+,respectively. In contrast to the spectrum of the N-7Ade adduct, the ion of mlz 302 is now more abundant. No ion of appreciable abundance is observed at mlz 327, owing to the difficulty in excising the ring carbon to form the (C24H13NC)*+species. The series of ions resulting from successive cleavages of the Ade sixmembered ring (as described above for the N-7Ade adduct) as well as the ions of mlz 287 and 289, resulting from excision of (2-10 and ring closure to a five-membered ring, are also observed. (E) DBP-10-MA. The NMR spectrum of the N6A adduct (Figure 5B) is similar to that of DBP-10-NedA (Figure 5A), with the exception of the resonances of the deoxyribose moiety. The resonances of the ribose moiety are assigned by 2D COSY and by comparison with the parent compound A (not shown). FAB of DBP-10-NGA produces (M+ H)+of mlz 568 and a fragment ion of mlz 436 by a fragmentation that is

+

560 Chem. Res. Toxicol., Vol. 6, No. 4, 1993

analogous to that observed for the dA adduct. CA of the (M + H)+ species again predominantly produces the ion of mlz 436 by elimination of the sugar moiety. Ions mlz 478 (M + H -go)+ and mlz 464 (M + H - 104)+result from ring cleavages of the sugar and are characteristic of ribosecontaining nucleosides (18). The portion of the CAD spectrum below mlz 436 is identical to that observed for the dA adduct. (F)DBP-10-CSG. The bond between the (2-10 of DBP and the C-8 of Gua is suggested by the lack of the sharp singlets corresponding to the proton resonances of the C-8 of Gua and the C-10 of DBP (Figure 5C). The same criteria adopted for elucidation of the structure of DBP10-C8Gua are used to determine the structure of DBP10-CSG. The chemical shifts of the ribose moiety are assigned by using 2D COSY and DzO-exchange experiments (not shown). The broad singlet at 6.73 ppm, exchangeable with D20 and integrating to two protons, is assigned as the NH2 of Gua. Upon CA in the tandem mass spectrometer, the (M + H)+species of mlz 584 gives the expected fragment of mlz 452 resulting from elimination of the sugar (Figure 3C). The ions of mlz 494 (M + H - 90)+ and mlz 480 (M + H - 104)+result from characteristic sugar cleavages of the ribose moiety. The lower mass portion of the CAD spectrum is consistent with substitution at the C-8 of Gua, as was discussed for the DBP-10-C8Gua adduct.

Conclusions As anticipated, DBP'+ reacts with the nucleophilic groups of purine bases regiospecifically at C-10. The most reactive nucleophilic groups for the Gua moiety are N-7 and C-8, whereas for Ade, they are N-7 and the 6-amino group. In accord with the corresponding BP adduct (11, 12), the DBP-10-CSdG adduct is efficiently transformed to DBP-10-CSGua by anodic oxidation, whereas DBP10-C8G is electrochemically very stable. Now that adducb formed by one-electron oxidation have been synthesized, it is necessary to obtain adducts from DBP diolepoxides. All of these adducts will serve as reference standards in the identification of DBP adducts formed in biological systems. Structure elucidation of biologically formed adducts will be conducted by fluorescence line narrowing spectroscopy (13,14,19,20) and/ or by FAB MS/MS (21), utilizing the characteristic fragmentation patterns of the various adducts.

Acknowledgment. This research was supported by US. Public Health Service Grants R01-CA49917and PolCA49210 from the National Cancer Institute. Core support at the Eppley Institute was from the National Cancer Institute (P30-CA36727). The four-sector tandem mass spectrometer was purchased with funds awarded by the former NSF regional instrumentation grant (CHE8620177) and the University of Nebraska-Lincoln. Additional support was provided by the NSF Biology Division (DIR 9017262). References (1) Cavalieri, E. L.,Rogan, E. G., Higginbotham, S.,Cremonesi, P., and Salmasi, S. (1989) Tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland of dibenzo[alpyrenes: The very potent activity of dibenzo[a,llpyrene. J. Cancer Res. Clin. Oncol. 115,67-72. (2) Cavalieri, E. L.,Higginbotham, S.,RamaKrishna, N. V. S., Devanesan, P. D., Todorovic, R., Rogan, E. G., and Salmasi, S. (1991)

RamaKrishna et al. Comparative dose-response tumorigenicity studies of dibenzola,llpyrene us 7,12-dimethylbenz[alanthracene,benzo[alpyrene and two dibenzo[a,l]pyrene dihydrodiols in mouse skin and rat mammary gland. Carcinogenesis 12, 1939-1944. (3) Higginbotham, S.,RamaKrishna, N. V. S.,Johansson, S., Rogan, E., and Cavalieri,E. (1993)Tumor-initiating activity and carcinogenicity of dibenzo[a,llpyrene uersus 7,12-dimethylbenz[alanthraceneand benzo[alpyrene at low doses in mouse skin. Carcinogenesis 14,875878. (4) Cavalieri,E., and Rogan, E. (1985)Role of radical cations in aromatic hydrocarbon carcinogenesis. Enuiron. Health Perspect. 64,64-84. (5) Cavalieri, E., and Rogan, E. (1992)The approach to understanding aromatic hydrocarbon carcinogenesis. The central role of radical cations in metabolic activation. Pharmacol. Ther. 55, 183-199. (6) Sims, P., and Grover, P. L. (1981)Involvement of dihydrodiols and diol epoxides in the metabolic activation of polycyclichydrocarbons other than benzo[alpyrene. In Polycyclic Aromatic Hydrocarbons and Cancer (Gelboin, H. V., and Ts'o, P. 0. P., Eds.) pp 117-181, Academic Press, New York. (7) Conney, A. H. (1982)Induction of microsomal enzymes by foreign chemicals and carcinogenesisby polycyclic aromatic hydrocarbons: G. H. A. Clowes Memorial Lecture. Cancer Res. 42,4875-4917. (8) Devanesan, P. D., Cremonesi, P., Nunally, J. E., Rogan, E. G., and Cavalieri, E. L. (1990) Metabolism and mutagenicity of dibenzo[a,elpyrene and the very potent environmental carcinogen dibenzo[a,llpyrene. Chem. Res. Toricol. 3, 580-586. (9) Cremonesi, P., Hietbrink, B., Rogan, E. G., and Cavalieri, E. L. (1992)One-electron oxidation of dibenzo[alpyrenes by manganic acetate. J. Org. Chem. 57, 3309-3312. (10) Cremonesi,P., Cavalieri,E. L., and Rogan, E. G. (1989)One-electron oxidation of 6-substituted benzo[alpyrenes by manganic acetate. A model for metabolic activation. J. Org. Chem. 54,3561-3570. (11) Rogan,E., Cavalieri,E.,Tibbels, S.,Cremonesi,P., Warner,C.,Nagel, D., Tomer, K., Cerny, R., and Gross, M. (1988) Synthesis and identification of benzo[al pyrene-guanine nucleoside adducts formed by electrochemical oxidation and horseradish peroxidase-catalyzed reactionof benzo[alpyrenewithDNA. J.Am. Chem. SOC. 110,40234029. (12) RamaKrishna, N. V.S., Gao, F., Padmavathi, N. S., Cavalieri, E. L., Rogan, E. G., Cerny, R. L., and Gross, M. L. (1992)Model adducts of benzo[alpyrene and nucleosides formed from its radical cation and diol epoxide. Chem. Res. Toricol. 5, 293-302. (13)Devanesan, P.D., RamaKrishna, N. V. S., Todorovic, R., Rogan, E. G., Cavalieri,E. L., Jeong, H., Jankowiak, R., and Small, G. 3. (1992) Identification and quantitation of benzo[a]pyrene-DNA adducts formed by rat liver microsomes in vitro. Chem. Res. Tozicol. 5 , 302-309. (14) Rogan, E. G., Devanesan, P. D., RamaKrishna, N. V. S.,Higginbotham, S.,Padmavathi, N. S., Chapman,K., Cavalieri,E. L., Jeong, H., Jankowiak, R., and Small, G. J. (1993) Identification and quantitationof benzo[alpyrene-DNAadductsformed inmouse skin. Chem. Res. Toxicol. 6,356-363. (15)NIH Guidelines for the Laboratory Use of Chemical Carcinogens (1981)NIH Publication No. 81-2385,U.S.Government Printing Office, Washington, DC. (16) RamaKrishna, N. V. S., Cavalieri, E. L., Rogan, E. G., Dolnikowski, G. G., Cerny, R. L., Gross, M. L., Jeong, H., Jankowiak, R., and Small, G. J. (1992)Synthesis and structure determination of the adducts of the potent carcinogen 7,12-dimethylbenz[a]anthracene and deoxyribonucleosides formed by electrochemical oxidation: Models for metabolic activation by one-electron oxidation. J.Am. Chem. SOC.114,1863-1874. (17) Cavalieri, E., and Calvin, M. (1971) Photochemical coupling of benzo[a]pyrene with 1-methylcytosine, photoenhancement of carcinogenicity. Photochem. Photobiol. 14,641-653. (18) Crow, F. W.,Tomer, K. B., Gross, M. L., McCloskey, J. A., and Bergstrom, D. E. (1984)Fast atom bombardment combined with tandem mass spectrometry for the determination of nucleosides. Anal. Biochem. 139,243-262. (19)RamaKrishna, N. V. S., Devanesan, P. D., Rogan, E. G., Cavalieri, E. L., Jeong, H., Jankowiak, R., and Small, G. J. (1992)Mechanism of metabolic activation of the potent carcinogen 7J2-dimethylben~[alanthracene. Chem. Res. Toricol. 5, 220-226. (20)Devanesan, P. D., RamaKrishna, N. V. S., Padmavathi, N. S., Higginbotham, S., Rogan, E. G., Cavalieri, E. L., Marsch, G. A., Jankowiak, R., andsmall, G. J. (1993)Identificationandquantitation of 7,12-dimethylbenz[a]anthracene-DNAadducts formed in mouse skin. Chem. Res. Toricol. 6,364-371. (21) Cavalieri, E. L.,Rogan, E. G., Devanesan, P. D., Cremonesi, P., Cerny, R. L., Gross, M. L., and Bodell, W. J. (1990)Binding of benzo[a]pyrenetoDNA by cytochromeP-450-catalyzedone-electron oxidation in rat liver microsomesand nuclei. Biochemistry 29,48204827.