Chem. Res. Toxicol. 1993,6, 837-845
837
Adducts of 6-Methylbenzo[a]pyrene and 6-Fluorobenzo[alpyrene Formed by Electrochemical Oxidation in the Presence of Deoxyribonucleosides N. V. S. RamaKrishna,? Kai-Ming Li,? Eleanor G. Rogan,? Ercole L. Cavalieri,*lt Mathai George,$Ronald L. Cerny,S and Michael L. Gross* Eppley Institute for Research in Cancer and Allied Diseases, 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 March 31, 199P Studies of the DNA adducts of benzo[alpyrene and selected derivatives are part of the strategy to elucidate mechanisms of tumor initiation by aromatic hydrocarbons. Reference adducts formed by reaction of deoxyribonucleosides with electrophilic intermediates of 6-fluorobenzo[alpyrene (6-FBP) and 6-methylbenzo[alpyrene (6-CHsBP) are investigated here because they are essential for identifying the structures of adducts formed in biological systems. Electrochemical oxidation of 6-FBP in the presence of deoxyribonucleosides led to adducts from the 6-FBP radical cation. With dG, a mixture of 6-FBP bound a t C-1 or C-3 to the N-7 of Gua was formed in 10% yield, whereas 6-FBP plus dC gave a mixture of 3-(6-FBP-l-yl)Cyt and 346FBP-3-y1)Cyt (15%). No adducts of 6-FBP were formed with dA or dT. Electrochemical oxidation of 6-CH3BP in the presence of dG produced 8-(BP-6-CHz-yl)dG ( 5 % ) and a mixture of 7-(6-CH3BP-l-yl)Gua and 7-(6-CH3BP-3-yl)Gua (23%). The only adduct formed with dA was 3-(BP-6-CHz-yl)Ade (9%). 6-CH3BP did not afford any adducts with dC or dT. The noncarcinogenic 6-C1BP and 6-BrBP did not produce adducts with dG, dA, dC, or dT. These results are consistent with the chemical properties of the 6-FBP and 6-CH3BP radical cations; that is, 6-FBP reacts a t C-1 and C-3, whereas 6-CH3BP reacts competitively a t C-1 and C-3, as well as a t the 6-CH3 position.
Introduction Two major mechanisms are involved in the metabolic activation of polycyclic aromatic hydrocarbons (PAH)' to initiate cancer: one-electron oxidation to give radical cations and monooxygenation with formation of bay-region diol epoxides (1). Some PAH are activated exclusively by one of these mechanisms, and others are activated by a combination of both. This knowledge has been provided by studies of the tumorigenicity, metabolism, and binding of PAH to DNA. Benzo[alpyrene (BP) forms DNA adducts in vitro and in vivo by one-electron oxidation with reaction of the BP radical cation (BP+)at C-6 (80% ) and by reaction of bay-region diol epoxides at C-10 (20%) (2, 3).
Substitution of BP at C-6 with fluorine or a methyl group to form 6-fluorobenzo[alpyrene (6-FBP) or 6-methylbenzo[a] pyrene (6-CH3BP) decreases the carcinogenic activity relatively to BP (443, and 6-chlorobenzo[alpyrene (6-ClBP) and 6-bromobenzo[alpyrene (6-BrBP) are virtually inactive (4, 5).
* To whom correspondence should be addressed.
+ University of Nebraska Medical Center.
University of Nebraska-Lincoln, Abstract published in Advance ACS Abstracts, October 1, 1993. 1 Abbreviations: BP, benzo[alpyrene; BP+, benzo[alpyrene radical cation;6-BrBP,6-bromobenzo[alpyrene;CA, collisionalactivation;CAD, collisionalactivationdecomposition;6-CHaBP,6-methylbenzo[alpyrene; 6-ClBP,6-chlorobenzo[a]pyrene; COSY, two-dimensionalchemicalshift DMF, correlationspectroscopy,DMBA, 7,12-dimethylbenz[alanthracene; dimethylformamide;FAB MS/MS, fast atom bombardmenttandem mass spectrometry;6-FBP,6-fluorobenzo[olpyrene;NOE,nuclear Overhauser effect,PAH, polycyclicaromatic hydrocarbon(s);PDA, photodiodearray. 8
0
Radical cations of 6-substituted derivatives of BP were obtained by one-electron oxidation with manganic acetate and trapped by acetate ion (7).Acetoxylation of 6-FBP+ occurs exclusively at C-6, with displacement of fluorine. For 6-C1BP+and 6-BrBP+,substitution with acetate ion occurs at C-1 and C-3, which are the positions of second highest charge density in the radical cations after C-6 (7, 8). For 6-CH3BP+, charge localization at C-6 activates the methyl group, rendering it the most reactive toward nucleophilic attack. Competitive acetoxylation also occurs at C-1 and (2-3. Analysis of the DNA adducts of 6-FBP and 6-CHaBP produced by activation with horseradish peroxidase or cytochrome P-450 shows that the profile of stable 6-FBP adducts is similar to that of the parent compound,whereas 6-CH3BP primarily forms adducts derived from its radical cation (9). In this paper, we report the synthesis and structure determination of adducts formed by electrochemical oxidation of 6-CH3BP and 6-FBP in the presence of deoxyribonucleosides. This serves two purposes. One is to generate model adducts that could be formed in biological systems by one-electron oxidation. The other is to gain more knowledge about the properties of 6-CH3BP+ and 6-FBP+ in their reaction with the weakly nucleophilic groups of DNA bases.
Experimental Section (A)General Procedures. (1) UV. UV absorbance spectra were recordedwitha Waters 990 photodiodearray (PDA) detector during HPLC with CHaOH/HzO or CH&N/HzO gradienta.
0893-228~193/2706-0837$04.00/ 0 0 1993 American Chemical Society
838 Chem. Res. Toxicol., Vol. 6, No.6,1993 (2) NMR. Proton and homonuclear two-dimensional chemical shift correlation spectroscopy (COSY) NMR spectra were recorded on a Varian XL-300 instrument at 299.938 MHz in MezSO-de at 30 "C. Chemical shifts are reported relative to tetramethylsilane, which was employed either as a primary internal reference or as a secondary reference relative to MezSO at 2.50 ppm, and the J values are given in hertz. Typical instrument parameters were as previously reported (10). Nuclear Overhauser effect (NOE) difference spectra were recorded by applying a presaturation pulse with a decoupler onresonance and subtracting the trace from the corresponding reference spectrum recorded under identical conditions but with the decoupler off-resonance. Spectra were typically obtained from at least 2560 transients. (3)Fast Atom Bombardment Tandem Mass Spectrometry (FAB MS/MS). Collisionally activated decomposition (CAD) spectra were obtained by using a VG ZAB-T, a four-sectortandem mass spectrometer of BEBE design (11). MS1 is a standard high-resolution double-focusing mass spectrometer (ZAB) of reverse geometry. MS2, also capable of high resolution, is a prototype Mattauch-Herzog-type design, incorporating a standard magnet and an inhomogeneousplanar 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-pointdetector was adequate. Samples were dissolved in 20 pL of MeZSO, and a 1-pLaliquot was placed on the probe along with 1pL of matrix, glycerol containing 1% CF&OOH. 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 70 % The collision cell was floated at 4 kV. MS1 was operated at a resolution of 1OOO; 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. (4)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 by using a YMC ODs-AQ 5-pm, 120-8, column (6.0 X 250 mm) (YMC, Overland Park, KS). After the column was eluted for 5 min with 30% CHsOH in HzO, a 70- min curvilinear gradient (CV5)to 100% CHsOH was run at 1.0 mL/min. Asecond solvent system consisted of elution for 5 min with 30% CHsCN in HzO, followed by a 75-min linear gradient (CV6)to 100% CHsCN, run at 1.0 mL/min. Preparative HPLC was conducted using a YMC ODs-AQ 5-pm, 120-A column (20 X 250 mm) at a flow rate of 6.0 mL/min. Both CH&N/HzO and CHsOH/HzO gradients were used for adduct purification. (5) Materials. 6-CHsBP (mp 215-216 "C) was available in our laboratory. 6-FBP was synthesized by a published procedure (7)and purified by medium-pressure liquid chromatography, mp 167-169 "C. The deoxyribonucleosidesdG, dA,dC, and d T were purchased from either Aldrich (Milwaukee, WI) or Sigma Chemical Co. (St. Louis, MO) and were desiccated over Pz06 under vacuum at 100 "C for 48 h prior to use. Commercially available dimethylformamide (DMF) (Aldrich) was purified by heating to reflux over CaH2, 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: Both 6-CHaPand 6-FBPare hazardous chemicalsand were handled according to NIH guidelines (12). (B) Electrochemical Synthesis of Adducts. Electrochemical syntheses were conducted with a previously described apparatus (10). The oxidation potentials used for the synthesis of adducts were 0.95 V for 6-CHsBP and 1.10 V for 6-FBP, which are slightly less than their anodic peak potentials of 1.10 and 1.16
.
RamaKrishna et al. Table I. Products of 6-CHaP or 6-FBP with Various Nucleosides Formed by Electrochemical Oxidation charge consumed time PAH nucleoside (electroneauiv) (h) producta (yield, 5% ) 6-CHsBP dG 1.5 2 BP-6-CHz-C8dG(5) mixture of 6-CH&P-1and -3-N7Gua (23) BP-bCHzOH (12) 6-CHsBP dA 1.4 2 BP-6-CHrN3Ade(9) BP-6-CH20H(12) 6-FBP dG 2.0 4 mixture of 6-FBP-1and -3-N7Gua (10) mixture of BP auinonesb (10) 6-FBP dC 1.5 4 miiture of 6-FBP-1and -3-N3Cyt(15) mixture of BP quinones (12) 6-CHsBP with dC and dT and 6-FBP with dA and dT afforded no detectable products. * The BP quinones are BP-l,g-dione and BP-3,6-dione. ~
~
~~~
V, respectively,as measured by cyclicvoltammetry (ModelCV27, Bioanalytical Systems, Lafayette, IN) in DMF. All of the individual deoxyribonucleosideshave anodic peak potentials of 21.36 V. Thus, during adduct synthesis at 0.95 and 1.10 V, none of the nucleosides were oxidized. Glassware, syringes,needles, electrochemicalcell, and platinum working and reference electrodes were dried at 150 "C prior to use. The electrochemical cell and working electrodes were assembled while hot and then cooled under argon. Coupling between 6-CHsBP or 6-FBP and the nucleophilic groups of nucleosides was accomplished by selective anodic oxidation of PAH in the presence of the nucleoside. Experimental conditions were as previously reported (13). In a typical preparation, DMF (35 mL) containing 0.5 M KClO, as the supporting electrolyte waspreelectrolyzedat +1.45 V, while argon was bubbled in the cell, until no appreciable current could be detected (ca. 30 min). The dG (374 pmol, 100 mg) or dA (797 pmol, 200 mg) was added, and stirring was continued until the solution was clear. Bubbling of argon was stopped, and a positive pressure was maintained. 6-FBP or 6-CHsBP (37 pmol, ca. 10 mg) was added as a solid; when it was dissolved the cell was switched on, and the electrode potential was gradually raised from 0 to 0.95 V for 6 - C H a P or 1.10 V for 6-FBP and kept constant at this value during the entire electrolysis. The reaction was stopped when the output current (i) had decreased to ca. 5% of the initial value and a charge 1.4-2.0 times the theoretical charge expected for a two-electron transfer had accumulated, 2-4 h (Table I). After the reaction was complete, DMF was removed under vacuum, the adducts were extracted four times from the solid (KC10,) by usinga solventmixture of ethanoYchloroform/acetone (2:1:1), and the resulting extract was filtered througha Whatman fluted fiiter paper. The combinedsolventmixture was evaporated under vacuum; the residue was dissolved in 3 mL of MezSO, filtered through a 0.45-pm filter, and analyzed by HPLC with the CHsCN/HzO gradient. Purification of the adducts was conducted by preparative HPLC in CHsCN/HzO, followed by the CH30H/H20gradient. The adducts isolated from the reaction of 6-CH3BP or 6-FBP with the nucleosides and their yields, as determined by monitoring absorbance at 254 nm during HPLC separation, are shown in Table I. 6-CHaP: UV, A,. (nm) 256, 266, 288, 298, 356, 374, 392; NMR, 6 3.22 (8, 3H, 6-CHs), 7.86-7.93 (m, 2H, 8-H, 9-H), 7.998.09 (m, 2H, 2-H, 4-H), 8.18 (d, lH, 3-H, J = 7.4 Hz), 8.31 (d, lH, 1-H,J = 7.7 Hz), 8.37 (d, lH, 12-H, J = 9.2 Hz), 8.43 (d, lH, 5-H, J = 9.7 Hz), 8.60-8.67 (m, lH, 7-H), 9.19-9.30 (m, 2H, lO-H, 11-H). BP-6-CHrC8dG. UV, A, (nm) 256,266,288,299,356,374, 392; NMR, 6 2.27 (m, lH, 2'-H), 2.73 (m, lH, 2'-H), 3.55 (m, 2H, 5'-Hz),3.88(m,lH,4'-H),4.42 (m,lH,3'-H),5.59(~,2H,&CHz), 6.35 (t, lH, 1'-H), 7.76-7.89 (m, 4H, 8-H, 9-H, 2-NHz[Gua], exchangedwith DzO), 8.02 (t, lH, 2-H, J = 8.1 Hz), 8.08-8.20 (m, 2H, 3-H, 4-H), 8.33 (d, lH, 1-H, J = 7.7 Hz), 8.42 (d, lH, 12-H,
Chem. Res. Toxicol., Vol. 6,No. 6, 1993 839
Deoxynucleoside Adducts of 6-CHfiP and 6-FBP
Scheme I. Electrochemical Oxidation of 6-CHsBP in the Presence of (A) dG or (B) dA
B
ANOOIC OXIDATION
5. =
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y 2
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0 9 5 V PI. 0 SM in DMF
NHZ
yorwnacm : 1
:
BP 6 CHIOH
BPb*CH,-N3A&
20
Scheme 11. Electrochemical Oxidation of 6-FBP in the Presence of (A) dG or (B) dC A OXIDATION
-
b, 1.lOV. PI, KCIO, 0 . W h DMF
F Mix. of B-FBP-1- and -347Gw
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10
B ANODIC OXIDATION
5,=
+
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F
Mix. of B-FBP-l-ad -343cVi
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:
20
J = 9.6 Hz), 8.66 (d, lH, 5-H, J = 9.8 Hz), 8.81 (m, lH, 7-H), 9.20-9.29 (m, 2H, IO-H, 11-H);MS, (M + H)+ CslHsNsOi calcd 532.1985; found 532.1987. Mixture of 6-CHsBP-1- and -3-N7Gua: UV, A, (nm) 204, 266, 299, 377, 398; NMR, 6 3.23 (s, 3H, 6-CH3) 6.35 (br 8, 2H, 2-NHz[Gua, exchanged with D20), 7.50 (d, 4-H[31), 7.55 (d,
4-H), 8.22-8.29 (m,2H, l-H, 3-H), 8.35-8.46 (m, 2H, 12-H, 5-H), 8.58 (m, 1H, 7-H), 9.20 (d, lH, 11-H, J 9.2 Hz), 9.28 (m, lH, 10-H). Mixture of 6-FBP-1- and -3-N7Gua: UV, A, (nm), 257, 265,287,298,363,379,394,412; NMR, 8 6.26 (br s,2H, 2-NHa[Gua], exchanged with DzO), 7.73 (d, 2-H[31), J = 7.7 Hz), 7.82
2-H[3]),7.69(d,2-H[1]),7.79(d, 12-H[ll),7.86-7.97(m,8-H[1,31,(d,12-H[1],J=9.5Hz),7.93-8.06(m,8-H[1,31,9-H[1,31,1-H[31, 12-H[3]), 8.10 (d, 2-H[11, J = 8.0 Hz), 8.15-8.22 (m,4-H[1,31), 9-H[1,31), 7.99-8.31 (m,5-H[31, 4-H[11, 3-H[ll), 8.33 (8, lH, 8.25-8.36 (m, 3-H[1], 5-H[1,3],8-H[Gual), 8.56 (m, 7-H[1,31), 8-H[Gua]), 8.36-8.58 (m, 1-H[3], 12-H[31, 5-H[ll), 8.64-8.72 9.18-9.36 (m, l@H[l,3], ll-H[1,31). (m, 7-H[1,3]), 9.11-9.41 (m, 10-H[1,3], ll-H[1,31, l-NHIGual, exchanged with D20);MS, (M + H)+CBHl&O calcd 416.1511; Mixture of 6-FBP-1- and -3-N3Cyt: UV, A- (nm),256,265, found 416.1497. 279,286, 291,297,303,375, 385,395,415; NMR, 8 6.17 (d, lH, BP-CCHzOH: UV, A, (nm) 224,266,288,298,354,374,392; B-H[Cyt]), 7.03 (br s, 2H, 4-NHz[Cyt], exchanged with DaO), NMR, 6 5.43 (bs, lH, OH, exchanged with DzO), 5.56 (8, 2H, 7.78-7.88 (m, 2-H[1,31, 6-H[Cytl), 7.W7.98 (m, &H[1,31, 6-CH2),7.85-7.93 (m, 2H, 8-H, 9-H),8.00-8.10 (m, 2H, 2-H, 4-H), 9-H[1,31), 8.08-8.16 (m, 4-H[11,5-H[31), 8.17-8.29 (m, 3-H[13, 8.20 (d, lH, 3-H, J = 7.5 Hz), 8.34 (d, lH, l-H, J = 7.8 Hz), 8.41 4-H[3], 12-H[11, 5-H[ll), 8.35-8.43 (m, 12-HC31, 1-H[31), 8.52 (d, lH, 12-H,J = 9.3 Hz), 8.51 (d, lH, 5-H, J = 9.5 Hz), 8.74-8.81 (m, 7-H[1,3]), 9.14-9.30 (m, 10-H[1,3], ll-H[1,3]); MS, (M + (m, lH, 7-H), 9.20-9.27 (m, 2H, 10-H, 11-H). H)+ CZrHlsN30F calcd 380.1199; found 380.1198. BP-6-CHz-N3Ade:UV, A, (nm) 204,266,290,302,376,396; NMR, 6 6.69 (8, 2H, 6-CHz), 7.06 (8, lH, 2-H[Adel), 7.30 (br s, Results 2H, 6-NHJAde1, exchanged with DzO), 7.79-7.93 (m, 2H, 8-H, 9-H), 8.07 (m, lH, 2-H), 8.14 (d, lH, 4-H, J = 9.8 Hz), 8.22 (8, Anodic oxidation of 6-CHsBP in the presence of dG lH, 8-H[Ade]), 8.22-8.28 (m, 2H, l-H, 3-H), 8.38-8.46 (m, 2H, yielded three adducts and one oxygenated derivative of 5-H, 7-H), 8.52 (d, lH, 12-H,J = 9.3 Hz),9.2+9.35 (m, 2H, 10-H, 6-CHsBP: BP-6-CH2-C8dG (5%), a mixture of 6-CH311-H);MS, (M + H)+CBHl& calcd 400.1562; found 400.1550. BP-1- and -3-N7Gua (23%), and BP-6-CH20H (12%) 6-FBP: UV, A, (nm), 254,264,286,296,353,370,387,407; (SchemeI). Anodic oxidationof 6-CH3BP in the presence NMR: 6 7.83-7.89 (m, 2H, 8-H, 9-H), 7.99-8.10 (m, 2H, 2-H,
RamaKrishna et al.
840 Chem. Res. Toxicol., Vol. 6, No. 6, 1993
i
9
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Figure 1. NMR spectra of (A) dG, (B) BP-6-CHz-C8dG and (C) BP-6-CHz-C8dG + D20. of dA yielded one adduct and one oxygenated derivative: BP-6-CHz-N3Ade (9 9% ) and BP-6-CH20H (12 % 1. The reaction of 6-CH3BP with dC or dT did not afford any detectable amounts of adducts. Anodic oxidation of 6-FBP in the presence of dG gave two adducts: a mixture of 6-FBP-1- and -3-N7Gua (10%) (Scheme 11). Anodic oxidation of 6-FBP in the presence of dC yielded two adducts: a mixture of 6-FBP-1- and -3-N3Cyt(15%)(Scheme11). The reaction of 6-FBP with dA or dT did not produce any adducts. The noncarcinogenic6-ClBP and 6-BrBP did not yield adducts with dG, dA, dC, or dT and were completely recovered. The products were first purified by two consecutive runs on preparative HPLC, with the CH&N/H20 gradient followed by the CH30H/H20 gradient. Finally, the purity of all adducts was independently checked by analytical HPLC in the above two solvent systems. Structure Elucidation of Adducts. Evidence for the structures of these adducts was obtained by a combination
of UV, NMR, and FAB MS/MS. Above 300 nm, spectra of the adducts showed absorbance maxima red-shifted by 3-11 nm, which is characteristic of substitution a t aromatic carbon atoms. Adducts in which substitution is at the 6-CH3 group did not show any appreciable red shift. Structure elucidation by NMR and MS is discussed below. BP-6-CH2-CSdG. The NMR spectrum of BP-6-CH2C8dG (Figure 1B) and the spectrum after D2O exchange (Figure 1C) are consistent with the assigned structure. The absence of the sharp singlet a t 8.20 ppm assigned to the C-8proton of the Guamoiety suggests that substitution occurred at this position in dG. The protons 1'-H, 2'-H, 3'-H, 4'-H, and 5'-Hz in the aliphatic region are unequivocally assigned by COSY. The aromatic proton resonances are assigned by using COSY and by comparing their chemicalshifts with those of the parent compound 6-CH3BP (see Figure 5A below). The singlet signal at 5.59 ppm, corresponding to two protons, is tentatively assigned as the 6-CH2 group. In an NOE experiment, irradiation of this singlet at 5.59 ppm enhanced the doublet a t 8.66 ppm
~
'
~
~
Chem. Res. Toxicol., Vol. 6, No. 6, 1993 841
Deoxynucleoside Adducts of 6 - C H d P and 6-FBP
'T'
100
60
20 4011:4
, ,
0 1 0 100
*
,
2 0
,,,
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.,,.I 1 ' , ' ; 6 ' ,
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399
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150
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Figure 2. (A) NMR spectrum of BP-6-CH2-C8dG,(B) NOE spectrum of BP-6-CH2-C8dG after irradiation at 5.59 ppm (corresponding to 6-CH2), (C) NMR spectrum of BP-6-CHzNBAde, and (D) NOE spectrum of BP-6-CH2-N3Ade after irradiation at 6.69 ppm (corresponding to CH2).
;;,
",
& 260
Figure 4. FAB CAD mass spectra of (M + H)+ions of (A) mlz 416 from BP-6-CH&8dG and (B) mlz 416 from the mixture of 6-CHsBP-1-and -3-N7Gua. match experiments is within 0.5 ppm of the theoretical value for C31H~6N~04 (all exact mass measurements are reported in the Experimental Section). The fragment of mlz 416 is produced by hydrogen transfer from the sugar to the base, followed by elimination of C~H803;the ion of mlz 265 is (CZOHI~-CHZ)+ (where C~oH12is BP). Collisional activation (CA) of the (M + H)+ ions of mlz 532 yields, as does the FAB-induced desorption, two major fragments of mlz 416 and mlz 265, along with two ions of lower abundance that are diagnostic of the structure of the adduct (Figure 3). The ions of mlz 152 (protonated Gua) and 164 (Gua-CHz)+ indicate that Gua was adducted and that the PAH is bound to dG via the CHz group. Moreover, the fragment ion of mlz 280 (dG-CHz H)+ and the large abundance of (CzoH11-CHz)+ are indicative of a dGCHzBP adduct. The CAD spectrum of the sourceproduced fragment ions of mlz 416 (Figure 4A) contains and 164 (Gua-CHz)+. ions of mlz 265 (CZOHII-CHZ)+ Mixture of 6-CHaBP-1- and -3-N7Gua. The NMR spectrum (Figure 5B) of this adduct mixture shows the presence of a sharp singlet a t 3.22 ppm (not shown) corresponding to the signal of the CH3 group of the 6-CH3BP moiety. This indicates that substitution did not occur at the methyl group. The two doublet resonances at 7.50 and 7.55 ppm are assigned as 4-H and 2-H of 6-CH3BP3-N7Gua, respectively, by using COSY. Similarly, the two doublet resonances at 7.69 and 7.79 ppm are assigned as the 2-H and 12-Hof 6-CH3BP-l-N7Gua,respectively. The 4-H and 12-H proton resonances are shielded due to the peri effect of Gua substituted at C-3 or C-1, respectively. The sharp singlet signal at 8.33 ppm is assigned as the C-8 proton of Gua, indicating that Gua is not substituted at C-8. The signal at 6.35 ppm, which disappears upon exchange with DzO, corresponds to the two protons of the NH2 of Gua and demonstrates that no substitution occurred at 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 characteristic of substitution of dG at N-7, which destabilizes the glycosidic bond, as was already observed for adducts of BP (10,14) and DMBA (13). The remaining aromatic protons are assigned by comparing their chemical shifts with those of the parent compound 6-CH3BP (Figure 5A) and by using COSY. The ratio of the two isomers, which is obtained by the integration values of the C-2 protons in both isomers (Figure 5B), was found to be 75: 25.
+
4
164 150
260
I, 250
: ,
, , , ,
360
, , , ,
350
,
460
442
1
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Figure 3. FAB CAD mass spectrum of (M + H)+ions of mlz 532 from BP-6-CH2-CSdG.
corresponding to 5-H and the multiplet at 8.78 ppm corresponding to 7-H (Figure 2B). Thus, the NOE difference experiment allows us to assign the 6-CHz resonance at 5.59 ppm and the 5-H and 7-H resonances at 8.66 and 8.78 ppm, respectively. The lack of the sharp singlet a t 8.2 ppm of the C-8 proton of Gua suggests that the covalent bond between dG and the 6-CHz group of 6-CH3BP occurs at the C-8 of dG. Furthermore, substitution does not occur in this adduct at the 2-NHz group of Gua because the signalcorresponding to the two NHz protons is part of the multiplet a t 7.8 ppm (Figure lB), as evidenced by exchange with DzO (Figure 1C). The downfield shift of the NH2 resonances of the Gua moiety with respect to dG (Figure 1A) is apparently due to interaction of the protons with the *-electrons of the aromatic ring of the 6-CH9BP moiety. A similar effect was observed for the analogous adduct of 7,12-dimethylbenz[alanthracene (DMBA) linked at the 12-methyl group to the C-8 of dG (13). The FAB mass spectrum of BP-6-CHz-CBdG has an (M + H)+ ion of mlz 532 and abundant fragment ions of mlz 416 and 265 (Figure 3). The exact mass for the protonated molecular ion as determined by using high-resolution peak
RamaKrishna et al.
842 Chem. Res. Toricol., Vol. 6, No. 6, 1993
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The FAB mass spectrum of a mixture of these two isomeric adducts shows the (M + H)+ion of mlz 416. Upon CA, the (M + H)+ ions fragment to produce a series of characteristic product ions (Figure 4B). The clusters of ions of mlz 263-265, and of mlz 290-292, indicate ringsubstituted PAH. Moreover, the CAD mass spectrum (Figure 4B) is drastically different from that of the mlz 416 fragment from the BP-6-CHz-C8dG adduct in which
substitution at the methyl group has occurred (Figure4A). Furthermore, the smaller abundance of mlz 290 ions with respect to that of mlz 263 ions is characteristic of substitution at N-7 rather than at C-8, as was demonstrated previously for the adducts of BP (10,14).The facile loss of NHs from the (M + HI+ ion to produce the fragment of m / z 399 suggests that adduction does not occur through the exocyclic nitrogen of Gua.
Deoxynucleoside Adducts of 6 - C H a P and 6-FBP
BP-6-CHz-N3Ade. The NMR spectrum (Figure 5C) shows the absence of the singlet signal at 3.22 ppm corresponding to the 6-CH3 group. The sharp singlet resonance at 6.69 ppm, which integrates to two protons, is assigned to 6-CH2. This indicates that substitution occurred at the methyl group. Loss of the deoxyribose moiety in this adduct is supported by the absence of resonances in the aliphatic region corresponding to the sugar protons. The presence of the two prominent singlets at 7.06 and 8.22 ppm, preliminarily assigned as the resonances of the 2-H and 8-H of Ade, indicates that these positions are not substituted. Furthermore, the two protons that give a resonance at 7.30 ppm are established as the 6-NH2 of Ade, as deduced by a D2O exchange experiment. Thus, no substitution occurred at the amino group. This indicates that substitution in the adduct took place at the N-7 or N-3 of the Ade moiety. To establish the site of bonding in Ade, a NOE experiment was conducted by irradiating the singlet signal at 6.69 ppm corresponding to the 6-CH2 group. The NOE difference spectrum (Figure 2D) shows enhancement of the signals corresponding to 5-H and 7-H and the singlet at 7.06 ppm corresponding to the 2-H of Ade. The enhancement of the signals corresponding to 5-H and 7-H unequivocally indicates that the 6-CH2 of 6-CH3BP is the position of linkage in the adduct. The enhancement of the signal corresponding to 2-H is proof that the N-3 of Ade is bound to the 6-CH2 of 6-CH3BP. On the other hand, the absence of enhancement of the NH2 signal in the NOE experiment clearly establishes that this adduct does not involve binding at the N-7 of Ade. A similar NOE study was conducted to establish the structures of N7Ade and N3Ade adducts of DMBA ( 13). The remaining protons in the aromatic region are designated by comparing their chemical shifts with those of the parent compound 6-CH3BP (Figure 5A) and by using COSY. FAB of BP-6-CHz-N3Ade yields the (M + H)+ ion of mlz 400, which upon CA (Figure 6A) gives predominantly ~ ) +absence . of other ions of mlz 265, ( C ~ O H ~ ~ - C HThe fragment ions is consistent with adduction at the CH2 group, as observed previously for adducts of DMBA (13). BP-6-CH20H. The structure of this compound is established by comparing the HPLC retention time, UV spectra, and NMR spectra with that of the authentic standard available in our laboratory. Mixture of 6-FBP-1- and -3-N7Gua. The NMR spectrum of this adduct mixture does not contain the proton resonances of the deoxyribose moiety in the aliphatic region (Figure 7B, not shown). This is in accord with substitution of dG at N-7, with destabilization of the glycosidic bond. By comparing the resonances of the aromatic region of the adduct with those of the parent compound 6-FBP (Figure 7A), it becomes evident that this is a mixture of the two isomers 6-FBP-1- and -3N7Gua. The aromatic proton resonances were assigned by using COSY and by comparing the chemical shifts of the adduct to those of the parent compound 6-FBP (Figure 7A) and dG (Figure lA), as described above for the structure elucidation of 6-CH3BP-1- and -3-N7Gua. The ratio of the 1-and 3-isomers of the 6-FBP-N7Gua adduct is 55:45, respectively, as obtained by comparing the integration values corresponding to the 2-H proton of the 1-isomer at 8.10 ppm and the 3-isomer at 7.73 ppm (Figure 7B). Upon FAB, the mixture of 6-FBP-l-N7Guaand 6-FBP3-N7Gua produces (M + HI+ ions of mlz 420. Upon CA,
Chem. Res. Toxicol., Vol. 6,No. 6,1993 843
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the (M + H)+ions dissociate to form three major fragment ions (Figure 6B). The fragment ion of mlz 403 is due to elimination of NH3, indicating that the binding of BP to Gua is not through the exocyclic nitrogen. The clusters of peaks a t mlz 268 and 295 are indicative of a ringsubstituted adduct. The relative abundances of the ions of mlz 268 and 295, however, are different from those of mlz 263 and 290 in the CAD spectrum of 6-CH3BP-1-and -3-N7Gua (Figure 4B), probably because of the electronwithdrawing properties of the fluoro substituent and the electron-donating properties of the 6-CH3 group. The minor peak of mlz 152 is protonated Gua. Mixture of 6-FBP-1-and -3-N3Cyt. The NMR spectrum of this compound (Figure 7C) contains a broad singlet signal at 7.03 ppm, for protons exchangeable with DzO. This corresponds to the amino protons of the Cyt moiety, indicating that this position is not substituted. The doublet at 6.17 ppm is assigned as 5-H of Cyt by comparing the chemical shift with that of dC (figure not shown). In COSY, this doublet at 6.17 ppm couples with the multiplet at 7.78 ppm, suggesting that the 6-H of Cyt is part of this multiplet. Upon comparison of this spectrum with that of the parent compound 6-FBP (Figure 7A), it is evident that this is a mixture of two isomers of Cyt adduct substituted a t C-1 or C-3 of 6-FBP. All of the aromatic proton resonances are assigned by using COSY. It is clear from the NMR spectrum that this molecule has lost the deoxyribose moiety. On the basis of the data, the bond in Cyt can be established as at N-3, thus destabilizing the glycosidic bond and allowing the sugar to be eliminated. The actual ratio of the two isomers could not be determined because resonances corresponding to the protons of the two isomers are not resolved. The FAB mass spectrum of the mixture of 6-FBP-1and -3-N3Cyt contains (M + HI+ ions of mlz 380. Upon CA, the (M + H)+ ions yield diagnostic ions of mlz 363 (M + H - NH3)+, mlz 311 CZoHloF-NCO+, and mlz 270
Deoxynucleoside Adducts of 6 - C H a P and 6-FBP
For the 6-CH3BP+ produced by manganic oxidation, charge localization a t C-6 renders the methyl group the most reactive toward attack by acetate ion after loss of the proton at the methyl group (7).Competitive acetoxylation of 6-CH3BP+also occurs to aminor extent at C-1 and C-3. Electrochemical oxidation of 6-CH3BP in the presence of dG produces an adduct with the methyl group bound at the C-8 of Gua, whereas it reacts at C-1 and C-3 with the N-7 of Gua. Electrochemical oxidation of 6-CH3BP with dA yields only the N3Ade adduct bound through the methyl group. BP-6-CHzOH is formed in both electrochemical oxidations, presumably derived from reaction of 6-CH3BP+ with traces of moisture that can enter the system during the reaction. The N7Ade and N7Gua adducts bound through the methyl group are not formed by electrochemical oxidation of 6-CH3BP. This contrasts with electrochemical oxidation of DMBA in the presence of dA and dG, in which both the N7Gua and N7Ade adducts are formed at the methyl groups. Analysis of the stable DNA adducts of 6-CH3BP formed in biological systems in vitro suggests that activation of this compound occurs predominantly by one-electron oxidation (9). Thus, the DNA adducts could be formed by this process, involving the methyl group and/or C-1 and C-3. If both types of adducts are identified, the mechanism of activation of 6-CHsBP could resemble in part that of unsubstituted PAH and in part that of mesoanthracenic methylated PAH such as DMBA.
Acknowledgment. This research was supported by
US. Public Health Grants R01-CA44686 and PO1CA49210 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 program (Grant CHE 8620177) and the University of Nebraska-Lincoln. Additional support was provided by the NSF Biology Division (DIR 9017262).
References (1) 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. (2) Devanesan, P. D., RamaKrishna, N. V. S., Todorovic, R., Rogan, E. G., Cavalieri,E. L., Jeong, H., Jankowiak, R., and Small, G. J. (1992) Identification and quantitation of benzo[alpyrene-DNA adducts formed by rat liver microsomes in vitro. Chem. Res. Toxicol. 5,302309. (3) 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
Chem. Res. Toxicol., Vol. 6, No. 6, 1993 846 quantification of benzo[a]ppene-DNA adducts formed in mouse skin. Chem. Res. Toricol. 6, 366-363. (4) Cavalieri, E., Roth, R., Grandjean, C., Althoff, J., Patil, K., Liakus, S., and Marsh, S. (1978)Carcinogenicity and metabolic profiies of 6-substituted benzo~alovrenederivatives on m o w skin. Chem.Biol. Interact. 22, 63Gf ( 6 ) Cavalieri, E., Roaan, E., Cremonesi, P., Higainbotham, S., and Salmasi, S. (1988,-T&origenicity of 6-haloge;ated derivativee of benzo[alpyrene in mouse skin and rat mammary gland. J. Cancer Res. Clin. Oncol. 114, 10-16. (6) Cavalieri, E., Rogan, E., Higginbotham, S., Cremonesi, P., and Salmasi, S. (1988) Tumor-initiating activity in m o w skin and carcinogenicity in rat mammary gland of fluorinated derivatives of benzo[alpyrene and 3-methylcholanthrene. J. Cancer Res. Clin. Oncol. 114,1622. (7) 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. 64, 3661-3670. (8) Sullivan, P. D., Bannoura, F., and Daub, G. (1986)13C and 1H EPR analysis of the benzo[a]pyrene cation radical. J. Am. Chem. SOC. 107,32-36. (9) Todorovic, R., Devanesan, P. D., Rogan, E. G.,and Cavalieri, E. L. (1993)3WPostlabeling analysh of theDNAadducts of &fluomheme [a]pyreneand6-methylbenzolalpyreneformed in vitro. Chem. Res. Toricol. 6, 630-634. (10)Rogan, E.,Cavalieri,E., Tibbels, S., Cremonesi,P., Warner, C., Nagel, D., Tomer, K., and Gross,M. (1988)Synthesis and identification of benzo[alpyrene-guanine nucleoside adducts formed by electrochemical oxidation and horseradish peroxidase-catalyzed reaction of benzo[alpyrene with DNA. J. Am. Chem. SOC. 110,4023-4029. (11) Gross, M. L. (1990) Tandem mass spectrometry: Multieector magnetic instruments. In Methods in Enzymology, Vol. 193, Mass Spectrometry (McCloskey,J. A., Ed.) pp 131-163,Academic Press, San Diego. (12)NZH Guidelines for the Laboratory Use of Chemical Carcinogens (1981)NIH Publication No. 81-2385,U.S.Government Printing Office, Washington, DC. (13) 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[alanthracene and deoxyribonucleosides formed by electrochemical oxidation: Models for metabolic activation by one-electron oxidation. J. Am. Chem. Soc. 114,1863-1874. (14) 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 ita radical cation and diol epoxide. Chem. Res. Toxicol. 5, 293-302. (16) Buhler, D. R., hlii, F., Thakker, D. R., Slaga, T. J., Conney, A. H., Wood, A.W., Chang, R. L., Levin, W., and Jerina, D. M. (1983)Effect of a 6-fluor0 substituent on the metabolism and biological activity of benzo[alpyrene. Cancer Res. 43,1541-1649. (16) Cavalieri, E., Rogan, E., Cremonesi, P., and Devanesan, P. (1988) Radical cations as precursors in the metabolic formation of quinones from benzo[alpyrene and 6-fluorobenzo[alpyrene: Fluoro substitution aa a probe for one-electron oxidation in aromatic substrates. Biochem. Phurmacol. 37, 2173-2182. (171 Cavalieri, E., Devanesan, P., and Rogan, E. (1988)Radical cations in the horseradishperoxidaw and prostaglandin H synthasemediated metabolism and binding of benzo[alpyrene to DNA. Biochem. Pharmacol. 37,2183-2188. (18)Cremonesi,P.,Rogan,E., and Cavalieri,E. (1992)Correlationetudiea of anodic peak potentials and conjugation potentials for polycyclic aromatic hydrocarbons. Chem. Res. Toxicol. 5, 346-366.
