Synthesis and Characterization of Bay Region Halohydrins Derived

Aug 15, 1998 - Qi Song,George R. Negrete,Alan R. Wolfe,Keshi Wang, andThomas Meehan*. Department of Biopharmaceutical Sciences and Division of ...
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Chem. Res. Toxicol. 1998, 11, 1057-1066

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Synthesis and Characterization of Bay Region Halohydrins Derived from Benzo[a]pyrene Diol Epoxide and Their Role as Intermediates in Halide-Catalyzed Cis Adduct Formation Qi Song, George R. Negrete,† Alan R. Wolfe, Keshi Wang, and Thomas Meehan* Department of Biopharmaceutical Sciences and Division of Toxicology, University of California, San Francisco, California 94143 Received March 25, 1998

The bay region epoxide of benzo[a]pyrene (anti-BPDE) alkylates DNA to form adducts with >98% trans stereochemistry. Halide ions catalyze this reaction; however, this pathway is characterized by the formation of adducts with altered cis stereochemistry. Bay region halohydrins are possible intermediates in these reactions, but are too unstable to be isolated from aqueous solutions. However, we successfully synthesized halohydrins in tetrahydrofuran (THF) by treatment of anti-BPDE with the corresponding lithium halide salt in the presence of acetic acid. Absorbance and CD spectroscopy clearly indicated the formation of chloro-, bromo-, and iodohydrins. The structure and stereochemistry of the chlorohydrin was established by NMR. Chloride addition is exclusively at the benzylic position of the epoxide, and the stereochemistry of the C-9 and -10 positions is trans. The long-wavelength absorbance band in the chloro-, bromo-, and iodohydrin is red-shifted 7, 13, and 22 nm, respectively, relative to the hydrolysis product of anti-BPDE. The ellipticity of the same absorbance band in CD spectra of enantiomerically pure halohydrins is opposite in sign compared to that of the corresponding anti-BPDE enantiomer. The relative stability of these derivatives is chlorohydrin > bromohydrin > iodohydrin. The chloro- and bromohydrins were isolated, but the iodohydrin decomposed during this manipulation. The addition of 500 mM chloride decreased the hydrolysis rate of the chlorohydrin 4-fold in 50% THF/buffer. Direct evidence for the transient formation of the iodohydrin in aqueous buffer/acetone mixtures was obtained by absorbance spectroscopy. At 1 M chloride, bromide, and iodide, alkylation of deoxyadenosine by antiBPDE in aqueous buffer yields 85, 91, and 92% cis adducts, respectively. In the absence of halide, alkylation of deoxyadenosine in buffer by anti-BPDE, the chlorohydrin, and the bromohydrin yields 32, 65, and 83% cis adducts, respectively.

Introduction (BaP)1

Benzo[a]pyrene is a potent carcinogen widely distributed in the environment. This and other polycyclic aromatic hydrocarbons (PAHs) are metabolically activated to form a set of stereoisomeric bay region epoxides (1). The most mutagenic and carcinogenic isomer derived from BaP activation is 7R,8S-dihydroxy-9S,10R-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE] (1* Corresponding author. † Present address: Division of Earth and Physical Sciences, University of Texas, San Antonio, TX 78249. 1 Abbreviations: PAHs, polycyclic aromatic hydrocarbons; BaP, benzo[a]pyrene; (()-anti-BPDE, racemic 7r,8t-dihydroxy-9t,10t-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene; (+)-anti-BPDE, 7R,8S-dihydroxy9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; (()-7r,8t,9t,10c- and (()-7r,8t,9t,10t-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene, racemic trans- and cis-tetrol, respectively; (()-trans-BPDCH, racemic 7r,8t,9t-trihydroxy-10c-chloro-7,8,9,10-tetrahydrobenzo[a]pyrene; (+)trans-BPDCH, 7S,8R,9R-trihydroxy-10R-chloro-7,8,9,10-tetrahydrobenzo[a]pyrene; (()-trans-BPDBH, racemic 7r,8t,9t-trihydroxy-10c-bromo7,8,9,10-tetrahydrobenzo[a]pyrene; (()-trans-BPDIH, racemic 7r,8t,9ttrihydroxy-10c-iodo-7,8,9,10-tetrahydrobenzo[a]pyrene; HRFABMS, highresolution-fast atom bombardment mass spectroscopy; LSIMS, liquidsecondary ion mass spectroscopy; CIMS, chemical ionization mass spectroscopy; ESIMS, electrospray ionization mass spectroscopy; HREIMS, high-resolution electron impact mass spectroscopy; THF, tetrahydrofuran; HEPES, N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid.

3). The activation and binding of PAHs to DNA have been extensively investigated (4-7). Evidence suggests that bay region epoxides alkylate DNA by an acidcatalyzed mechanism, with the intermediate formation of a benzylic carbocation. BPDE alkylates DNA primarily at the N-2 position of deoxyguanosine, and to successively lesser degrees, the N-6 and N-4 positions of deoxyadenosine and deoxycytidine, respectively (8-13). The nucleophile can attack the C-10 position of BPDE from above or below the benzo ring, resulting in adducts with the nucleophile either trans or cis to the C-9 hydroxyl group of the hydrocarbon moiety. Alkylation of DNA by anti-BPDE through the acid-catalyzed pathway generates >98% trans adducts (12-14). The carbocation also reacts with water to form a set of stereoisomeric hydrolysis products, whose composition is also >90% trans (15, 16). These product stereoselectivities are unusual for SN1 reactions. The stereochemistry of anti-BPDE hydrolysis (15, 17) and alkylation reactions (12) has been explained in terms of an equilibrium between two BPDE conformers which specifically act as precursors for either cis or trans products. We have described a new, halide-catalyzed route in the formation of covalent adducts between activated BaP and

S0893-228x(98)00056-3 CCC: $15.00 © 1998 American Chemical Society Published on Web 08/15/1998

1058 Chem. Res. Toxicol., Vol. 11, No. 9, 1998 Chart 1. Structures of the (+)-Enantiomer of anti-BPDE and the Corresponding Bay Region Trans Halohydrins

Song et al. Scheme 1. Lithium Halide Method for Synthesis of Halohydrins from anti-BPDEa

a Syntheses were carried out with racemic anti-BPDE or with resolved enantiomers.

DNA (14, 16, 18, 19). Initially, we observed that halide ions catalyze the hydrolysis of (()-anti-BPDE (14). We also showed that chloride catalyzes the formation of covalent adducts between anti-BPDE and nucleosides, nucleotides, RNA, and DNA (16, 18, 19). The effectiveness of halides at catalyzing the hydrolysis reaction follows the order I- > Br- > Cl- . F- (14). This pathway generates the same hydrolysis products and covalent adducts formed by anti-BPDE in the absence of halides, but the stereoselectivity is modified in favor of cis products. Kinetic and product studies on halide-catalyzed covalent adduct formation support a mechanism where the halide attacks the C-10 position of anti-BPDE in the first step, generating a trans halohydrin (18). This intermediate then undergoes SN2 attack by an exocyclic amino group of deoxyadenosine, displacing the halide. The net result is a double inversion of configuration at C-10 of BPDE with formation of a cis adduct. In aqueous media, the bay region chlorohydrin of BPDE forms predominately cis adducts with deoxyadenosine. However, the chlorohydrin may also lose chloride to generate a carbocation, which reacts to form primarily trans adducts with most alkylation targets. We synthesized bay region halohydrins from antiBPDE to establish whether they are formed in aqueous alkylation reactions and whether they are intermediates in the halide-catalyzed activation pathway. Initial attempts to demonstrate the formation of halohydrins in halide-catalyzed reactions were unsuccessful because of their instability in aqueous solution. We, therefore, synthesized the halohydrins in tetrahydrofuran (THF). In this paper, we report the structures, stereochemistry, and spectroscopic properties of the synthetic chloro-, bromo-, and iodohydrins of anti-BPDE (Chart 1). This spectroscopic data was used to identify the transient formation of an iodohydrin intermediate in aqueous iodide-catalyzed reactions. Finally, we demonstrate that the stereochemistry of adducts formed by alkylation of deoxyadenosine with BPDE is dependent on the identity and concentration of any halide(s) present. Similarly, if alkylation is carried out with a synthetic halohydrin, the product stereochemistry also varies depending on which one is used.

Experimental Procedures Hazardous Materials. PAH epoxides are known animal carcinogens and suspected human carcinogens, and should be handled with caution. The biological activities of the BPDE halohydrins are not known at this time; however, they are mutagenic in the Ames assay (unpublished).

General. Synthesis of the halohydrins (Scheme 1) was carried out by an adaptation of the method described by Bajwa and Anderson (20). 1H and 13C NMR spectra were recorded on GE GN-500 MHz, QE-300 MHz, and Bruker AMX-300 MHz spectrometers. Chemical shift data are reported in parts per million downfield from tetramethylsilane as an internal standard with coupling constants (J) in hertz. UV/visible spectra were recorded on a Hewlett-Packard 8452A diode array or Varian Cary 3E spectrophotometer. Mass spectra were obtained on VG 7070SE and Kratos MS-50 mass spectrometers. CD spectra were recorded on a JASCO J-500A spectropolarimeter. HPLC. Separations were carried out with an Altex HPLC system. Samples were detected with a Rainin Dynamax UV-D II absorbance detector and profiles analyzed using Rainin Dynamax MacIntegrator software. A Rainin Microsorb silica column (5 µm, 4.6 mm × 250 mm) was used for normal phase separations of products from the halohydrin syntheses. A Waters Associates semipreparative silica column (Prep NovaPak HR Si, 6 µm, 7.8 mm × 300 mm) was used for halohydrin purification. Alkylation of Deoxyadenosine. Reactions between deoxyadenosine and anti-BPDE (or the halohydrins) and analysis of the adducts formed by reverse phase HPLC were carried out as described previously (18). Chemicals. Racemic and resolved enantiomers of antiBPDE were synthesized as described (21, 22). THF was distilled from CaH2 or CaH2/NaBH4. Lithium halides were obtained from Sigma and dried under vacuum in an oil bath at a temperature of more than 110 °C for at least 2 h before they were used. Acetic acid was distilled in the presence of 10% acetic anhydride. Preparation of 7r,8t,9t-Trihydroxy-10c-chloro-7,8,9,10tetrahydrobenzo[a]pyrene (Trans-BPDCH) by the LiCl Route. Reaction mixtures contained either (+)-, (-)-, or (()anti-BPDE as substrates. In a typical synthesis, (()-anti-BPDE (19.8 mg, 65.3 µmol) was dissolved in 4 mL of dry THF under a N2 atmosphere. LiCl (39.6 mg, 934 µmol) was added, followed immediately by 1 mL of acetic acid. The reaction mixture was stirred at room temperature under N2 for 1.5-2 h and the reaction monitored by normal phase HPLC (50% THF/hexane). Due to the lability of the halohydrins, purification procedures were carried out below room temperature whenever possible. Two purification methods were used. In method A, 12 mL of toluene was added, followed by 6 mL of H2O. After extraction, the aqueous phase was removed and the organic phase washed with 4 mL of H2O three more times. The organic phase was dried (MgSO4), filtered through a small silica Sep-Pak cartridge, to remove traces of inorganic salts, and evaporated to dryness in a Speed Vac cooled with dry ice. At this stage, about 95% of the product was racemic trans-BPDCH, as indicated by HPLC analysis (Figure 1). Further purification was carried out by normal phase HPLC (47% THF/hexane). The product was collected from the column and the solvent removed under vacuum at or below room temperature. In general, yields were about 80%. In method B, THF and acetic acid were removed from the reaction mixture under vacuum (∼3 mmHg) and the residue was dissolved in THF. The product was purified by flash chromatography on silica (55% THF/hexane). Fractions containing the product were concentrated, and the product was precipitated by the addition of hexanes, collected by centrifuga-

Halohydrin Intermediates in Cis Adduct Formation

Chem. Res. Toxicol., Vol. 11, No. 9, 1998 1059 Table 1. Spectroscopic Properties of anti-BPDE Bay Region Halohydrinsa absorbance λmax (nm) M (M-1 cm-1) CD (L - R) (M-1 cm-1)

BPDCH

BPDBH

BPDIH

350 33300 (+) 15.3

356 27300 (+) 28

364 21000 (+) 51

a The absorbance and CD extinction coefficient values reported are for the long-wavelength band of the indicated halohydrin. Values were obtained in THF. In each case, the 7S,8R,9R,10Rhalohydrin enantiomer [i.e., the one derived from (-)-anti-BPDE] was used for CD measurements.

Figure 1. Normal phase HPLC trace of the products obtained by reaction of (()-anti-BPDE with LiCl/acetic acid. A 0.46 cm × 25 cm Rainin Microsorb silica (5 µm) column was used for this separation, with 47% THF/hexane as the mobile phase at a flow rate of 0.67 mL/min. The sample was detected by absorbance at 345 nm. tion, and dried under vacuum (∼3 mmHg). Yields were similar with both methods, but a higher purity of the trans-BPDCH was achieved with method B. Compounds were stored at -80 °C. UVTHF (λ max): 350, 333, 282, 271, 249 nm. IR (KBr): 3346, 3050, 2945, 1727, 1604, 1431, 1295, 1270, 1209, 1091, 1073, 1029, 887, 838, 721, 430 cm-1. 1H NMR (500 MHz, DMSO-d6): δ 6.40 (d, H-10, J10e,9e ) 3.0), 4.72 (dd, H-9, J9e,10e ) 3.0, J9e,8a ) 2.0), 4.56 (dd, H-8, J8a,9e ) 2.0, J8a,7a ) 9.0), 5.29 (d, H-7, J7a,8a ) 9.0), 8.05-8.50 (m, overlapped aromatic protons). 13C NMR (75 MHz, DMSO-d6): δ 58.08 (C-10), 73.95 (C-9), 69.83 (C-7, C-8 overlapped), 115.00-138.73 (overlapped aromatic carbons C-1-C-6, and C-11-C-20). LSIMS (negative mode): m/z 393 [(M - 1 - HCl + glycerol)-], 301 [(M - 1 - HCl)-], 283 [(M 1 - HCl - H2O)-]. HRFAB-MS: M+ calcd for C20H15O3Cl 338.07097, found 338.07110. Anal. Calcd for C20H15O3Cl: C, 70.91; H, 4.46; Cl, 10.46. Found: C, 71.08; H, 4.42; Cl, 10.26%. LSIMS (negative mode) of the minor product: m/z 361 [(M 1)-], 301 [(M - 1 - CH3COOH)-], 283 [(M - 1 - CH3COOH H2O)-], 265 [(M - 1 - CH3COOH - 2H2O)-]. Preparation of (()-7r,8t,9t-Trihydroxy-10c-bromo-7,8,9,10-tetrahydrobenzo[a]pyrene [(()-Trans-BPDBH]. Conditions for the synthesis of (()-trans-BPDBH were similar to those described for trans-BPDCH except that LiBr replaced LiCl as the halide source. Due to product instability, this reaction was carried out at a lower temperature (0 or -80 °C gave similar results). The reaction was complete in 1 h. Workup was similar to that described for trans-BPDCH using method A. However, the organic phase was only extracted twice with H2O, and it was filtered with Celite. Dry ice was used to cool the SpeedVac prior to and during evaporation of the organic phase. The product was purified further by normal phase HPLC (47% THF/ hexane). The retention time of trans-BPDBH was similar to that of the chlorohydrin. Although the bromohydrin was fairly stable in dilute solution, it decomposed when the solvent was evaporated. UVTHF (λmax): 356, 341, 284, 274, 249 nm. Preparation of (()-7r,8t,9t-Trihydroxy-10c-iodo-7,8,9,10-tetrahydrobenzo[a]pyrene [(()-Trans-BPDIH]. The synthesis of (()-trans-BPDIH was attempted with LiI/Amberlyst 15 and LiI/acetic acid at -80 °C. HPLC of the reaction mixtures showed that a higher proportion of the desired product was obtained with LiI/acetic acid. Reagent concentrations were similar to those described for the chlorohydrin. HPLC was used to monitor the reaction which indicated the formation of BPDIH in the first 15 min of the reaction. Beyond this time, some

degradation products began to appear. The iodohydrin was extremely unstable, and attempts to purify it were unsuccessful. UVTHF (λmax): 364, 348.5, 289 nm. UV and CD Spectroscopy. Spectroscopic data obtained on the long-wavelength bands of the halohydrins are summarized in Table 1. Absorption spectra of trans-BPDCH and transBPDBH (in THF) were obtained using purified samples. Due to the instability of trans-BPDIH, its spectrum was obtained from a freshly generated dilute reaction mixture. CD spectra were obtained from enantiomerically pure halohydrin samples prepared from (-)-anti-BPDE. Dilute reaction mixtures were used in each case. The dilute reaction mixtures (1 mL for absorption spectra, 4 mL for CD spectra) consisted of THF, freshly distilled from CaH2/NaBH4, containing 100-200 mM lithium halide, 100 mM acetic acid (distilled from 10% acetic anhydride), 1-3 mM ascorbic acid for BPDBH or BPDIH (to prevent Br2 or I2 formation), and 25-35 µM anti-BPDE. (The ascorbic acid makes absorbance measurements below 280 nm difficult.) A low acetic acid/halide ratio was used to minimize side product formation. The reaction (at room temperature) reached completion immediately in the case of BPDIH, and within a few minutes in the case of BPDBH (as determined by monitoring the spectra). The BPDCH reaction was allowed to stand for several hours. Extinction coefficients of the halohydrins were determined by measuring the amount of tetrol produced after hydrolysis of these derivatives. BPDCH and BPDE (in the presence of halides) are quantitatively hydrolyzed to tetrol, in the absence of nucleophiles. BPDCH was converted to tetrol by adding aliquots of a standard solution to aqueous samples and allowing the compound to hydrolyze completely. After evaporation of the samples, the product was dissolved in methanol. The amount of BPDCH initially present was obtained from the known extinction coefficient of tetrol in methanol (23). The extinction coefficients of BPDBH and BPDIH were obtained similarly. Onetenth of a volume of water was added to freshly prepared reaction mixtures of these halohydrins. The hydrolysis reaction was followed to completion, and (if necessary) the spectrum was extrapolated back to time zero. Because these samples were in 90% THF solutions, we measured the extinction coefficient of tetrol in this solvent and obtained the same value as in methanol (M,344nm ) 44 400 M-1 cm-1).

Results Synthesis of Halohydrins. Treatment of anti-BPDE with LiCl produced one major and one minor product which were readily separated by HPLC (Figure 1). The liquid secondary ion mass spectrum (LSIMS, negative mode) of the major product isolated by method A (extraction and HPLC) was consistent with a chlorohydrin. However, a molecular ion was not observed. This sample was also analyzed by several other techniques, including LSIMS (positive mode), CIMS, ESIMS, and HREIMS. Only [M - HCl] and [M - HCl - H2O] fragments were detected by these methods. When BPDCH was prepared by method B, where the final product was precipitated, a molecular ion (m/z

1060 Chem. Res. Toxicol., Vol. 11, No. 9, 1998

Song et al.

Table 2. Calculated Atomic Ratios from the Single Mass Search of m/z 338.0711 Observed in the HRFAB Mass Spectrum of BPDCH calculated ratioa

deviation (ppm)

12C

1H

16O

35Cl

-6.1 0.3 6.8

26 20 14

10 15 20

1 3 5

0 1 2

a

The expected formula for the molecular ion of 9,10-chlorohydrin 2 is C20H15O3Cl.

Scheme 2. Proposed Structure and Mass Spectral Fragmentation Pattern for the Minor Acetate Adduct Formed in the Synthesis of BPDCH by the LiCl Method (see Figure 1)

338.071) was observed by HRFAB MS. A single mass search of this ion provided the expected formula C20H15O335Cl, within a deviation of 0.3 ppm. These data are summarized in Table 2. An isotopic peak for this ion at M + 2 was observed, and the ratio of the M and M + 2 peaks was 2.8:1. The relative abundance of the molecular ion observed for BPDCH was small. Minor Product in the Synthesis of BPDCH. The minor product (Figure 1) formed by LiCl treatment of BPDE was identified by mass spectrometry as an adduct between acetic acid and the hydrocarbon. LSIMS of this derivative showed a molecular ion at m/z 362. The proposed structure and fragmentation pattern for this product are shown in Scheme 2. Because chloride and acetic acid compete as nucleophiles for the C-10 position of BPDE, the critical variable is the ratio of chloride to acetic acid. BPDCH made up ∼95% of the product at an acetic acid:salt ratio of 20:1. The relative yield of acetate adduct increased with an increase in this ratio. Other proton donors were tried, but acetic acid was the most effective, particularly at high ratios of chloride to acetic acid. NMR Spectroscopy. 1H and 13C NMR spectra (Supporting Information) and derivatization (see below) firmly established the regiochemistry of epoxide ring opening and the stereochemistry of the alicyclic region of BPDCH. The appearance of a new doublet downfield (δ 6.40) with a coupling constant J of 3.0 Hz in the proton NMR spectrum of BPDCH established that chloride added to the C-10 benzylic position of anti-BPDE. This assignment was supported by the presence of the doublet at δ 5.29 with a large coupling constant J of 9.0 Hz, which

Table 3. Assignment of Cross-Peaks in the 300 MHz Heteronuclear Correlation NMR Spectrum of trans-BPDCHa

1H,13C

1H

δ (ppm)

coupling (Hz)

13C

δ (ppm)

H-7 H-8 H-9 H-10

5.29 4.56 4.72 6.40

J7,8 ) 9 J8,9 ) 2; J7,8 ) 9 J9,10 ) 3; J8,9 ) 2 J9,10 ) 3

C-7 C-8 C-9 C-10

69.83 69.83 73.95 58.08

aSee

Experimental Procedures for details.

can be assigned to H-7. The other two alicyclic protons of the cyclohexenyl ring were assigned to H-9 (dd, δ 4.72, J9,10 ) 3.0 Hz, J9,8 ) 2.0 Hz) and H-8 (dd, δ 4.56, J8,9 ) 2.0 Hz, J8,7 ) 9.0 Hz) on the basis of chemical shift comparison with (()-7r,8t,9t,10c- and (()-7r,8t,9t,10ttetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (racemic trans- and cis-tetrol, respectively) (12, 24). The H-10 proton of the BPDCH is shifted downfield by about 0.65 ppm relative to the H-10 proton of trans-tetrol. The equatorial protons of cyclohexane carbons with hydroxyl or chloride substituents differ in chemical shift by about 0.45 ppm (25). Three alicyclic carbon resonances appear in the 13C NMR spectrum of BPDCH at δ 58.08, 69.83, and 73.95. The intensity at δ 69.83 is approximately double that of the other two resonances, suggesting it results from two overlapping carbons. The 13C resonances were partly established by selective heteronuclear decoupling (26). Selective saturation at H-10 (δ 6.40) resulted in the collapse of a doublet at δ 57.07-59.15, while other doublets at δ 73.61-74.39, 69.66-70.15, and 69.66-70.33 remained unchanged. Therefore, δ 58.08 is assigned to C-10. Similarly, selective irradiation of H-7 (δ 5.29) changed one of the doublets around δ 69.83 into a broad singlet, while the three other doublets were unchanged. Thus, one of the two overlapping carbons at δ 69.83 is C-7. Unambiguous assignments of the remaining 13C resonances were established by a two-dimensional (2D) 1 H,13C-heteronuclear correlation NMR experiment (27, 28). This experiment corroborated the selective heteronuclear decoupling results and also provided chemical shift assignments for C-8 and C-9 of BPDCH. The carbons at δ 69.83 are correlated with the H-7 and H-8 proton resonances, which verifies the previous assignment that one of these overlapped signals is due to C-7. The cross-peaks observed between C-9 and H-9 and C-10 and H-10 gave final assignments of C-10 and C-9 to δ 58.08 and 73.95, respectively (27). These data are summarized in Table 3. Assignment of Stereochemistry. The geometry of the alicyclic region of BPDCH is closely related to that of substituted cyclohexenes, which adopt either a halfchair or a boat conformation (29). The coupling constants between methine hydrogens (e.g., J8,9 ) 2.0 Hz) in the alicyclic region of BPDCH suggest that the half-chair conformation is favored. The C-10 substituent will be quasi-axial to avoid adverse steric interaction with the bay region hydrogen at C-11. Because the relative stereochemistry of the 7-, 8-, and 9- hydroxyl groups in BPDCH should remain the same as in BPDE, the C-9 substituent will be quasi-axial, while the C-7 and C-8 substituents are quasi-diequatorial. The large coupling constant observed for J7,8 (9 Hz) in BPDCH and the small coupling constants for J9,10 (3.0 Hz) and J8,9 (2.0 Hz) favor this argument (24). The small coupling constant for J9,10

Halohydrin Intermediates in Cis Adduct Formation

Chem. Res. Toxicol., Vol. 11, No. 9, 1998 1061

Scheme 3. A Trans, but Not Cis, Halohydrin Is Reconverted into an Epoxide by Treatment with Basea

a

Conditions are given in Experimental Procedures.

(3 Hz) also supports a quasi-axial conformation for the C-10 halide substituent. The dihedral angles between H-9 and H-10 protons are very similar in both cis and trans isomers, so the value of J9,10 cannot be diagnostic for the assignment of relative stereochemistry at C-10. In addition, the spatial distances between H-7a-H-10a and H-7a-H-10e are similar, which makes differentiating between an axial or equatorial C-10 proton by an NOE experiment difficult. Epoxide opening by the lithium halide reaction is expected to give trans products, as exemplified by the clean conversion of cyclohexene oxide to trans-2-halocyclohexanol (20, 30). NMR studies on the structurally related stereoisomeric cis- and trans-tetrol of BPDE provide support for this expectation (12, 24). The coupling constants of the four alicyclic protons in trans-tetrol are nearly identical to the corresponding values obtained for BPDCH, which is consistent with the trans assignment of the 9- and 10-substituents in the chlorohydrin. Because NMR experiments cannot be used to unequivocally differentiate between C-10 cis and trans substitution, we employed another approach. Trans chlorohydrins, but not cis chlorohydrins, undergo favorable backside intramolecular attack at the halide-substituted carbon by the adjacent alkoxide generated under basic conditions, resulting in conversion to an epoxide (Scheme 3). We subjected BPDCH to pH g12 for 20 min in an aqueous-organic solution. The presence of organic solvent in the reaction mixture serves to inhibit the competitive BPDCH hydrolysis reaction. Samples of untreated and treated BPDCH and anti-BPDE were then analyzed by HPLC. As shown in Figure 2, treatment of BPDCH with base resulted in the formation of a product which coeluted with BPDE. This product also exhibited a UV spectrum identical to that of BPDE. Thus, lithium chloride opening of the epoxide in anti-BPDE generates a bay region chlorohydrin with C-9-C-10 trans stereochemistry. Regio- and Stereochemistry of BPDBH and BPDIH. NMR data were not obtained on the less stable bromo- and iodohydrin derivatives. However, regio- and stereochemistry were deduced from other evidence. Reaction of BPDE occurs primarily by breakage of the bond between the epoxide oxygen and the C-10 position. If substitution had occurred at C-9, no appreciable change in the UV spectrum of the pyrene moiety would be

Figure 2. Comparison of HPLC chromatograms of (A) antiBPDE, (B) trans-BPDCH, (C) the product of trans-BPDCH (same quantity as used in sample B) after treatment with NaOH for 20 min (reaction mixture was 1:8:2 THF/acetone/20 mM aqueous NaOH, with a 5:1 NaOH:BPDCH ratio), and (D) coinjection of the samples from A and C. HPLC conditions are the same as those described in Figure 1, except that the flow rate was 0.8 mL/min.

Figure 3. UV absorbance spectra of trans halohydrins, obtained as described in Experimental Procedures. Spectra were recorded at a halohydrin concentration of 10 µM in THF for purified samples of BPDCH or BPDBH or at the same concentration in the LiI reaction mixture for BPDIH.

expected (31). On the basis of the observed red shifts in their UV spectra (below), bromide and iodide attacked the C-10 position of BPDE. The stereochemistry of deoxyadenosine alkylation by the three halohydrins (below) supports trans halide addition in each case. UV Spectra. Relative to that of tetrol (UV maxima at 247, 268, 279, 328, and 344 nm), the UV spectra of BPDCH, BPDBH, and BPDIH display progressively increasing bathochromism (red shift) and hypochromism (reduced absorption), which is consistent with increasing polarizability of the pyrenyl system with attachment of successively larger halides (Figure 3). There is also a progressive broadening of the absorption bands. The Ba absorption band (230-250 nm), Bb absorption band (260300 nm), and La absorption band (310-370 nm) result from transitions parallel to the long, short, and long axes of the pyrenyl moiety, respectively (32). For a given halohydrin, the red shift is smallest for the Ba band, intermediate for the Bb band, and largest for the La band,

1062 Chem. Res. Toxicol., Vol. 11, No. 9, 1998

Song et al. Scheme 4. Mechanism of Adduct Formation in Aqueous Buffera

Figure 4. CD spectra of trans halohydrins, recorded directly from the lithium halide reaction mixtures as described in Experimental Procedures. The substrate for these reactions was (-)-anti-BPDE, whose spectrum is also shown. Table 4. Effect of Chloride on the Hydrolysis Rate of trans-BPDCHa [NaCl] (mM)

K (min-1)

t1/2

0 10 25 50

1.12 0.99 1.14 0.75

37 42 36 56

[NaCl] (mM)

K (min-1)

t1/2

100 250 500

0.46 0.41 0.27

91 102 153

a Hydrolyses were carried out at room temperature in 2.5 mM HEPES (pH 7.6), containing 50% THF.

roughly in a 1:2:4 ratio. For a given absorption band, the red shift is smallest for BPDCH, intermediate for BPDBH, and largest for BPDIH, also roughly in a 1:2:4 ratio. CD Spectra. The Cotton effects seen in the CD spectra of the halohydrins are much more intense than those of anti-BPDE, and those of the longest-wavelength La absorption band are opposite in sign from the BPDE ellipticity (Figure 4). The sign of the rotation by these samples at the D line (by convention the sign of the enantiomers) was not measured, but would be the same as the sign of the La band ellipticity. The relative values of these Cotton effects are -1.0, +12, +22, and +40 for the 7S,8R isomers of anti-BPDE, BPDCH, BPDBH, and BPDIH, respectively. The BPDCH:BPDBH:BPDIH ratio of Cotton effects is close to 1:2:4, the same ratio obtained for the relative red shifts in the absorbance spectra. In the halohydrins, the ellipticity at the λmax of the Bb absorption band is opposite in sign from that of the La absorption band. This is not true of anti-BPDE. The magnitude of the Bb band Cotton effect relative to that of the La band signal (a ratio of 0.21, 0.58, and 0.79 for BPDCH, BPDBH, and BPDIH, respectively) also increases with the size of the halide. Chlorohydrin Stability in the Presence of Chloride. The t1/2 of trans-BPDCH at room temperature in a mixture of 50% THF/2.5 mM HEPES buffer (pH 7.6), and with no halide present, is ∼40 s (Table 4). The addition of 500 mM chloride increases the t1/2 more than

a In the absence of halides, BPDE forms a C-10 carbocation that alkylates deoxyadenosine to generate a trans adduct as the major product by the SN1 pathway. In the presence of halide, a fraction of the BPDE (by an SN2 reaction) or BPDE carbocation (by an SN1 reaction) is diverted into the formation of a trans halohydrin. The halohydrin intermediate undergoes SN2 nucleophilic attack by deoxyadenosine to generate a cis adduct. Alternatively, the halohydrin can lose halide to form a carbocation, which accounts for the formation of some trans adducts by this intermediate. Trans adduct formation from the halohydrin is reduced by adding halide, which increases the rate of carbocation reconversion back to the halohydrin. At high concentrations of halide, trans adduct formation reaches a minimum of bromohydrin > iodohydrin, which correlates inversely with the leaving group abilities of the halides. Halohydrins are more stable in the reaction mixture than in pure THF. This is probably due to the presence of high concentrations of halide, which stabilize the halohydrin. In support of this view, we found that the rate of trans-BPDCH hydrolysis in buffer is markedly decreased by high concentrations of chloride. We observed formation of the BPDE iodohydrin in aqueous solution at pH 2, and in smaller amounts at pH 7. At pH 2, all of the BPDE is immediately protonated and converted to the C-10 carbocation, which is then attacked by water or iodide to form tetrol or BPDIH, respectively. At neutral pH, hydrolysis of BPDE is comparatively slow, so BPDIH forms by iodide attack on either the epoxide or carbocation (16). Once formed, hydrolysis of BPDIH occurs at a rate that is dependent on iodide and solvent concentration. Because BPDIH formation is slower at pH 7 while the rate of its disappearance is similar at both high and low pH, a higher concentration occurs at the lower pH. Transient absorption changes were not observed with BPDE reaction mixtures containing chloride or bromide. There is a large difference in the rate of cis-tetrol formation from BPDE during halide-catalyzed hydrolysis, depending on the halide used, and the order of effectiveness is I- > Br- > Cl- . F- (16). Under aqueous conditions, iodide is the most nucleophilic halide and its SN2 attack on BPDE is more rapid than that of the other halides (16). Iodide is also more effective at trapping the C-10 BPDE carbocation by SN1 attack than the other halides. Thus, reaction of BPDE with iodide at either acidic or neutral pH leads to the formation of higher transient concentrations of halohydrin than reaction with chloride or bromide. Because iodide is also the best leaving group among the halides, BPDIH is the least stable halohydrin in the absence of halides. We have previously reported that reaction of BPDCH with deoxyadenosine resulted in the formation of the same four covalent adducts that are formed by antiBPDE (18). The only difference between the two alkylating agents is in the relative amounts of cis and trans

Song et al.

adducts formed. Adding chloride to this reaction increases the proportion of cis adducts. At high chloride concentrations, the proportions of cis adducts formed by trans-BPDCH and anti-BPDE approach the same value (∼88% cis), a result which strongly implicates intermediate formation of a trans halohydrin in halide-catalyzed alkylation reactions (18). Iodide catalyzes the formation of significantly more cis adducts than either bromide or chloride, reflecting the higher concentration of halohydrin it generates. This difference is greatest at low concentrations. Going from 0 to 1 mM halide, the increase in the percentage of cis adduct formation is 6 times greater for iodide than for bromide, while chloride has no detectable effect. Iodide is a better leaving group than chloride. However, this factor would have little effect on the stereochemical outcome of alkylation reactions, because an increase in leaving group ability by replacement of chloride with iodide would increase the rate of SN1 and SN2 pathways to approximately the same extent. It is well established that anti-BPDE forms primarily trans adducts with DNA by an SN1 pathway (Scheme 4). What has not been recognized is that the SN1 pathway competes with an SN2 pathway in the presence of halide. The stereochemical outcome of BPDE-DNA adduct formation is determined by the competition between these pathways. Physiological concentrations of chloride are sufficient to generate significant amounts of cis adducts with deoxyadenosine (18). Conformational studies on PAH-modified ODNs have provided important information on differing properties of various types of adducts. Trans adducts derived from either (+)-anti- or (-)-anti-BPDE, when in normal basepaired sequences, are in a solvent-exposed environment in the minor groove and minimally disrupt the secondary structure of DNA (33). The hydrocarbon moiety of the cis(+)- or cis(-)-anti-BPDE adduct is intercalated between the base pairs flanking the modified site, with the substituted dGuo base displaced into the minor or major groove (34, 35). This conformation is more disruptive to the DNA duplex. Under certain circumstances, the disruptive cis adduct could be more mutagenic than the trans adduct. In support of this expectation, bulky and disruptive adducts are known to be more effective at blocking DNA replication than less bulky lesions (36), and work with BPDE has shown that the cis adduct is a better blocking agent for these enzymes than trans adducts (37). Rates of DNA repair between cis and trans adducts also differ. There is a 10-fold greater human nucleotide excision repair rate for cis versus trans BPDE-dG adducts in normal duplex sequences (opposite dC) (38). The cis repair rate decreases to that of the trans adduct when the modified dG is opposite dA. Our results have shown that chlorohydrins can form in aqueous solution at physiological concentrations of salt. But whether chlorohydrins formed in serum or other extracellular fluids would undergo cellular uptake and survive transit to a cell nucleus is not known. Chlorohydrins that form intracellularly may be more likely to alkylate DNA. The concentration of chloride ion in typical cells is only 5-15 mM. However, the natural targets of PAH carcinogens are epithelial tissues. Many types of epithelial cells contain 30-50 mM chloride, while secretory epithelial cells contain 60-90 mM chloride (3941). The degree to which the BPDE chlorohydrin would form, and alkylate intracellular targets in various cell

Halohydrin Intermediates in Cis Adduct Formation

types, remains to be determined. However, the chloridecatalyzed pathway could operate at a level high enough to influence stereochemistry and levels of adducts in epithelial cells, and therefore might influence PAH mutagenicity and tumorigenicity, as well as tissue susceptibility.

Acknowledgment. This work was supported in part by grants from NIH (CA40598, ES06869, and AI39152) and the California State Toxic Substances Research & Teaching Program. HRFAB mass spectroscopy was carried out at the Washington University MS Resource (NIH Grant P41RR0954); other spectra were obtained at the University of California, San Francisco, National MS Facility. Elemental analysis was carried out by M-H-W Labs (Phoenix, AZ). We thank Dr. Ignacio Rodriguez for assistance in obtaining CD spectra. Supporting Information Available: The 2D spectrum of the BPDCH and additional evidence supporting the formation of BPDIH in aqueous solution (2 pages). Ordering information is given on any current masthead page.

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