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Reactions of Benzene Oxide with Thiols Including Glutathione Alistair P. Henderson,*,†,‡ Martine L. Barnes,† Christine Bleasdale,† Richard Cameron,† William Clegg,† Sarah L. Heath,† Andrew B. Lindstrom,§ Stephen M. Rappaport,‡ Suramya Waidyanatha,‡ William P. Watson,| and Bernard T. Golding*,† School of Natural SciencessChemistry, Bedson Building, University of Newcastle upon Tyne, Newcastle upon Tyne, NEI 7RU, United Kingdom, Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7431, National Exposure Research Laboratory, United States Environmental Protection Agency, MD-205-05, Research Triangle Park, North Carolina 27711, and Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, SK10 4TJ, United Kingdom Received August 9, 2004
S-Phenylmercapturic acid is a minor metabolite of benzene used as a biomarker for human benzene exposures. The reaction of intracellular glutathione with benzene oxide-oxepin, the initial metabolite of benzene, is presumed to give 1-(S-glutathionyl)-cyclohexa-3,5-dien-2-ol, which undergoes dehydration to S-phenylglutathione, the precursor of S-phenylmercapturic acid. To validate the proposed route to S-phenylglutathione, reactions of benzene oxide-oxepin with glutathione and other sulfur nucleophiles have been studied. The reaction of benzene oxide with an excess of aqueous sodium sulfide, followed by acetylation, gave bis-(6-trans-5acetoxycyclohexa-1,3-dienyl)sulfide, the structure of which was proved by X-ray crystallography. Reactions of benzene oxide-oxepin in a 95:5 (v/v) mixture of phosphate buffer in D2O with (CD3)2SO were monitored by 1H NMR spectroscopy. In the absence of glutathione, the halflife of benzene oxide-oxepin was ca. 34 min at 25 °C and pD 7.0. The half-life was not affected in the range of 2-15 mM glutathione in the presence and absence of a commercial sample of human glutathione S-transferase (at pH 7.0, 8.0, 8.5, or 10.0). The adduct 1-(S-glutathionyl)cyclohexa-3,5-diene-2-ol was identified in these reaction mixtures, especially at higher pH, by mass spectrometry and by its acid-catalyzed decomposition to S-phenylglutathione. Incubation of benzene oxide with N-acetyl-L-cysteine at 37 °C and pH 10.0 and subsequent mass spectrometric analysis of the mixture showed formation of pre-S-phenylmercapturic acid and the dehydration product, S-phenylmercapturic acid. The data validate the premise that benzene oxide-oxepin can be captured by glutathione to give (1R,2R)- and/or (1S,2S)-1-(S-glutathionyl)cyclohexa-3,5-dien-2-ol, which dehydrate to S-phenylglutathione. The capture is a relatively inefficient process at pH 7 that is accelerated at higher pH. These studies account for the observation that the metabolism of benzene is dominated by the formation of phenol. The pathway leading to S-phenylmercapturic acid is necessarily minor on account of the low efficiency of benzene oxide capture by glutathione at pH 7 vs spontaneous rearrangement to phenol.
Introduction Despite more than a century of research, the molecular mechanism of benzene carcinogenesis is not understood (1, 2). It may be presumed that the answer to the problem lies in the complex metabolism of benzene that is initiated by cytochrome P450 (CYP450)1 oxidation to benzene oxide-oxepin (1) (3-5). A substantial part of 1 rearranges to phenol, the major metabolite of benzene (6, 7). It has * To whom correspondence should be addressed. (A.P.H.) Tel: 1 + 919 + 9667319. Fax: 1 + 919 + 9664711. E-mail: alistair@ email.unc.edu. (B.T.G.) Tel: 44 + 191 + 2226647. Fax: 44 + 191 + 2226929. E-mail:
[email protected]. † University of Newcastle upon Tyne. ‡ University of North Carolina at Chapel Hill. § United States Environmental Protection Agency. | Syngenta Central Toxicology Laboratory. 1 Abbreviations: GSH, glutathione; GST, glutathione S-transferase; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; m-CPBA, m-chloroperbenzoic acid; CYP450, cytochrome P450.
long been known that S-phenylmercapturic acid (2) is a minor metabolite of benzene (8, 9), which is now used as a biomarker for human benzene exposures (10, 11). Compound 2 is presumed to be derived by the reaction of intracellular glutathione (GSH) with 1. The product of this thiol-epoxide reaction is presumed to be 1-(Sglutathionyl)-cyclohexa-3,5-dien-2-ol, shown as diastereoisomers 3a,b in Scheme 1, which is assumed to undergo dehydration to S-phenylglutathione (4), further metabolism of which gives 2 (Scheme 1). However, the intermediates 3a,b in this sequence have never been definitively characterized (9). Analogous intermediates to 3a,b have been reported from the metabolism of naphthalene and halobenzenes. For naphthalene, the reaction of naphthalene 1,2-oxide with GSH in the presence of glutathione S-transferase (GST) gave both 1-hydroxy-2-glutathionyl1,2-dihydronaphthalene and 1-glutathionyl-2-hydroxy-
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Scheme 1. Reaction of Benzene Oxide with GSH and Subsequent Degradative Steps
1,2-dihydronaphthalene, which underwent further metabolism to S-(1-hydroxy- and 2-hydroxy-1,2-dihydronaphthalenyl)mercapturic acids (12). For 1,2-dichlorobenzene, CYP450 oxidation in the rat was proposed to lead to both 3,4- and 4,5-dichlorobenzene oxide. This was inferred by observation of the GSH conjugates 2,3-, 3,4-, and 4,5-dichloro-6-glutathionyl-cyclohex-2,4-dienol in bile and the corresponding dichlorophenylmercapturic acids in urine (13). Previously, 3,4-dichlorophenylmercapturic acid was isolated and characterized as a metabolite of 1,2-dichlorobenzene in rabbits (14). Similar results have been reported for bromo- (15) and chlorobenzene (16) and recently for the antiepileptic drug lamotrigine, a 2,3dichlorophenyl derivative (17). However, none of the nonaromatized GSH conjugates referred to above was fully characterized and not at all with respect to their stereochemistry, and their intermediacy and derivation from an arene oxide precursor remain a speculative, albeit reasonable, hypothesis. The formation of 3a,b and hence 4 and 2 competes with other reactions of 1, including the rearrangement of benzene oxide to phenol, the epoxide hydrolase-mediated reaction to cyclohexa-3,5-diene-1,2-diol (9), and the proposed oxidation of oxepin to (Z,Z)-muconaldehyde (1820). We have considered the possibility that 3a,b could also lead to phenol by elimination of GSH in preference to water (21). To explore this possibility and to validate the proposed route to 4 and ultimately 2, we have performed a comprehensive study of the reactions of 1 with GSH and other sulfur nucleophiles. To enable this study, we have also investigated the stability of 1 in aqueous organic mixtures. The implications of these results for benzene metabolism are discussed.
Materials and Methods Caution: Benzene oxide-oxepin must be handled in a wellventilated hood by an operator wearing appropriate protective clothing. Instruments. The 1H NMR spectra were run on a Bruker WP-200E (200 MHz) or a JEOL (500 MHz) spectrometer. Residual proton signals from the deuterated solvents were used as references [acetonitrile (1.95 ppm), D2O (4.81 ppm), d6-DMSO (2.50 ppm), and chloroform (7.25 ppm)]. 13C spectra were recorded on a Bruker WP-200E (50.3 MHz) or a JEOL (125 MHz) spectrometer, and the residual 13C signal from the
Henderson et al. deuterated solvent was used as a reference [acetonitrile (1.2 and 117.8 ppm), d6-DMSO (39.7 ppm), and chloroform (77.0 ppm)]. All coupling constants were measured in Hertz. All infrared spectra were recorded on a Nicolet 20-PC Fourier Transform IR spectrophotometer as either potassium bromide disks or Nujol mulls. Mass spectra were recorded on a Kratos MS80 RF spectrometer in +EI mode. Combustion analysis was performed using a Carlo Erba 1106 CHN analyzer. MS/MS analyses were conducted using a Sciex API3000 triple quadrupole mass spectrometer (Applied Biosystems/MDS Sciex, Foster City, CA). Analytes were injected via infusion mode at a rate of 10 µL/min. The mass spectrometer was operated in Q1 scan mode to determine parent ions and then in multiple reaction monitoring mode to assess resulting daughter ions. The system was operated with negative and positive turbo ion spray atmospheric pressure ionization. Chemicals, Solvents, and Enzymes. Chemicals and solvents were either AnalaR grade, which were used directly, or laboratory reagent grade purified further where appropriate and were commonly available. GSH (reduced) and GST from equine liver (50-100 units per mg protein) and human placenta (66 units per mg protein) were obtained from the Sigma-Aldrich Company. The human enzyme was used for kinetic studies with benzene oxide, whereas the equine enzyme was used for experiments in which mass spectrometry was used to monitor reactions of benzene oxide. The activity of the human enzyme was checked using the manufacturer’s assay with 1-chloro-2,4dinitrobenzene. trans-4,5-Dibromocyclohexene (5). To 1,4-cyclohexadiene (21.2 g, 25.0 mL, 0.264 mol) in dichloromethane (200 mL) stirred and maintained below 10 °C was added a 2.9 M solution of bromine in dichloromethane dropwise until an orange color persisted (ca. 90 mL of the solution). The resulting solution was concentrated to give a white solid that was dried in vacuo (61.4 g, 96%). mp 32-34 °C, lit. (20), mp 32-34 °C. Found: C, 30.06; H, 3.12%. C6H8Br2 requires C, 30.03; H, 3.36. 1H NMR (200 MHz, CDCl3): 5.65 (2H, m, H-1 and H-2), 4.50 (2H, m, H-4 and H-5), 2.62 (4H, m, H-3 and H-6). trans-4,5-Dibromocyclohexene Oxide (6). Purified mchloroperbenzoic acid (m-CPBA) (22) (37.3 g, 0.216 mol) and trans-4,5-dibromocyclohexene (43.2 g, 0.18 mol) in dichloromethane (300 mL) were heated at reflux for 22 h. The resulting solution was cooled to 0 °C and filtered, and the solid was discarded. The filtrate was washed with 20% sodium hydrogen sulfite (100 mL) and saturated sodium hydrogen carbonate (3 × 50 mL) and dried (MgSO4). Concentration in vacuo gave a white solid (34.0 g, 74%). mp 59-61 °C, lit (20) 58-59 °C. Found: C, 28.16; H, 3.00%. C6H8Br2O requires C, 28.13; H, 3.13. 1H NMR (200 MHz, CDCl3): 4.31 (2H, m, H-4 and H-5), 3.22 (2H, m, H-1 and H-2), 2.94 (2H, m, H-3), 2.65 (2H, m, H-6). m/z (+EI): 258 (M+, 30%), 256 (64), 254 (28), 177 (15), 175 (14), 139 (100). Benzene Oxide-Oxepin (1). To trans-4,5-dibromocyclohex1-ene oxide (26.1 g, 0.102 mol) in dry diethyl ether (200 mL) at room temperature was added, dropwise with stirring, 1,8diazabicyclo[5.4.0]undec-7-ene (DBU) (65.1 g, 64.0 mL, 0.428 mol). After 3 days, the resulting yellow mixture containing precipitated DBU hydrobromide was washed with saturated sodium hydrogen carbonate (4 × 100 mL) and saturated brine (100 mL) and dried (MgSO4). After filtration, the ethereal solution was fractionally distilled through a Vigreux column, first at atmospheric pressure to remove ether and then at ca. 15 mmHg (bath temperature ca. 60 °C) to afford 1 as a yellow liquid containing ca. 2% ether but free of phenol (12.9 g, 98%). 1H NMR (200 MHz, d -acetonitrile): 6.33 (2H, m, H-4 and H-5), 3 6.01 (2H, m, H-1 and H-2), 5.23 (2H, m, H-3 and H-6). 13C NMR (50.3 MHz, d3-acetonitrile): 135.0, 126.4, and 122.9. Racemic trans,trans-(7a) and meso-bis-(2-Hydroxycyclo-3,5-hexadienyl)sulfide (7b). Compound 1 (0.35 g, 3.72 mmol) was stirred with Na2S‚9H2O (5.0 g, 20.6 mmol) in water (15 mL) at 0 °C for 45 min. The mixture was washed with diethyl ether (6 × 50 mL), and the combined ether extracts were
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Table 1. Kinetic Data for Reactions of 1 in the Absence and Presence of GSH and GST concn of GSH (mM)
pD
temp (°C)
rate constant (× 104 s-1)
half-life (min) of 1
0a 2a 2 (+ GST)a 6a 15a 0a 0b 0b 0b 0b 0c 0d
7.0 7.0 7.0 7.0 7.0 7.0 7.2 9.0 5.3 4.1 7.0 7.0
25 25 25 25 25 37 37 37 37 37 37 37
3.4 3.5 2.9 3.2 3.4 13.6 0.21 0.19 0.23 0.73 18 0.27
34 33 40 36 34 8.5 550 600 500 160 6.3 420
a 95:5 v/v D O-(CD ) SO. b 50:50 v/v D O-(CD ) SO. c 95:5 v/v 2 3 2 2 3 2 D2O-CD3CN. d 50:50 v/v D2O-CD3CN.
dried (MgSO4) and concentrated under reduced pressure to give a crystalline solid. The solid was washed with pentane and dried (MgSO4) to afford a mixture of the sulfides 7a,b (0.13 g, 31% combined yield). mp 77-80 °C, lit. (23) mp 75-79 °C. 1H NMR (200 MHz, d6-acetone): 6.08-5.88 (8H, m, 8 × dienyl-H), 4.534.24 (2H, br m, 2 × CHOH), 4.12 (1H, d, J 6.6 Hz, CHS), 4.06 (1H, d, J 6.4 Hz, CHS), 3.83-3.58 (2H, br, 2 × OH). bis-(6-trans-5-Acetoxycyclohexa-1,3-dienyl)sulfide (8a). A solution of freshly distilled acetic anhydride (0.26 g, 2.13 mmol) in dry ether (4 mL) was added to a solution of the sulfides 7a,b (0.13 g, 0.59 mmol), pyridine (95 µL, 1.17 mmol), and 4-N,N-(dimethylamino)pyridine (5 mg, 0.04 mmol) in dry ether (10 mL) at 0 °C. After complete addition of the acetic anhydride, the mixture was stirred at 0 °C for 2 h and allowed to warm to room temperature. After 60 h, the reaction was quenched by addition of saturated aqueous NaHCO3 (50 mL) and the layers were separated. The ethereal layer was washed sequentially with saturated NaHCO3 (3 × 20 mL) and water (3 × 10 mL) and dried (MgSO4). Removal of the solvent gave a solid that was recrystallized from ether to give 8a (0.13 g, 73%). mp 114117 °C; lit. (23), mp 115-117 °C. Found: C, 62.43; H, 5.73%. C16H18O4S requires C, 62.73; H, 5.92. 1H NMR (200 MHz, CDCl3): 6.05-6.26 (4H, m, 4 × dienyl-H), 5.75-5.94 (4H, m, 4 × dienyl-H), 5.51 (2H, d, J 5.8 Hz, 2 × CHOAc), 3.80 (2H, d, J 5.4 Hz, 2 × CHS), 2.02 (6H, s, 2 × Me). νmax cm-1 (KBr): 3046, 2968, 2925, 1730 (CdO), and 1650 (CdC). m/z (+EI): 306 (M+, 20%), 246 (20), 186 (68), 137 (20), 110 (85), 95 (100), 78 (62), 43 (88). Stability of 1 in Aqueous Media. Kinetic studies were performed in duplicate for 15 mM solutions of 1 in mixtures of D2O (containing appropriate quantities of potassium dihydrogen phosphate + disodium hydrogen phosphate to give the desired pD) and CD3CN or (CD3)2SO. The ratios of the solvent mixtures were 50:50 and 95:5 (water-organic, v/v). The reactions were monitored at 37 °C by observing the resonances in the range δ 6.4-7.3 ppm by 1H NMR. The conditions are shown in Table 1. Reactions of 1 in the Presence of GSH. These reactions were studied in a manner similar to those described above for buffered 95:5 (v/v) D2O-(CD3)2SO solutions (containing appropriate quantities of potassium dihydrogen phosphate + disodium hydrogen phosphate to give the desired pD) of 1 at 25 °C and pD 7.0. The concentrations of GSH used were 0, 2, 6, and 15 mM. The conditions are given in Table 1. Reactions of 1 in the Presence of GSH and GST. Compound 1 (15 mM) in buffered 95:5 (v/v) D2O-(CD3)2SO was incubated at 25 °C with 2 mM GSH in the presence and absence of 0.5 units of GST. Kinetic studies were performed as described above. MS-MS Analysis of Reactions of 1 with GSH in the Presence and Absence of GST. Compound 1 (6.6 mM) was incubated with GSH (6.6 mM) in the presence and absence of GST (1 mg) at 37 °C for 30 min in ammonium acetate buffer (5 mL, 0.1 M, various pH), and the resulting solution was analyzed
Scheme 2. Preparation of 1
by MS-MS using a Sciex API3000 triple quadrupole mass spectrometer. A portion of the reaction mixture from the reaction at pH 8.5 was adjusted to pH 1 by addition of concentrated formic acid and incubated at 37 °C for 30 min. The resulting solution was analyzed by MS-MS spectroscopy. Reaction of 1 in Aqueous Media with N-Acetyl-L-Cysteine Methyl Ester and N-Acetyl-L-Cysteine. Compound 1 (10.6 mM) was incubated with either N-acetyl-L-cysteine methyl ester or N-acetyl-L-cysteine (1 mol equiv) at 37 °C for 30 min in ammonium acetate buffer (5 mL, 0.1 M, pH 7.0, 8.5, or 10.0), and the solution was analyzed by MS-MS using a Sciex API3000 triple quadrupole mass spectrometer. A portion of the reaction mixture from the reaction at pH 10 was adjusted to pH 1 by addition of formic acid and incubated at 37 °C for 30 min. The resulting solution was analyzed by MS-MS spectroscopy. X-ray Crystallography. Crystals of 8a were examined on a Bruker SMART CCD diffractometer with Mo KR radiation (λ ) 0.71073 Å). C16H18O4S, M ) 306.4, monoclinic, I2/a, a ) 13.0259(3) Å, b ) 8.4599(2) Å, c ) 14.1034(4) Å, β ) 90.754(2)°, V ) 1554.03(7) Å3, Z ) 4, and R ) 0.034.
Results and Discussion Preparation of 1. Although the preparation of 1 has been reported (24, 25), we have optimized its preparation to ensure the absence of phenol and a very high level of purity. 1,4-Cyclohexadiene was reacted with 1 equiv of bromine to give trans-4,5-dibromocyclohexene (5), which was epoxidized to the crystalline trans-4,5-dibromocyclohexene oxide (6), a stable and convenient precursor of 1. Treatment of 6 with an excess of DBU gave highly pure 1 after fractional distillation (Scheme 2). Reaction of 1 with Sodium Sulfide. The stereospecific trans-1,2-opening of 1 with a series of sulfur nucleophiles has been previously examined (23). The reaction of 1 with an excess of Na2S in water at 0 °C was reported to give a mixture of the diastereoisomers, racemic trans, trans- and meso-bis(2-hydroxycyclo-3,5-hexadienyl)sulfide (7a) and (7b), respectively (Figure 1). The mixture of diastereoisomers was acetylated using acetic anhydride in pyridine. One of the two possible diacetates, 8a or 8b, was crystallized from ether and analyzed spectroscopically. In the absence of a crystal structure, the authors could not determine which of the diacetate structures, 8a or 8b, represented the crystalline diastereoisomer. We
Figure 1. Isomers from the reaction of benzene oxide with sodium disulfide followed by acetylation.
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Figure 2. Molecular structure of 8a in the crystalline state, with 50% probability ellipsoids for nonhydrogen atoms. The leftand right-hand sides of the molecule are related by a crystallographic 2-fold rotation axis, and both enantiomers are present in the racemic crystal structure.
have repeated this sequence of reactions to obtain a crystalline diacetate in 23% overall yield starting from 1. A structure investigation of this diacetate using X-ray crystallographic analysis established that the stereochemistry of the molecule is represented by the diacetate structure 8a (Figure 2). This experiment serves to validate the mode of ring opening of benzene oxide with a sulfur nucleophile, i.e., direct attack of the nucleophile at an epoxide carbon, with the inversion of configuration at this carbon indicating a SN2 mechanism. Molecules of 8a have crystallographic 2-fold rotation symmetry. The six-membered rings have C(2), C(3), C(4), and C(5) almost in a plane [rms deviation, 0.037 Å; C(2)C(3)-C(4)-C(5) torsion angle, 12.0°], with the saturated carbon atoms C(1) and C(6) lying on opposite sides of this plane, to give a torsion angle of 30.6° for C(2)-C(1)C(6)-C(5) and pseudoaxial positions for sulfur and the acetate substituent. Reactions of 1 in Aqueous Media in the Presence and Absence of GSH. Previous studies have shown that 1 is unstable in aqueous media (26-29), especially at lower pH. The rearrangement to phenol occurs spontaneously but is substantially accelerated in acidic media (26, 27, 29). We desired an aqueous medium for monitoring reactions of 1 with GSH by 1H NMR spectroscopy and therefore examined mixtures of D2O with (CD3)2SO and CD3CN (Table 1). We have found that a 95:5 (v/v) mixture of phosphate buffer in D2O with (CD3)2SO was suitable. In the absence of GSH, the half-life of 1 was ca. 34 min at 25 °C at pD 7.0 (pD ) pH + 0.4). In the presence of different concentrations of GSH, the half-life was not significantly affected in the range of 2-15 mM GSH. Raising the GSH concentration to 150 mM did reduce the half-life (to ca. 3 min), but under these conditions, pD stability could not be maintained. No effect was observed when GST was added in an experiment employing 2 mM GSH. In summary, in all experiments with 1 and GSH at pD 7, the major product that was observed was phenol and the presence of GSH with or without GST did not accelerate its formation. MS-MS spectroscopic analysis of the reaction between benzene oxide with GSH in the presence and absence of GST was carried out at different pH values (7.0, 8.0, 8.5,
Henderson et al.
and 10.0), and the products were analyzed by mass spectrometry in the negative ion mode. The ion m/z 400.2 (M - 1) corresponding to addition of GSH to benzene oxide was observed as shown in Figure 3 (Scheme 1). The spectrum also showed a significant peak at m/z 92.9 assigned as phenol arising from rearrangement of benzene oxide (Figure 3). Phenol was confirmed by extraction of the reaction mixture and subsequent GC-MS analysis using the method of Lindstrom et al. (4, 5), which showed phenol but not benzene oxide. The adduct of mass 400.2 was assigned the structure 3a,b by observation of its acid-catalyzed decomposition to 4 and by analogy with the reaction of benzene oxide with sulfide ion (see above). The product fragmentation of the peak at m/z 400.2 produced daughter ions at m/z 382.2, corresponding to 4, arising from dehydration/aromatization of the ring, whereas a signal corresponding to phenol was not observed from m/z 400.2. Partial fragmentation of the tripeptide backbone of m/z 400.2 was also observed. Reaction of benzene oxide with GSH at various pH values (7.0, 8.5, and 10.0) showed increasing formation of the adduct 3a,b with increasing pH. There appeared to be no difference in the amount of adducts 3a,b formed with and without GST at each pH value. There was only a trace of product at pH 7, while at pH 8.5 and pH 10, it was found to increase, in the ratio 1:20:25, pH 7:8.5:10. When an excess of benzene oxide was added to GSH at pH 8.5, either with or without GST, increased formation of 3a,b was observed. The reaction did not go to completion, as GSH was still present at the end of the experiment, possibly due to the competing conversion of benzene oxide to phenol. Attempts to detect 3a,b by 1H NMR spectroscopy under the conditions described were unsuccessful, phenol being the only product observed as detailed above. Presumably the yield of 3a,b was below the limit of detection by NMR spectroscopy (ca. 1%). It is interesting that a yield of 15% was determined for the reaction of 1 mM benzene oxide with 6 mM GSH in the presence of the soluble fraction from rat liver (9). Inactivation of the putative enzyme activity in the rat liver fraction reduced the yield of 3a,b to
