Reactive Intermediate Formation from the 2-(Fluoromethoxy)-1,1,3,3,3

601 Elmwood Avenue, Box 711, Rochester, New York 14620. Received September 11, 2001. 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A) ...
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Chem. Res. Toxicol. 2002, 15, 623-628

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Reactive Intermediate Formation from the 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (Compound A)-Derived Cysteine S-Conjugate S-[2-(Fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine in Pyridoxal Model Systems Zeen Tong† and M. W. Anders* Department of Pharmacology and Physiology, University of Rochester, 601 Elmwood Avenue, Box 711, Rochester, New York 14620 Received September 11, 2001

2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A) is a degradation product of the anesthetic sevoflurane and undergoes cysteine conjugate β-lyase-dependent bioactivation to nephrotoxic metabolites in rats. The present experiments were designed to identify reactive intermediates formed from S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine, a compound A-derived cysteine S-conjugate, in two pyridoxal model systems, namely Cu2+/ pyridoxal and N-dodecylpyridoxal in cetyltrimethylammonium micelles. S-[2-(Fluoromethoxy)1,1,3,3,3-pentafluoropropyl]-L-cysteine was incubated in the model systems with benzyl bromide, pentafluorobenzyl bromide, aniline, and o-phenylenediamine as trapping agents. The products were purified by TLC and identified by 19F and 1H NMR spectroscopy and by GC/MS. In the absence of trapping agents, 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid and 3,3,3-trifluorolactic acid, which have been identified previously in biotransformation studies, were formed. With the chemical models, 2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropanethiolate, the expected first intermediate, was not trapped with benzyl bromide. Rather, the dehydrofluorination product 2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenylthiolate was trapped with benzyl bromide to give benzyl 2-(fluoromethoxy)-3,3,3-trifluoropropanethioate, which was formed in both chemical models. When pentafluorobenzyl bromide was used as a trapping agent, GC/ MS analysis showed that the expected thiolate was trapped to give pentafluorobenzyl 2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl sulfide in the N-dodecylpyridoxal model. In both chemical models, 2-(fluoromethoxy)-3,3,3-trifluorothioacyl fluoride was trapped with aniline to give N-phenyl 2-(fluoromethoxyl)-3,3,3-trifluoropropanethioamide, which cyclized to give 3-phenyl-4-thiono-5-(trifluoromethyl)-1,3-oxazolane. The results demonstrate that most of the reactive intermediates and products formed by the β-lyase-catalyzed biotransformation of compound A-derived cysteine S-conjugates are also formed in the two chemical systems studied. Some products were, however, formed in chemical systems that have not been observed in previous in vivo and in vitro studies; it is not known whether these products are formed in biological systems and whether they contribute to the observed nephrotoxicity of cysteine S-conjugates.

Introduction (β-lyase)1

The cysteine conjugate β-lyase pathway has been established as a mechanism for the bioactivation of a range of nephrotoxic haloalkenes in rodents (2, 3). Haloalkene-derived cysteine S-conjugates are biotransformed by renal cytosolic and mitochondrial β-lyases to reactive intermediates, including thioacyl fluorides (4-6), thioketenes (7, 8), R-thiolactones (9), and 2,2,3trihalothiiranes (10-12). Compound A, 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro1-propene, 1 (Figure 1), is the major degradation product of the anesthetic sevoflurane formed in anesthesia circuits equipped with carbon dioxide scrubbers (13-15). * To whom correspondence should be addressed. Phone: (585) 2751678. Fax: (585) 273-2652. E-mail: [email protected]. † Present address: Wyeth Research, 500 Arcola Road, Collegeville, PA 19426. 1 Abbreviations: β-lyase, cysteine conjugate β-lyase

Compound A is nephrotoxic in rats (16-20) and undergoes bioactivation by the β-lyase pathway (1, 20-22). Metabolites indicative of the β-lyase-dependent bioactivation of compound A are excreted in the urine of both rats given compound A and human subjects anesthetized with sevoflurane and are formed by incubation of cysteine S-conjugates of compound A with kidney homogenates (1, 23, 24). The pathway for the glutathione- and β-lyasedependent bioactivation of compound A is shown in Figure 1. The present experiments were designed to identify and characterize reactive intermediates formed in pyridoxal model systems that mimic the β-lyase-dependent bioactivation of cysteine S-conjugates of compound A. Accordingly, reactive-intermediate and product formation from S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine, 4, was studied in two pyridoxal model systems.2 We report here evidence for the formation of thiolate 6,

10.1021/tx010148b CCC: $22.00 © 2002 American Chemical Society Published on Web 04/05/2002

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Figure 1. Glutathione- and β-lyase-dependent bioactivation pathway of compound A. 1, 2-fluoromethoxy-1,1,3,3,3-pentafluoro-1-propene (compound A); 2, S-[2-(fluoromethoxy)-1,1,3,3,3pentafluoropropyl]glutathione; 3, S-[2-(fluoromethoxy)-1,3,3,3tetrafluoro-1-propenyl]glutathione; 4, S-[2-(fluoromethoxy)1,1,3,3,3-pentafluoropropyl]-L-cysteine; 5, S-[2-(fluoromethoxy)1,3,3,3-tetrafluoro-1-propenyl]-L-cysteine; 6, 2-(fluoromethoxy)1,1,3,3,3-pentafluoropropanethiolate; 7, 2-(fluoromethoxy)-1,3,3,3tetrafluoro-1-propenethiolate; 8, 2-(fluoromethoxy)-3,3,3-trifluorothiopropanoyl fluoride; 9, 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid; 10, 3,3,3-trifluorolactic acid. GST, glutathione transferase; GSH, glutathione; GGT, γ-glutamyltransferase; DP, dipeptidases; β-lyase; cysteine conjugate β-lyase.

thioacyl fluoride 8, and acids 9 and 10 from cysteine conjugate 4 in the two model systems.

Materials and Methods Chemicals. Aniline, benzyl bromide, pentafluorobenzyl bromide, benzyl mercaptan, o-phenylenediamine, cetyltrimethylammonium chloride, EDTA disodium dihydrate, triethylamine, and copper (II) chloride dihydrate were purchased from Aldrich Chemical Co. (Milwaukee, WI). Pyridoxal 5′-phosphate was obtained from Sigma Chemical Co. (St. Louis, MO). Compound A was supplied by Abbott Laboratories (Abbott Park, IL). Instrumental Analyses. 1H and 19F NMR spectra were acquired with a Bruker 270 MHz spectrometer operating at 270 2 Reactive intermediate formation from the vinylic cysteine Sconjugate of compound A, S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1propenyl)-L-cysteine 5 (Figure 1), was not studied because of its rapid cyclization (1).

Tong and Anders MHz for 1H and 254 MHz for 19F. Chemical shifts (δ) are referenced to tetramethylsilane (0.0 ppm) for 1H NMR spectra (CDCl3) or to trifluoroacetamide (0.0 ppm) for 19F NMR spectra (D2O or CDCl3). GC/MS analyses were performed with a Hewlett-Packard 5790 gas chromatograph (25 m × 0.2 mm, 0.5µm film thickness, HP-1 cross-linked methyl silicon column, splitless injection) coupled to a Hewlett-Packard 5970B mass selective detector. The injector and transfer line temperatures were 240 and 285 °C, respectively. The samples were analyzed with a temperature program of 70 °C for 1 min followed by a linear gradient of 10 °C/min to 250 °C and then held at 250 °C for 3 min. Syntheses. S-[2-(Fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine, 4, and N-dodecylpyridoxal bromide were obtained by synthesis (1, 25). Benzyl 2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl sulfide, 12 (see Supporting Information). Benzyl 2-fluoromethoxy-1,3,3,3-tetrafluoro-1-propenyl sulfide, 13 (see Supporting Information). Incubation of S-[2-(Fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine, 4, in a Cu2+/Pyridoxal Model. The Cu2+/pyridoxal model described by Thomas et al. (26) was used. Reaction mixtures contained 4 mM cysteine conjugate 4, 2 mM pyridoxal 5′-phosphate, 1 mM CuCl2, and 4 mM benzyl bromide or aniline (when included) in 40 mM phosphate buffer (pH 7.0 or pH 8.0) containing 10% D2O in a total volume of 1 mL; controls lacked CuCl2. The progress of the reaction was measured by recording 19F NMR spectra after 0, 1, and 3 h of incubation at 37 °C. After 3 h of incubation, the reaction mixture was extracted twice with 2 mL of ethyl ether. The ether extracts were pooled and concentrated under a stream of dry nitrogen. The products were purified by preparative TLC (silica gel) with hexanes/ethyl acetate (4:1) as the eluent and analyzed by GC/ MS. Incubation of S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine, 4, with a N-dodecylpyridoxal Model. The model system described by Kondo et al. (25) was used. Reaction mixtures contained 0.25 mM N-dodecylpyridoxal bromide, 3 mM cetyltrimethylammonium chloride, 4 mM cysteine conjugate 4, 4 mM benzyl bromide, pentafluorobenzyl bromide, or aniline (when included), and 1 mM EDTA in 40 mM phosphate buffer (pH 8.0) containing 10% D2O in a total volume of 1 mL; controls lacked N-dodecylpyridoxal bromide. The progress of the reaction was monitored by recording 19F NMR spectra after 0, 2, and 6 h of incubation at 37 °C. After 6 h of incubation, the reaction mixture was extracted twice with 2 mL of ethyl ether. The ether extracts were pooled and concentrated under a stream of dry nitrogen. The trapped products were purified by preparative TLC (silica gel) with hexanes/ethyl acetate (4:1) as the eluent and analyzed by GC/MS.

Results Cu2+/Pyridoxal Model. The formation of reactive intermediates from cysteine conjugate 4 was studied in a Cu2+/pyridoxal model. 19F NMR spectroscopic analysis of reaction mixtures in the complete model system conducted at pH 7.0 showed the formation of 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid 9 and the appearance of an unidentified major resonance at 2.44 ppm (Figure 2B in the Supporting Information). After incubation at pH 8.0, the intensity of the resonance at 2.44 ppm decreased sharply compared with reaction mixtures incubated at pH 7.0, whereas the intensity of the resonances assigned to acid 9 increased. Resonances assigned to 3,3,3-trifluorolactic acid 10 were also observed after incubation at pH 8.0 (Figure 2C in the Supporting Information). Little conversion of conjugate 4 to products was observed in control incubation mixtures that lacked CuCl2 (Figure 2A in the Supporting Information).

Compound A-Derived Reactive Intermediates Scheme 1

Chem. Res. Toxicol., Vol. 15, No. 5, 2002 625 Scheme 3

Scheme 2

Benzyl bromide was used as a trapping agent in the Cu2+/pyridoxal model at pH 7.0. 19F NMR spectroscopic analysis showed the formation of one predominant product in reaction mixtures that contained both benzyl bromide and cysteine conjugate 4 (Figure 3B in the Supporting Information). The amount of product trapped decreased as the pH of the buffer was increased from 7.0 to 8.0 (data not shown). GC/MS analysis showed the formation of a product with a retention time of 14.3 min and a molecular ion of m/z 282, which is 22 mass units less than the expected product formed by reaction of benzyl bromide with propanethiolate 6 (Figure 1), i.e., benzyl 2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl sulfide 12. The trapped product was purified by TLC and was identified as benzyl 2-(fluoromethoxy)-3,3,3-trifluoropropanethioate 11 (Scheme 1): 1H NMR (CDCl3) δ 7.3 (m, 5H), 5.39 (dd, 2H, JFH ) 54), 4.53-4.64 (m, 1H), 4.19 (s, 2H); 19F NMR (CDCl3) δ 2.15 (d, 3F), -78.10 (t, 1F, JFH ) 54); MS, m/z (relative abundance): 65 (11.1%), 91 (100%, C6H5CH2), 123 (3.1%, C6H5CH2S), 131 [3.8%, CH(OCH2F)CF3], 282 (6.3%, M+). A minor product formed from cysteine conjugate 4 in the Cu2+/pyridoxal model and trapped by reaction with benzyl bromide was identified by GC/MS and by comparison with synthetic sulfide 13 as benzyl 2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl sulfide 13 (Scheme 2). Thioacyl fluorides are formed as reactive intermediates of fluoroalkene-derived cysteine S-conjugates (4). Hence, studies were conducted to determine whether 2-(fluoromethoxy)-3,3,3-trifluorothiopropanoyl fluoride 8 (Figure 1) was formed from cysteine conjugate 4 and could be trapped with aniline. With the Cu2+/pyridoxal model, the conversion of cysteine conjugate 4 to products was complete in 1 h (Figure 3C in the Supporting Information). Three products trapped by reaction with aniline were observed by GC/MS analysis, and two were purified by TLC. The major product formed in the Cu2+/pyridoxal model system had a retention time of 14.6 min and was identified as N-phenyl-2-(fluoromethoxy)-3,3,3-trifluoropropanethioamide 14 (Scheme 3): 1H NMR (CDCl3) δ

7.34-7.90 (m, 5H), 5.58 (d of d of d, 2H, JFH ) 54), 5.095.19 (m, 1H); 19F NMR (CDCl3) δ 1.79 (d, 3F), -74.92 (t, 1F, JFH ) 54); MS m/z (relative abundance): 65 (19.5%), 77 (100%), 93 (14.2%, C6H5NH2), 109 (49.6%), 110 [47.8%, CH(OCH2F)CF3-HF-H], 136 (85.7%, C6H5NHCS), 166 (10.9%, M+-CF3-S), 186 (12.5%, M+-OCH2F-S), 198 (14.1%, M+-CF3), 216 (29.7%, M+-HF-S), 218 (34.9%, M+OCH2F), 219 (31.1%, M+-OCH2F+H), 267 (48.0%, M+). A minor product derived from cysteine conjugate 4 and trapped with aniline in the Cu2+/pyridoxal model had a retention time of 14.4 min and was identified as 3-phenyl4-thiono-5-trifluoromethyl-1,3-oxazolane 15 (Scheme 3), the cyclization product of thioamide 14: 1H NMR (CDCl3) 6.98-7.45 (m, 5H), 5.42-5.59 (m, 2H), 4.93-5.05 (m, 1H); 19F NMR (CDCl ) 2.26 (d); MS m/z (relative abundance) 3 77 (75%), 93 (6.6%), 104 (96.8%), 135 (9.8%), 149 (23%), 247 (100%, M+). About 90% of the cysteine conjugate 4 lost could be accounted for by the formation of thioamide 14 and oxazolane 15 (Scheme 3). Although oxazolane 15 was isolated and identified by 1H and 19F NMR spectroscopy from the incubation mixture, injection of purified thioamide 14 onto the GC column also resulted in the formation of oxazolane 15, indicating that cyclization may occur in reaction mixtures or on the GC column, or both. Another minor product formed from cysteine conjugate 4 and trapped with aniline had a retention time of 15.6 min and a molecular ion of m/z 247, but was not identified. Thioamide 14 was the predominant trapped species in the Cu2+/pyridoxal model system at pH 7.0 (Figure 3C in the Supporting Information), but less was formed in reaction mixtures at pH 8.0 (data not shown). o-Phenylenediamine was also used to trap reactive intermediates formed from cysteine conjugate 4 in the Cu2+/pyridoxal model system. 19F NMR spectroscopic analysis showed the appearance of two new resonances after incubation overnight, whereas GC/MS analysis showed the formation of several products formed by reaction with o-phenylenediamine. The major product had a retention time of 14.0 min and a molecular ion of m/z 248 and was tentatively identified as 2-[2,2,2trifluoro-1-(fluoromethoxy)ethyl]benzimidazole 17 (Scheme 4). A second abundant product had a retention time of 17.8 min and a molecular ion of m/z 262 and was tentatively identified as N-(2-aminophenyl)-4-thiono-5(trifluoromethyl)oxazolane 18 (Scheme 4). Two minor

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Chem. Res. Toxicol., Vol. 15, No. 5, 2002 Scheme 4

products were also trapped: one had a retention time of 17.2 min and a molecular ion of m/z 282, and the other had a retention time of 14.4 min and a molecular ion of m/z 228. The former compound was tentatively identified from its mass spectrum as N-(2-aminophenyl)-2-(fluoromethoxy)-3,3,3-trifluorothiopropanamide 16 and the latter as oxazolane 19. N-Dodecylpyridoxal Model. Fewer experiments were conducted with the N-dodecylpyridoxal model system because yields were low, which made identification of products difficult. The results obtained with the Cu2+/ pyridoxal model were, however, confirmed in the Ndodecylpyridoxal model system when benzyl bromide, aniline, and o-phenylenediamine were used as the trapping agents (data not shown). Pentafluorobenzyl bromide was also used as a trapping agent in the N-dodecylpyridoxal model system. GC/MS analysis showed that propanethiolate 6 was apparently trapped by pentafluorobenzyl bromide to yield pentafluorobenzyl 2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl sulfide, which had a molecular ion of m/z 394 and a base ion of m/z 191 (Figure 4 in the Supporting Information), although the trapping efficiency was low and the structure of the product was not confirmed by synthesis or by NMR spectroscopy.

Discussion The objective of the present study was to investigate reactive intermediate and product formation from the compound A-derived cysteine S-conjugate S-(2-fluoromethoxy-1,1,3,3,3-pentafluoropropyl)-L-cysteine, 4, in two chemical models, Cu2+/pyridoxal and N-dodecylpyridoxal, that mimic β-lyase-catalyzed β-elimination reactions from cysteine S-conjugates (25, 26). The Cu2+/ pyridoxal model catalyzes transamination reactions more

Tong and Anders

slowly than β-elimination reactions at pH 7.0 (26), whereas the N-dodecylpyridoxal model catalyzes both transamination and β-elimination reactions, but β-elimination is favored at pH 8.0 (25). The catalytic mechanism of pyridoxal-dependent βelimination reactions of S-haloethyl-L-cysteine conjugates involves Schiff base or aldimine formation, R-proton abstraction, and β-elimination. Accordingly, the expected initial product of a β-elimination reaction from a Shaloalkyl-L-cysteine conjugate is an R-haloalkylthiolate. 2-Chloro-1,1,2-trifluoroethanethiolate, which is formed by β-elimination from S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine, was trapped by reaction with benzyl bromide to yield benzyl 2-chloro-1,1,2-trifluoroethyl sulfide (4). Similarly, incubation of cysteine conjugate 4 with benzyl bromide in the chemical models would be expected to give benzyl 2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl sulfide 12. This product was not, however, observed by 19F NMR spectroscopic or GC/MS analyses in either chemical model. Rather, thiolester 11 (Scheme 1) and benzyl sulfide 13 (Scheme 2) were obtained in both chemical models, although the relative amounts differed. Thiolester 11 may be formed from thioacyl fluoride 8 (Scheme 1): hydrolysis of thioacyl fluoride 8 would give a thioacid or thiolacid. The conjugate base of the thiolacid may displace bromide from benzyl bromide to give thiolester 11. Benzyl sulfide 13 may be formed from propanethiolate 6 through an intermediate R-fluoropropenethiolate 7 formed by loss of HF (Scheme 2). Reaction of R-fluoropropenethiolate 7 with benzyl bromide would give benzyl sulfide 13 (Scheme 2). With pentafluorobenzyl bromide as the trapping agent in the N-dodecylpyridoxal model system, propanethiolate 6 was trapped as shown by GC/ MS analysis, although little product was formed and trapped. The results indicate that propanethiolate 6 was formed by a β-elimination reaction from cysteine conjugate 4, but readily lost HF to give propenethiolate 7 and thioacyl fluoride 8 before reaction with benzyl bromide or pentafluorobenzyl bromide. The thioacyl fluoride formed from S-(2-chloro-1,1,2trifluoroethyl)-L-cysteine was trapped with diethylamine in previous studies (4). To facilitate product detection by UV irradiation during TLC separation, aniline was used to trap thioacyl fluoride 8, which was formed by loss of fluoride from propanethiolate 6 (Scheme 3). Thioamide 14 and oxazolane 15 (Scheme 3), along with an unidentified minor product, were obtained in both the Cu2+/pyridoxal and N-dodecylpyridoxal models. The cyclization of thioamide 14 to oxazolane 15 may occur by displacement of fluoride from the fluoromethoxy group by the nucleophilic nitrogen of the propanethioamide 14 (Scheme 3). Commandeur et al. used o-phenylenediamine to trap thioacyl fluorides formed from S-haloethyl-L-cysteine conjugates (11). An advantage of this trapping agent is that it may allow discrimination between thioacyl fluoride and 2,2,3-halothiiranes. o-Phenylenediamine was used in the present study to trap the thioacyl fluoride 8 (Scheme 4). The same products were observed in the two chemical models by 19F NMR spectroscopic and GC/MS analyses, but the structures of the trapped intermediates were not confirmed by synthesis or 1H NMR spectroscopy. On the basis of the results obtained with aniline as the trapping agent, a mechanism can be proposed for the formation of the trapped products (Scheme 4). The major

Compound A-Derived Reactive Intermediates

product, which had a tR of 14 min and a molecular ion of m/z 248, was tentatively identified as 2-[1-(fluoromethoxy)2,2,2-trifluoroethyl]benzimidazole 17, which may be formed by cyclization of thioamide 16. The other major product, which had a tR of 17.8 min and a molecular ion of m/z 262, was tentatively identified as 3-(2-aminophenyl)-4thiono-5-trifluoromethyl-1,3-oxazolane 18, which may be formed by cyclization of thioamide 16. Two minor products were also formed: one, which had a tR of 17.2 min and a molecular ion of m/z 282, was tentatively identified as N-2-aminophenyl-2-(fluoromethoxy)-3,3,3-trifluoropropanethioamide 16. The second minor product, which had a tR of 14.4 min and a molecular ion of m/z 228, was tentatively identified as benzimidazole 19, which may be formed by loss of HF from benzimidazole 17 or loss of H2S from oxazolane 18. No 2-mercaptoquinoxaline or 2-fluoroquinoxaline was observed by GC/MS, indicating that conjugate 4 did not form a thiirane intermediate. The present study confirms and expands mechanisms for the formation of reactive intermediates and products from compound Α-derived cysteine conjugates. The findings demonstrate that cysteine S-conjugate 4 is converted in chemical models to many of the same products that are formed from compound A and compound A-derived cysteine S-conjugates both in vivo and in vitro and shows that chemical models can be used to help identify metabolites of cysteine S-conjugates. R-Fluoropropanethiolate 6 is the expected β-lyase product and was formed in both chemical models and trapped by reaction with pentafluorobenzyl bromide and benzyl bromide after loss of HF (Scheme 2). Thioacyl fluoride 8 was trapped by aniline and o-phenylenediamine to give thioamides 14 and 16 (Schemes 3 and 4). In the absence of a trapping agent, thioacyl fluoride 8 underwent hydrolysis to acids 9 and 10, both of which have been identified as metabolites of compound A (1, 23, 24). These studies demonstrate that, although chemical models can be used to mimic the β-lyase-catalyzed biotransformation of cysteine S-conjugates, reactive intermediates and products were formed in the chemical models that have not been described in previous in vivo and in vitro experiments. Moreover, the toxicity of the products formed in the chemical models but not in vivo or in vitro has not been investigated; hence, it is not known whether these products are formed in biological systems and whether they may contribute to the observed nephrotoxicity of cysteine S-conjugates.

Acknowledgment. This research was supported by National Institutes of Environmental Health Sciences Grant ES03127 and by Abbott Laboratories. The authors thank Gail Johnson for her assistance in preparing the manuscript. Supporting Information Available: Experimental details of the syntheses of benzyl 2-(fluoromethoxy)-1,1,3,3,3pentafluoropropyl sulfide, 12, and benzyl 2-fluoromethoxy1,3,3,3-tetrafluoro-1-propenyl sulfide, 13, and Figures 2, 3, and 4. This material is available free of charge via the Internet at http://pubs.acs.org.

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