Computational and Experimental Studies on the Distribution of

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Computational and Experimental Studies on the Distribution of Addition and Substitution Products of the Microsomal Glutathione Transferase 1-Catalyzed Conjugation of Glutathione with Fluoroalkenes Larry J. Jolivette† and M. W. Anders* Department of Pharmacology and Physiology, University of Rochester, 601 Elmwood Avenue, Box 711, Rochester, New York 14642 Received September 25, 2002

The glutathione transferase-catalyzed reaction of glutathione with haloalkenes results in the formation of addition or substitution products or both. Glutathione conjugates of haloalkenes may be metabolized and excreted at different rates, may follow different metabolic pathways, and may exhibit different toxicities. Microsomal glutathione transferase 1 (MGST1)-catalyzed conjugation of chlorotrifluoroethene, hexafluoropropene, and 2-(fluoromethoxy)-1,1,3,3,3pentafluoro-1-propene results in differing proportions of addition and substitution products. The aim of the present study was to develop a computational model to predict the outcome of the MGST1-catalyzed reaction of glutathione with haloalkenes. An ab initio computational study of the reaction of ethanethiolate, a surrogate for glutathione, with the chlorotrifluoroethene, hexafluoropropene, and 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene was conducted. An empirical study was also conducted to quantify the distribution of addition and substitution products that resulted from the MGST1-catalyzed reaction of glutathione with these fluoroalkenes. The results show that this computational model accurately predicted the distribution of the addition and substitution products that result from the MGST1-catalyzed reaction of glutathione with these fluoroalkenes.

Introduction Haloalkenes are widely used in industry and commerce, and some are carcinogenic in rodents. Experimental evidence supports a glutathione-dependent bioactivation pathway that leads to selective nephrotoxicity or nephrocarcinogenicity or both (1). The mechanism of bioactivation of haloalkenes includes hepatic glutathione transferase-catalyzed glutathione S-conjugate formation, enzymatic hydrolysis by γ-glutamyltransferase and aminopeptidase M or cysteinylglycine dipeptidase to the corresponding cysteine S-conjugates (2), and finally, bioactivation of the cysteine S-conjugates to reactive metabolites by cysteine conjugate β-lyase (3). Glutathione S-conjugates of haloalkenes may exist as regioisomers and geometric isomers (4-6). The different glutathione S-conjugates formed from haloalkenes may be metabolized and excreted at different rates, may follow different metabolic pathways, and may exhibit different toxicities (7). The glutathione transferase-catalyzed reaction of glutathione with haloalkenes is a SNV1 reaction that involves the formation of a carbanionic intermediate (8-10). Two reaction mechanisms have been proposed for the SNV reaction: the first mechanism is characterized by an out* To whom correspondence should be addressed. Tel: 585-275-1678. Fax: 585-273-2652. E-mail: [email protected]. † Present address: Preclinical Drug Discovery, Cardiovascular and Urogenitary Center for Excellence in Drug Discovery, GlaxoSmithKline, UW2725, 709 Swedeland Road, King of Prussia, PA 19406. 1 Abbreviations: S V, nucleophilic vinylic substitution; MGST1, N microsomal glutathione transferase 1.

of-plane, π-orbital approach that can be a multistep process and that is distinguished by the formation of a true carbanionic intermediate on the potential energy surface or a single-step process that is distinguished by the formation of a transient carbanionic intermediate (11). The second mechanism is characterized by an inplane, σ-orbital approach that is a single-step process (12). These reaction mechanisms were investigated in a computational study of the reaction of methanethiolate with trichloroethene that demonstrated that the energetically favored reaction mechanism is an out-of-plane, π-orbital approach that involves the formation of a distinct carbanionic intermediate (13). Glutathione conjugation of fluoroalkenes may give addition products (14, 15) or a mixture of addition and substitution products (16, 17). MGST1-catalyzed conjugation of chlorotrifluoroethene is regioselective and stereoselective, whereas the cytosolic glutathione transferasecatalyzed reaction is regioselective but not stereoselective (4). The ratio of addition products to substitution products that results from the reaction of glutathione with hexafluoropropene and 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro1-propene (compound A) differs depending on whether the reaction is catalyzed by rat liver cytosolic glutathione transferases or by rat liver microsomal glutathione transferase (16, 17). These findings show that the product distribution of glutathione S-conjugates of fluoroalkenes differs depending on whether the reaction is catalyzed by cytosolic or microsomal glutathione transferases. The objective of the present study was to determine if the product distribution of glutathione S-conjugates that

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resulted from the purified rat liver MGST1-catalyzed reaction of glutathione with the fluoroalkenes could be modeled by computation. Three fluoroalkenes were chosen for study: chlorotrifluoroethene, hexafluoropropene, and 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene. The reaction of ethanethiolate with the fluoroalkenes was modeled as a multistep, out-of-plane, π-orbital approach wherein the first step of the reaction determines the regioselectivity of the reaction and the second step determines the distribution of addition and substitution products. The reaction was modeled in the gas phase to simulate an active site that lacks aqueous solvent, and a single water molecule was included as a proton source for the addition reactions. Transition-state theory was applied to compute the theoretical product distribution of the modeled reactions. These computationally determined data were compared with the empirically determined product distributions of glutathione S-conjugates of chlorotrifluoroethene, hexafluoropropene, and 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene that resulted from the purified rat liver MGST1-catalyzed reaction to evaluate the predictive potential of this computational approach for the MGST1-catalyzed reaction of glutathione with halogenated alkenes.

Materials and Methods Materials. Hydroxyapatite and glutathione were obtained from Sigma Chemical Co. (St. Louis, MO). Carboxymethyl sepharose was obtained from BioRad Inc. (Hercules, CA). Male Sprague-Dawley rats weighing 300-350 g were obtained from Charles River (Wilmington, MA). Hexafluoropropene was obtained from Lancaster Synthesis (Windham, NH). 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene was provided by Abbott Laboratories (Abbott Park, IL). MGST1 was purified as previously described (18). S-(1,2,2-Trichlorovinyl)glutathione was synthesized as previously described (19). S-(2-[Fluoromethoxy]-1,1,3,3,3-pentafluoropropyl)glutathione was synthesized as described by Iyer et al. (20). Both S-(1,2,2-trichlorovinyl)glutathione and S-(2-[fluoromethoxy]-1,1,3,3,3-pentafluoropropyl)glutathione were purified by recrystallization from 85% ethanol as previously described by Dohn et al. (4). The purity (>95%) and identification of synthetic glutathione S-conjugates were determined with atmospheric pressure chemical ionization (APCI) LC/MS. Construction of Reaction Profiles. Full geometry optimization and frequency calculations were performed with the Gaussian 98 software application (21) running on an SGI O2 workstation (Mountain View, CA). Starting geometries were prepared with the MOPAC module of Chem3D (version 6.0, CambridgeSoft Inc., Cambridge, MA). Transitional vibrational modes were animated with the SPARTAN molecular modeling software (version 5.1, Wavefunction Inc., Irvine, CA). All electronic energy results were corrected for zero-point energy. Thermodynamic properties were evaluated at 310.15 K and 1 atm pressure. Geometry optimization of reactant and product van der Waals complexes, carbanionic intermediates, and transition structures was computed at the HF/6-31+G* level of theory as described previously (13). Three-dimensional structures of each of the carbanionic intermediates were generated, and Gaussian Zmatrixes for the carbanionic intermediates were generated with the AIGOUT keyword in a single point energy computation within the MOPAC module of Chem3D. The resulting Gaussian Z-matrixes were used to create input files for geometry optimization with the Gaussian 98 molecular modeling program. Multiple starting geometries of each of the transition structures were generated with the QST2 methodology contained within the Gaussian 98 molecular modeling program. Reactant and product input geometries for this methodology were generated

Jolivette and Anders by manipulation of the bond length of the bond of interest within the Gaussian Z-matrixes obtained from the output file of the geometry-optimized carbanionic intermediates. The starting geometries for the reactant and product van der Waals complexes as well as for carbanionic intermediate-water van der Waals complexes were generated from IRC computations of the appropriate transition structures. Computation of the Theoretical Distribution of Products. The theoretical distribution of products was computed based on transition-state theory (22). Activation energies were calculated as the difference in potential energies of the transition structure and the reactant van der Waals complex for the first step of the reaction and the difference in potential energies of the transition structure and the carbanionic intermediate or the carbanionic intermediate-water van der Waals complex for the second step of the reaction. The values of the translational, rotational, and vibrational partition functions were obtained from frequency computations on transition structures, reactant van der Waals complexes, carbanionic intermediates, and carbanionic intermediate-water van der Waals complexes. Computation of the theoretical distribution of products was performed as previously described (23, 24). Biosynthesis of Glutathione S-Conjugates of Hexafluoropropene and 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro1-propene. Glutathione was incubated with 2-(fluoromethoxy)1,1,3,3,3-pentafluoro-1-propene or hexafluoropropene in the presence of purified rat liver MGST1. Reaction mixtures contained 0.1 mM EDTA, 1 mM glutathione, 15 µg of purified rat liver MGST1, and 4 mM hexafluoropropene or 2-(fluoromethoxy)1,1,3,3,3-pentafluoro-1-propene in a final volume of 1 mL of 0.1 M potassium phosphate buffer (pH 7.4) that contained 0.1% Triton X-100 and were incubated at 37 °C for 20 min. The reactions were terminated by addition of 40 µL of 70% trichloroacetic acid. The precipitated proteins were removed by centrifugation, and the supernatant was collected for LC/MS analysis. Separation and Quantification of Glutathione S-Conjugate Formation. The reaction products were fractionated with an Agilent 1100 LC/MS system fitted with a Waters NovaPak C18 column (2 mm × 150 mm). Forty microliter samples of the supernatants were injected onto the column. The reaction products were eluted with 15% acetonitrile in water containing 0.1% trifluoroacetic acid. The glutathione S-conjugates were detected by monitoring UV absorbance at 202 nm and by APCI LC/MS operating in positive scan mode (150-550 m/z). The mass spectrophotometer operating conditions were as follows: fragmentor voltage, 120 V; nebulizer pressure, 60 psi; corona current, 4 µA; capillary voltage, 3500 V; and drying gas temperature and flow rate, 350 °C and 5 mL/min, respectively. Chromatogram peak areas were integrated with the software provided with the LC/MS system. The glutathione S-conjugates were quantified by comparison of the UV absorbance (202 nm) chromatogram peak area with that of standard curves generated from glutathione, S-(1,2,2-trichlorovinyl)glutathione, and S-(2[fluoromethoxy]-1,1,3,3,3-pentafluoropropyl)glutathione, all of which yielded nearly identical individual calibration curves. The identity of the glutathione S-conjugates was determined by comparison of the mass spectral data with reported structures of the glutathione S-conjugates of 2-(fluoromethoxy)-1,1,3,3,3pentafluoro-1-propene and hexafluoropropene (16, 17).

Results Computation of the Theoretical Distribution of Products. Gas-phase potential energy profiles were generated for the reaction of ethanethiolate with chlorotrifluoroethene, hexafluoropropene, and 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene. The reaction mechanism considered was a two-step process in which the first step involves an out-of-plane attack at the vinylic carbon by ethanethiolate followed by addition of a proton

Fluoroalkene GSH Conjugation

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Table 1. Comparison of the Theoretical and Experimental Distribution of Addition and Substitution Products that Resulted from the Reaction of Either Glutathione or Ethanethiolate with Chlorotrifluoroethene, Hexafluoropropene, and 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene substrate

experimental product distributiona (addition:substitution)

theoretical product distributionb (addition:substitution)

chlorotrifluoroethene hexafluoropropene 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene

100:0 41.5:58.5 19.9:80.1

100:0 42.8:57.2 20.2:79.8

a Glutathione was incubated with hexafluoropropene or 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene in the presence of purified MGST1 as described in Materials and Methods. The products that resulted from the reactions were identified and quantified by LC/UV/ MS analyses. The distribution of products that resulted from the MGST1-catalyzed reaction of glutathione with chlorotrifluoroethene is from Hargus et al. (6). b The reaction of ethanethiolate with chlorotrifluoroethene, hexafluoropropene, and 2-(fluoromethoxy)-1,1,3,3,3pentafluoro-1-propene was modeled for an out-of-plane, two-step π-orbital approach at the HF/6-31+G* level of theory. The theoretical distributions of products that resulted from the reactions of ethanethiolate with chlorotrifluoroethene, hexafluoropropene, and 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene were determined as described in Materials and Methods.

Scheme 1. Modeled Reaction Scheme of the Two Step Reaction of Ethanethiolate with Chlorotrifluoroethene that Results in the Formation of Addition and Substitution Products

resulting in an addition product or by the loss of fluoride resulting in a substitution product. The theoretical distributions of addition and substitution products were determined for each of the modeled reactions with the application of transition state theory and are summarized in Table 1. Thermodynamic properties of transition structures and stationary points along the reaction pathway for the reactions of ethanethiolate with chlorotrifluoroethene, hexafluoropropene, and 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene are listed in Tables 2-4, respectively. (1) Chlorotrifluoroethene. The reaction of ethanethiolate with chlorotrifluoroethene was modeled at the HF/ 6-31+G* level of theory, and the reaction is shown in Scheme 1. The activation energies and rotational, vibrational, and translational partition functions for the van der Waals complex of ethanethiolate with chlorotrifluoroethene, intermediate 1, and transition structures TS1, TS2, TS3, TS5, and TS7 (Scheme 1) are shown in Table 2. The activation energy for the transition structure for the first step in the reaction in which the thiolate ion of ethanethiolate attacked the vinylic carbon bearing two fluorine atoms of chlorotrifluoroethene was 10.10 kcal/

mol, whereas the attack of the thiolate ion of ethanethiolate on the vinylic carbon bearing the chlorine and fluorine atoms had an activation energy of 20.42 kcal/ mol. The rotational, vibrational, and translational partition functions for TS1, TS2, and the ethanethiolatechlorotrifluoroethene van der Waals complex were computed under the conditions of 310.15 K and 1 atm pressure and are listed in Table 2. The theoretical rates of reaction through these half reactions and the theoretical distribution of intermediates 1 and 2 were computed based on the computed activation energies and partition functions as described in Materials and Methods. The theoretical rates of formation of intermediates 1 and 2 were 6.95 × 102 and 2.43 × 10-5 s-1, respectively. The theoretical distribution of the carbanionic intermediates that resulted from the first step of this reaction was 100% intermediate 1. The second step of the reaction of ethanethiolate with chlorotrifluoroethene through intermediate 1 was modeled (Scheme 1). The activation energy to TS3 from intermediate 1 that led to the formation of ethyl (E)-2chloro-1,2-difluoroethenyl sulfide 3 was 24.76 kcal/mol. The activation energy to TS5 from intermediate 1 that led to the formation of ethyl (Z)-2-chloro-1,2-difluoroethenyl sulfide 5 was 25.98 kcal/mol. The activation energy to TS7 from the carbanionic intermediate 1-water van der Waals complex that led to the formation of ethyl 2-chloro-1,1,2-trifluoroethyl sulfide 7 was 5.25 kcal/mol. The theoretical rates of reaction through these half reactions and the theoretical distribution of conjugates 3, 5, and 7 were computed as described in Materials and Methods. The theoretical rates of formation of conjugates 3, 5, and 7 were 2.19 × 10-5, 2.71 × 10-6, and 9.96 × 107 s-1, respectively. The overall theoretical product distribution of conjugates 3, 5, and 7 was 0, 0, and 100%, respectively. (2) Hexafluoropropene. The modeled reaction of ethanethiolate with hexafluoropropene is shown in Scheme 2. The activation energies and rotational, vibrational, and translational partition functions for the van der Waals complex of ethanethiolate with hexafluoropropene, intermediate 9, and transition structures TS9, TS10, TS11, TS13, and TS15 (Scheme 2) are shown in Table 3. The activation energy for the transition structure in the first step of the reaction in which the thiolate ion of ethanethiolate attacked the vinylic carbon bearing two fluorine atoms of hexafluoropropene was 6.05 kcal/mol, whereas the attack of the thiolate ion of ethanethiolate on the carbon bearing the trifluoromethyl group and a fluorine atom had an activation energy of 33.70 kcal/mol. The theoretical rates of reaction through the half reac-

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Table 2. Thermodynamic Properties of Transition States and Intermediates for the Reaction of Ethanethiolate with Chlorotrifluoroethene partition functionsb

structurea reactant complex intermediate 1 intermediate 1 complex TS1 TS2 TS3 TS5 TS7

activation energy (kcal/mol)

10.10 20.42 24.76 25.98 5.26

vibrational

rotational

translational

6.43E + 20 2.49E + 17 1.83E + 20

1.51E + 06 9.70E + 05 1.57E + 06

1.02E + 08 1.02E + 08 1.18E + 08

1.21E + 18 8.12E + 17 2.31E + 17 2.00E + 17 1.58E + 19

1.13E + 06 1.10E + 06 9.86E + 05 1.03E + 06 1.42E + 06

1.02E + 08 1.02E + 08 1.02E + 08 1.02E + 08 1.18E + 08

a The structures in this table that are not referred to explicitly in Scheme 1 are as follows: reactant complex refers to the ethanethiolatechlorotrifluoroethene van der Waals complex and intermediate 1 complex refers to the intermediate 1-water van der Waals complex. b Thermodynamic properties were determined at the HF/6-31+G* level of theory at 310.15 K and 1 atm.

Scheme 2. Modeled Reaction Scheme of the Two Step Reaction of Ethanethiolate with Hexafluoropropene that Results in the Formation of Addition and Substitution Products

tions that led to the formation of carbanionic intermediates 9 and 10 and the theoretical distribution of intermediates 9 and 10 were computed as described in Materials and Methods. The theoretical rates of formation of intermediates 9 and 10 were 1.26 × 106 and 1.05 × 10-14 s-1, respectively. The theoretical distribution of the intermediates that resulted from the first step of this reaction was almost exclusively intermediate 9. The second step of the reaction of ethanethiolate with hexafluoropropene through intermediate 9 was modeled. The activation energy to TS11 from intermediate 9 that led to the formation of ethyl (E)-1,2,3,3,3-pentafluoro-1propenyl sulfide 11 was 14.59 kcal/mol. The activation energy to TS13 from intermediate 9 that led to the formation of ethyl (Z)-1,2,3,3,3-pentafluoro-1-propenyl sulfide 13 was 14.59 kcal/mol. The activation energy to TS15 from the intermediate 9-water van der Waals complex that led to the formation of ethyl 1,1,2,3,3,3hexafluoropropyl sulfide 15 was 12.75 kcal/mol. The rotational, vibrational, and translational partition functions for the transition structures and intermediates

(reactants of these half reactions) are listed in Table 3. The theoretical rates of formation of conjugates 11, 13, and 15 from intermediate 9 and the theoretical distribution of these conjugates were computed as described in Materials and Methods. The theoretical rates of formation of conjugates 11, 13, and 15 were 5.22 × 102, 5.23 × 102, and 7.81 × 102 s-1, respectively. The overall theoretical distribution of conjugates 11, 13, and 15 was 28.6, 28.6, and 42.8%, respectively. (3) 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene. The modeled reaction of ethanethiolate with 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene is shown in Scheme 3. The activation energies and rotational, vibrational, and translational partition functions for the van der Waals complex of ethanethiolate with 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene, intermediate 16, and transition structures TS16, TS17, TS19, TS21, and TS23 (Scheme 3) are shown in Table 4. The activation energy for the transition structure that led to the formation of carbanionic intermediate 17 (reaction occurring at the vinylic carbon bonded to two fluorine atoms) was 5.12 kcal/mol, whereas the attack of the thiolate ion of ethanethiolate on the carbon bearing the fluoromethoxy and trifluoromethyl groups had an activation energy of 22.13 kcal/mol. The theoretical rates of formation and the theoretical distribution of intermediates 16 and 17 were computed as described in Materials and Methods. The theoretical rates of formation of intermediates 16 and 17 were 6.50 × 10-7 and 6.95 × 106 s-1, respectively. The theoretical distribution of the intermediates that resulted from the first step of the reaction was exclusively carbanionic intermediate 17. The second step of the reaction of ethanethiolate with 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene through intermediate 17 was modeled. The activation energy to TS19 from intermediate 17 that led to the formation of ethyl (E)-2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl sulfide 19 was 14.52 kcal/mol. The activation energy to TS21 from intermediate 17 that led to the formation of ethyl (Z)-2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl sulfide 21 was 12.45 kcal/mol. The activation energy to TS23 from the intermediate 17-water van der Waals complex that led to the formation of ethyl 2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl sulfide 23 was 12.38 kcal/mol. The theoretical rates of formation of products from intermediate 17 and the theoretical distribution of conjugates 19, 21, and 23 were computed as described in Materials and Methods. The theoretical



Table 3. Thermodynamic Properties of Transition States and Intermediates for the Reaction of Ethanethiolate with Hexafluoropropene partition functionsb

structurea reactant complex intermediate 9 intermediate 9 complex TS9 TS10 TS11 TS13 TS15


6.05 33.70 14.59 14.59 12.75

vibrational

rotational

translational

1.05E + 22 5.40E + 18 2.51E + 21

2.28E + 06 1.57E + 06 2.38E + 06

1.33E + 08 1.33E + 08 1.50E + 08

4.67E + 19 1.31E + 19 7.87E + 18 7.87E + 18 3.27E + 20

1.85E + 06 1.67E + 06 1.67E + 06 1.67E + 06 2.15E + 06

1.33E + 08 1.33E + 08 1.33E + 08 1.33E + 08 1.50E + 08

a The structures in this table that are not referred to explicitly in Scheme 2 are as follows: reactant complex refers to the ethanethiolatehexafluoropropene van der Waals complex and intermediate 9 complex refers to the intermediate 9-water van der Waals complex. b Thermodynamic properties were determined at the HF/6-31+G* level of theory at 310.15 K and 1 atm.

Scheme 3. Modeled Reaction Scheme of the Two Step Reaction of Ethanethiolate with 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene that Results in the Formation of Addition and Substitution Products

rates of formation of conjugates 19, 21, and 23 were 2.88 × 102, 2.39 ×104, and 6.13 × 103 s-1, respectively. The overall theoretical distribution of conjugates 19, 21, and 23 was 1.0, 78.8, and 20.2%, respectively. Quantification of Glutathione S-Conjugates of Hexafluoropropene and 2-(Fluoromethoxy)-1,1,3,3,3pentafluoro-1-propene. Hexafluoropropene and 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene were incubated with glutathione in the presence or absence of purified rat liver MGST1. The distribution of glutathione S-conjugates that resulted from addition and substitution reactions was quantified. The enzyme-catalyzed distributions of addition and substitution products are summarized in Table 1 and are described below. Enzymecatalyzed product distributions were corrected for nonenzymatic product formation. (1) Hexafluoropropene. Incubation of hexafluoropropene with glutathione in the presence of purified MGST1 resulted in the formation of two conjugates that

eluted at 5.9 and 6.3 min and were detected by both UV and APCI mass spectral analyses. The mass spectrum of the product that eluted at 5.9 min showed a parent (MH+) ion at m/z 438, indicating that the conjugate was a substitution product that resulted from the reaction of glutathione with hexafluoropropene. The mass spectrum of the conjugate that eluted at 6.3 min showed a MH+ ion at m/z 458, indicating that the conjugate formed was an addition product that resulted from the reaction of glutathione with hexafluoropropene. These conjugates were, therefore, identified as S-(1,2,3,3,3-pentafluoro-1propenyl)glutathione (m/z 438) and S-(1,1,2,3,3,3-hexafluoropropyl)glutathione (m/z 458) based on the structural assignments of the glutathione S-conjugates that resulted from the reaction of glutathione with hexafluoropropene as reported by Koob and DeKant (16). The products were quantified as described above. The substitution product accounted for 58.5 ( 1.0% (n ) 3, mean ( SD) of the glutathione S-conjugate formed, and the remaining 41.5 ( 1.0% (n ) 3, mean ( SD) of product formed was the addition product. (2) 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene. Incubation of 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene with glutathione in the presence of purified rat liver MGST1 resulted in the formation of two conjugates that eluted at 6.9 and 8.4 min and were detected by both UV and APCI mass spectral analyses. The mass spectrum of the conjugate that eluted at 6.9 min showed a MH+ ion at m/z 488, indicating that the conjugate was an addition product that resulted from the reaction of glutathione with 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro1-propene. The mass spectrum of the conjugate that eluted at 8.4 min showed a MH+ ion at m/z 468, indicating that the conjugate was a substitution product that resulted from the reaction of glutathione with 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene. These conjugates were, therefore, identified as S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]glutathione (m/z 488) and S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]glutathione (m/z 468) based on the structural assignments of the glutathione S-conjugates that resulted from the reaction of glutathione with 2-(fluoromethoxy)1,1,3,3,3-pentafluoro-1-propene as reported by Jin et al. (17). The products were quantified as described above. The substitution product accounted for 80.1 ( 2.3% (n ) 3, mean ( SD) of the glutathione S-conjugate formed, and the remaining 19.9 ( 2.3% (n ) 3, mean ( SD) of product formed was the addition product.

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Table 4. Thermodynamic Properties of Transition Structures and Intermediates for the Reaction of Ethanethiolate with 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene partition functionsb

structurea


reactant complex intermediate 17 intermediate 17 complex TS16 TS17 TS19 TS21 TS23

22.13 5.12 14.52 12.45 12.38

vibrational

rotational

translational

6.18E + 23 1.53E + 20 2.56E + 22

3.28E + 06 2.54E + 06 3.56E + 06

1.62E + 08 1.62E + 08 1.81E + 08

3.19E + 20 3.03E + 21 1.28E + 20 3.25E + 20 1.38E + 22

2.51E + 06 2.93E + 06 2.45E + 06 2.61E + 06 3.33E + 06

1.62E + 08 1.62E + 08 1.62E + 08 1.62E + 08 1.81E + 08

a The structures in this table that are not referred to explicitly in Scheme 3 are as follows: reactant complex refers to the ethanethiolatechlorotrifluoroethene van der Waals complex and intermediate 17 complex refers to the intermediate 17-water van der Waals complex. b Thermodynamic properties were determined at the HF/6-31+G* level of theory at 310.15 K and 1 atm.

Discussion Glutathione transferases catalyze the reaction of glutathione with haloalkenes. The reaction is a prototypical SNV reaction in which the glutathione S-conjugates that result from the reaction of glutathione with haloalkenes may be addition or addition-elimination, i.e., addition or substitution, or both, products (8, 9). The product distribution of addition and substitution products that resulted from the reaction of glutathione with hexafluoropropene and 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro1-propene is dependent on whether the reaction is catalyzed by cytosolic or microsomal glutathione transferases (16, 17). The glutathione S-conjugates of haloalkenes exist as regioisomers and geometric isomers (4-6). Different glutathione conjugates of a haloalkene may be metabolized and excreted at different rates, may follow different metabolic pathways, and may exhibit different toxicity (7). The lack of human risk-assessment tools for haloalkenes has attracted interest in predicting the distribution of products that result from reaction of glutathione with haloalkenes (25). The goal of the this study was to compute the theoretical distribution of addition and substitution products that result from the reaction of ethanethiolate with chlorotrifluoroethene, hexafluoropropene, and 2-(fluoromethoxy)1,1,3,3,3-pentafluoro-1-propene and to compare this theoretical distribution with the experimentally determined distribution of the addition and substitution products that are formed from the purified rat liver MGST1catalyzed reaction of glutathione with these haloalkenes. The reaction was modeled as a multistep, out-of-plane, π-orbital reaction wherein the first step of the reaction determines the regioselectivity of the reaction and the second step determines the distribution of addition and substitution products. This reaction mechanism is derived from chemical studies on the mechanism of the SNV reaction. The reaction of methanethiolate with β-halostyrenes results in products that retain the stereochemical configuration of the reactant β-halostyrene as well as an “element effect,” which supports a multistep, out-of-plane attack by the sulfur nucleophile on the vinylic carbon of the β-halostyrene (26). Additionally, carbanionic intermediates were observed that resulted from the reactions of thiolate anions with R-nitro-β-substituted stilbenes and methoxybenzylidene (27, 28). Further evidence is derived from a theoretical study of the reaction of methanethiolate with trichloroethene, which demonstrated that the in-plane, σ-orbital and the single-step, out-of-plane,

π-orbital attacks by methanethiolate are higher energy processes than the two-step, out-of-plane, π-orbital attack by methanethiolate (13). The reactions of ethanethiolate with chlorotrifluoroethene, hexafluoropropene, and 2-(fluoromethoxy)-1,1,3,3,3pentafluoro-1-propene were modeled at the HF/6-31+G* level of theory. The diffuse function was chosen to model the thiolate anion. The results of transition-state theory computations indicated that the modeled reactions proceeded through the attack of ethanethiolate at the vinylic carbon bearing two fluorine atoms of chlorotrifluoroethene, hexafluoropropene, and 2-(fluoromethoxy)-1,1,3,3,3pentafluoro-1-propene with the subsequent loss of fluoride or the addition of a proton to form terminal products. The geometries for the transition structures and intermediates along these reaction paths indicated key features that are consistent with the pathway postulated in the reaction schemes shown in Schemes 1-3. An example of the key features of these geometries is presented in Figure 1, which shows the transition state and carbanionic intermediate for the reaction of ethanethiolate with chlorotrifluoroethene. Each modeled reaction path indicated that the transition structure for the first step of the reaction leading to the carbanionic intermediates showed a loss of planarity (Figure 1A) and significant localization of negative charge on the carbon atom in the β-position to the site of nucleophilic attack. The carbanionic intermediates showed further movement from planarity, increased “s-character” of the carbon atoms as compared with the initial transition structure, and shortening of the carbon-sulfur bond (Figure 1B). The transition structures that led to substitution products showed movement to planarity, increased “pcharacter” of the carbon atoms as compared with the carbanionic intermediate, lengthening of the carbonnucleofuge bond, rotation of the leaving halogen about the carbon-carbon bond toward alignment with the β-carbon π-orbital, and loss of localized negative charge on the β-carbon atom (Figure 1C,D). The transition structures that led to the addition products showed loss of localized negative charge on the β-carbon atom, retention of s-character of the carbon atoms that was observed in the carbanionic intermediate structures, and a linear arrangement of the atoms involved in the transfer of the proton from the water molecule to the β-carbon atom (Figure 1E). Thermodynamic properties, i.e. partition functions, at 310.15 K and 1 atm were computed for transition



Figure 1. Structures of intermediate 1 and transition states TS1, TS3, TS5, and TS7 (Scheme 1) optimized at the HF/6-31+G* level of theory for the reaction of ethanethiolate with chlorotrifluoroethene. (A) Transition structure, TS1, of the first step of the addition-elimination reaction of ethanethiolate with chlorotrifluoroethene. The loss of planarity of the haloalkene indicated localization of negative charge on the carbon β to the site of attack. (B) Carbanionic intermediate, intermediate 1, of the substitution reaction of ethanethiolate with chlorotrifluoroethene. This structure was characterized by further loss of planarity of the halogenated alkene reactant that accompanied localization of negative charge on the carbon β to the site of attack and an increase of s-character relative to TS1. (C) Transition structure, TS3, that led to the formation of ethyl (E)-2-chloro-1,2-difluorovinyl sulfide 3 (Scheme 1). This structure was characterized by an elongated carbon-nucleofuge bond, positioning of the nucleofuge antiperiplanar to the antibonding π-orbital of the β-carbon atom, and reduced negative charge on the β-carbon atom, which was indicated by the planar arrangement of atoms relative to the carbanionic intermediate 1. (D) Transition structure, TS5, that led to the formation of ethyl (Z)-2-chloro1,2-difluorovinyl sulfide 5 (Scheme 1). This structure was characterized by an elongated carbon-nucleofuge bond, positioning of the nucleofuge antiperiplanar to the antibonding π-orbital of the β-carbon atom, and reduced negative charge on the β-carbon atom, which was indicated by the planar arrangement of atoms relative to the carbanionic intermediate 1. (E) Two views of the transition structure, TS7, that led to the formation of ethyl 2-chloro-1,1,2-trifluoropropyl sulfide 7 (Scheme 1). This structure demonstrated the linear arrangement the β-carbon atom of intermediate 1 with the hydrogen and oxygen atoms of the proton donor water molecule, loss of negative charge on the β-carbon atom, and retention of the s-character observed with intermediate 1.

structures, carbanionic intermediates, and reactant van der Waals complexes (Tables 2-4). The transition structures were evaluated for relative contribution to product formation via transition-state theory to obtain theoretical product distributions. The theoretical product distribution of addition and substitution products for the gasphase reaction of ethanethiolate with chlorotrifluoroethene, hexafluoropropene, and 2-(fluoromethoxy)-1,1,3,3,3pentafluoro-1-propene and experimentally determined product distributions of addition and substitution reaction-derived glutathione S-conjugates formed by purified rat liver MGST1-catalyzed reactions of glutathione with chlorotrifluoroethene (6), hexafluoropropene, and 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene were in excellent agreement, and they are listed in Table 1. In summary, the theoretical product distributions of the addition and substitution products that resulted from the computationally modeled gas-phase reactions of ethanethiolate with chlorotrifluoroethene, hexafluoropropene, and 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1propene were in close agreement with the experimentally determined product distributions of addition and substitution products that resulted from the purified rat liver MGST1-catalyzed reactions of glutathione with chlorotrifluoroethene, hexafluoropropene, and 2-(fluoromethoxy)1,1,3,3,3-pentafluoro-1-propene (Table 1). This finding

indicates that the chemical reaction mechanism or the chemical conditions, or most likely both, within the active site of MGST1 play a major role in determining the product outcome of the MGST1-catalyzed reaction of glutathione with fluoroalkenes and possibly other haloalkenes. The reaction mechanism is likely an out-ofplane, π-orbital approach by glutathione thiolate anion that results in the formation of a carbanionic intermediate that collapses with loss of a halide ion to yield a substitution product or accepts a proton to yield an addition product. Furthermore, the computational results presented herein indicate that the thermodynamic properties of the competing transition structures determine the product distribution of the competing addition and substitution reactions, which suggests that the participation of the MGST1 enzyme in the catalysis of the reaction of glutathione with fluoroalkenes did not involve preferential stabilization of any of the transition structures to the extent that the relative distribution of addition and substitution products did not differ from those that were computationally predicted. The findings of this study demonstrated the applicability of computational chemical techniques to the MGST1-catalyzed reaction of glutathione with fluoroalkenes and likely other haloalkenes. Computational chemical modeling may prove to be an important tool for the rationalization and prediction of

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the glutathione-dependent metabolism and bioactivation of haloalkenes.

Acknowledgment. This research was supported by the National Institutes of Environmental Health Sciences Grant ES03127 (M.W.A.) and National Institutes of Environmental Health Sciences Training Grant ES07026 (L.J.J.). We thank Dr. Ann M. Richard for expert advice and assistance and Agilent Technologies (Palo Alto, CA) for the use of an LC/MS during the conduct of this research.

References (1) Anders, M. W., and DeKant, W. (1998) Glutathione-dependent bioactivation of haloalkenes. Annu. Rev. Pharmacol. Toxicol. 38, 501-537. (2) Elfarra, A. A., and Anders, M. W. (1984) Renal processing of glutathione conjugates. Role in nephrotoxicity. Biochem. Pharmacol. 33, 3729-3732. (3) Lash, L. H., Elfarra, A. A., and Anders, M. W. (1986) Renal cysteine conjugate β-lyase. Bioactivation of nephrotoxic cysteine S-conjugates in mitochondrial outer membrane. J. Biol. Chem. 261, 5930-5935. (4) Dohn, D. R., Quebbemann, A. J., Borch, R. F., and Anders, M. W. (1985) Enzymatic reaction of chlorotrifluoroethene with glutathione: 19F NMR evidence for stereochemical control of the reaction. Biochemistry 24, 5137-5143. (5) Commandeur, J. N., and Vermeulen, N. P. (1990) Identification of N-acetyl-(2,2-dichlorovinyl)- and N-acetyl(1,2-dichlorovinyl)-Lcysteine as two regioisomeric mercapturic acids of trichloroethylene in the rat. Chem. Res. Toxicol. 3, 212-218. (6) Hargus, S. J., Fitzsimmons, M. E., Aniya, Y., and Anders, M. W. (1991) Stereochemistry of the microsomal glutathione S-transferase catalyzed addition of glutathione to chlorotrifluoroethene. Biochemistry 30, 717-721. (7) Commandeur, J. N., Boogaard, P. J., Mulder, G. J., and Vermeulen, N. P. (1991) Mutagenicity and cytotoxicity of two regioisomeric mercapturic acids and cysteine S-conjugates of trichloroethylene. Arch. Toxicol. 65, 373-380. (8) Boyland, E., and Chasseaud, L. F. (1967) Enzyme-catalysed conjugations of glutathione with unsaturated compounds. Biochem. J. 104, 95-102. (9) Chasseaud, L. F. (1979) The role of glutathione and glutathione S-transferases in the metabolism of chemical carcinogens and other electrophilic agents. Adv. Cancer Res. 29, 175-274. (10) Rappoport, Z., and Gazit, A. (1985) Nucleophilic attacks on carbon-carbon double bonds. 31. Complete and partial stereoconversion in vinylic substitution of (E)- and (Z)-β-chloro-Rphenylcinnamaldehydes and (E)-2-iodo-1,2-diphenyl-1-nitroethylene by nucleophiles. J. Org. Chem. 50, 3184-3194. (11) Rappoport, Z. (1981) Nucleophilic vinylic substitution. A single or multistep process? Acc. Chem. Res. 14, 7-15. (12) Glukhovtsev, M. N., Pross, A., and Radom, L. (1994) Is SN2 substitution with inversion of configuration at vinylic carbon feasible? J. Am. Chem. Soc. 116, 5961-5962. (13) Shim, J. Y., Boone, P. F., and Richard, A. M. (1999) Theoretical study of the SNV reaction of trichloroethylene (TCE) and CH3Sas a model for glutathione conjugation of TCE. Chem. Res. Toxicol. 12, 308-316. (14) Dohn, D. R., and Anders, M. W. (1982) The enzymatic reaction of chlorotrifluoroethylene with glutathione. Biochem. Biophys. Res. Commun. 109, 1339-1345.

Jolivette and Anders (15) Odum, J., and Green, T. (1984) The metabolism and nephrotoxicity of tetrafluoroethylene in the rat. Toxicol. Appl. Pharmacol. 76, 306-318. (16) Koob, M., and DeKant, W. (1990) Metabolism of hexafluoropropene. Evidence for bioactivation by glutathione conjugate formation in the kidney. Drug Metab. Dispos. 18, 911-916. (17) Jin, L., Davis, M. R., Kharasch, E. D., Doss, G. A., and Baillie, T. A. (1996) Identification in rat bile of glutathione conjugates of fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether, a nephrotoxic degradate of the anesthetic agent sevoflurane. Chem. Res. Toxicol. 9, 555-561. (18) Weinander, R., Mosialou, E., DeJong, J., Tu, C.-P. D., Dypbukt, J., Bergman, T., Barnes, H. J., Hoog, J.-O., and Morgenstern, R. (1995) Heterologous expression of rat liver microsomal glutathione transferase in simian COS cells and Escherichia coli. Biochem. J. 311, 861-866. (19) DeKant, W., Martens, G., Vamvakas, S., Metzler, M., and Henschler, D. (1987) Bioactivation of tetrachloroethylene-Role of glutathione S-transferase catalyzed conjugation versus cytochrome P-450 dependent phospholipid alkylation. Drug Metab. Dispos. 15, 702-709. (20) Iyer, R. A., Baggs, R. B., and Anders, M. W. (1997) Nephrotoxicity of the glutathione and cysteine S-conjugates of the sevoflurane degradation product 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1propene (Compound A) in male Fischer 344 rats. J. Pharmacol. Exp. Ther. 283, 1544-1551. (21) Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A., Cheeseman, J. R., Zakrzewski, V. G., Montgomery, J. A. J., Stratmann, R. E., Burant, J. C., Dapprich, S., Millam, J. M., Daniels, A. D., Kudin, K. N., Strain, M. C., Farkas, O., Tomasi, J., Barone, V., Cossi, M., Cammi, R., Mennucci, B., Pomelli, C., Adamo, C., Clifford, S., Ochterski, J., Peterson, G. A., Ayala, P. Y., Cui, Q., Morokuma, K., Malick, D. K., Rabuck, A. D., Raghavachari, K., Foresman, J. B., Cioslowski, J., Ortiz, J. V., Baboul, A. G., Stefanov, B. B., Liu, G., Liashenko, A., Piskorz, P., Komaromi, I., Gomperts, R., Martin, R. L., Fox, D. J., Keith, T., Al-Laham, M. A., Peng, C. Y., Nanayakkara, A., Gonzalez, C., Challacombe, M., Gill, P. M. W., Johnson, B., Chen, W., Wong, M. W., Andres, J. L., Gonzalez, C., Head-Gordon, M., Replogle, E. S., and Pople, J. A. (1998) Gaussian 98 Revision A.7, Gaussian, Inc., Pittsburgh, PA. (22) Steinfeld, J. I., Francisco, J. S., and Hase, W. L. (1989) Chemical Kinetics and Dynamics, Prentice Hall Inc., Englewood Cliffs, NJ. (23) Liotta, D., and Jones, D. K. (1995) in Advances in Molecular Modeling (Liotta, D., Ed.) pp 1-19, JAI Press Inc., London. (24) Jones, D. K., and Liotta, D. (1995) in Advances in Molecular Modeling (Liotta, D., Ed.) pp 67-98, JAI Press Inc., London. (25) Shim, J.-Y., and Richard, A. M. (1996) Conformational aspects of glutathione conjugates of chlorinated alkenes: a computational study. Chem. Res. Toxicol. 9, 667-675. (26) Chen, X., and Rappoport, Z. (1998) Substitution of β-halostyrenes by MeS-. J. Org. Chem. 63, 5684-5686. (27) Bernasconi, C. F., Fassberg, J., Killion, R. B. J., and Rappoport, Z. (1990) Kinetics of reactions of thiolate ions with R-nitro β-substituted stilbenes in 50% Me2SO-50% water. Observation of the intermediate in nucleophilic vinylic substitution reactions. J. Am. Chem. Soc. 112, 3169-3177. (28) Bernasconi, C. F., Ketner, R. J., Chen, X., and Rappoport, Z. (1998) Kinetics of the reactions of methoxybenzylidene Meldrum’s acid with thiolate ions, alkoxide ions, OH-, and water in aqueous DMSO. Detection and kinetic characterization of the SNV intermediate. J. Am. Chem. Soc. 120, 7461-7468.

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Computational and Experimental Studies on the Distribution of

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