Theoretical Study of the SNV Reaction of Trichloroethylene (TCE) and

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Chem. Res. Toxicol. 1999, 12, 308-316

Articles Theoretical Study of the SNV Reaction of Trichloroethylene (TCE) and CH3S- as a Model for Glutathione Conjugation of TCE Joong-Youn Shim, Phillip F. Boone, and Ann M. Richard* National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711 Received November 4, 1998

Trichloroethylene (TCE), a major environmental pollutant, is activated to mutagenic and nephrotoxic intermediates through a glutathione (GSH) conjugation pathway. Three product isomers of GSH-TCE conjugation, having potentially different toxicities, are theoretically possible: cis- or trans-S-(1,2-dichlorovinyl)glutathione (cis- or trans-1,2-DCVG, respectively) or 2,2-DCVG. This study involved application of ab initio molecular orbital theory to computing potential energy profiles (PEPs) and predicting product outcome of the reaction of CH3S- with TCE as a model for GSH-TCE conjugation in biological systems. A goal of this study was to determine the extent to which a body of chemical knowledge pertaining to nucleophilic vinylic substitution (SNV) reactions, of which the GSH-TCE conjugation is a representative example, is relevant to this biological conjugation problem. PEPs were computed for all studied species at the HF/6-31+G* level of theory; electron correlation effects were estimated at the MP2/631+G* and MP4/6-31+G* levels, and the influence of solvation was estimated using the PSGVB solvation model. Multiple proposed reaction pathways were considered, including conjugation at the C1 or C2 site on TCE, by in-plane (σ) or out-of-plane (π) approach of the nucleophile. Some aspects of the MP2 and HF PEPs were found to differ significantly. However, on the basis of comparison of activation barriers, calculations at all levels of theory predict preference for C2 conjugation over C1 conjugation and formation of the trans-1,2-DCVM product over the cis-1,2-DCVM product. These predictions are consistent with GSH-TCE conjugation results from in vivo experiments. In contrast, relative product energies appear to be a poor indicator of the product outcome for this system. Hence, theoretical consideration of the reaction chemistry in the vicinity of the site of nucleophilic addition appears to be necessary and sufficient to predict the outcome of the enzyme-mediated GSH-TCE conjugation.

Introduction Trichloroethylene (TCE1), a common industrial solvent and widespread environmental contaminant, undergoes bioactivation to nephrotoxic and mutagenic intermediates through a glutathione (GSH) conjugation pathway (15) catalyzed by glutathione S-transferase (GST, EC 2.5.1.18). In theory, GSH conjugation of TCE can produce three experimentally discernible isomers, with reaction at C1 yielding cis- and trans-S-(1,2-dichlorovinyl)gluta* To whom correspondence should be addressed: MD-68, US-EPA, Research Triangle Park, NC 27711. Phone: (919) 541-3934. Fax: (919) 541-0694. E-mail: [email protected]. 1 Abbreviations: 1,2-DCVC, S-(1,2-dichlorovinyl)cysteine; 1,2DCVG, S-(1,2-dichlorovinyl)glutathione; 1,2-DCVM, S-(1,2-dichlorovinyl)methanethiolate; DFT, density functional theory; GSH, glutathione; GST, glutathione S-transferase; HCS, hyperconjugation stabilization; HF, Hartree-Fock; INT, intermediate; IRC, intrinsic reaction coordinate; LG, leaving group; MeS-, methanethiolate; MP, Møller-Plesset; Nu-, nucleophile; PEP, potential energy profile; PR, product; PS-GVB, pseudo-spectral generalized valence bond; PX, product complex; RE0, reactant; REX, reactant complex; SNV, nucleophilic vinylic substitution; sp, single-point; TCE, trichloroethylene; TS, transition state; ZPE, zero point energy.

thione (cis- and trans-1,2-DCVG, respectively) conjugates and reaction at C2 yielding the 2,2-DCVG conjugate. Experimental studies have provided evidence for the production of two predominant cysteine conjugate product isomers formed from GST-mediated GSH-TCE conjugation in vivo (6, 7). Using chemical standards, the major product was assigned as S-(2,2-dichlorovinyl)cysteine (2,2-DCVC) and the minor product as trans-1,2DCVC; no cis-1,2-DCVC was found (7, 8). Product isomer outcome, in turn, has biological significance since different GSH-TCE conjugate isomers have been shown to undergo different rates of metabolism in vivo and to produce different toxicological outcomes (6, 9). Two factors that determine the product isomer outcome of GSH-TCE conjugation in vivo are the local reaction chemistry at the site of conjugation and the possible modulating or controlling influence of the surrounding media and enzyme. In this study, we attempted to address the following question. To what extent is knowledge of the local reaction chemistry of the thiolate-TCE interaction necessary and sufficient for determining the

10.1021/tx9802419 CCC: $18.00 © 1999 American Chemical Society Published on Web 03/10/1999

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Scheme 1. SNV Reaction Pathways Leading to Retention and Inversion Products from the Out-of-Plane, π-Approach of the Nucleophile

conjugation outcome in the biological system in the absence of knowledge of the larger enzyme interaction? To answer this question, it is useful and appropriate to draw on the available body of chemical knowledge relevant to the particular type of reaction being studied. The GSH-TCE conjugation reaction is representative of a well-studied, mechanistically varied class of reactions known as nucleophilic vinylic substitutions (SNV) (10). SNV reaction mechanisms have been shown to depend on steric factors, the electronic properties of the nucleophile (Nu-), the ease of expulsion of the leaving group (LG), and the electron-withdrawing properties of substituents on Cβ, adjacent to the site of nucleophilic addition (10-13). Two main SNV mechanisms have been proposed: (1) an out-of-plane, π-approach reaction that can be either multistep, characterized by a true carbanionic intermediate on the reaction potential energy surface, or single-step, involving formation of a transient carbanion (11), and (2) an in-plane, σ-approach reaction (14). The mechanism, in turn, determines the stereochemical outcome of the SNV reaction; an in-plane reaction leads to stereochemical inversion in the products, whereas a π-approach pathway leads to either retention or inversion in the products (Scheme 1) depending on the lifetime and stability of the intermediate carbanion (12, 15). To rationalize the spectrum of possible SNV mechanisms, a variable transition state has been proposed that assumes a prominent role for a hyperconjugative stabilization (HCS) effect (11, 12, 15, 16). The HCS effect is postulated to provide a driving force for the LG expulsion step and is maximized by antiperiplanar alignment of the CR LG(σ*) orbital to the Cβ(π) orbital. As shown in Scheme 1, this alignment can be achieved through either a -120° rotation about the CsC bond or a +60° rotation followed by pyramidal inversion of the carbanionic charge (17). The possibility of in-plane reaction leading to inversion of configuration (not shown) has been assumed to be a high-energy alternative to reaction involving the out-of-plane, π-approach of the Nu- (8, 10). However, a theoretical study of the reaction of H2CdC(H)Cl and Cl(14) showed that a single-step, σ-approach pathway is energetically preferred. On the basis of the above considerations, a total of eight possible SNV pathways, illustrated in Scheme 2, could be considered for determining the stereochemical outcome of the GSH-TCE conjugation reaction. The σ1 pathway involving in-plane attack at C1 is sterically

hindered. In addition, the present system does not satisfy the conditions to produce a sufficiently long-lived carbanion for 120° rotation in the π1c, π1d, and π2d pathways (8, 17-19). This leaves consideration of four plausible pathways for GSH-TCE conjugation: π1a, π1b, π2a, and σ2. Pathways π1a and π1b would yield experimentally distinguishable products (cis- and trans-1,2-DCVG isomers, respectively), whereas the products of pathways π2a and σ2 (2,2-DCVG) would be experimentally indistinguishable. It is of interest to evaluate the relative plausibility of these four pathways to gain insight into key factors governing the stereochemical outcome of the GSH-TCE conjugation reaction. In a previous computational study, we predicted the product isomer outcome of the GSH-TCE conjugation from conformational analysis of the partially constrained GSH bound to TCE, and based on approximate thermodynamic considerations (i.e., comparison of relative product energies) (20). This study represents a refinement of that work, with a narrower focus on the reactive thiolate portion of GSH allowing for more rigorous treatment of the reaction mechanism underlying conjugation. Prior theoretical studies of SNV reaction mechanisms, many supported by experimental results, were used to guide and constrain model development in this study. On the basis of evidence that the major catalytic function of GST in facilitating GSH conjugation is to stabilize the reactive deprotonated GSH, i.e., GS- (21-23), methanethiolate (MeS-) was chosen to approximate GSH. Pathways were considered for both C1 and C2 conjugation reactions of MeS- and TCE, involving either in-plane or out-of-plane attack of the MeS-. Intermediate levels of ab initio theory, chosen for a reasonable description of electronic structure, were used to compute detailed potential energy profiles (PEPs) for this model system, whereas the highest levels of ab initio theory that could be practically achieved were used to a limited extent to evaluate convergence of results. Predicted product outcomes based on these PEPs are compared with the results of in vivo experiments and the results of our earlier computational study.

Methods Reaction profiles for the TCE + MeS- conjugation were constructed at the HF/3-21+G* and HF/6-31+G* levels of theory, with extended basis functions considered necessary for treatment of the diffuse S anionic species (24). Full geometry optimization and frequency calculations were carried out using

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Scheme 2. Possible SNV Pathways for GSH-TCE Conjugation

the SPARTAN molecular modeling program (version 5.0, Wavefunction, Inc., Irvine, CA) on an SGI workstation. Geometries of reactant and product complexes were obtained by optimization of the nearest transition state for both C1 and C2 conjugation reactions, and all results were zero point energy (ZPE) corrected. The following stationary points were computed: separated reactants TCE + MeS- (RE0), reactant complex TCE*MeS- (REX), transition states (TS1, TS2_cis, TS2_trans, TS2, and TS′), intermediates (INT), product complex Cl(H)Cd C(Cl)SCH3*Cl- or Cl2CdC(H)SCH3*Cl- (PX12_cis, PX12_trans, and PX22), and products Cl(H)CdC(Cl)SCH3 + Cl- or Cl2Cd C(H)SCH3 + Cl- (PR12_cis, PR12_trans, and PR22). Several approaches were employed for locating transition states (TSs) of the TCE + MeS- reaction profiles. TS geometries of the previously published H2CdC(H)Cl + Cl- system (14), computed at a high level of ab initio theory [a G2(+) procedure with MP2/6-31+G* geometries and HF/6-31+G* frequencies], were essentially reproduced by us at the HF/3-21+G* level and used to obtain TS1 geometries (from REX to INT) by direct substitutions of desired atoms and subsequent TS optimization. The in-plane TS for the single-step C2 conjugation reaction, denoted TS′, was also obtained by this approach. Initial TS2 structures for the chloride elimination step were constructed with the developed anionic charge localized anti to the MeS group, similar to the nonplanar intermediates anticipated for the out-of-plane C1 or C2 conjugation reactions (paths π1 and π2 in Scheme 2). In all cases, TS structures were optimized and verified as having a single imaginary vibrational frequency and normal mode consistent with the anticipated reaction coordinate. For the additional conformation of TSs, forward and backward intrinsic reaction coordinate (IRC) calculations at the HF/3-21+G* level were performed with Gaussian 94 (25). HF/ 3-21+G* TS geometries were used as starting points for TS optimizations at the higher HF/6-31+G* level. To estimate the influence of electron correlation on the PEP results, single-point (sp) energy calculations were carried out at the MP2/6-31+G* level for all species, and at the MP4/631+G* level for selected species using Gaussian 94 on a Cray C90 computer. In addition, sp solvation energies at both the

HF/6-31+G* and MP2/6-31+G* levels were computed using the PS-GVB (pseudo-spectral generalized valence bond) theory (26) as implemented in Jaguar (version 3.0, Schrodinger, Inc., Portland, OR) on an SGI workstation. Geometry optimization at the MP2/6-31+G* level using Gaussian 94 was attempted, within the limits of available computational resources, for a few selected stationary points of C1 and C2 conjugation and the structures and energies were examined after partial or full optimization.

Results Calculated gas-phase PEPs for the C1 and C2 conjugations are presented in Figures 1 and 2, with the corresponding aqueous phase PEPs presented in Figures 3 and 4. Geometries of transition states and intermediates corresponding to the gas-phase C1 and C2 conjugations, computed at the HF/6-31+G* level, are presented in Figures 5 and 6. Absolute reference energies and solvation energies (∆Esolv), sufficient to reconstruct the PEPs in Figures 1-4, are provided in Tables 1 and 2. To facilitate cross comparison of methods, Table 2 also lists energies for all C1 and C2 conjugation PEP species computed relative to REX for each method of calculation. Gas-Phase HF/6-31+G* Results. HF/3-21+G* results were performed as a first stage in the construction of the gas-phase HF/6-31+G* PEPs. The HF/3-21+G* PEP geometries and energies (not shown) were essentially reproduced at the HF/6-31+G* level (to within 1 kcal/mol or 10%, whichever is smaller), indicating convergence of the Hartree-Fock (HF) theory. According to the HF/6-31+G* results presented in Figure 1, C1 conjugation proceeds through a multistep pathway involving formation of a distinct intermediate, INT. Two possible pathways (paths π1a and π1b in Scheme 2) branch from INT to yield either a cis or a trans

Theoretical Study of GSH-TCE Conjugation

Figure 1. Gas-phase PEPs (kilocalories per mole) corresponding to the C1 conjugation reaction of TCE with MeS-, computed at the HF/6-31+G*, MP2/6-31+G* (parentheses), and MP4/631+G* (brackets) levels based on HF/6-31+G* geometries; energies represent differences between adjacent states.

Figure 2. Gas-phase PEPs (kilocalories per mole) and energy differences corresponding to the C2 conjugation reaction of TCE with MeS-, computed at the HF/6-31+G*, MP2/6-31+G* (parentheses), and MP4/6-31+G* (brackets) levels based on HF/ 6-31+G* geometries; energies represent differences between adjacent states.

product, depending on the direction of internal rotation about the CsC bond. The TS2 barrier for trans product formation (1.02 kcal/mol) is predicted to be smaller than for cis product formation (4.82 kcal/mol). Interestingly, the predicted relative stabilities for the corresponding product complexes and products are reversed, with the cis product having a slightly lower energy than the trans product. HF/6-31+G* computations failed to locate an inplane pathway (path σ1 in Scheme 2) for C1 conjugation; every attempt yielded a structure corresponding to TS1 for the out-of-plane MeS- approach. This is likely due to the steric crowding and large electronic repulsion experienced by MeS- approaching C1 in the plane containing the two chlorines. For C2 conjugation, HF/6-31+G* calculations yielded two distinct pathways (paths π2a and σ2 in Scheme 2), with the PEP results shown in Figure 2. However, the single-step pathway corresponding to the in-plane, σ-approach of MeS- through a planar transition state (see

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Figure 3. PEPs (kilocalories per mole) corresponding to the C1 conjugation reaction of TCE with MeS- using the PS-GVB solvation model (based on HF/6-31+G* geometries) to compute energies at the HF/6-31+G* and MP2/6-31+G* (parentheses) levels.

Figure 4. PEPs (kilocalories per mole) corresponding to the C2 conjugation reaction of TCE with MeS- using the PS-GVB solvation model (based on HF/6-31+G* geometries) to compute energies at the HF/6-31+G* and MP2/6-31+G* (parentheses) levels. Table 1. Reference Energies (arbitrary units) Used To Compute Relative Energies for Reaction Profiles in Figures 1-4 MeSTCE REXc Cltrans-1,2-DCVMd cis-1,2-DCVM 2,2-DCVM

HF/6-31+G*a

MP2/6-31+G*b

-437.0881224 -1454.694446 -1891.799633 -459.539661 -1432.303270 -1432.305550 -1432.302466

-437.384641 -1455.376402 -1892.761043 (-1892.889756) -459.671145 -1433.152450 -1433.154812 -1433.150509

a Energies evaluated for fully geometry-optimized structure at the indicated level with ZPE correction. b Single-point (sp) energy of the HF/6-31+G* structure evaluated at the MP2/6-31+G* level; the MP4/6-31+G* result is shown in parentheses for REX. c All energies reported in Figures 1-4 were computed relative to the corresponding REX energy (with and without solvation). d trans1,2-DCVM t trans-S-(1,2-dichlorovinyl)methanethiol, etc.

TS′ in Figure 6) is predicted to have a significantly higher activation barrier (26 kcal/mol) than the corresponding

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Figure 5. Geometries of transition states and the intermediate optimized at the HF/6-31+G* level for the C1 conjugation reactions of TCE with MeS- in the gas phase, with the following parameters listed: C-Cl, C-C, and C-S bond lengths, Cl-Cβ-H bond angle, H-C-C-Cl(gauche) and Cl-C-C-Cl(gauche) dihedral angles, Mulliken charge of Cβ (parentheses), and distance of Cβ from the plane of its neighboring atoms (brackets).

Figure 6. Geometries of transition states and the intermediate optimized at the HF/6-31+G* level for the C2 conjugation reactions of TCE with MeS- in the gas phase, with the following parameters listed: C-Cl, C-C, and C-S bond lengths, Cl-Cβ-Cl bond angle, Cl-C-C-Cl(gauche) and Cl-C-C-H(gauche) dihedral angles, Mulliken charge of Cβ (parentheses), and distance of Cβ from the plane of its substituents (brackets).

out-of-plane, π-approach pathway through a nonplanar intermediate (see INT in Figure 6). Geometries of TS1, INT, TS2_cis, and TS2_trans for the C1 conjugation computed at the HF/6-31+G* level, shown in Figure 5, indicate a number of features consistent with the π1a and π1b pathways postulated in Scheme 2. TS1 shows some loss of planarity and significant localization of negative charge on Cβ upon addition of MeS-. TS1 proceeds to INT with lengthening of the Cs C and CsCl bonds, shortening of the CsS bond, and increasing nonplanarity. Finally, INT proceeds to TS2_trans or TS2_cis with simultaneous CsC bond shortening, CRsCl(LG) bond lengthening, and rotation

of Cl(LG) about the CsC bond toward alignment with the Cβ(π) orbital. At the HF/6-31+G* level, TS1 had a normal mode consistent with CsS bond formation and CdC bond stretching, while TS2_cis and TS2_trans had normal modes consistent with CdC bond formation and CsCl(LG) bond cleavage. HF/6-31+G*-optimized geometries of TS1, INT, and TS2 for the C2 conjugation π2a path, shown in Figure 6, are similar in most respects to the corresponding structures for C1 conjugation shown in Figure 5. Calculated imaginary frequencies and normal modes of the transition states are consistent with CsS bond formation and CdC bond elongation for TS1, CdC bond formation and

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Table 2. PEP Energies Relative to REX Energy for Each Level of Theory, and PS-GVB Solvation Energies (kilocalories per mole) HF/6-31+G*a TCE MeSCltrans-1,2-DCVMe cis-1,2-DCVM 2,2-DCVM REO REXf C1 conjugation TS1 INT TS2_trans TS2_cis PX_trans PX12_cis PR12_trans PR12_cis C2 conjugation TS1 INT TS2 TS′ PX22 PR22

PS-GVBb HF/6-31+G*

MP2/6-31+G*c

MP4/6-31+G*c

∆Esolvd from HF/6-31+G*

∆Esolvd from MP2/6-31+G*

3.17 0.00

0.88 -76.13 -76.37 -0.76 -0.83 -1.60 -75.25 -62.43

1.34 -73.97 -74.84 -0.22 -0.20 -0.90 -72.63 -61.00

PS-GVBb MP2/6-31+G*

10.71 0.00

-2.12 0.00

14.79 0.00

29.51 21.18 22.20 26.01 -34.13 -40.26 -27.17 -28.60

40.21 25.96 30.02 33.40 -40.69 -39.44 -41.87 -43.38

9.20 9.45 6.59 9.56 -33.06 -40.75 -24.46 -25.94

9.96 9.07 6.70 9.38

7.45 14.35 17.20 20.31 -38.93 -40.05 -38.51 -39.98

-51.72 -57.65 -54.60 -55.03 -68.98 -61.61 -77.13 -77.20

-62.74 -56.11 -50.39 -50.26 -66.88 -60.30 -75.07 -75.05

23.23 9.88 14.54 49.40 -39.93 -26.67

32.33 15.52 21.87 55.06 -38.81 -42.21

7.68 2.62 5.06 41.13 -40.33 -23.24

8.02 1.73 4.40 36.31

15.97 8.04 13.28 50.49 -39.24 -37.98

-53.32g -56.79 -55.09 -56.77 -61.31 -77.97

-52.71g -55.58 -52.79 -51.64 -59.92 -75.74

0.00

a Energies evaluated for full geometry-optimized structures with zero point energy corrections. b PS-GVB solvation model used to compute single-point (sp) energies for HF/6-31+G* structures at the indicated level of theory, relative to that of REX. c Energies of HF/6-31+G* structures (sp) evaluated at MP2/ or MP4/6-31+G* levels, relative to that of REX. d ∆Esolv ) E(gas phase) - E(solution), from corresponding PS-GVB sp calculation at the indicated level of theory based on HF/6-31+G* structures. e trans-1,2-DCVM t trans-S-(1,2-dichlorovinyl)methanethiol, etc. f Absolute reference energies in arbitrary units reported in Table 1. g PS-GVB calculations for TS1 failed to converge at the HF/ or MP2/6-31+G* levels, so results were computed at the HF/ and MP2/6-31G* levels, respectively.

CsCl(LG) bond cleavage for TS2, and CsS bond formation and CsCl(LG) bond cleavage for TS′. Gas-Phase sp MP2/6-31+G* and MP4/6-31+G* Results. To approximately account for the effects of electronic correlation on the C1 and C2 conjugation PEPs, sp MP2/6-31+G* energies were computed for all PEP reaction species and sp MP4/6-31+G* energies were computed for a subset of these; both were based on the HF/6-31+G*-optimized geometries. These energies are reported in Figures 1 and 2, with reference energies listed in Table 1 and energies relative to REX reported in Table 2. MP2 and HF results predict similar overall exothermicities of the C1 and C2 conjugation reactions but differ in important respects. In particular, MP2 predicts significant reduction in activation barriers for both the C1 and C2 conjugation reactions. In addition, for C1 conjugation, MP2, but not MP4, predicts that there is no longer a barrier to INT formation, and both MP2 and MP4 predict TS2_trans to be a lower-energy form than INT. Hence, the MP2 results call into question the nature of the C1 conjugation profile, as a single-step or multistep reaction. MP2 results for the C2 reaction, in contrast, largely reproduce the shape of both the in-plane and outof-plane approach PEPs. MP4 results for transition states and intermediates of C1 and C2 conjugation indicate reasonable convergence of the Møller-Plesset (MP) theory results. Although calculation of fully optimized geometries and frequencies at the MP2/6-31+G* level for all PEP species was beyond the computational feasibility of this study, an attempt was made to locate stable intermediates of C1 and C2 conjugation by MP2/6-31+G* optimization of the corresponding HF/6-31+G* INT structures. This attempt was unsuccessful in the case of C1 conjugation, yielding a

PX12_trans structure resembling the HF/6-31+G* geometry, whereas a stable intermediate closely resembling INT was found in the case of C2 conjugation. Attempts to refine the TS1 structure for C1 conjugation with transition state optimization at the MP2/6-31+G* level were also unsuccessful and ultimately limited by available computational resources. The computational infeasibility of performing a more thorough search of the MP2/ 6-31+G* potential energy surface may have prevented us from locating these stationary points at the MP2 level of theory. However, Zheng and Ornstein (27) also reported loss of the first transition state, predicted to be present at the HF/6-31+G** level, when MP2/6-31+G** sp energies were computed for a model reaction of MeSwith 1-chloro-2,4-dinitrobenzene. Solution-Phase Results. Solvation energies were computed using the PS-GVB model, which is based on a quantum mechanical description of the molecule coupled to a continuum solvent model (26). This model has been shown to be more accurate than the reaction field models (e.g., SCI-PCM) in reproducing experimental solvation energies of anions, such as Cl- and HS- (27, 28). Gasphase geometries are generally considered reasonable and computationally feasible approximations to the solution-phase geometries for studying overall trends and PEP features. Additional support for the use of gas-phase geometries in this work is provided by a recent modeling study of nucleophilic addition of hydroxyl anion to N,Ndimethylacetamide, where reaction profile species that were allowed to fully relax in the presence of solvent varied little from the HF/6-31+G**-optimized geometries (29). PS-GVB solvation energies, computed at both the HF/ 6-31+G* and MP2/6-31+G* levels of theory (based on HF/6-31+G* geometries), are listed in Table 2. The

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solvent-adjusted PEPs for C1 and C2 conjugation (obtained by adding solvation energies, ∆Esolv, to the gasphase energies) are shown in Figures 3 and 4. The major effect of inclusion of solvation on all C1 and C2 conjugation PEPs is a dramatic lowering of reactant and product energies with respect to the corresponding reactant and product complex energies due to the large energy of solvation for the Cl- and MeS- anions (see Table 2). In addition, due to the greater localization of charge in the loose-ion complexes than in the transition states, the effect of solvation at the HF/6-31+G* level is to effectively increase the TS1 and TS2 activation barriers. In most other respects, the HF/6-31+G* PEPs in Figures 3 and 4 reproduce the major features of the corresponding gasphase PEPs. MP2/6-31+G* solution-phase results for C2 conjugation also show significant increases in TS barrier heights relative to the gas-phase results, yet these PEPs retain the main features of the corresponding gas-phase PEP, including a well-defined intermediate. For C1 conjugation, in contrast, problems observed with the definition of TS1 and INT in the gas-phase C1 conjugation appear to be accentuated in the solution-phase conjugation. For most other reaction species listed in Table 2, solvation energies for the two calculation methods vary by less than a few kilocalories per mole.

Discussion PEPs for a model GSH-TCE system, postulated to incorporate key elements of the in vivo biochemical reaction system, were computed for this study at the highest levels of ab initio theory deemed feasible for obtaining geometries, frequencies, and barrier heights and for estimating solvation effects. Despite a number of tools in molecular modeling programs for facilitating TS determination, the characterization of reaction profiles remains a computationally intensive and nonroutine undertaking. Such calculations often require knowledge of the proper reaction coordinate and a reasonably accurate guess for the TS geometry to be successful. In addition, due to lack of experimental data on TS geometries and activation enthalpies for many types of reaction systems, there tends to be a limited consensus on the necessary level of treatment for such calculations (30). In this study, we relied heavily on previously published reports of similar model systems to guide our efforts (8, 14, 17, 27, 29). The HF/6-31+G* extended basis set was chosen to adequately model the sulfur anion, whereas the MP2/631+G* method is the lowest level of corresponding ab initio theory to account for electron correlation effects. Although full geometry optimization and frequency calculation at the MP2/6-31+G* level is generally recommended, it was beyond present computational capabilities and was attempted for only a few equilibrium geometries (REX and INT). When the discrepancies in HF and MP2 results are considered, it is interesting to note that two prior studies on model SNV reaction systems (involving charged nucleophiles) also reported MP2/6-31+G** sp results that significantly underestimated the HF/631+G** results (27, 29), and in one of these, modeling hydroxide anion addition to N,N-dimethylacetamide, the HF/6-31+G** results agreed more closely with experimental activation enthalpies (29). To attempt to further resolve these discrepancies, we also explored the use of

Shim et al.

density functional (DFT) methods (pBP/DN** sp energy calculations in SPARTAN, and B3LYP geometry optimization with frequencies in Gaussian 94; results not shown). However, difficulties in obtaining convergence in the B3LYP calculations, the lack of consistency in sp results (with corresponding MP2 and HF energies), reports of the inaccuracy of such methods for anionic compounds containing sulfur (31), and the lack of literature reports in the use of such methods for computing reaction profiles for similar SNV systems led us to focus exclusively on the HF and MP2 results in this report. We face the following question. Given current computational constraints, discrepancies in the HF and MP2 results, and the lack of experimental validation criteria for assessing the accuracy of the computed PEPs, can useful conclusions be extracted from the present theoretical analysis that can be informative with respect to the biological conjugation problem? Clearly, we cannot estimate with any degree of certainty absolute activation enthalpies for the various modeled pathways of C1 and C2 conjugation. However, we can attempt to extract conclusions from the larger patterns in the PEP results that are reproduced at most or all levels of theoretical description and that are consistent with proposed SNV reaction mechanisms and prior theoretical studies. All three C1 and C2 conjugation modeled pathways computed at all levels of theory, with and without solvation, are predicted to be highly exothermic (approximately 40 kcal/mol). Evidence that the nucleophilic addition step is rate-limiting for SNV reactions of halogenated vinylic compounds has been provided in previous published reports (10, 11). Consistent with these observations, the HF/6-31+G* and MP4 results (and the MP2/ 6-31+G* results for C2 conjugation) predict larger barriers to nucleophile (MeS-) addition than Cl- elimination for the π-approach C1 and C2 conjugation reactions. Results computed at all levels of theory, with and without solvation, also indicate that the out-of-plane, π-approach of MeS- is clearly preferred for both C1 and C2 conjugation. The HF/6-31+G* PEP results for both C1 and C2 conjugation reactions of TCE with MeS- are consistent with the multistep, π-approach SNV reaction mechanism postulated by Rappoport (10-12) and others (8, 17). For both C1 and C2 multistep pathways, the anionic charge is localized on Cβ antiperiplanar to the incoming MeS- (as evidenced by the direction of nonplanarity in Figures 5 and 6). A minimum rotation coupled with inversion of the carbanion at Cβ enables the anionic charge to be aligned antiperiplanar to the σ* orbital of Cl(LG) at CR to maximize the HCS effect (16, 17). The TS2 geometries in Figures 5 and 6 provide evidence of a progression toward this optimal alignment to yield elimination products with retained configuration for both C1 and C2 conjugation reactions. Although a σ-approach, single-step pathway for C2 conjugation was successfully identified in this study, it clearly provides a much higher-energy and less probable alternative to the π-approach, multistep pathway. This is most likely due to the greater ability of Cβ, with two attached chlorines, to stabilize the negative charge of a presumed anionic intermediate, in contrast to the H2CdC(H)Cl + Clsystem, where an in-plane, σ-approach was reportedly favored (14). A primary goal of this analysis was to use the PEPs in Figures 1-4 to approximately infer the product isomer outcome of the TCE + MeS- reaction. For comparison of

Theoretical Study of GSH-TCE Conjugation

Chem. Res. Toxicol., Vol. 12, No. 4, 1999 315

C1 and C2 conjugation reactions, it is necessary to consider the overall barriers to reaction. For all modeled conjugation reactions at all levels of theory, with and without solution, the barrier to PR22 formation is predicted to be lower than the barrier to PR12_cis or PR12_trans formation. Hence, all calculations predict preference for the C2 conjugation pathway over the C1 conjugation pathway, consistent with formation of the 2,2-DCVG retention product of the π2a pathway in Scheme 2. This outcome agrees with the results of in vivo experiments, in which 2,2-DCVC was assigned as the major cysteine conjugate product resulting from GSHTCE conjugation (6, 7). (Note that rapid hydrolysis of the GSH conjugates to the corresponding cysteine conjugates, preserving the GSH conjugate product ratio, is assumed.) To predict the product outcome of C1 conjugation, the relative activation barriers to formation of TS2_cis and TS2_trans must be considered. At all levels of theory, with and without solvation, the pathway through TS2_trans is preferred by 2-3 kcal/mol over the pathway through TS2_cis. Hence, all calculations predict a preference for formation of the trans-1,2-DCVG retention product, corresponding to the -60° rotation π1b path in Scheme 2. This result is further supported by the finding that geometry optimization of INT at the MP2/6-31+G* level relaxed to the PX12_trans structure. This result also agrees with the results of in vivo experiments, in which trans-1,2-DCVC was assigned as the minor cysteine conjugate product resulting from GSH-TCE conjugation (6, 7), whereas no cis-1,2-DCVC product was detected in that study. Hammond’s postulate, which assumes a late, ratedetermining transition state resembling products, provided a rationale for the use of relative product energies to predict the preferred product outcome in our earlier computational study of GSH-TCE conjugation (20). Clearly, PEPs generated for this study do not support this assumption. On the basis of comparison of relative product energies, C1 conjugation would be predicted (at all levels of theory, with and without solvation) to be preferred over C2 conjugation by a small energy margin (1-3 kcal/mol). In addition, preference for the PR12_cis outcome would be predicted by a small energy margin (∼1-2 kcal/mol) over the PR12_trans outcome in all cases. By this measure, product outcome probabilities would be in the order PR12_cis > PR12_trans ≈ PR22, a prediction which is at odds both with the experimental in vivo results and with predictions based on comparison of PEP activation barriers.

teraction can serve to define and constrain the modeling problem in useful ways. Where such reaction chemistry has been well-characterized, it provides an alternate source of experimental validation and a means for illuminating the study of biological interactions. In this study, the GSH-TCE conjugation reaction, whose stereochemical outcome has possible toxicological consequences, was modeled by replacement of GSH with MeS-, a simplified nucleophile. We neglected the larger influence of the GST enzyme, whose major catalytic function is to stabilize the deprotonated thiol of GSH for nucleophilic reaction, in favor of focusing more rigorously on the chemistry underlying conjugation at this reactive thiolate. For this purpose, we drew on a body of knowledge pertaining to the richly variegated SNV reaction mechanisms of vinyl halides. Although the highest level of theoretical treatment was inadequate for precise activation energy determinations, there was sufficient consistency in the patterns of PEP results to allow useful insights into the reaction mechanism. On the basis of comparison of PEP activation energy barriers, predicted product outcome probabilities were ordered 2,2-DCVC > trans-1,2-DCVC > cis-1,2-DCVC and were in agreement with results of an in vivo experimental study of GSHTCE conjugation. Prediction of the product outcome based on relative product energies, in contrast, disagreed both with experimental results and with the predictions based on relative PEP activation energy barriers. Hence, we conclude that consideration of the reaction mechanism local to the site of GSH conjugation appears to be both necessary and sufficient for predicting the product outcome for this class of GST-mediated SNV reactions in biological systems.

Conclusion

References

Clearly, major simplifying assumptions must be made to model the reaction outcome in a biological system, particularly when the reaction is known to be mediated by an enzyme interaction. The challenge is in choosing an appropriate tradeoff in terms of adequacy of chemical description (i.e., molecule size, extent of solvation, temperature, etc.) and theoretical treatment (electronic description, product energies, reaction profiles, etc.), within practical computational constraints. In all such efforts, theory must ultimately be anchored by experimental data. However, we are often severely constrained by the lack of relevant data pertaining to the biological interactions of interest. In these cases, knowledge of the probable reaction chemistry underlying a biological in-

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Acknowledgment. We thank James Rabinowitz and Ya-Jun Zheng for helpful comments and Stephen Little for technical assistance. This work was carried out while J.-Y.S. held a postdoctoral fellowship in the UNC Curriculum of Toxicology, funded by the EPA/UNC Toxicology Research Program, Training Agreement T901915. Gaussian 94 calculations were performed on a Cray C90 computer located at the EPA-National Environmental Supercomputer Center in Bay City, MI. This manuscript has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

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