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Bioactivation Mechanisms of Haloalkene Cysteine S-Conjugates Modeled by Gas-Phase, Ion-Molecule Reactions Nganha C. Luu,†,‡ Ramaswamy A. Iyer,§,| M. W. Anders,§ and Douglas P. Ridge*,† Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, and Department of Pharmacology and Physiology, University of Rochester Medical Center, 601 Elmwood Avenue, Box 711, Rochester, New York 14620 Received October 28, 1999
Glutathione conjugate formation plays important roles in the detoxification and bioactivation of xenobiotics. A range of nephrotoxic haloalkenes undergo bioactivation that involves glutathione and cysteine S-conjugate formation. The cysteine S-conjugates thus formed may undergo cysteine conjugate β-lyase-catalyzed biotransformation to form cytotoxic thiolates or thiiranes. In the studies presented here, cysteine conjugate β-lyase-catalyzed biotransformations were modeled by anion-induced elimination reactions of S-(2-bromo-1,1,2-trifluoroethyl)-N-acetylL-cysteine methyl ester, S-(2-chloro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine methyl ester, and S-(2-fluoro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine methyl ester in the gas phase. Examination of these processes in the gas phase allowed direct observation of the formation of cysteine S-conjugate-derived thiolates and thiiranes, whose formation is inferred from condensed-phase results. The cysteine S-conjugates of these haloethenes exhibit distinctive patterns of mutagenicity that are thought to be correlated with the nature of the products formed by their cysteine conjugate β-lyase-catalyzed biotransformation. In particular, S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine is mutagenic, whereas the chloro and fluoro analogues are not. It has been proposed that the mutagenicity of S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine is correlated with the greater propensity of the bromine-containing cysteine S-conjugate to form a thiirane compared with those of the chlorine- or fluorine-containing conjugates. The ease of thiirane formation is consistent with the gas-phase results presented here, which show that the brominecontaining conjugate has a greater propensity to form a thiirane on anionic base-induced elimination than the chloro- or fluoro-substituted analogues. The blocked cysteine S-conjugates were deprotonated by gas-phase ion-molecule reactions with hydroxide, methoxide, and ethoxide ions and then allowed to decompose. The mechanisms for these decompositions are discussed as well as the insights into the bioactivation of these cysteine S-conjugates provided by the further decompositions of thiolate intermediates.
Introduction The glutathione-dependent biotransformation of xenobiotics is historically associated with the detoxification reaction (1). In addition to its role in xenobiotic detoxification, glutathione conjugate formation is also an important bioactivation mechanism for several groups of compounds, including geminal and vicinal haloalkanes and haloalkenes (2-4). A range of nephrotoxic haloalkenes undergo glutathione- and cysteine conjugate β-lyase (β-lyase)1-dependent bioactivation; the glutathione transferase-catalyzed reaction of haloalkenes with glutathione affords glutathione S-conjugates, which may undergo γ-glutamyltransferase-catalyzed hydrolysis to cysteinylglycine S-conjugates. The cysteinylglycine S-conjugates may undergo dipeptidase-catalyzed hydrolysis to cysteine S-conjugates. The cysteine S-conjugates thus formed may * To whom correspondence should be addressed. † University of Delaware. ‡ Present address: DuPont Co., Deepwater, NJ 08203. § University of Rochester Medical Center. | Present address: Bristol-Myers Squibb Co., Princeton, NJ 08543. 1 Abbreviations: β-lyase, cysteine conjugate β-lyase; CID, collisioninduced dissociation.
undergo β-lyase-dependent bioactivation to give ammonia, pyruvate, and unstable thiols or N-acetyltransferase-catalyzed N-acetylation to give mercapturic acids, which can be either excreted or hydrolyzed by aminoacylases to cysteine S-conjugates. The β-lyase-dependent bioactivation of cysteine S-conjugates affords reactive, electrophilic products. The objective of these studies was to investigate the fate of cysteine S-conjugates of fluorine-containing monomers, some of which are used in the commercial production of polymers (5). The three monomers of interest are bromotrifluoroethylene, chlorotrifluoroethylene, and tetrafluoroethylene. The formation and fate of the glutathione S-conjugates of the study haloalkenes have been reviewed (4). In the study presented here, gas-phase models of the β-lyase-catalyzed biotransformation of cysteine S-conjugates were investigated. The structures of the N-acetyl-L-cysteine S-conjugate methyl esters examined in this study, namely, S-(2-bromo-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine methyl ester 1, S-(2-chloro1,1,2-trifluoroethyl)-N-acetyl-L-cysteine methyl ester 2, and S-(2-fluoro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine methyl ester 3, are shown in Scheme 1. The reactions of
10.1021/tx990179v CCC: $19.00 © 2000 American Chemical Society Published on Web 06/29/2000
Bioactivation Mechanisms Scheme 1
Chem. Res. Toxicol., Vol. 13, No. 7, 2000 611 Table 1. Fractional Distribution of Products Formed by the Gas-Phase Reaction of Hydroxide, Methoxide, and Ethoxide with Blocked Cysteine S-Conjugates 1-3 Conjugate 1 products formed (%) base
thiolate (RS-)
bromide (Br-)
45 43 42
55 57 58
HO-
CH3OC2H5O-
anionic Brønsted bases with the blocked cysteine Sconjugates were used as models of the enzymatic reaction. Cysteine S-conjugates are substrates for the pyridoxal phosphate-dependent β-lyase, and all conjugates that are substrates for β-lyase share the common chemical property of possessing a good leaving group on the β-carbon atom (6). The mechanism of the β-lyasecatalyzed reaction involves deprotonation of the R-carbon, thereby facilitating elimination of the leaving group on the β-carbon. A number of relatively simple eliminations involving alkyl halides (7-13), ethers (14), and thioethers (15, 16) have been examined and characterized in some detail. These studies indicate that the mechanisms of these simple, gas-phase eliminations closely resemble the mechanisms of similar condensed-phase processes. This indicates that gas-phase studies may be useful in elucidating mechanisms of elimination reactions involving more complex species. Anionic bases typically react with esters to form enolates as terminal products (17, 18). Recently, however, we have found that reactions of hydroxide and alkoxide with blocked cysteine S-conjugates leads to the formation of transient enolic intermediates that eliminate thiolates, thus supporting the postulated mechanism of the β-lyase-catalyzed bioactivation of cysteine S-conjugates (19, 20). We report herein the results of studies of the elimination reactions of blocked cysteine S-conjugates 1-3 (Scheme 1). The blocked cysteine S-conjugates were reacted with anionic bases hydroxide, methoxide, and ethoxide in the gas phase to mimic the enzymatic reaction. This study provides direct evidence for the formation of several reactive intermediates, which are unstable in aqueous solutions.
Materials and Methods Syntheses. S-(2-Bromo-1,1,2-trifluorethyl)-N-acetyl-L-cysteine, S-(2-chloro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine, and S-(1,1,2,2-tetrafluoroethyl)-N-acetyl-L-cysteine were prepared as described previously (21). S-(2-Bromo-1,1,2-trifluoroethyl)-Nacetyl-L-cysteine methyl ester 1, S-(2-chloro-1,1,2-trifluoroethyl)N-acetyl-L-cysteine methyl ester 2, and S-(1,1,2,2-tetrafluoroethyl)-N-acetyl-L-cysteine methyl ester 3 were prepared by methylation of the N-acetyl-L-cysteine S-conjugates (21) with diazomethane (Caution: Diazomethane is toxic and mutagenic and should be used with care in an efficient fume hood). The methyl esters thus obtained were used directly without further purification. FT-ICR-MS (see below) mass spectra of the esters indicated that they had the expected composition and structures and were free of significant impurities. FT-ICR-MS Experiments. Experiments were performed with a dual-cell Fourier transform ion cyclotron resonance mass spectrometer (FTMS 2000, Finnigan, Madison, WI). Water, methanol, and ethanol mixtures were introduced into the source cell of the mass spectrometer through the batch inlet system. A 0.1 s, 7 eV electron beam pulse on ca. 7 × 10-7 Torr of H2O or a mixture of H2O and methanol or ethanol produced hydroxide, methoxide, and ethoxide ions, respectively. The cysteine S-
Conjugate 2 products formed (%) base
RS-
Cl-
(RS - HF)-
([M] - H)-
HOCH3OC2H5O-
73 78 76
8 6 5
10 11 14
9 5 5
Conjugate 3 products formed (%) base
RS-
([M] - H)-
([M] - H (SC2F3H))-
HOCH3OC2H5O-
87 86 78
8 4 7
5 10 15
conjugates were introduced on a probe through a vacuum lock to a pressure of approximately 2 × 10-7 Torr and reacted with hydroxide, methoxide, and ethoxide ions. The reactant ion was isolated by ejecting all other ions from the trap after the electron beam pulse. The time evolution of reactant and product ions was observed by obtaining mass spectra of the contents of the ion trap at various delay times after the isolation event. CID experiments were performed with either a two-cell or a one-cell procedure. In the two-cell procedure, a thiolate ion formed by ion-molecule reactions in the source cell was transferred to the analyzer cell of the mass spectrometer where it was isolated and accelerated to various center-of-mass collision energies and allowed to collide with Ar pulsed into the apparatus with a computer-controlled solenoid valve. In the onecell procedure, 10-6 Torr of Ar was added to the source cell, and CID spectra of thiolate ions were obtained by allowing enough reaction time for the original reactant ions to disappear, ejecting all the ions from the trap but the ion of interest, exciting the ion of interest to a selected kinetic energy, allowing time for collisions, and obtaining a spectrum of the parent ion and fragments. The two methods gave consistent results, indicating that the CID results are not complicated by the presence of the neutral blocked cysteine S-conjugates in the source cell. Figure 2 shows the results for conjugate 1 obtained with the two-cell procedure, and Figures 3 and 4 show results for conjugates 2 and 3 obtained with the one-cell procedure.
Results Table 1 shows the distribution of primary products from the ion-molecule reactions of conjugates 1-3 with hydroxide, methoxide, and ethoxide. The distribution was computed by dividing the relative intensity for each product ion (including all isotopes) by the sum of the relative intensities of the signals for all the ions, giving results that were reproduced to within (0.02. For conjugates 2 and 3, the major product was an ion with C2HF3XS- stoichiometry, which corresponds to an alkyl thiolate (RS-). The thiolate ion was also a major product for conjugate 1, but halide ion was observed as the primary product for conjugate 1. The deprotonated conjugates ([M] - H-) were also observed as primary products for conjugates 2 and 3. An ion [(RS - HF)-] corresponding to the loss of HF from the alkyl thiolate
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Figure 1. Reaction of OH- with conjugate 1. Hydroxide was produced from H2O by a 0.1 s, 7 eV electron beam pulse on 1 × 10-6 Torr of H2O. The cysteine S-conjugate was introduced on a probe through a vacuum lock to a pressure of approximately 10-7 Torr and reacted with OH- to form RS-, Br-, and [RSCysBr]-. Table 2. Relative Rate Constants for the Ion-Molecule Reactions of Cysteine S-Conjugates 1-3 with Hydroxide, Methoxide, and Ethoxide cysteine S-conjugate
OH-
CH3O-
C2H5O-
conjugate 1 conjugate 2 conjugate 3
[1.00] [1.00] [1.00]
0.28 0.46 0.16
0.18 0.28 0.05
RS- was observed as a minor product with conjugate 2. With conjugate 3, a minor product corresponding to the loss of 2,2,3-trifluorothiirane from the deprotonated conjugate [([M] - H - (SC2F3H))-] was observed. Typical kinetic data are shown in Figure 1. The base OH- disappeared exponentially as the abundance of the primary products increased. The relative magnitudes of the pseudo-first-order rate constants for the disappearance of base as a result of reaction with the conjugates are given in Table 2. The pressure of each conjugate was held constant in the analyzer cell, while kinetic data, such as those shown in Figure 1, were obtained for each of the bases. Conjugate pressure was regulated by controlling the probe temperature and monitoring the electronimpact positive-ion mass spectrum of the conjugate. The relative pseudo-first-order rate constants are thus proportional to the relative rate constants for the reaction between the base and the conjugate. Figure 1 shows the formation of a secondary product, namely, Br- clusters with the neutral conjugate (RSCysBr-), from conjugate 1. The formation of halide ions from the thiolate ions was verified by CID, as shown in Figures 2-4.
Figure 2. Collision-induced dissociation of thiolate (RS-, m/z 193) formed as shown in Figure 1. The thiolate was transferred to the analyzer cell, isolated, accelerated to various kinetic energies, and allowed to collide with CO2 gas pulsed to the source cell with a computer-controlled solenoid valve. Spectra a-c show the residual RS- and collision-produced fragment ions that were obtained at kinetic energies of 0.21, 0.45, and 0.98 eV, respectively.
Discussion The results presented above are most readily interpreted in terms of a mechanism that begins with transfer of an R-proton from the blocked cysteine S-conjugate to the ionic base forming an enolate, as indicated in Scheme 1. In the absence of solvent, the exothermicity of the initial proton transfer is partitioned into the ion and the neutral product. The energetic enolate may then decompose rapidly and not appear in the spectra. No stable enolate ion [(M - H)-] was observed for conjugate 1, and only small amounts were observed for conjugates 2 and 3 (Table 1); however, observation of any stable enolate
Figure 3. Collision-induced dissociation of thiolate (RS-, m/z 149) formed from conjugate 2. The thiolate was transferred to the analyzer cell, isolated, accelerated to various kinetic energies, and allowed to collide with CO2 gas added to the source cell with a computer-controlled solenoid valve. Spectra a-c show the residual RS- and collision-produced fragment ions that were obtained at center-of-mass kinetic energies of 0 (isolation), 0.33, and 1.69 eV, respectively.
supports the intermediacy of an enolate in the formation of other products.
Bioactivation Mechanisms
Chem. Res. Toxicol., Vol. 13, No. 7, 2000 613 Scheme 3
Scheme 4
Scheme 5
(RS-,
Figure 4. Collision-induced dissociation of thiolate m/z 133) formed from conjugate 3. The thiolate was transferred to the analyzer cell, isolated, accelerated to various kinetic energies, and allowed to collide with CO2 gas added to the source cell with a computer-controlled solenoid valve. Spectra a-c of the residual RS- and collision-produced fragment ions were obtained at center of mass kinetic energies of 0 (isolation), 0.32, and 0.94 eV, respectively.
Scheme 2
The energetic enolate may decompose to form thiolate. A fraction of the thiolate ions retains enough energy to lose halide, whereas the balance is observed in the mass spectrum as a stable product. Scheme 2 presents a mechanism for halide loss resulting in the formation of a thiirane. The postulate that the thiolate from conjugate 1 may lose Br- to form thiirane was tested by CID experiments. CID of the thiolate (m/z 193) produced Br-, confirming that the thiolate readily loses bromide, probably to form the mechanistically and thermodynamically accessible thiirane. These CID results substantiate further the postulated initial proton transfer from the R-carbon, because deprotonation of the R-carbon leads readily to the cleavage of the carbon-sulfur bond prior to formation of the thiolate. As shown in Table 1, bromide loss is the dominant outcome for conjugate 1 upon reaction with all three bases, which indicates that thiirane formation is facile and requires little energy. Conjugates 1 and 2 react similarly with the bases studied except that HF elimination from the thiolate is
important with compound 2, especially for the low-energy bases. A decomposition mechanism is shown in Scheme 3 that is initiated by transfer of the enolic proton to the reacting bases. The resulting enolate may decompose to a thiolate ion, which loses chloride to form the thiirane. Competing with chloride loss, the thiolate ion may also undergo a 1,2-elimination of HF to form a “thia-enolate” ion. CID of the thiolate shows the loss of chloride which gives presumably a thiirane, which supports the suggested mechanism. HF loss is much less important than Cl- loss in the CID of the thiolate, perhaps because Clloss is simpler than HF loss and is thus entropically favored. It could also be that Cl- loss is energetically favored, but as indicated in Table 1, HF loss is favored somewhat by the lower-energy bases. A mechanism for the reactions of conjugate 3 is suggested in Scheme 4. As mentioned above, the formation of the stable enolate ion is more important for conjugate 3 than for conjugates 1 and 2. The fraction of enolate ion formed with sufficient energy to decompose forms thiolate, which does not decompose further. CID of the thiolate (m/z 133) shows a fragment at m/z 113, which corresponds to the loss of HF from the thiolate, but there was no evidence for the formation of the m/z 113 fragment in the ion-molecule reaction spectra, indicating an energy barrier for HF elimination. The ion-molecule reaction product at m/z 162 ([M] - H (SC2F3H))- could correspond elimination of 2,2-difluorothioacyl fluoride from the enolate as shown in Scheme 5. More complicated elimination mechanisms could produce species with other structures, but nothing in the results requires a more complicated mechanism. The relative rate constants shown in Table 2 follow the same trend for all three cysteine S-conjugates. The reactions are fastest for the strongest base (OH-) and slowest for weakest base (C2H5O-). This reactivity is consistent with a mechanism involving deprotonation. The variation of product branching ratio with base strength suggests that weaker bases slightly favor thiirane formation in conjugate 1 and HF elimination in conjugate 2. Any interpretations of the branching ratios should be attempted with caution. The effects are small,
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and the branching ratios are uncertain by approximately (0.02. In addition, the distribution of energy in the proton transfer reaction is not necessarily statistical. Long-range proton transfer to the base, more likely for stronger bases, could result in highly excited OH bonds in the neutral product of the proton transfer. H2O could, for example, carry off a larger portion of the exothermicity of the proton transfer than would be expected statistically, leaving the enolate with less energy than would be expected. The deprotonation of the R-carbon of the cysteine S-conjugates and the formation of thiolates or thiiranes, or both, in the gas phase appear to resemble the β-lyasecatalyzed, condensed-phase reactions that lead to toxicity. The toxicity of the cysteine S-conjugates is dependent on β-lyase-catalyzed bioactivation. β-Lyase is a pyridoxal phosphate-dependent enzyme that catalyzes elimination reactions of amino acids by mechanisms that involve deprotonation of the R-carbon in condensed phases. β-Lyase-catalyzed β-elimination reactions do not occur with cysteine S-conjugates that lack a proton on the R-carbon, such as R-methyl analogues, which are not toxic (22-25). 1,1-Dichloroalkene-derived cysteine S-conjugates are mutagenic in the Ames test, whereas 1,1difluoroalkene-derived cysteine S-conjugates, such as conjugates 2 and 3, are not mutagenic (26-28). Recent studies show, however, that bromine-containing, 1,1difluoroalkene-derived cysteine S-conjugates, and specifically conjugate 1, are mutagenic in the Ames test and require β-lyase-dependent bioactivation for the expression of mutagenicity (29). The facile formation of thiirane from compound 1 in the studies presented here distinguishes it from compounds 2 and 3, indicating that thiirane formation may play a role in mutagenicity and, perhaps, cytotoxicity. Thiirane formation from haloalkene-derived cysteine S-conjugates has been proposed previously (30, 31), although direct evidence for their formation was lacking. Recently, however, experimental evidence for formation of thiiranes from S-(2-halo-1,1,2-trifluoroethyl)-L-cysteines was presented (32, 33). Also, computational studies indicate a preference for thiirane formation from brominecontaining S-(2,2-dihalo-1,1-difluoroethyl)-L-cysteines over thiirane formation from bromine-lacking S-(2,2-dihalo1,1-difluoroethyl)-L-cysteines (34). These results actually constitute direct observation of the formation of thiolate from the S-conjugate 1 and the subsequent elimination of Br - from the thiolate in the formation of a neutral species with C2HF3S stoichiometry. The CID spectra in Figure 2 are plotted in terms of ion fragment mass but could quite appropriately be plotted in terms of neutral fragment mass. In that case, the peak at ion mass 79 would represent a neutral fragment at mass 114 corresponding to C2HF3S stoichiometry. The peak thus represents direct observation by mass spectrometry of C2HF3S formed from C2HF3SBr-. The fragmentation occurs efficiently at all collision energies, and no competing fragmentation is observed. Together, these observations support a simple, concerted fragmentation process that leads to a thiirane rather than a process involving 1,2 shifts of H or F atoms that would be required to produce other ions with C2HF3S stoichiometry. In the FTICR-MS experiment, there is no solvent, so there are no solvolysis reactions to make the elementary steps in the reaction mechanisms. In the kinetic studies, the time required to obtain a mass spectrum of the ions in the
Luu et al.
trap is only a few milliseconds compared to the reaction time and ion-molecule collision time of seconds. Under these conditions, the steps in the reactions (deprotonation to form an enolate, which loses the thiolate, which, in turn, eliminates Br - to form thiirane) reveal themselves directly. Similarly, the kinetic data reveal that the sequences of elementary processes following deprotonation of those conjugates produce thiirane as a minor product in the case of conjugate 2 and not at all in the case of conjugate 3. Although the biotransformation, computational, and CID data all provide evidence for thiirane formation from S-(2,2-dihalo-1,1-difluoroethyl)-L-cysteines, the role of 2,2fluoro-3-halotrihalothiirane formation in the toxicity of fluoroalkene-derived cysteine S-conjugates has not been established. Moreover, other steps in the bioactivation of S-(2,2-dihalo-1,1-difluoroethyl)-L-cysteines, including initial rates of glutathione S-conjugate formation and conversion to cysteine S-conjugates, N-acetylation and deacetylation, transport, and β-lyase activity, will all contribute to the in vivo toxicity of cysteine S-conjugates.
Conclusions Deprotonation of conjugates 1-3 leads to the formation of thiolates that, in turn, may form reactive intermediates such as thiiranes. Thiirane formation is the major product formed from conjugate 1 on reaction with hydroxide, methoxide, and ethoxide. Substantially less thiirane formation was observed with conjugates 2 and 3. The deprotonation mechanism proposed above is supported by the reaction kinetics and by the CID results. The observation that conjugate 1 is mutagenic in the Ames test, whereas conjugates 2 and 3 are not, may indicate an association between thiirane formation and mutagenicity. Although thiirane may be the key reactive intermediate associated with the mutagenicity of conjugate 1, additional studies are warranted to define the role of thiirane formation in the mutagenicity and cytotoxicity of cysteine S-conjugates.
Acknowledgment. This research was supported in part by National Institutes of Environmental Sciences Grant ES03127 to M.W.A.
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