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Chem. Res. Toxicol. 1996, 9, 140-146
Reaction of Trifluoroacetaldehyde with Amino Acids, Nucleotides, Lipid Nucleophiles, and Their Analogs Hequn Yin, Robert J. Crowder, Jeffrey P. Jones, and M. W. Anders* Department of Pharmacology, University of Rochester, 601 Elmwood Avenue, Box 711, Rochester, New York 14642 Received April 27, 1995X
Trihaloacetaldehydes are used as sedatives, are key intermediates in the metabolism of 1,1,1,2-tetrahaloethanes, some of which are chlorofluorocarbon substitutes, and are metabolites of trihaloethanols, which are intestinal and bone marrow toxins. In the present study, trifluoroacetaldehyde was used as a model to examine the reactions of trihaloacetaldehydes with cellular nucleophiles, including amino acids, nucleotides, and lipid components. Reaction of trifluoroacetaldehyde hydrate (10 mM) with amino acids (100 mM) in buffer at pH 7.0 and 30 °C showed that only L-cysteine formed stable adducts, which were identified as (2R,4R)and (2S,4R)-2-(trifluoromethyl)thiazolidine-4-carboxylic acid. The absolute stereochemistry of (2R,4R)- and (2S,4R)-2-(trifluoromethyl)thiazolidine-4-carboxylic acid was determined by homonuclear Overhauser effect experiments. The diastereoisomers were formed in a 2.8:1 ratio at 37 °C and in a 1:4.0 ratio at 80 °C. Trifluoroacetaldehyde also reacted with L-cysteine methyl ester and 2-mercaptoethylamine to form stable thiazolidine derivatives, but did not react with N-acetyl-L-cysteine. The reaction of trifluoroacetaldehyde with the amino groups of ATP, GMP, CMP, L-citrulline, and urea resulted in the formation of stable imines. TMP, which lacks an exocyclic amino group, did not react. Glutathione reacted with trifluoroacetaldehyde to form (2R,5R)- and (2S,5R)-5-amino-6-[(carboxymethyl)imino]-2-(trifluoromethyl)1,3-oxathiane, whose formation was accompanied by simultaneous cleavage of the glutamyl moiety. The reactivity of nucleophilic groups with trifluoroacetaldehyde follows the order SH > NH2 > OH. The results of the present study indicate that trifluoroacetaldehyde covalently modifies cellular nucleophiles. The biological significance of these reactions warrants further investigation. The reaction of trifluoroacetaldehyde with L-cysteine and glutathione may afford routes for the stereoselective synthesis of cysteine prodrugs and five- or six-membered heterocyclic compounds.
Introduction Trihaloacetaldehydes, which exist predominantly as hydrates in aqueous solution, are used as sedatives (1), are metabolites of trihaloethanols (2), which are intestinal and bone marrow toxins (2), and are key intermediates in the metabolism of 1,1,1,2-tetrahaloethanes, some of which are chlorofluorocarbon substitutes (3, 4). Trifluoroacetaldehyde is a metabolite of trifluoroethanol (2) and 2-chloro-1,1,1-trifluoroethane (HCFC-133a)1 (3) and is a putative metabolite of 1,1,1,2-tetrafluoroethane (HFC-134a) (4-6). Chlorodifluoroacetaldehyde hydrate, the key intermediate in 1,2-dichloro-1,1-difluoroethane (HCFC-132b) metabolism (7), is a slow-binding inhibitor of acetylcholinesterase, pseudocholinesterase, and carboxylesterase (8). 1,1,1,2-Tetrachloroethane, which is presumably biotransformed to 1,2,2,2-tetrachloroethanol and then to trichloroacetaldehyde by cytochrome P450 oxidation, covalently modifies proteins and nucleotides in the liver, lung, kidney, and stomach of rats given [14C]1,1,1,2-tetrachloroethane (9). Recent studies have shown that oxidation of HCFC-133a by cytochrome P450 generates trifluoroacetaldehyde as the key intermediate (3). Covalent adducts of trifluoroacetaldehyde with urea and an unknown cellular nucleophile were detected by 19F Abstract published in Advance ACS Abstracts, December 1, 1995. Abbreviations: HCFC-133a, 2-chloro-1,1,1-trifluoroethane; HFC134a, 1,1,1,2-tetrafluoroethane; HCFC-132b, 1,2-dichloro-1,1-difluoroethane; ATP, adenosine 5′-triphosphate; CMP, cytosine 5′-monophosphate; GMP, guanosine 5′-monophosphate; TMP, thymidine 5′monophosphate; NOE, nuclear Overhauser effect. X 1
0893-228x/96/2709-0140$12.00/0
NMR spectroscopy in the urine of rats given HCFC-133a or trifluoroacetaldehyde hydrate (3). These findings indicate that trihaloacetaldehydes may modify cellular nucleophiles and, therefore, contribute to their toxicities or those of precursor compounds. In the present study, the reaction of trifluoroacetaldehyde with amino acids, nucleotides, lipid nucleophiles, and their analogs was examined. 19F NMR spectroscopy was used to follow the course and kinetics of the reactions and to determine the structures of intermediate and terminal products. We report herein the results of studies on the reaction of trifluoroacetaldehyde with L-cysteine and glutathione to form stable cyclic adducts and with ATP, CMP, and GMP to form stable imines; no reaction was observed between trifluoroacetaldehyde and other amino acids and TMP.
Experimental Procedures Materials. Trifluoroacetaldehyde hydrate was purchased from Fluorochem Limited (Derbyshire, England) and purified by distillation; 19F and 1H NMR spectroscopic analysis indicated good purity. Amino acids, their derivatives, glutathione, Lcitrulline, ATP, CMP, GMP, and TMP were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI) and were of the highest grade commercially available. Instrumental Analyses. 19F NMR spectra were acquired with a Bruker WP-270 spectrometer operating at 254.18 MHz with D2O as the lock signal. The acquisition parameters were as follows: excitation pulse width, 7 µs; relaxation delay, 0.51.0 s for qualitative analysis and 1.7 s for quantitative analysis;
© 1996 American Chemical Society
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Chem. Res. Toxicol., Vol. 9, No. 1, 1996 141
Figure 1. Proposed reaction mechanism for the formation of (2S,4R)- and (2R,4R)-2-(trifluoromethyl)thiazolidine-4-carboxylic acid by the reaction of trifluoroacetaldehyde with L-cysteine. sweep width (SW), 5000-20 000 Hz; number of scans, 1001000. Chemical shifts were referenced externally to a 5.0 mM solution of trifluoroacetamide in D2O (δtrifluoroacetamide ) 0 ppm). Spectra were recorded at room temperature in a 5-mm tube with the sample spinning at ∼25 rpm. Reactant to product ratios were determined by integration of resonances. Variations in longitudinal relaxation times (T1) of the organofluorine compounds were corrected by choosing relaxation times that allowed full relaxation of all the fluorine nuclei. 1H NMR spectra were acquired on the same instrument with a proton probe operating at 270.13 MHz. Proton chemical shifts were referenced to residual DHO in the deuterium oxide solution, which was set at 4.67 ppm. Mass spectra were recorded with an HP-5970 mass spectrometer (70 eV, electron impact) coupled to a Hewlett-Packard 5880A gas chromatograph equipped with an HP-1 capillary column (dimethylsilicone gum, 25 m × 0.2 mm × 0.5 µm film thickness). Conditions for GC/MS analyses were as follows: splitless injection; injector temperature, 250 °C; initial column temperature, 30 °C for 0.5 min; program rate, 10 °C/min to 200 °C; final column temperature, 200 °C for 3 min; interface temperature, 250 °C; carrier gas, He. Synthesis of (2S,4R)- and (2R,4R)-2-(Trifluoromethyl)thiazolidine-4-carboxylic Acids (2 and 3). L-Cysteine (0.5 g, 4.1 mmol), trifluoroacetaldehyde hydrate (0.7 g, 6.0 mmol), 2 mL of water, and a magnetic stir bar were placed in a 20-mL glass vial that was sealed with a Teflon-lined crimp-top septum and placed in a heating block on a magnetic stirrer. The septum was pierced with a small needle to provide pressure relief. After 6 h of heating and stirring at 80 °C, a sample of the mixture was withdrawn and analyzed for the loss of trifluoroacetaldehyde hydrate by 19F NMR spectroscopy. After the reaction was complete, the mixture was lyophilized. Five milliliters of water was added to the yellowish-white solid material obtained. The poorly soluble product was removed by filtration, and the crystals were washed twice with a small amount of water and air-dried. 1H and 19F NMR spectral analysis showed the presence of thiohemiacetals 2 or 3 or both. The filtrate was concentrated to about 2 mL and cooled in a refrigerator. After cooling, the solution was seeded with a crystal of the previously obtained solid to induce crystallization. Crystals were collected by filtration, washed twice with a small volume of cold water, and dried in vacuo. 1H and 19F NMR spectroscopic analysis showed the presence of only isomer 2 (yield, 66%). Isomer 3 was not purified, and its spectral data were obtained by comparing those of a mixture of thiazolidines 2 and 3 with those of pure isomer 2. Spectral data of both diastereomers are listed in Table 1. Methyl esters of thiazolidines 2 and 3 were prepared by reaction of the free acids with diazomethane in ether and gave retention times of 12.0 and 11.7 min, respectively, by GC/ MS. Their mass spectral fragmentation patterns [m/z (relative abundance)] were identical: 215 (4.7, molecular ion), 156 (100, M - COOMe), 86 (36, CH2CHCOOMe), 59 (62, COOMe), 195 (11.8, M - HF), 146 (10.8, M - CF3), 69 (5.9, CF3). Homonuclear NOE Experiments. Homonuclear NOE experiments were conducted with a mixture of thiazolidines 2 and 3 (Figure 1) with a GE400 NMR spectrometer. D2O was used as the lock signal. Acquisition parameters were as follows: excitation pulse width, 6 µs; spectral width, 3505 Hz; decoupling irradiation power, 10-15 mW; irradiation time, 10 s; preacquisition decay, 1 ms; acquisition time, 4.68 s; pulse
Figure 2. Definition of equilibrium constants (Keq) for the reaction of trifluoroacetaldehyde with nucleophiles. Since the concentration of trifluoroacetaldehyde was not determined, the relative reactivity of different nucleophiles was compared to that of water (Keq ) 1 when Nu: is water). delay, 3 s; number of scans, 16. Difference spectra were obtained by subtracting control spectra irradiated at a -2 ppm offset. Decoupling Studies. Homonuclear decoupling 1H NMR studies were used to simplify the ABX spectral system in the thiazolidine derivatives. Experiments were conducted with pure isomers as well as with mixtures of diastereomers. Proton signals corresponding to HX signals in thiazolidines 2 or 3 were irradiated at low decoupling power. The decoupled spectra of HX and HY in thiazolidines 2 and 3 exhibited an AB system, and the chemical shifts and coupling constants of HA and HB were calculated. Interatomic Distance Calculations. Interatomic distances between HX and HY in isomers 2 and 3 were calculated with the MOPAC program (93 version). Molecular structures were built and input with the SYBYL program (Tripos Inc., St. Louis, MO). Energy minimization was carried out with the semiempirical AM1 Hamiltonian, with the COSMO salvation model (esp ) 78.4), and precise criteria. The interatomic distances between HX and HY were measured in the energyminimized structures. 19F NMR Studies on the Reaction of Trifluoroacetaldehyde with Nucleophiles. Reaction mixtures containing 10 mM trifluoroacetaldehyde hydrate, 100 mM nucleophile, and 100 mM potassium phosphate buffer (pH 7.0) were incubated at 37 or 80 °C for various times. The 19F NMR spectra were recorded at the beginning and end of the incubation periods. Determination of Equilibrium Constants, Keq. Reaction mixtures containing 10 mM trifluoroacetaldehyde hydrate, 100 mM nucleophile, and 100 mM potassium phosphate buffer (pH 7.0) were prepared in 5-mm NMR tubes and incubated for 2 h at 37 °C in a shaking water bath. 19F NMR spectra were recorded at the end of incubation. Equilibrium constants, Keq, were calculated as described in Figure 2. The water concentration was taken as 55 M. The trifluoroacetaldehyde concentration at equilibrium changed by ATP > GMP. The 19F NMR chemical shifts of their products were -5.0 (J value not determined), -5.0 (3JHF ) 5.5 Hz), and -5.2 ppm (3JHF ) 4.9 Hz), respectively. Reaction of Trifluoroacetaldehyde with Glutathione. Glutathione reacted with trifluoroacetaldehyde to form diastereomeric thiohemiacetals with 19F NMR chemical shifts of -0.74 (3JHF ) 6.8 Hz) and -0.82 ppm (3JHF ) 6.8 Hz), respectively (Figure 8). Two stable products, 4 and 5 (Figure 9), with 19F NMR chemical shifts of 1.01 (3JHF ) 7.3 Hz) and 0.52 ppm (3JHF ) 7.5
Yin et al.
Hz), respectively, were formed in a 1:4 ratio after prolonged heating at 80 °C, but were not formed at 37 °C. Products 4 and 5 were identified as (2R,5R)- and (2S,5R)-5-amino-6-[(carboxymethyl)imino]-2-(trifluoromethyl)-1,3-oxathiane, respectively, by conversion to their methyl esters 6 and 7 and by spectral analysis of the purified derivatives. Characterization of compounds 6 and 7 by mass spectrometry and 1H NMR unequivocally showed the loss of a γ-glutamyl moiety, which shows that compounds 4 and 5 lack γ-glutamyl groups. Compounds 6 and 7 are diastereomers, and their absolute stereochemistry was established by the observation that the two amino protons (HX and HY, see Figure 9) are distinct because of intramolecular hydrogen bonding between HX and the ring oxygen in isomer 6, but not in isomer 7, when acetone-d6 was used as a solvent. Such intramolecular hydrogen bonding in isomer 6 was disrupted by DMSO, a solvent that competitively formed intermolecular hydrogen bonds with the amino protons and resulted in the merging of the two amino protons at 3.65 ppm, a chemical shift indicative of hydrogen bonding (in the absence of hydrogen bonds, amino protons, such as in compound 7, resonate at about 2.88 ppm). The loss of a γ-glutamyl moiety is probably the result of hydrolysis after ring formation between trifluoroacetaldehyde, the thiol group of the cysteinyl moiety, and the carbonyl oxygen of the glycinyl moiety. Reaction of Trifluoroacetaldehyde with Other Nucleophiles. The amino group in urea and the terminal primary amino group in L-citrulline reacted with trifluoroacetaldehyde to give imines (Table 3); their spectral data are summarized in Table 4. The thiol group in 2-mercaptoethanol reacted reversibly with trifluoroacetaldehyde to give a thiohemiacetal with a 19F NMR chemical shift of -0.85 ppm (3JHF ) 6.9 Hz). Hemiacetal formation was not observed with the hydroxyl group in ethanolamine at 37 °C. Comparison of the Reactivities of Different Functional Groups with Trifluoroacetaldehyde. The relative reactivities of sulfhydryl, amino, and hydroxyl groups with trifluoroacetaldehyde were determined by the integration of 19F NMR resonances after a 2-h incubation at 37 °C (Table 5). The reactivities of sulfhydryl and amino groups with trifluoroacetaldehyde were (5.0 × 102)-(4.8 × 104)- and 1-44-fold higher than that of water, respectively. The reactivity of each functional group was also dependent on the reaction conditions. At pH 7.0 and 37 °C, only L-cysteine, L-cysteine methyl ester, 2-mercaptoethylamine, L-citrulline, urea, ATP, CMP, and GMP reacted to form detectable amounts of stable products. Thiol groups in all compounds tested reacted reversibly with trifluoroacetaldehyde to give thiohemiacetals, but hydroxyl groups in L-serine, ethanolamine, and mercaptoethanol did not yield detectable hemiacetal formation with trifluoroacetaldehyde.
Discussion The reaction of trifluoroacetaldehyde, as a model trihaloacetaldehyde, with various nucleophiles, including amino acids, nucleotides, lipid components, and their analogs, was investigated. Stable products were formed with L-cysteine, L-cysteine methyl ester, L-citrulline, urea, ATP, GMP, and CMP. Reversible product formation was observed with N-acetyl-L-cysteine, glutathione, and 2-mercaptoethanol under physiological conditions (37 °C, pH 7.0). At 80 °C, stable products were also formed with glutathione and 2-mercaptoethylamine.
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Chem. Res. Toxicol., Vol. 9, No. 1, 1996 145
Figure 9. Reaction of glutathione with trifluoroacetaldehyde to give 1,3-oxathianes 4 and 5 and their methylation to esters 6 and 7. Table 4. Summary of
19F
nucleophile
reversible hemiacetal -0.61 ppm (3JHF ) 6.8 Hz) -0.71 ppm (3JHF ) 6.8 Hz) -0.76 ppm (3JHF ) 6.8 Hz) -0.85 ppm (3JHF ) 6.8 Hz) -0.53 ppm (3JHF ) 6.8 Hz) -0.60 ppm (3JHF ) 6.8 Hz) -0.78 ppm (3JHF ) 6.8 Hz) -0.85 ppm (3JHF ) 6.9 Hz) -0.74 ppm (3JHF ) 6.8 Hz) -0.82 ppm (3JHF ) 6.8 Hz)
L-cysteine
N-acetyl-L-cysteine L-cysteine
of Trifluoroacetaldehyde with Various Nucleophilesa,b
methyl ester
2-mercaptoethylamine 2-mercaptoethanol glutathione L-citrulline
urea ATP GMP CMP water
stable product formation (S,R) 0.76 ppm (3JHF ) 7.3 Hz) (R,R) 1.51 ppm (3JHF ) 7.3 Hz) (S,R) -0.71 ppm (3JHF ) 6.7 Hz) (R,R) 1.39 ppm (3JHF ) 7.3 Hz) 1.83 ppm (3JHF ) 7.8 Hz) 0.52 ppm (3JHF ) 7.5 Hz) 1.01 ppm (3JHF ) 7.3 Hz) 1.67 ppm (3JHF ) 6.7 Hz) -5.53 ppm (3JHF ) 5.2 Hz) -5.43 ppm (3JHF ) 5.2 Hz) -5.00 ppm (3JHF ) 5.5 Hz) -5.20 ppm (3JHF ) 4.9 Hz) -5.00 ppm
-8.51 ppm (3JHF ) 3.6 Hz)
a All 19F spectral data were recorded in 100 mM potassium phosphate buffer (pH 7.0). Chemical shifts are sensitive to ionic strength, pH, and solvent. b Chemical shifts were referenced externally to 5 mM trifluoroacetamide in D2O (δtrifluoroacetamide ) 0 ppm).
Table 5. Relative Reactivity of Different Nucleophiles with Trifluoroacetaldehyde Hydrate nucleophiles
functional group
Keq
N-acetyl-L-cysteine L-cysteine L-cysteine methyl ester 2-mercaptoethanol 2-mercaptoethylamine glutathione L-citrulline urea CMP ATP GMP water TMP
SH SH SH SH SH SH NH2 NH2 NH2 NH2 NH2 H 2O control
4.8 × 104 1.4 × 103 6.2 × 102 5.5 × 102 5.5 × 102 5.0 × 102 4.4 × 101 2.0 × 101 5.2 2.8 1 1 0
The reaction of trifluoroacetaldehyde with L-cysteine initially resulted in the reversible and concentrationdependent formation of diastereomeric thiohemiacetals in equal amounts. Subsequent ring closure of the thiohemiacetals lead to the stereoselective formation of diastereomeric thiazolidine derivatives. The stereoselectivity of thiazolidine formation was temperaturedependent. A kinetically controlled reaction at 37 °C favored the formation of isomer 3, whereas a thermodynamically controlled reaction at 80 °C favored the formation of isomer 2. The carboxylic group in L-cysteine was
important in determining the stereoselective course of the reaction: L-cysteine methyl ester did not exhibit any stereoselectivity in the formation of diastereomeric thiazolidine derivatives, perhaps due to the influence of hydrogen bonding between the R-amino group and the carboxylic group in the transition state of the reaction. N-Acetyl-L-cysteine failed to form a thiazolidine derivative, indicating that a free amino group is necessary for ring closure of the thiohemiacetal intermediates. These reactions, in addition to their biological implications, may also have application in the stereoselective synthesis of five-membered heterocyclic compounds. The thiazolidine compounds formed from trifluoroacetaldehyde and Lcysteine may also possibly be used as cysteine prodrugs (10). The reaction of trifluoroacetaldehyde with glutathione at 37 °C showed reversible thiohemiacetal formation, whereas reaction at 80 °C resulted in the stereoselective formation of stable 1,3-oxathianes 4 and 5. Moreover, ring closure was accompanied by loss of the glutamyl group. Further research will be needed to determine whether similar reactions occur with proteins that lead to cleavage of peptides at the N-terminus of L-cysteine residues. The oxygen atom in 1,3-oxathianes is most likely derived from the carbonyl group in the L-glycine moiety. The lone electron pair on nitrogen in L-glycine
146 Chem. Res. Toxicol., Vol. 9, No. 1, 1996
may be donated to form an “enolate-like” structure, and the oxygen anion may then attack the thiohemiacetal carbon, leading to ring closure. Another possible mechanism of ring closure is attack of the hemiacetal hydroxyl group on the amide carbonyl carbon followed by the loss of water to form an imine. This latter mechanism should, however, lead to the cleavage of L-glycine, which was not observed. Naylor et al. (11) studied the reaction of glutathione with [13C]formaldehyde and reported the formation of a six-membered γ-thio-δ-lactam on the basis of the mass spectrum of the products. Analogous products were not observed in our experiments. The reaction of trifluoroacetaldehyde with glutathione may also have use in the stereoselective synthesis of six-membered heterocyclic compounds. The formation of stable imine products by the reaction of trifluoroacetaldehyde with nucleotides may provide an explanation for the observed covalent modification of DNA and RNA by metabolites of 1,1,1,2-tetrahaloethanes (9). The biotransformation of 1,1,1,2-tetrahaloethanes by cytochrome P450 will putatively lead to the formation of trichloroacetaldehyde, which may also react to form a stable imine. Previous studies with formaldehyde showed that the conjugated imine formed by reaction with nucleotides may react with another nucleotide or with protein nucleophiles and lead to cross-links (12-15). It is not clear at this moment whether trihaloacetaldehydes cause cross-linking. The relative reactivities of various cellular nucleophiles, including sulfhydryl, amino, and hydroxy groups, with trifluoroacetaldehyde were compared at pH 7.0 and 37 °C. The results are summarized in Tables 1-5. In general, the reactivity followed the order SH > NH2 > OH. Sulfhydryl groups reacted readily and reversibly with trifluoroacetaldehyde to form thiohemiacetals, stable thiazolidine, or 1,3-oxathianes when a neighboring functional group, such as in L-cysteine, 2-mercaptoethylamine, and glutathione, was present. Amide nitrogens, as in urea and L-citrulline, or exocyclic amino groups in ATP, CMP, and GMP reacted with trifluoroacetaldehyde to form stable, conjugated imine adducts. Primary amines, as in 2-aminoethanol and 2-mercaptoethylamine, did not react. The hydroxyl groups in either water or ethanol showed the lowest reactivity toward trifluoroacetaldehyde. The minor metabolite (19F NMR chemical shift 2.45 ppm, J ) 7.3 Hz) found in the urine of rats given HCFC133a was not identified. The information obtained from the present study, however, indicated that it may be an adduct formed by reaction of trifluoroacetaldehyde with a cellular nucleophile (3). The same adduct was detected in the urine of rats given trifluoroacetaldehyde (3). The chemical shift indicates that it may contain a (trifluoromethyl)ethylene moiety attached to two heteroatoms (N, S, or O) in a ring system. The 19F NMR chemical shifts in related open-chain systems are as follows: trifluoroacetaldehyde [CF3CH(OH)(OH)], ca. -9 ppm; L-cysteine thiohemiacetal [CF3C(SR)(OH)], ca. -0.61 to -0.71 ppm; GMP [CF3CHdN+HR], ca -5.0 ppm. These chemical shift data indicate that the unknown metabolite may not be any of these analogs, but rather possibly has a CF3 moiety attached to a ring structure. In fact, the coresonance 19F NMR spectra of urine samples from HCFC-133a-treated rats mixed with trifluoroacetaldehyde-L-cysteine adducts showed that the chemical shift of (2S,4R)-2-(trifluoromethyl)thiazolidine-4-carboxylic acid (2.27 ppm, J ) 7.3 Hz) was only 0.18 ppm upfield of the
Yin et al.
unknown metabolite. Further experiments ruled out trifluoroacetaldehyde-glutathione adducts as the unknown metabolite. In conclusion, trifluoroacetaldehyde was shown to be capable of reacting with cellular nucleophiles. The reaction of trifluoroacetaldehyde with these nucleophiles may have biological implications, as well as potential use in chemical synthesis.
Acknowledgment. We thank Sandra E. Morgan for her assistance in the preparation of the manuscript and Dr. Scott Kennedy for providing access to the University of Rochester Cancer Center NMR facility (supported in part by grant NCI CA11198) and for his help in conducting NOE experiments. H.Y. was supported by a fellowship from the Pharmaceutical Research and Manufacturers of America Foundation, Inc. This research was supported by National Institute of Environmental Health Science Grant ES05407 to M.W.A.
References (1) Rall, T. W. (1990) Hypnotics and sedatives; ethanol. In Goodman and Gilman’s The Pharmacological Basis of Therapeutics (Gilman, A. G., Rall, T. W., Nies, A. S., and Taylor, P., Eds.) pp 345382, Pergamon Press, New York. (2) Kaminsky, L. S., Fraser, J. M., Seaman, M., and Dunbar, D. I. (1992) Rat liver metabolism and toxicity of 2,2,2-trifluoroethanol. Biochem. Pharmacol. 44, 1829-1837. (3) Yin, H., Jones, J. P., and Anders, M. W. (1995) Metabolism of 1-fluoro-1,1,2-trichloroethane, 1,2-dichloro-1,1-difluoroethane, and 2-chloro-1,1,1-trifluoroethane. Chem. Res. Toxicol. 8, 262-268. (4) Olson, M. J., Reidy, C. A., and Johnson, J. T. (1990) Defluorination of 1,1,1,2-tetrafluoroethane (R-134a) by rat hepatocytes. Biochem. Biophys. Res. Commun. 166, 1390-1397. (5) Olson, M. J., Kim, S. G., Reidy, C. A., Johnson, J. T., and Novak, R. F. (1991) Oxidation of 1,1,1,2-tetrafluoroethane (R-134a) in rat liver microsomes is catalyzed primarily by cytochrome P450IIE1. Drug Metab. Dispos. 19, 298-303. (6) Olson, M. J., Reidy, C. A., Johnson, J. T., and Pederson, T. C. (1990) Oxidative defluorination of 1,1,1,2-tetrafluoroethane (R134a) by rat liver microsomes. Drug Metab. Dispos. 18, 992-998. (7) Harris, J. W., and Anders, M. W. (1991) Metabolism of the hydrochlorofluorocarbon 1,2-dichloro-1,1-difluoroethane. Chem. Res. Toxicol. 4, 180-186. (8) Yin, H., Jones, J. P., and Anders, M. W. (1993) Slow-binding inhibition of carboxylesterase and other serine hydrolases by chlorodifluoroacetaldehyde. Chem. Res. Toxicol. 6, 630-634. (9) Colacci, A., Bartoli, S., Bonora, B., Buttazzi, C., Lattanzi, G., Mazzullo, M., Niero, A., Turina, M. P., and Grilli, S. (1989) Covalent binding of 1,1,1,2-tetrachloroethane to nucleic acids as evidence of genotoxic activity. J. Toxicol. Environ. Health 26, 485-495. (10) Roberts, J. C., Nagasawa, H. T., Zera, R. T., Fricke, R. F., and Goon, D. J. W. (1987) Prodrugs of L-cysteine as protective agents against acetaminophen-induced hepatotoxicity. 2-(Polyhydroxyalkyl)- and 2-(polyacetoxyalkyl)thiazolidine-4(R)-carboxylic acids. J. Med. Chem. 30, 1891-1896. (11) Naylor, S., Mason, R. P., Sanders, J. K. M., and Williams, D. H. (1988) Formaldehyde adducts of glutathione: Structure elucidation by two-dimensional N.M.R. spectroscopy and fast-atombombardment tandem mass spectrometry. Biochem. J. 249, 573579. (12) Solomon, M. J., and Varshvavsky, A. (1985) Formaldehydemediated DNA-protein crosslinking: A probe for in vivo chromatin structure. Proc. Natl. Acad. Sci. U.S.A. 82, 6470-6474. (13) Hashmi, M., Dechert, S., Dekant, W., and Anders, M. W. (1994) Bioactivation of [13C]dichloromethane in mouse, rat, and human liver cytosol: 13C Nuclear magnetic resonance spectroscopic studies. Chem. Res. Toxicol. 7, 291-296. (14) Goldmacher, V. S., and Thilly, W. G. (1983) Formaldehyde is mutagenic for cultured human cells. Mutat. Res. 116, 417-422. (15) Casanova, M., Deyo, D. F., and Heck, H. D. (1989) Covalent binding of inhaled formaldehyde to DNA in the nasal mucosa of Fischer 344 rats: Analysis of formaldehyde and DNA by highperformance liquid chromatography and provisional pharmacokinetic interpretation. Fund. Appl. Toxicol. 12, 397-417.
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