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Chem. Res. Toxicol. 1998, 11, 794-799
Thietanium Ion Formation from the Food Mutagen 2-Chloro-4-(methylthio)butanoic Acid Larry J. Jolivette,† Andrew S. Kende,‡ and M. W. Anders*,† Department of Pharmacology and Physiology, University of Rochester, 601 Elmwood Avenue, Box 711, Rochester, New York 14642, and Department of Chemistry, University of Rochester, Hutchinson Hall, River Campus, Rochester, New York 14627-0216 Received January 26, 1998
2-Chloro-4-(methylthio)butanoic acid (1) is a direct-acting mutagen and suspected gastric carcinogen isolated from fish preserved with salt and nitrite. The reactive intermediates formed from acid 1 that may be associated with its mutagenicity have not been identified. A candidate reactive intermediate is proposed in this work. 1-Methyl-2-thietaniumcarboxylic acid may result from internal displacement of chloride by neighboring-group participation of sulfide sulfur in the solvolysis of acid 1. Evidence for the formation of 1-methyl-2-thietaniumcarboxylic acid was derived from experiments in which 4-chlorophenol and aniline were included to react with electrophilic intermediates formed from acid 1. Incubation of acid 1 in the absence of the aniline or 4-chlorophenol resulted in the formation of 2,4-bis(methylthio)butanoic acid. Incubation of acid 1 with 4-chlorophenol or aniline gave adducts that were identified by 1H NMR spectroscopy and GC/MS as 2-(4-chlorophenoxy)-4-(methylthio)butanoic acid and 4-(4chlorophenoxy)-2-(methylthio)butanoic acid or 2-anilino-4-(methylthio)butanoic acid and 4-anilino2-(methylthio)butanoic acid, respectively. The structures of these adducts indicate the intermediate formation of 1-methyl-2-thietaniumcarboxylic acid as a reactive intermediate derived from acid 1 that may be associated with its observed mutagenicity.
Introduction 2-Chloro-4-(methylthio)butanoic acid (1), which was isolated from salt- and nitrite-treated fish and similarly treated methionine, is a direct-acting mutagen in the Ames test and is a suspected gastric carcinogen (1, 2). The reactive intermediates associated with the observed mutagenicity of acid 1 have not been identified. Consideration of candidate structures for reactive intermediates of acid 1 led to the proposal that 1-methyl2-thietaniumcarboxylic acid (2) (Scheme 1) may be formed by the internal displacement of chloride by neighboring-group participation of sulfide sulfur. Precedent for the formation of thietanium compounds by neighboring-group participation has been established by experiments in which thietanium ions reacted with oxygen nucleophiles during solvolysis of 3-(alkylthio)- and 3-(arylthio)propyl p-toluenesulfonates (3). The objective of the present study was to investigate whether the solvolysis of acid 1 leads to the formation of 1-methyl-2-thietaniumcarboxylic acid. Incubation of acid 1 with 4-chlorophenol or aniline led to the formation of adducts, which were identified by 1H NMR spectroscopic and mass spectral analysis, that indicate the formation of 1-methyl-2-thietaniumcarboxylic acid.
Materials and Methods Materials. Acid 1 was obtained by synthesis, as described below. HF‚pyridine (70/30, w/w), 4-chlorophenol, and aniline were purchased from Aldrich Chemical Co. (Milwaukee, WI). * Address correspondence to: M. W. Anders. Tel: 716-275-1681. Fax: 716-244-9283. E-mail:
[email protected]. † Department of Pharmacology and Physiology. ‡ Department of Chemistry.
Synthesis of 2-Chloro-4-(methylthio)butanoic Acid, 1. 2-Chloro-4-(methylthio)butanoic acid was synthesized according to the procedure of Olah et al. (4). L-Methionine (2.24 g, 15 mmol) and potassium chloride (2.24 g, 30 mmol) were added to 20 g of a 48:52 mixture of HF‚pyridine, prepared by diluting HF‚pyridine with dry pyridine, and sodium nitrite (2.07 g, 30 mmol) was added to the mixture in three portions over 10 min. The reaction mixture was stirred under nitrogen at room temperature for 3 days. The reaction mixture was poured into 50 mL of ice water, and the aqueous mixture was extracted with diethyl ether (3 × 75 mL). The combined organic layers were extracted with 1.2 M HCl (6 × 50 mL), dried over anhydrous MgSO4, and concentrated to yield crude acid 1. The product was purified by flash chromatography on silica gel with 20% (v/v) ethyl acetate in hexane as the eluent. The yield was 1.10 g (44%), and the product was characterized by 1H NMR spectroscopy. A sample of the product was derivatized with diazomethane (Caution: diazomethane is toxic and mutagenic and should be used with care in an efficient fume hood) to form the methyl ester of acid 1 and analyzed by GC/MS, which indicated that the product (methyl ester) was about 91% pure: 1H NMR (CDCl ) δ 4.63-4.70 (dd, 1 H), 2.74-2.82 (m, 2 H), 3 2.29-2.40 (m, 2 H), 2.19-2.21 (s, 3 H); GC/MS m/z (%) 184 (5.9), 182 (15.4), 123 (3.3), 121 (9.6), 110 (10.4), 108 (33.5), 75 (61.1), 61 (100), 59 (34.2). Instrumental Analyses. GC/MS analyses were performed with a Hewlett-Packard 5790 gas chromatograph (30 m × 0.2 mm, 0.33-µm film thickness, HP-1 cross-linked methyl silicone column; splitless injection) coupled to a Hewlett-Packard 5970B mass selective detector; the injector and transfer-line temperatures were 240 and 285 °C, respectively. The samples were analyzed with a temperature program of 50 °C for 1 min followed by a linear gradient of 10 °C/min to 250 °C, which was maintained for 5 min. 1H NMR spectra were recorded with a Bruker 270-MHz spectrometer operating at 270.13 MHz. Chemical shifts are expressed in ppm downfield from tetramethylsilane (δ ) 0).
S0893-228x(98)00016-2 CCC: $15.00 © 1998 American Chemical Society Published on Web 05/30/1998
Thietanium Ion Formation
Chem. Res. Toxicol., Vol. 11, No. 7, 1998 795 Scheme 1
Resonances were assigned to protons by comparison of observed chemical shifts with chemical shifts extracted or calculated from published tables based on Shoolery’s rules (5). HPLC. Adducts were purified with a Hewlett-Packard 1090 HPLC system coupled to a Waters µ-Bondapak C18 column (7.8 × 300 mm). The column was eluted with 70:30 methanol/water (adjusted to pH 2.0 with trifluoroacetic acid) for 4-chlorophenol adducts or with 60:40 methanol/water (adjusted to pH 2.0 with trifluoroacetic acid) for aniline adducts at a flow rate of 2 mL/ min. The absorbance of the eluate was measured at 220, 250, and 280 nm with a Hewlett-Packard 1040A diode-array detector. Incubations. Acid 1 (50 µmol) was added to 0.2 M sodium bicarbonate buffer (pH 8.1) in D2O to make 1 mL of a 50 mM solution. 1H NMR spectra were recorded at room temperature each hour for 11 h to determine the stability of acid 1 in aqueous solution. Reaction mixtures were incubated at 37 °C for 24 h in 1.5mL microcentrifuge tubes containing 200 mM sodium bicarbonate buffer (pH 8.1) and 21 mM acid 1 in a final volume of 1 mL. Aniline or 4-chlorophenol was added to the reaction mixture at a final concentration of 21 mM, and the reaction mixtures were incubated for 4 and 24 h. At the end of the incubation period, the incubation mixtures were brought to pH 2 by addition of HCl and extracted three times with 2 mL of ethyl acetate. The organic phases were combined, concentrated under a stream of N2, and analyzed by GC/MS. Isolation of Aniline and 4-Chlorophenol Adducts of Acid 1. Acid 1 (0.735 mmol) was incubated at 80 °C for 14 h in 35 mL of 0.2 M sodium bicarbonate buffer (pH 8.1) in the presence of aniline or 4-chlorophenol (0.735 mmol). The incubation mixtures were brought to pH 2 by addition of HCl and extracted three times with 50 mL of ethyl acetate. The organic layers were combined and extracted with 35 mL of 1 M sodium bicarbonate buffer (pH 7.9). The aqueous layer was brought to pH 2 with HCl and extracted three times with 50 mL of ethyl acetate. The organic layer was dried over anhydrous MgSO4, concentrated in vacuo, and dissolved in methanol sufficient to yield a concentration of 1 mg of solute/20 µL of solvent. The adducts were resolved by HPLC, and the eluate fractions containing peaks of interest were collected and analyzed by GC/ MS and 1H NMR spectroscopy.
Results Fate of Acid 1 in Aqueous Solution. Acid 1 was incubated in bicarbonate buffer (pH 8.1) and analyzed
by 1H NMR spectroscopy hourly for 11 h. A new resonance was observed at 2.17 ppm after 1 h, and the strength of this signal increased over the next 10 h (data not shown), indicating that acid 1 is not stable in an aqueous solution. Acid 1 was incubated at 37 °C for 24 h in 0.2 M bicarbonate buffer (pH 8.1), and the reaction mixture was extracted with ethyl acetate, derivatized with diazomethane, and analyzed by GC/MS. A product that eluted at 14.7 min (the methyl ester of acid 1 eluted at 12.5 min) was observed in the total ion chromatogram and increased in area over the 24-h incubation time (Figure 1A). The mass spectrum of the compound that eluted at 14.7 min indicated the formation of 2,4-bis(methylthio)butanoic acid (4) (Figure 2A). The product was isolated by HPLC, and its assignment as acid 4 was confirmed by its 1H NMR spectrum, which lacked aromatic hydrogens but showed aliphatic chemical shifts that were consistent with the predicted chemical shifts (Table 1, Figure 3A). A product that eluted at 11.6 min was tentatively identified from its mass spectrum as 2,4dihydroxybutanoic acid (10) (data not shown). Reaction of Acid 1-Derived Thietanium Ions with 4-Chlorophenol. 4-Chlorophenol was included with acid 1 in incubation mixtures to react with the putative thietanium ion intermediates to form stable compounds. Workup of reaction mixtures containing acid 1 and 4-chlorophenol that were incubated at 37 °C for 24 h showed that about 20% of acid 1 was lost and that two new products, in addition to those observed in the absence of 4-chlorophenol, were observed (Figure 1B). The products formed in the presence of 4-chlorophenol plus those formed in its absence, i.e., acids 4 and 10, accounted for about 70% of the acid 1 lost. The mass spectra (Figure 2B,C) of the products formed were the methyl esters of the expected products formed by reaction of 1-methyl-2-thietaniumcarboxylic acid with 4-chlorophenol. The products were identified as 2-(4-chlorophenoxy)-4-(methylthio)butanoic acid (5) and 4-(4chlorophenoxy)-2-(methylthio)butanoic acid (6). Acids 4, 5, 6, and 10 were formed in the ratio 2:1:1:2, based on their relative peak areas in the total ion chromatogram. Acids 4, 6, and 10 amount to 5/6 (about 83%) of the
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Figure 1. Reaction of acid 1-derived reactive intermediates with 4-chlorophenol or aniline. Acid 1 was incubated in sodium bicarbonate buffer (pH 8.1) for 0 and 24 h at 37 °C in the absence or presence of 4-chlorophenol or aniline. The incubation mixtures were extracted with ethyl acetate, derivatized with diazomethane, and analyzed by GC/MS, as described in Materials and Methods. A: Total ion chromatogram of extracts of incubation mixture incubated for 0 and 24 h in the absence of 4-chlorophenol or aniline. The mass spectrum of the product that eluted at 14.7 min is shown in Figure 2A. B: Total ion chromatogram of extracts of incubation mixture incubated for 24 h in the presence of 4-chlorophenol. The mass spectra of the products that eluted at 21.5 and 22.1 min are shown in Figure 2B,C. C: Total ion chromatogram of extracts of incubation mixture incubated for 24 h in the presence of aniline. The mass spectra of the products that eluted at 21.2 and 22 min are shown in Figure 2D,E. The product that eluted at 14.7 min in Figure 1A is the same product that eluted at 15 min in Figure 1B and at 15.5 min in Figure 1C. The change in elution times was due to a decrease in the effective column length as the column aged.
products formed. This indicates that at least 60% of acid 1 formed thietanium ion 2, which yielded observed acids 4, 6, and 10. Acid 1 and 4-chlorophenol were incubated at 80 °C for 14 h, at which time acid 1 was no longer detectable, to increase the amount of thietanium ion formed and, thereby, to facilitate product identification. HPLC analysis showed that the adducts that were formed by reaction of 4-chlorophenol with acid 1 eluted at 13.3 and 14.5 min (data not shown). The adducts were collected as they
Jolivette et al.
eluted from the column, dissolved in acetone-d6, and analyzed by GC/MS and 1H NMR spectroscopy. The mass spectra of the purified adducts were identical with those observed in the experiments described above (Figure 2B,C). The 1H NMR spectrum of the product that eluted at 13.3 min from the HPLC column showed chemical shifts consistent with the structural assignment of 2-(4-chlorophenoxy)-4-(methylthio)butanoic acid (5) (Table 1, Figure 3B). The product that eluted at 14.5 min was identified as 4-(4-chlorophenoxy)-2-(methylthio)butanoic acid (6) (Table 1, Figure 3C). Reaction of Acid 1-Derived Thietanium Ions with Aniline. Aniline was included with acid 1 in incubation mixtures to react with the putative thietanium ion intermediates to form stable compounds. After workup of the reaction mixtures as described above, two new products, in addition to those observed in the absence of aniline, were observed (Figure 1C). The mass spectra (Figure 2D,E) of the products formed, after derivatization with diazomethane, were the methyl esters of the expected products formed by reaction of 1-methyl-2-thietaniumcarboxylic acid with aniline. The products were identified as 2-anilino-4-(methylthio)butanoic acid (7), 4-anilino-2-(methylthio)butanoic acid (8), and acids 4 and 10. The products formed in the presence of aniline, i.e., acids 7 and 8, plus those formed in its absence, i.e., acids 4 and 10, accounted for nearly all of the acid 1 lost, which amounted to 45%. Acids 4, 7, 8, and 10 were formed in a ratio of 1.5:8:5:1, based on integration of peak areas in the total ion chromatogram. The formation of acids 4, 8, and 10 amounted to about 50% of the products formed, which indicates that at least 50% of acid 1 formed thietanium ion 2, which gave rise to observed acids 4, 8, and 10. Acid 1 and aniline were incubated at 80 °C for 14 h, at which time acid 1 was no longer detectable, to increase product formation. HPLC analysis of the reaction mixture showed the formation of a product formed by reaction of aniline with acid 1 that eluted at 12.7 min (data not shown). The product formed was collected as it eluted from the HPLC column and analyzed by GC/ MS, which showed that the peak that eluted at 12.7 min represented a mixture of two compounds (data not shown). The later-eluting product was detected only after derivatization with diazomethane, whereas detection of the earlier-eluting product was not altered by derivatization with diazomethane. The two products were separated by pH-dependent extraction. The mass spectrum of the earlier-eluting compound was identical with that of acid 7 (Figure 2E). The 1H NMR spectrum showed chemical shifts that were consistent with the predicted chemical shifts for acid 7 (Table 1, Figure 3D). The mass spectrum (Figure 2F) of the later-eluting product indicated that the compound is 3-(methylthio)1-phenyl-2-pyrrolidone (9), the cyclization product of acid 8. This assignment was confirmed by 1H NMR analysis: the proton chemical shifts of the product were consistent with the predicted chemical shifts for pyrrolidone 9 (Table 1, Figure 3E). In addition, acid 10 was also detected by GC/MS analysis (data not shown).
Discussion The objective of the present study was to investigate the formation of acid 1-derived reactive intermediates that may contribute to its mutagenicity. R-Chloro acids
Thietanium Ion Formation
Chem. Res. Toxicol., Vol. 11, No. 7, 1998 797
Figure 2. Mass spectra of the products formed after incubation of acid 1 in the absence or presence of 4-chlorophenol or aniline. Acid 1 was incubated in sodium bicarbonate buffer (pH 8.1) for 24 h at 37 °C in the absence or presence of 4-chlorophenol or aniline. The incubation mixtures were extracted with ethyl acetate, derivatized with diazomethane, and analyzed by GC/MS, as described in Materials and Methods. A: Mass spectrum of the product that eluted at 14.7 min (see Figure 1A) after incubation of acid 1 in buffer. B: Mass spectrum of the product that eluted at 21.5 (see Figure 1B) after incubation of acid 1 with 4-chlorophenol. C: Mass spectrum of the product that eluted at 22.1 min (see Figure 1B) after incubation of acid 1 with 4-chlorophenol. D: Mass spectrum of the product that eluted at 21.2 min (see Figure 1C) after incubation of acid 1 with aniline. E: Mass spectrum of the product that eluted at 22 min (see Figure 1C) after incubation of acid 1 with aniline. F: Mass spectrum of the cyclization product of acid 8 (see Scheme 3) which was formed during HPLC fractionation of the products resulting from the reaction of acid 1 with aniline.
are not, in general, mutagenic: Chen et al. (1) reported that conversion of a range of protein R-amino acids to their respective R-chloro acids did not result in the formation of mutagenic species. In addition, 2-chloropropanoic acid, at concentrations up to 8000 nmol/plate, is not mutagenic in the Ames test.1 In contrast, the R-chloro analogue of methionine, acid 1, is mutagenic (1). Thus, R-chloro substitution alone is not sufficient to impart mutagenicity to alkanoic acids. Consideration of the structure of acid 1, particularly the presence of the thioether functionality, indicated the possibility of neighboring-group participation in cyclization of acid 1. Internal displacement of the R-chlorine 1
S. Vamvakas and I. Herminghaus, personal communication.
by the thioether sulfur may result in formation of 1-methyl-2-thietaniumcarboxylic acid (2) (Scheme 1). Neighboring-group effects in organosulfur compounds are well-known, particularly with 2-haloalkyl thioethers (6). The transition state for thioether participation requires that the thioether sulfur, the R-carbon bearing the leaving group, and the leaving group are in a linear antiperiplanar array; thus, a general requirement for thioether neighboring-group participation is a structure that is sufficiently flexible to accommodate the anti-periplanar transition state or a rigid ring system that holds the participating atoms in the proper configuration. For an acyclic structure of the type RS-(CH2)nX, where X is the leaving group, it is expected that for n ) 2 the neighboring-group participants would be in approximately the
798 Chem. Res. Toxicol., Vol. 11, No. 7, 1998
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Table 1. Predicteda and Observed Chemical Shifts (no. of protons, splitting) of the Acid 1 Adducts Isolated and Analyzed by 1H NMR Spectroscopy (Figure 3)
a Resonances were assigned to protons by comparison of observed chemical shifts with chemical shifts extracted or calculated from published tables based on Shoolery’s rules (5). b The number of protons of these two signals integrated as 5, the sum number of protons predicted for the two signals.
same plane and for n ) 4 and 5 there would be sufficient flexibility in the structure to allow thioether neighboringgroup participation. When n ) 3, however, a planar configuration is not expected because the formation of planar four-membered rings is disfavored enthalpically and entropically. Hence, although thietanium ion formation is not favored, Eliel and Knox demonstrated thietanium ion formation resulting from thioether neighboringgroup participation in 3-(alkylthio)- and 3-(arylthio)propyl p-toluenesulfonates (3). The results presented herein demonstrate that acid 1 in aqueous solution cyclizes to form 1-methyl-2-thietaniumcarboxylic acid (2) (Scheme 1). The formation of a thietanium ion intermediate was established by its reaction with sulfur (acid 1), oxygen (4-chlorophenol), and nitrogen (aniline) nucleophiles, which gave products that resulted from the attack of the nucleophile at the carbons R to the sulfonium sulfur. The reaction of thietanium ion 2 with acid 1 may be expected to give sulfonium ion [3-carboxy-3-(methylthio)propyl](3-carboxy-3-chloropropyl)methylsulfonium (3) (Scheme 1). Attack of the carboxylate oxygen of the 3-carboxy-3-(methylthio)propyl on the carbon R to the sulfonium ion would result in the formation of acid 1 and R-(methylthio)butyrolactone, but hydrolysis products of the lactone were not observed (Scheme 1, reaction a). Attack of the carboxylate oxygen of the 3-carboxy-3chloropropyl group on the carbon R to the sulfonium ion may result in loss of R-chlorobutyrolactone, which may undergo hydrolysis to give 2,4-dihydroxybutanoic acid (10) and 2,4-bis(methylthio)butanoic acid (4) (Scheme 1, reaction b).
Figure 3. 1H NMR spectra of products formed after incubation of acid 1 in the presence or absence of 4-chlorophenol or aniline (see Figure 1): A, 2,4-bis(methylthio)butanoic acid (4); B, 2-(4chlorophenoxy)-4-(methylthio)butanoic acid (5); C, 4-(4-chlorophenoxy)-2-(methylthio)butanoic acid (6); D, 2-anilino-4(methylthio)butanoic acid (7); E, 3-(methylthio)-1-phenyl-2pyrrolidone (9). Numbers correspond to chemical shifts shown in Table 1. Spectra A and E were recorded in CDCl3, spectra B and C were recorded in CD3COCD3, and spectrum D was recorded in CD3CD2OD.
The incubation of acid 1 in the presence of 4-chlorophenol resulted in the formation of 2-(4-chlorophenoxy)-4(methylthio)butanoic acid (5) and 4-(4-chlorophenoxy)2-(methylthio)butanoic acid (6) (Scheme 2). The formation of acids 5 and 6 can be explained by the attack of 4-chlorophenol on the carbons R to the sulfonium ion of thietanium ion 2 (Scheme 2, pathways a and b, respectively). Acid 5 could arise by the direct displacement of chloride in acid 1 by 4-chlorophenol, but a direct displacement reaction would not be expected to give observed acid 6. Similarly, incubation of acid 1 in the presence of aniline gave 2-anilino-4-(methylthio)butanoic acid (7) and 4-anili-
Thietanium Ion Formation Scheme 2
Scheme 3
Chem. Res. Toxicol., Vol. 11, No. 7, 1998 799
diazomethane. These data indicate that thietanium ion 2 formed from acid 1 does not serve as a methylating agent. The finding that thietanium ion 2 undergoes ringopening reactions with sulfur and oxygen nucleophiles is in agreement with previous studies that showed that the reaction of thietanium ions with nucleophiles leads to ring-opened products (9, 10). Methyl group transfer, which is a common reaction in biological systems, was not observed with thietanium ion 2 but is not a common reaction in chemical systems, apparently because of the requirement for an enzyme-stabilized linear transition state (11). In summary, the data presented herein provide evidence for the formation of a thietanium ion intermediate from acid 1. Because of the known reactivity of sulfonium ions, further studies are warranted to investigate the role of thietanium ion formation in the mutagenicity of acid 1.
Acknowledgment. This research was supported by National Institutes of Environmental Health Sciences Grant ES03127 (M.W.A.) and National Institute of General Medical Sciences Grant T32 GM08427 (L.J.J.). The authors thank Ms. Sandra Morgan for her assistance in preparing the manuscript.
References
no-2-(methylthio)butanoic acid (8) (Scheme 3). Acids 7 and 8 may be formed by the attack of aniline on the carbons R to the sulfonium ion of thietanium ion 2 (Scheme 3, pathways a and b, respectively). Acid 7 may also be formed by the direct displacement of chloride from acid 1 by aniline, but a direct displacement reaction does not account for the formation of acid 8. When acid 1 was incubated in the presence of aniline, 3-(methylthio)-1phenyl-2-pyrrolidone (9) was formed (Scheme 3). γ-Lactam 9, which was formed during workup of the reaction mixture, may arise by the cyclization of acid 8, which provides further evidence for the formation of acid 8 and its precursor thietanium ion 2. Thietanium ion 2 may also act as a spurious methyl donor, as demonstrated for other sulfonium ions (7, 8). Although sought, no evidence for methyl group transfer was obtained: 4-chloroanisole could not be detected by GC/MS analysis prior to derivatization with diazomethane. Similarly, neither 2-thietanecarboxylic acid or its methyl ester nor the methyl ester of acid 1 was observed by GC/MS analysis before derivatization with
(1) Chen, W., Weisburger, J. H., Fiala, E. S., Spratt, T. E., Carmella, S. G., Chen, D., and Hecht, S. S. (1996) Gastric carcinogenesis: 2-Chloro-4-methylthiobutanoic acid, a novel mutagen in salted, pickled sanma hiraki fish, or similarly treated methionine. Chem. Res. Toxicol. 9, 58-66. (2) Furihata, C., Oka, M., Amin, S., Krzeminski, J., Weisburger, J. H., Kobayashi, K., and Tatematsu, M. (1996) Effect of 2-chloro4-methylthiobutanoic acid in a rapid bioassay for gastric carcinogens. Cancer Lett. 108, 129-135. (3) Eliel, E. L., and Knox, D. E. (1985) Neighboring group participation by sulfur involving four-membered-ring intermediates (RS4). J. Am. Chem. Soc. 107, 2946-2952. (4) Olah, G. A., Shih, J., and Prakash, G. K. S. (1983) Preparation of R-bromo- and R-chlorocarboxylic acids from R-amino acids. Helv. Chim. Acta 66, 1028-1030. (5) Silverstein, R. M., Bassler, G. C., and Morrill, T. C. (1991) Spectrometric Identification of Organic Compounds, Wiley, New York. (6) Gundermann, K. D. (1963) Neighboring group and substituent effects in organosulfur compounds. Angew. Chem., Int. Ed. Engl. 2, 674-683. (7) Barrows, L. R., and Magee, P. N. (1982) Nonenzymatic methylation of DNA by S-adenosylmethionine in vitro. Carcinogenesis 3, 349-351. (8) Hoffman, J. L. (1994) Bioactivation by S-adenosylation, Smethylation, or N-methylation. Adv. Pharmacol. 27, 449-477. (9) Dittmer, D. C., and Patwardhan, B. H. (1981) Cyclic sulphonium salts. In The Chemistry of the Sulphonium Group (Stirling, C. J. M., Eds.) pp 387-522, Wiley, New York. (10) Dittmer, D. C., and Sedergran, T. C. (1985) Four-Membered Sulfur Heterocycles. In Small Ring Heterocycles, Part 3 (Hassner, A., Eds.) pp 437-768, Wiley, New York. (11) Knipe, A. C. (1981) Reactivity of sulphonium salts. In The Chemistry of the Sulphonium Group (Stirling, C. J. M., Eds.) pp 314-385, Wiley, New York.
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