Thietanium ion formation from the food mutagen 2-chloro-4- (methylthio)butanoic acid

Article abstract of DOI:10.1021/tx980016r

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-(4-chlorophenoxy)- 2-(methylthio)butanoic acid or 2-anilino-4-(methylthio)butanoic acid and 4- anilino-2-(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.

Full text of DOI:10.1021/tx980016r

794
Chem. Res. Toxicol. 1998, 11, 794-799
Th ieta n iu m Ion F or m a tion fr om th e F ood Mu ta gen
2-Ch lor o-4-(m eth ylth io)bu ta n oic Acid
Larry J . J olivette,† 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 J anuary 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.
1
Incubation of acid 1 with 4-chlorophenol or aniline gave adducts that were identified by H
NMR spectroscopy and GC/MS as 2-(4-chlorophenoxy)-4-(methylthio)butanoic acid and 4-(4-
chlorophenoxy)-2-(methylthio)butanoic acid or 2-anilino-4-(methylthio)butanoic acid and 4-anilino-
2-(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.
Syn th esis of 2-Ch lor o-4-(m eth ylth io)bu ta n oic Acid , 1.
2-Chloro-4-(methylthio)butanoic acid was synthesized according
In tr od u ction
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-methyl-
2-thietaniumcarboxylic acid (2) (Scheme 1) may be
formed by the internal displacement of chloride by
neighboring-group participation of sulfide sulfur. Pre-
cedent 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.
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 (Ca u tion : 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 (CDCl3) δ 4.63-4.70 (dd, 1 H), 2.74-2.82 (m, 2 H),
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).
In str u m en ta l An a lyses. 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 temper-
atures 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.
Ma ter ia ls a n d Meth od s
Ma ter ia ls. 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: anders@pharmacol.rochester.edu.
† Department of Pharmacology and Physiology.
‡ Department of Chemistry.
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
Sch em e 1
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 aro-
matic 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 2,4-
dihydroxybutanoic acid (10) (data not shown).
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).
HP LC. 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.
In cu ba tion s. 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 1.5-
mL microcentrifuge tubes containing 200 mM sodium bicarbon-
ate 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.
Isola tion of An ilin e a n d 4-Ch lor op h en ol Ad d u cts 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 incuba-
tion 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.
Rea ction of Acid 1-Der ived Th ieta n iu m Ion s w ith
4-Ch lor op h en ol. 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-chlo-
rophenol. The products were identified as 2-(4-chlo-
rophenoxy)-4-(methylthio)butanoic acid (5) and 4-(4-
chlorophenoxy)-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
Resu lts
F a te of Acid 1 in Aqu eou s Solu tion . Acid 1 was
incubated in bicarbonate buffer (pH 8.1) and analyzed

796 Chem. Res. Toxicol., Vol. 11, No. 7, 1998
J olivette 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).
Rea ction of Acid 1-Der ived Th ieta n iu m Ion s w ith
An ilin e. 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 ex-
pected products formed by reaction of 1-methyl-2-thieta-
niumcarboxylic 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 mix-
ture 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 detec-
tion 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
F igu r e 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 mix-
tures were extracted with ethyl acetate, derivatized with
diazomethane, and analyzed by GC/MS, as described in Materi-
als 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.
1
with that of acid 7 (Figure 2E). The H 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
1
8. This assignment was confirmed by H NMR analy-
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 analy-
sis 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
sis: the proton chemical shifts of the product were
consistent with the predicted chemical shifts for pyrroli-
done 9 (Table 1, Figure 3E). In addition, acid 10 was
also detected by GC/MS analysis (data not shown).
Discu ssion
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
F igu r e 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-chloro-
propanoic 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 cycliza-
tion of acid 1. Internal displacement of the R-chlorine
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 anti-
periplanar array; thus, a general requirement for thio-
ether 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 neighbor-
ing-group participants would be in approximately the
1
S. Vamvakas and I. Herminghaus, personal communication.

798 Chem. Res. Toxicol., Vol. 11, No. 7, 1998
J olivette et al.
Ta ble 1. P r ed icted a a n d Obser ved Ch em ica l Sh ifts (n o.
of p r oton s, sp littin g) of th e Acid 1 Ad d u cts Isola ted a n d
An a lyzed by 1H NMR Sp ectr oscop y (F igu r e 3)
a
Resonances were assigned to protons by comparison of ob-
served chemical shifts with chemical shifts extracted or calculated
b
from published tables based on Shoolery’s rules (5). 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 neighboring-
group 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 forma-
tion is not favored, Eliel and Knox demonstrated thieta-
nium ion formation resulting from thioether neighboring-
group 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-thieta-
niumcarboxylic 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.
F igu r e 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-(4-
chlorophenoxy)-4-(methylthio)butanoic acid (5); C, 4-(4-chlo-
rophenoxy)-2-(methylthio)butanoic acid (6); D, 2-anilino-4-
(methylthio)butanoic acid (7); E, 3-(methylthio)-1-phenyl-2-
pyrrolidone (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 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-3-
chloropropyl 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).
The incubation of acid 1 in the presence of 4-chlorophe-
nol 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, respec-
tively). Acid 5 could arise by the direct displacement of
chloride in acid 1 by 4-chlorophenol, but a direct dis-
placement reaction would not be expected to give ob-
served 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
Sch em e 2
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 ring-
opening 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 evi-
dence for the formation of a thietanium ion intermediate
from acid 1. Because of the known reactivity of sulfo-
nium ions, further studies are warranted to investigate
the role of thietanium ion formation in the mutagenicity
of acid 1.
Sch em e 3
Ack n ow led gm en t. 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.
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314-385, Wiley, New York.
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)-1-
phenyl-2-pyrrolidone (9) was formed (Scheme 3). γ-Lac-
tam 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 diaz-
omethane. 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
TX980016R