Formation of 4,4-dialkoxycyclohexa-2,5-dienone N-(Thiol-S-yl)imine during reaction of 4-alkoxynitrosobenzenes with Thiols in alcoholic solvents

Article abstract of DOI:10.1021/tx980088i

During the interaction of nitrosoarenes with glutathione in aqueous media, intermediate generation of a highly resonance-stabilized sulfenamide cation has been repeatedly suggested. Most intermediates and end products could be explained by reactions of this sulfenamide cation with different nucleophiles such as excess thiol, solvent water, and metabolically produced arylamine. The present paper presents evidence for adduct formation of the sulfenamide cation with solvent alcohol at neutral pH. Sulfenamide cations generated from 4-nitrosophenetole and 4-nitrosoanisole, respectively, are strongly suggested to form the metastable ketals 4-ethoxy-4-methoxycyclohexa- 2,5-dienone N-(glutathion-S-yl)imine and 4,4-dimethoxycyclohexa-2,5-dienone N-(glutathion-S-yl)imine, respectively, during reaction with solvent methanol. Reaction of the two sulfenamide cations in ethanol yielded 4,4- diethoxycyclohexa-2,5-dienone N-(glutathion-S-yl)imine and 4-ethoxy-4- methoxycyclohexa-2,5-dienone N-(glutathion-S-yl)-imine, respectively. Although the metastability of the ketals did not allow isolation of pure solid material, chromatographic and chemical behavior as well as tandem MS fragmentation substantiate a ketal structure of these intermediates. To confirm the proposed structure, new compounds, 2,6-dimethyl-4- nitrosophenetole, 2,6-dimethyl-4-nitrophenetole, 2,6-dimethyl-4-phenetidine, and N-(glutathion-S-yl)-N-hydroxy-4-aminoacetophenone, were synthesized and included in supportive experiments. In summary, the detection of ketals corroborates once more the occurrence of a sulfenamide cation which obviously not only reacts with Soft nucleophiles such as GSH but, to a limited extent, also reacts with hard nucleophiles. The toxicological significance of this result is discussed.

Full text of DOI:10.1021/tx980088i

Chem. Res. Toxicol. 1998, 11, 1423-1433
1423
F or m a tion of 4,4-Dia lk oxycycloh exa -2,5-d ien on e
N-(Th iol-S-yl)im in e d u r in g Rea ction of
4-Alk oxyn itr osoben zen es w ith Th iols in Alcoh olic
Solven ts
Dieter Gallemann,* Anke Greif, Peter Eyer, J ohannes Dasenbrock,†
Elmar Wimmer,† J ohann Sonnenbichler,‡ Isolde Sonnenbichler,‡
Wolfram Scha¨fer,‡ and Ingrid Buhrow‡
Walther-Straub-Institut, Ludwig-Maximilians-Universita¨t Mu¨nchen,
Nussbaumstrasse 26, D-80336 Mu¨nchen, Germany
Received April 28, 1998
During the interaction of nitrosoarenes with glutathione in aqueous media, intermediate
generation of a highly resonance-stabilized sulfenamide cation has been repeatedly suggested.
Most intermediates and end products could be explained by reactions of this sulfenamide cation
with different nucleophiles such as excess thiol, solvent water, and metabolically produced
arylamine. The present paper presents evidence for adduct formation of the sulfenamide cation
with solvent alcohol at neutral pH. Sulfenamide cations generated from 4-nitrosophenetole
and 4-nitrosoanisole, respectively, are strongly suggested to form the metastable ketals
4-ethoxy-4-methoxycyclohexa-2,5-dienone N-(glutathion-S-yl)imine and 4,4-dimethoxycyclohexa-
2,5-dienone N-(glutathion-S-yl)imine, respectively, during reaction with solvent methanol.
Reaction of the two sulfenamide cations in ethanol yielded 4,4-diethoxycyclohexa-2,5-dienone
N-(glutathion-S-yl)imine and 4-ethoxy-4-methoxycyclohexa-2,5-dienone N-(glutathion-S-yl)-
imine, respectively. Although the metastability of the ketals did not allow isolation of pure
solid material, chromatographic and chemical behavior as well as tandem MS fragmentation
substantiate a ketal structure of these intermediates. To confirm the proposed structure, new
compounds, 2,6-dimethyl-4-nitrosophenetole, 2,6-dimethyl-4-nitrophenetole, 2,6-dimethyl-4-
phenetidine, and N-(glutathion-S-yl)-N-hydroxy-4-aminoacetophenone, were synthesized and
included in supportive experiments. In summary, the detection of ketals corroborates once
more the occurrence of a sulfenamide cation which obviously not only reacts with soft
nucleophiles such as GSH but, to a limited extent, also reacts with hard nucleophiles. The
toxicological significance of this result is discussed.
plausibly explained as resulting from interactions of the
resonance-stabilized sulfenamide cation with various
nucleophiles. Some of these products still await struc-
tural elucidation (8), one of them being characterized in
this paper.
During the inceptive studies on NOPt/GSH reactions,
Klehr tried to isolate the remarkably unstable semimer-
captal [for structure, see Figure 1 in the preceding paper
(8)] and finally performed his experiments at -40 °C in
In tr od u ction
Reactions of nitrosoarenes with biological thiols have
gained increasing interest among toxicologists when it
became clear that nitrosoarenes and their redox couples
N-hydroxyarylamines are involved in toxic, allergic,
mutagenic, and carcinogenic effects (1). The rapid reac-
tions with cellular thiols, leading mainly to unreactive
metabolites, have been considered to be important for
detoxication of nitrosoarenes. During the investigations
of the underlying reaction mechanisms, a variety of
observations pointed to the intermediate generation of
an N-sulfenylnitrenium ion (also termed “sulfenamide
cation”). Major hints at the occurrence of this reactive
species came from kinetic investigations of nitrosoarene/
GSH interactions (2), and from detailed studies of the
metabolic pattern observed during the reaction of the
phenacetin metabolite 4-nitrosophenetole (NOPt1) with
GSH (3-8). The multiplicity of products has been most
1
Abbreviations: API, atmospheric pressure ionization; CID, colli-
sion-induced dissociation; EI-MS, electron impact mass spectrometry;
Et/Me-ketal, 4-ethoxy-4-methoxycyclohexa-2,5-dienone N-(glutathion-
S-yl)imine; Et/d3Me-ketal, 4-ethoxy-4-trideuteriomethoxycyclohexa-2,5-
dienone N-(glutathion-S-yl)imine; Et2-ketal, 4,4-diethoxycyclohexa-2,5-
dienone N-(glutathion-S-yl)imine; FAB-MS, fast atom bombardment
mass spectrometry; GS-EPQDI, N-(4-ethoxyphenyl)-N′-(glutathion-S-
yl)-1,4-benzoquinone diimine; GS-MPQDI, N-(4-methoxyphenyl)-N′-
(glutathion-S-yl)-1,4-benzoquinone diimine; GS-NHAn, N-(glutathion-
S-yl)-4-anisidine; GS-NHPt, N-(glutathion-S-yl)-4-phenetidine; GSO-
NHAn, N-(glutathion-S-yl)-4-anisidine S-oxide; GSO-NHPt, N-(gluta-
thion-S-yl)-4-phenetidine S-oxide; GS-NAn+, N-(glutathion-S-yl)-4-
anisidine cation; GS-Nd3An+, N-(glutathion-S-yl)-4-trideuteriomethoxy-
benzene cation; GS-NPt+, N-(glutathion-S-yl)-4-phenetidine cation; GS-
QI, N-(glutathion-S-yl)-1,4-benzoquinonimine; LC-MS/MS, liquid chro-
matography-tandem mass spectrometry; Me2-ketal, 4,4-dimethoxy-
cyclohexa-2,5-dienone N-(glutathion-S-yl)imine; NH2An, 4-anisidine;
NH2Pt, 4-phenetidine; NOAn, 4-nitrosoanisole; NOPt, 4-nitrosophene-
tole; tR, retention time; VR, retention volume.
* To whom correspondence should be addressed. Telephone: 49 89
5160-7281. Fax: 49 89 5160-7224. e-mail: gallemann@lrz-muenchen.de.
† Present address: Merck KGaA, Institute of Pharmacokinetics and
Metabolism, Am Feld 32, D-85567 Grafing, Germany.
‡ Present address: Max-Planck-Institut fu¨r Biochemie, Am Klopfer-
spitz 18a, D-82152 Martinsried, Germany.
10.1021/tx980088i CCC: $15.00 © 1998 American Chemical Society
Published on Web 12/01/1998

1424 Chem. Res. Toxicol., Vol. 11, No. 12, 1998
Gallemann et al.
F igu r e 1. Supposed formation and decay reactions of alcohol adducts formed during reaction of 4-alkoxynitrosoarenes with GSH
in alcoholic solvents.
the presence of 80% MeOH (5, 6). However, instead of
capturing the desired semimercaptal, he observed an-
other metastable product in low yields (6). Tracer
experiments using [u-ring-14C]-NOPt and [Gly-2-3H]-GSH
indicated this compound to be a 1:1 adduct of ni-
trosoarene and thiol (4). Later experiments on the NOPt/
GSH interaction showed that this metabolite was neither
generated in pure aqueous reaction mixtures nor gener-
ated when MeOH was replaced by CH3CN. These results
fed the idea that MeOH may have added to the reactive
sulfenamide cation, especially since analogous nitrenium
ions are known to be captured by alcohols (9-14).
The scarce results reported hitherto on the reactivity
of sulfenamide cations indicate the ring positions to react
primarily with soft nucleophiles (7, 8). The only reaction
with a hard nucleophile, solvent water, is reported in the
preceding paper (8). Therefore, proof of adduct formation
with MeOH would be a further example of sulfenamide
cation reactions with hard nucleophiles, thus also sug-
gesting reactions with hard nucleophilic sites of biological
macromolecules (e.g., DNA bases). To get more insight
into the reactivity of sulfenamide cations, we tried to
prove the presumed adduct formation with alcohols.
Because of the metastability and low yields of the
adducts, however, direct structural proof was not pos-
sible. However, different kinds of circumstantial evi-
dence strongly supported the reversible formation of an
alcohol adduct. The experimental strategy was based on
trapping the sulfenamide cations of NOPt and 4-ni-
trosoanisole (NOAn) with MeOH and EtOH, respectively.
Both educts should yield the same Et/Me-ketal that
should be observable by HPLC methods and LC-MS/MS.
Since the Et/Me-ketal may reversibly release one alcohol
group, NOPt descendants should be observed from NOAn
and vice versa. Figure 1 sketches the supposed reactions.
For the sake of simplicity, the sulfenamide cations (shown
in brackets) are presented only in the most important
resonance structure as deduced from the product distri-
bution {Table 1 of the preceding paper (8)].
Exp er im en ta l P r oced u r es
Ch em ica ls. 4-Anisidine, calcium hydride, 4,4-dimethoxycy-
clohexa-2,5-dienone (96%), 2,6-dimethylphenol, ethyl iodide,
methanol-d4 (99.8 atom % D), nitrosonium tetrafluoroborate,
NH2Pt, and sodium methylate were purchased from Aldrich
Chemie (D-89552 Steinheim). GSH was obtained from Boe-
hringer (D-68305 Mannheim), 2-mercaptoethanol from Fluka
Chemie (D-89231 Neu-Ulm), and ascorbic acid from Sigma
Chemie (D-82041 Deisenhofen). All other reagents were pur-
chased from Merck (D-64293 Darmstadt) at the purest grade
available. NOPt (15) and N-(glutathion-S-yl)-4-benzoquinon-
imine (8) (GS-QI) were prepared as described earlier. 2,6-
Dimethylphenetole was synthesized by reaction of 2,6-dimeth-
ylphenol with ethyl iodide according to standard methods (16).
4-Nitrosoacetophenone and N-hydroxyacetophenone had been
synthesized previously (3).
Ca u tion : Nitrosoarenes bearing π-donating substituents have
tested positive in the Ames mutagenicity assay with S. typh-
imurium without activation (17-21).
1
In str u m en ta tion a n d An a lytica l Meth od s. For H NMR,
EI-MS, FAB-MS, and analytical/semipreparative HPLC, see the
preceding paper (8). IR spectra were registered with a Perkin-
Elmer IR spectrometer, UV/vis spectra by a Shimadzu UV-

Alcohol Adducts of Sulfenamide Cations
Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1425
of the ketal HPLC cuts was proved by diode array detection
comparing the base-line-corrected UV/vis spectra at the begin-
ning, top, and end of the respective HPLC peaks.
LC-MS and LC-MS/MS were performed on a Finnigan MAT
triple quadrupole mass spectrometer TSQ 7000 (Finnigan MAT,
San J ose, CA), equipped with an atmospheric pressure ioniza-
tion (API) system. The API source was operated in the positive
electrospray ionization mode. Measurements were accom-
plished using a capillary temperature of 200 °C, an electrospray
ionization high voltage of 4.5 kV, nitrogen as auxiliary and
sheath gas, and without sheath liquid. Quadrupoles were
scanned with a scan time varying from 1 to 3 s over the mass
range of interest. Collision-induced dissociation (CID) was
performed using argon as the collision gas at a pressure of
approximately 2 mTorr. To obtain product ion spectra, the
collision energy was kept at -13 eV. Product ion abundances
are expressed relative to the most abundant product rather than
to the transmitted precursor. LC separation was performed on
Lichrospher 100 RP 18 (125 × 4 mm, Merck) and gradient
elution with MeOH/ammonium acetate buffer (0.01 M, pH
6.5): 0-10 min, 20-40% MeOH; 10-14 min, 40% MeOH; 14-
16 min, 40-90% MeOH; 16-20 min, 90% MeOH; flow, 0.7 mL/
min.
Syn th eses. (A) 4-Nitr osoa n isole (NOAn) was prepared
and purified according to the procedure described for NOPt (15).
Sky-blue crystals, mp 18.5-19.7 °C [lit. 21-23 °C (22, 23)], yield
44% of theory, chromatographically pure. 1H NMR in CDCl3:
δ 3.97 (3H, s, -OCH3), 7.04 (2H), and 7.93 (2H) (ABCD system
of the aromatic protons) [cf. (23)]. EI-MS [70 eV, cf. (23)]: m/z
137 (100%, [M]+•), 107 (36%, [M-NO]+•), 92 (33%, [M-NO-
CH3]+•). To determine ꢀ (UV/vis) of NOAn, [ring-u-14C]-NOPt
(4) [specific activity 8.5 Bq/nmol, log ꢀ342 ) 4.26 (MeOH) (24)]
was subjected to alkaline methanolysis (10 mM nitrosoarene,
50 mM sodium methylate in MeOH; complete reaction after 22
h at 37 °C under an argon atmosphere). After neutralization,
NOAn was separated from its nitro derivative by RP-HPLC. UV/
vis: λmax ) 340 nm, log ꢀ340 ) 4.22 (MeOH).
(B) N-(Glu ta th ion -S-yl)-4-p h en etid in e S-Oxid e (GSO-
NHP t). One volume of GSH (50 mM; in ammonium bicarbonate
buffer, 50 mM, pH 6.5) was added dropwise to a vigorously
stirred solution of NOPt (50 mM; in ammonium bicarbonate
buffer, 50 mM, pH 6.5/MeOH (1:1 v/v)]. After ether extraction
of lipophilic metabolites, the batch was allowed to stand at
ambient temperature overnight to allow decay of a hitherto
unknown product which on HPLC separation coeluted with the
desired sulfinamide. HPLC separation was performed the next
day under the following conditions: Prep Nova-Pak C18 (7.8 ×
300 mm, Waters); flow, 3 mL/min; elution with ammonium
bicarbonate buffer (10 mM, pH 6.5)/MeOH; gradient: 0-14 min,
25-40% MeOH; 14-15 min, 40% MeOH. For 1H NMR spec-
troscopy, the substance was used as obtained after lyophiliza-
tion. For elemental analysis, the product was separated from
buffer salts by gel chromatography on Superdex Peptide HR 10/
30 (Pharmacia Biotech, D-79111 Freiburg) with water as eluent,
following lyophilization to constant weight. Colorless powder
F igu r e 2. HPLC chromatograms of 4-alkoxynitrosobenzenes
reacted with GSH in alcoholic solutions (90%); stoichiometry
5:1; reaction time 20 min; HPLC separation on Prep Nova-Pak
(300 × 7.8 mm). (a) NOPt + GSH in EtOH; (b) NOPt + GSH in
MeOH; (c) NOAn + GSH in EtOH; (d) NOAn + GSH in MeOH.
For compound identification, see Figure 4 and Figure 1 of the
preceding paper (8).
(yield: 3% of theory). UV/vis (6): λmax ) 233/285 nm, log ꢀ233
)
3.82 (MeOH/phosphate buffer, pH 6.5). 1H NMR in D2O: δ 1.38
(3H, t, J ) 7.0 Hz, OCH2CH3), 2.1 (2H, m, Glu-â-CH2), 2.5 (2H,
m, Glu-γ-CH2), 3.51 (1H, dd, J ) 9 Hz/14 Hz, Cys-â1), 3.59 (1H,
dd, J ) 5 Hz/14 Hz, Cys-â2), 3.7 (1H, m, Glu-R), 3.80 (2H, s,
Gly-R), 4.13 (2H, q, J ) 7.0 Hz, OCH2CH3), 4.89 (1H, dd, J ) 5
Hz/9 Hz, Cys-R), 7.03 (2H), and 7.15 (2H) (A2B2 system of the
aromatic protons). FAB-MS (glycerol) (6): m/z 459 (43%,
[M+H]+), 322 (100%, [GSO]+). Elemental analysis was unsuc-
cessful as HPLC buffer salts could not completely be separated
from the acid- and alkali-sensitive (1, 25) tripeptide derivative.
However, the above-mentioned physical properties and chemical
reactivity of this derivative were in line with other well-
characterized sulfinamides (1).
265 spectrophotometer. Analytical separation of the ketals was
carried out on Lichrospher 100 RP 18 (4 × 125 mm; Merck) with
MeOH/sodium phosphate buffer (10 mM, pH 6.5) gradients and
detection at 230 and 340 nm (bandwidth 4 nm). Alternatively,
separations were performed on Prep Nova-Pak C18 (7.8 × 300
mm, Waters, Milford, MA) using a MeOH/sodium phosphate
buffer (10 mM, pH 6.5) gradient: 0-15 min, 5-50% MeOH; 15-
19 min, 50% MeOH; 19-20 min, 50-90% MeOH; 20-23 min,
90% MeOH; 23-26 min, 90-5% MeOH; flow 3.0 mL/min. Peaks
were identified by comparing the UV/vis spectra (220-500 nm)
and retention times with authentic standards. In addition,
glutathione sulfinamides were recognized by their characteristic
double peaks (cf. Figure 2) and sulfenamides by their slow decay
with formation of the corresponding arylamine (1). Peak purity
(C) N-(Glu ta th ion -S-yl)-4-a n isid in e S-Oxid e. GSO-NHAn
was prepared as described above for GSO-NHPt. HPLC separa-

1426 Chem. Res. Toxicol., Vol. 11, No. 12, 1998
Gallemann et al.
tion was performed with ammonium bicarbonate buffer (50 mM,
pH 6.5)/MeOH; gradient: 0-10 min, 10-30% MeOH; 10-12
min, 30% MeOH. Colorless powder (yield: 16% of theory). UV/
vis: λmax ) 233/285 nm (MeOH/phosphate buffer, pH 6.5). 1H
NMR in D2O: δ 2.2 (2H, m, Glu-â-CH2), 2.6 (2H, m, Glu-γ-CH2),
3.52 (1H, dd, J ) 9 Hz/13 Hz, Cys-â1), 3.60 (1H, dd, J ) 5 Hz/
13 Hz, Cys-â2), 3.79 (1H, t, J ) 6.5 Hz, Glu-R), 3.88 (2H, Gly-
R), 3.86 (3H, s, OCH3), 4.91 (1H, dd, J ) 5 Hz/9 Hz, Cys-R),
7.04 (2H), and 7.18 (2H) (A2B2 system of the aromatic protons).
FAB-MS (glycerol): m/z 445 (85%, [M+H]+), 322 (100%, [GSO]+).
As already discussed above, this derivative was not amenable
to elemental analysis. Again, chemical reactivity corresponded
to the sulfinamide structure.
to an aqueous solution of excess potassium ferricyanide. The
organic compound was extracted with pentane and applied
again to RP-HPLC to remove impurities which coeluted with
the N-hydroxy derivative and could not be separated by the final
sublimation. After this treatment, the nitroso compound was
chromatographically pure. Sublimation (p ) 0.05 mbar, T1
)
20 °C, T2 ) -35 °C) delivered sky-blue crystals. Yield: 10% of
theory; mp 20.7-21.3 °C. IR: 2981 (m), 2930 (m), 1594 (w),
1489 (st), 1420 (m), 1388 (w), 1269 (st), 1220 (st), 1088 (st), 1029
(st), 962 (m), 899 (m), 745 (m) cm-1. UV/vis [cf. 2,6-dimethyl-
4-nitrosoanisole (29)]: λmax ) 230/327 nm, log ꢀ327 ) 4.05
(MeOH). 1H NMR in CDCl3 [cf. 2,6-dimethyl-4-nitrosoanisole
(23, 29)]: δ 1.47 (3H, t, J ) 7.2 Hz, OCH2CH3), 2.41 (6H, s,
Ar-CH3), 3.97 (2H, q, J ) 7.2 Hz, OCH2CH3), 7.61 (2H, s, ArH).
13C NMR in CDCl3: δ 16.4 (OCH2CH3), 17.2 (Ar-CH3), 69.0
(OCH2CH3), 123.1 (C3 and C5), 132.8 (C2 and C6), 163.5 and
164.6 (C1/C4). EI-MS (70 eV): m/z 179 (100%, [M]+•), 151 (58%,
[M-C2H4]+•), 121 (51%, [M-C2H4-NO]+•), 91 (62%), 77 (51%).
(D) N-(Glu t a t h ion -S-yl)-N-h yd r oxy-4-a m in oa cet op h e-
n on e was obtained by rapidly mixing 45 µL of 4-nitrosoac-
etophenone (22 mM in CH3CN) with 5 µL of GSH (100 mM, in
ammonium acetate buffer, 10 mM, pH 7 adjusted with am-
monia). In view of the instability of this compound (almost
complete decay within 0.5 h), no attempts at isolation were
undertaken. Instead, 10 µL of the solution was applied to LC-
MS/MS within 10 s and separated on Lichrospher 100 RP 18
(125 × 4 mm, Merck) with isocratic elution (70% ammonium
acetate buffer, 10 mM, pH 6.5/30% MeOH). The first intense
UV peak eluting after Rv ) 1.6 mL exhibited an MS typical for
semimercaptals (1): m/z 457 (100%, [M+H]+), 439 (76%, [M+H-
H2O]+), 152 (11%, [M+H-GS]+). Chemical reactivity of the
separated compound was in line with the presumed semimer-
captal structure, too (1): Acidification (pH 2 for 0.5 h) liberated
the corresponding arylamine as indicated by comparison with
an authentic standard. Reaction with excess GSH yielded
N-hydroxy-4-aminoacetophenone exclusively. During incuba-
tion at room temperature for 0.5 h, the compound degraded
under formation of 4-nitrosoacetophenone, N-hydroxy-4-ami-
noacetophenone, and presumably N-(glutathion-S-yl)-4-ami-
noacetophenone S-oxide. The UV/vis spectrum of the semimer-
captal, however, was atypical (1): λmax ) 309 nm (eluent);
conceivably, this effect may result from the strong electron
acceptor para-substituent.
Anal.: C, 66.99; H, 7.35; N, 7.86; O, 17.8. Calcd for C10H13
NO2: C, 67.02; H, 7.31; N, 7.82; O, 17.85.
-
(F ) 2,6-Dim eth yl-4-n itr op h en etole was prepared by oxida-
tion of unpurified 2,6-dimethyl-4-nitrosophenetole with Caro’s
acid (30) in MeOH/water (2:1 v/v; reaction at room temperature
for 3 days). The product was extracted with dichloromethane
and recrystallized from hexane. The compound was chromato-
graphically pure and did not form any nitroso metabolites on
mixing with GSH. Yellowish needles, mp 55.8-56.5 °C. UV/
vis: λmax ) 289 nm, log ꢀ289 ) 3.90 (MeOH). 1H NMR in CDCl3:
δ 1.45 (3H, t, J ) 7.2 Hz, OCH2CH3), 2.36 (6H, s, ArCH3), 3.92
(2H, q, J ) 7.2 Hz, OCH2CH3), 7.93 (2H, s, ArH). EI-MS (70
eV): m/z 195 (100%, [M]+•), 167 (92%, [M-C2H4]+•), 137 (54%,
[M-C2H4-NO]+•). High-resolution EI-MS: m/z 195.09029,
C10H13NO3 requires 195.08954.
(G) 2,6-Dim eth yl-4-p h en etid in e was prepared by reduction
of unpurified 2,6-dimethyl-4-nitrosophenetole with excess GSH
(at pH 7) and subsequent hydrolysis of the major product
sulfenamide (1) (pH 3, adjusted with phosphoric acid). Fifteen
minutes later, the mixture was neutralized with sodium hy-
droxide and chromatographed on a Sep Pak C18 cartridge
(Waters). Polar compounds such as GSH and GSSG were eluted
with water before the desired arylamine was eluted with 0.1 N
hydrochloric acid. After neutralization of the acidic fractions,
the arylamine was extracted with peroxide-free ether. The
reddish brown powder was chromatographically pure. UV/vis:
λmax ) 233/287 nm (MeOH). 1H NMR in CDCl3: δ 1.39 (3H, t,
J ) 7.2 Hz, OCH2CH3), 2.21 (6H, s, Ar-CH3), 3.77 (2H, q, J )
7.2 Hz, OCH2CH3), 4.6 (2H, broad, -NH2), 6.46 (2H, s, ArH).
EI-MS (70 eV): m/z 165 (35%, [M]+•), 136 (100%, [M-C2H5]+•).
High-resolution EI-MS: m/z 165.11587, C10H15NO requires
165.11536. Identical material was formed during the slow decay
of the sulfenamide (1) and by reduction of 2,6-dimethyl-4-
nitrosophenetole with sodium dithionite.
(E) 2,6-Dim eth yl-4-n itr osop h en etole. To avoid any aque-
ous acidic conditions which could hydrolyze the 4-nitrosobenzene
ether (26), 2,6-dimethylphenetole was nitrosated with nitroso-
nium tetrafluoroborate (3-fold excess) in dry acetonitrile as
described recently (23). The reddish brown reaction mixture
was poured into a vigorously stirred mixture (1:1 v/v) of
n-pentane/sodium phosphate buffer (0.5 M, pH 8) in order to
scavange the protons formed during hydrolysis of excess ni-
trosonium tetrafluoroborate. The organic layer was washed
with sodium phosphate buffer and twice with water. After
drying over sodium sulfate, the solvent was removed by vacuum
and the viscous residue applied to flash chromatography on a
short column of silica gel 60 (Merck) with hexane/dichlo-
romethane (3:2 v/v) as eluent. The turquoise-green fraction
containing 2,6-dimethyl-4-nitrosophenetole was collected. Re-
moving the solvent by vacuum delivered a sky-blue, viscous
liquid. RP-HPLC showed one single peak in the chromatogram,
suggesting a pure product. However, on complete reduction of
the nitroso compound with excess GSH or ascorbate, about 10%
of the HPLC peak remained with a UV maximum shifted to
shorter wavelength. Comparison with an authentic standard
revealed the impurity to be 2,6-dimethyl-4-nitrophenetole (see
below). In fact, formation of nitroaromatics is known during
the reaction of activated arenes with surplus nitrosating agent
(16). Besides, several nitroso- and nitroaromatics are known
to be scarcely separated by any separation method (27, 28).
(H) 4-Eth oxy-4-m eth oxycycloh exa -2,5-d ien on e N-(Glu -
ta th ion -S-yl)im in e (Et/Me-k eta l). GSH (20 µL, 20-50 mM
in sodium phosphate buffer, 0.2 M, pH 7.4) was rapidly added
to a vigorously stirred solution of NOPt (180 µL, 11 mM in
MeOH). It took almost 20 min to withdraw the alcohol in a
Speed Vac concentrator (p ) 0.05 mbar, 20 °C) to ensure
favorable HPLC separation. The solid residue (partly depleted
from the volatile nitrosoarene and arylamine) was dissolved in
200 µL of water and applied to HPLC. (To chromatograph
incubates of shorter reaction times, the reaction mixture was
attenuated with 800 µL of water to obtain the composition of
the HPLC eluent. Due to the high volume, this procedure
caused significant loss of resolution.) Yield: about 0.02 µmol
To get the pure nitroso compound, the mixture was slowly
fed in a solution of excess ascorbate (pH 7, H2O/MeOH ) v/v).
After the insoluble azoxy compound had been filtered off, the
N-hydroxy derivative was separated from the nitroarene by
semipreparative RP-HPLC and collected at -70 °C under argon.
The nitrosoarene was obtained by adding this fraction dropwise
(2% of theory) at an estimated log ꢀ340
≈ 4.2. [Due to the
nm
metastability of Et/Me-ketal, determination of ꢀ was not pos-
sible. The order of magnitude indicated is estimated from that
of N-(glutathion-S-yl)benzoquinonimines described in the pre-
ceding paper (8). Et/Me-ketal was obtained in distinctly lower
yields when NOAn was reacted with GSH in EtOH. In this

Alcohol Adducts of Sulfenamide Cations
Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1427
educt ratio from 1:0.2 to 1:1 (NOPt:GSH) resulted in
higher yields, but caused a more rapid product decay
from 1:0.5 ratios upward. Unfortunately, increased educt
concentrations did not elevate the amount of the desired
product. Thus, the maximum yield from a single HPLC
separation topped at about 0.02 µmol (for detailed reac-
tion conditions, see Experimental Procedures).
Sta bility. Immediate rechromatography of the HPLC-
isolated Et/Me-ketal revealed a distinct decay with
formation of the known NOPt metabolites N-(glutathion-
S-yl)-4-benzoquinonimine (GS-QI) (8) and GSO-NHPt (5)
(Figure 4) and a few unknown minor products. Immedi-
ate freezing of the collected HPLC peak at -70 °C could
not prevent decomposition. When the frozen HPLC cut
was lyophilized overnight, the compound completely
decomposed under formation of the quinonimine GS-QI,
the sulfinamide GSO-NHPt, and the sulfinamide GSO-
NHAn. Both sulfinamides were identified by their HPLC
double peaks (1), by their UV/vis spectra, and by sample
spiking with authentic standards. These findings appear
to be in agreement with mechanistic considerations
presented in Figure 4: The sulfenamide cation resulting
from the interaction of NOPt with GSH (GS-NPt+) adds
to solvent MeOH to yield a metastable ketal (Et/Me-
ketal). Thereupon, liberation of the ipso-methoxy and
-ethoxy groups, respectively, generates two different
sulfenamide cations (GS-NPt+ and GS-NAn+), giving rise
to formation of the sulfinamides GSO-NHPt and GSO-
NHAn.
F igu r e 3. UV/vis spectrum of 4-ethoxy-4-methoxycyclohexa-
2,5-dienone N-(glutathion-S-yl)imine (Et/Me-ketal) [MeOH/
sodium phosphate buffer (10 mM, pH 6.5), 1:1 v/v] obtained by
diode array detection.
experiment, the ketal could only be detected after 20 min
reaction time using a 5-fold excess of NOAn.
(I) 4,4-Dieth oxycycloh exa -2,5-d ien on e N-(glu ta th ion -S-
yl)im in e (Et2-ketal) was detected during interaction of a 5-fold
excess of NOPt with GSH in EtOH after 20 min reaction time
(for details, see synthesis of Et/Me-ketal).
(J ) 4,4-Dim eth oxycycloh exa -2,5-d ien on e N-(glu ta th ion -
S-yl)im in e (Me2-ketal) was prepared according to Et/Me-ketal
using NOAn instead of NOPt.
Ch em ica l r ea ctivity of th e k eta ls was investigated by
adding 0.01 mL of reactant (GSH, ascorbate, and sodium
dithionite, respectively; 0.1 M solutions in 0.2 M sodium
phosphate buffer, pH 7 readjusted) to a freshly prepared HPLC
cut of the ketal (approximately 0.5 mL). Acid hydrolysis was
performed by adjusting to pH 1 with 1 M HCl in freshly
prepared HPLC cuts. After 10 min reaction at ambient tem-
perature (and neutralization of acid cuts), the whole incubate
was reinjected into the HPLC system.
Since the marked instability of Et/Me-ketal did not
1
allow characterization by H NMR or FAB-MS spectros-
copy, more stable derivatives were intended to be syn-
thesized. As previously reported by Fernando et al. (31),
inclusion of two methyl groups ortho to the ipso-position
should result in an increased stability of an ipso-
compound, since steric interaction complicates the ipso-
residue turning back into a coplanar position. To this
end, a synthetic route to 2,6-dimethyl-4-nitrosophenetole
was developed. When this NOPt derivative was reacted
with GSH (90% MeOH, 20 min at 0 °C), the chromato-
gram of the mixture exhibited a small peak with the same
UV/vis spectrum as Et/Me-ketal. As expected, this
compound was distinctly more stable than its alkyl-
deficient derivative, but not stable enough to yield pure
Resu lts
During reaction of 10 mM NOPt with 2-5 mM GSH
in solutions of 90% MeOH/10% sodium phosphate buffer
(0.2 M, pH 7.4) at room temperature, a low-yield product
was formed (Et/Me-ketal, Figure 2b) exhibiting a UV
spectrum (λmax ) 340 nm; Figure 3) uncommon to the
nitrosoarene metabolites known hitherto (1). Et/Me-
ketal was already formed within 0.5 min and was
detectable in the reaction mixture even 20 min later. On
RP-HPLC separation, Et/Me-ketal was eluted similar to
other monocyclic glutathione conjugates (Figure 2b),
indicating incorporation of the polar GSH into the ketal.
Et/Me-ketal was not observed in pure aqueous reaction
mixtures. When MeOH was replaced by CH3CN (90%)
in order to mimic the solvent effect of MeOH, Et/Me-ketal
was not formed either. However, when MeOH was
replaced by EtOH, the HPLC chromatogram of the
reaction mixture showed a very small peak immediately
after the sulfenamide (Et2-ketal; Figure 2a) exhibiting the
same UV/vis spectrum as Et/Me-ketal (Figure 3). Thus,
adduct formation between NOPt, GSH, and the solvent
alcohol was presumed.
1
solid samples for H NMR or FAB-MS. Upon lyophiliza-
tion, a significant decay was noticed, but this derivative
could still be detected in the dry powder kept at -20 °C
for 2 days.
Ch em ica l Rea ctivity. Since synthesis of a stable
ketal derivative failed, HPLC cuts of the Et/Me-ketal
were used for investigation of chemical reactivity. When
the HPLC cut was supplemented with excess GSH, slow
formation of N-(glutathion-S-yl)-4-phenetidine (GS-NHPt)
and subsequent NH2Pt was observed (cf. Figure 4). The
decay product GSO-NHPt was observed as well while GS-
QI was not detected, possibly because of further reactions
(8). Reaction of isolated Et/Me-ketal with excess ascor-
bate resulted in the same product pattern. The strong
reductant sodium dithionite immediately formed NH2Pt
with traces of NH2An (cf. Figure 4). Acid hydrolysis of
Et/Me-ketal yielded the quinonimine GS-QI, NH2Pt, and
traces of NH2An.
To isolate Et/Me-ketal in amounts sufficient for struc-
tural elucidation, factors affecting product formation were
examined. Variation of the reaction temperature (-20
°C to +20 °C), pH shifts between 9 and 6, and increase
of the MeOH portion in the reaction mixture did not
essentially alter the yield of Et/Me-ketal. Increasing the
To gain additional evidence for the presumed structure
of Et/Me-ketal, an alternative synthetic route was pur-
sued: NOAn was reacted with GSH in EtOH (cf. Figure

1428 Chem. Res. Toxicol., Vol. 11, No. 12, 1998
Gallemann et al.
F igu r e 4. Formation of different ketals during reaction of 4-alkoxynitrosobenzenes with GSH in alcoholic solutions and their
consequent reactions.
4). In fact, the HPLC chromatogram of this reaction
mixture (Figure 2c) showed a small peak at the same
retention time as Et/Me-ketal from reaction mixtures of
NOPt and GSH in MeOH (Figure 2b). When both
reaction mixtures were chromatographed on another C18
column (see Experimental Procedures), the respective
peaks eluted again at the same retention time. The UV/
vis spectra of both peaks were congruent. Because of the
extremely small yields during syntheses in EtOH, the
NOAn-derived Et/Me-ketal was only subjected to acid
hydrolysis, resulting in formation of NH2An and NH2Pt
with traces of quinonimine GS-QI.
In vestigation by LC-MS/MS. To unequivocally prove
the structure of the presumed Et/Me-ketal, the reaction
mixture of NOPt and GSH (in 90% MeOH, stoichiometry
1:0.5, 2 min reaction at ambient temperature) was
investigated by LC-MS/MS. The quinonimine GS-QI, the
sulfinamide GSO-NHPt, and the sulfenamide GS-NHPt
emerging in the reaction mixture were identified by their
retention times and relative peak height at the UV/vis
detector which was included between the LC and the MS/
MS unit. In addition, identity was confirmed by the
respective mass spectra. GS-QI: m/z 435 (22%, [M+Na]+),
413 (100%, [M+H]+). GSO-NHPt: m/z 459 (100%,
[M+H]+), 322 (45%, [GSO]+). GS-NHPt: m/z 443 (100%,
[M+H]+), 306 (37%, [GS]+) (1, 8). The presumed Et/Me-
ketal eluting immediately in front of the sulfenamide GS-
NHPt (Figure 2b) was detected by its UV absorption at
340 nm. The corresponding mass spectrum exhibited
intense peaks at m/z 441 (100%) and 427 (36%), most
probably reflecting the sulfenamide cations GS-NPt+ and
GS-NAn+ generated by protonation of one ketal oxygen
and immediate loss of MeOH and EtOH, respectively
(Figure 5). To prove these presumed sulfenamide cation
structures, the product ion spectra of m/z 441 and 427
were recorded (Figure 6a,b). Both spectra exhibited
seven intense peaks with the same mass while five other
peaks showed the characteristic difference between the
ethoxy and the methoxy group of 14 units (Table 1).
NOAn was also reacted with GSH in a MeOH-contain-
ing solvent. Expectedly, a peak exhibiting the known
UV/vis spectrum was detected at a shorter retention time
(Me2-ketal, Figure 3d). This ketal displayed the same
instability as the above-described derivatives and yielded
the analogous decay products (GS-QI and GSO-NHAn),
the corresponding sulfenamide GS-NHAn, and the aryl-
amine NH2An. Mild reduction with excess GSH deliv-
ered the sulfenamide GS-NHAn. The only products
found during acid hydrolysis were NH2An and the
quinonimine GS-QI. A variety of other 4,4-disubstituted
2,5-cyclohexadienone imines have been observed to split
off the imino group upon acidification, yielding the
respective cyclohexadienone derivative. However, forma-
tion of 4,4-dimethoxycyclohexa-2,5-dienone or its subse-
quent hydrolysis product 1,4-benzoquinone (32, 33) could
not be traced during acid hydrolysis of Me2-ketal, as
proved with authentic standards. Another ketal deriva-
tive, Et2-ketal, resulting from reaction of NOPt with GSH
in EtOH, liberated only NH2Pt and the quinonimine GS-
QI upon acidification. Attempts to obtain ketal deriva-
tives of higher alcohols such as i-PrOH failed.
An additional LC-MS/MS experiment was undertaken
using CD3OD instead of CH3OH as reaction solvent. The
MS spectrum recorded during elution of the deuterated
derivative Et/d3Me-ketal again exhibited the intense peak
at m/z 441 (100%). The peak at m/z 427 (10%), however,
was attended by a more intense peak at m/z 430 (41%),
probably corresponding to a trideuteriomethoxy sulfen-
amide cation GS-Nd3An+ (Figure 5). Upon CID, m/z 441

Alcohol Adducts of Sulfenamide Cations
Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1429
F igu r e 5. Presumed fragmentation of the ketal derivatives upon protonation.
Ta ble 1. Com p a r ison of th e P r od u ct Ion s of Su bstitu ted Su lfen a m id e Ca tion s
substituents
C2H5O-
CH3O-
CD3O-
CH3CO-
fragments
precursor ion
proposed structure
(Figure 6a)
(Figure 6b)
(Figure 6c)
(Figure 6d)
M+
441
427
430
439
aryl substituent
containing product ions
[M-H2O]+
423
295
245
168
153
409
281
231
154
139
412
284
234
157
142
421
293
-
[M-GluNH]+
[ArNHS]+
[ArS]+
-
-
aryl substituent deficient
product ions
[GS-2H]+
304
272
254
245
175
153
145
304
272
254
245
175
153
145
304
272
254
245
175
153
145
304
272
254
245
175
153
145
[GS-2H-S]+
[GS-2H-S-H2O]+
[GS-2H-CH2CO2H]+
[GS-2H-Glu]+
[GS-2H-S-(GlyCO)-OH]+
[GluNH]+
produced the same product ion spectrum as m/z 441 from
the undeuterated ketal. The product ion scan of m/z 430
(Figure 6c) revealed a shift of 3 units of the methoxy-
containing fragments, but not of the alkoxy-deficient
fragments (Table 1).
Some effort was undertaken to prove the sulfenamide
cation structure of the three fragments m/z 441, 427, and
430. To this end, we synthesized a glutathione semi-
mercaptal since this family is known to yield the corre-
sponding sulfenamide cation during FAB-MS fragmen-
tation (1, 5, 6, 25, 34). Semimercaptals are unstable,
especially in the case of donor aryl substituents (1), so
that the 4-methoxy or 4-ethoxy derivative was not
amenable to LC-MS/MS analysis. [The only stable glu-
tathione semimercaptals described at all have been
obtained from the acceptor substituted 3- and 4-nitroni-
trosobenzene, respectively (34).] Hence, we reacted the
acceptor substituted 4-nitrosoacetophenone with glu-
tathione to obtain the respective semimercaptal N-(glu-
tathion-S-yl)-N-hydroxy-4-aminoacetophenone (eq 1) which
proved to be stable enough for separation and detection
by LC-MS/MS.
In fact, this semimercaptalsupon protonation inside
the ion sourcesdelivered an intense fragment at m/z 439
corresponding to the respective sulfenamide cation (eq
1). This sulfenamide cation was further fragmented by
CID, delivering the product ion spectrum shown in Figure
6d. Again, seven substituent-deficient fragments were
formed which had already been observed in the product
spectra of the precursors m/z 441, 427, and 430 (Table
1). The acetyl-containing fragments [M-H2O]+ (m/z 421)
and [M-GluNH]+ (m/z 293) were observed likewise, while
the corresponding product ions m/z 243, 166, and 151
were lacking (Table 1).

1430 Chem. Res. Toxicol., Vol. 11, No. 12, 1998
Gallemann et al.
F igu r e 7. Tentative fragmentation pattern of the glutathione
sulfenamide cations from Figure 6 and Table 1, respectively.
The fragments indicated by lower case letters refer to (37, 38,
58, 59). Neutral loss fragments are designated by the respective
molecular masses.
be assumed. Second, incorporation of solvent alcohol was
further proven when carrying out NOPt/GSH reactions
in CD3OD which resulted in fragments with masses 3
units higher than observed in MeOH. Third, typical end
products such as the sulfinamides both of NH2Pt and of
NH2An were observed, indicating the intactness of the
N-S linkage of the glutathione moiety and the exchange
of the alcohol group.
The LC-MS/MS experiments did not allow detection
of the molecular ion of the ipso-adducts but showed
fragments due to the loss of one alcohol group. As
evidenced by molecular orbital calculations, the most
probable sites of protonation are the two alkoxy oxygen
atoms. In fact, the ab initio Mulliken net atomic charge
distribution for the structural analogue 4-hydroxy-4-
ethoxycyclohexa-2,5-dienone N-acetylimine revealed both
ketal oxygen atoms as the most negatively charged
reaction centers (35). Hence, the ketals once protonated
at one of both sites will immediately lose the respective
alcohol due to the extensive resonance stabilization of the
resulting sulfenamide cations (Figure 5). To corroborate
this mechanism, considerable effort was undertaken to
actually prove the sulfenamide cation structure of the
API fragments m/z 441, 430, and 427, particularly as
their product ion spectra were recognized to be remark-
ably different from C-glutathione derivatives reported in
the literature (36-41). Subsequent investigation of
different C-S and N-S conjugated glutathione deriva-
tives revealed the fragmentation patterns to be highly
dependent on the nature of glutathione binding and the
residue.2 The glutathione sulfenamide cations exhibited
particular interesting product ion spectra which may be
rationalized by the fragmentations indicated in Figure
7. The hitherto unknown glutathione fragments m/z 304,
F igu r e 6. Product ion spectra (CID at -13 eV) of the sulfe-
namide cations (a) m/z 441 (R ) OC2H5), (b) m/z 427 (R ) OCH3),
(c) m/z 430 (R ) OCD3), and (d) m/z 439 (R ) COCH3) (for
details, see Experimental Procedures).
Discu ssion
The results presented in this paper indicate formation
of an alcohol adduct during reaction of 4-alkoxyni-
trosobenzenes with GSH in alcoholic solvents. Even if
direct identification of the alcohol adducts by NMR and
MS failed due to the high lability of the compounds,
numerous circumstantial evidence supports their inter-
mediate existence. First, NOPt in MeOH and NOAn in
EtOH yielded the same adduct (HPLC, UV) upon reaction
with GSH. Hence, alcohol addition in position 4 has to
2 D. Gallemann, J . Dasenbrock, and E. Wimmer (1996). unpublished
result.

Alcohol Adducts of Sulfenamide Cations
Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1431
272, 254, 245, 175, and 153 suggest dehydrogenation
during fragmentation corresponding to the reactive and
oxidative character of the nitrenium ion moiety. Finally,
the sulfenamide cation generated from a synthetic semi-
mercaptal exhibited almost the same fragmentation as
the presumed sulfenamide cations formed from the
ketals3 (cf. Figure 6 and Table 1). These findings
substantiate not only the ipso-structure of the alcohol
adducts, but also the arylamine nitrogen atom as the
binding site of the glutathionyl residue as well. Hence,
despite the lack of a molecular ion in the MS and other
spectroscopic proof of the entire alcohol adducts, all the
experimental results strongly suggest the ketal structure
4,4-dialkoxycyclohexa-2,5-dienone N-(glutathion-S-yl)-
imine.
Formation of such ketals is clearly corroborated by
mechanistic considerations of the NOPt/GSH interaction.
As evidenced previously, an intermediate reactive sulfe-
namide cation originates during reactions of nitrosoare-
nes with alkylthiols (1, 2, 6-8). This sulfenamide cation
was recently shown to undergo ring addition reactions
not only with soft nucleophiles such as GSH and ary-
lamines (7), but also to a limited extent with the hard
nucleophile H2O (8). Therefore, addition of alcohols to
sulfenamide cations forming an ipso-adduct is reasonable,
particularly as a substantial positive partial charge is
located at the 4-position (8), i.e., the obvious site of alcohol
attack.
The reported addition of solvent alcohol and water,
respectively, are the only hitherto known examples of
ring addition reactions of hard nucleophiles to sulfena-
mide cations (also termed N-sulfenylnitrenium ions4) (1).
Other arylnitrenium ions are well-known to react with
alcohols, and this interaction was frequently used for
trapping these reactive cations (9-14, 26, 42-48). Simi-
lar to the results reported here, these arylnitrenium ions
gave lower adduct yields with solvent EtOH compared
to MeOH (11, 47). During alcohol adduct formation, the
occurrence of similar ipso-adducts has been proved by
structural elucidation of some metastable derivatives
(10-12, 46-48). In contrast to the formation of ipso-
adducts of 4-alkoxysulfenamide cations reported here, the
ipso-adducts of arylnitrenium ions follow different decay
pathways depending on their aryl substituents (Figure
8).
Basically, both types of ipso-adducts can be protonated
at two different positions: the imino nitrogen atom and
the alkoxy oxygen atom. ipso-Adducts derived from
arylnitrenium ions apparently are protonated at the
imino nitrogen atom, delivering the positive charge into
the ring (48, 49). If the ipso-substituent R1 is a hardly
migrating group, there is considerable chance for solvent
methanol and water to add at the 3- or 1-position,
respectively (Figure 8a,b). Subsequent leaving of metha-
nol and amine, respectively, yields stable end products,
i.e., 3-methoxyaniline (13, 14, 46, 48) and cyclohexa-2,5-
dienone derivatives (12, 46, 48, 49).
F igu r e 8. Decay possibilities of metastable/unstable ipso-
adducts formed from various arylnitrenium ions in MeOH [R1
) Me (10, 12, 46), Ph (10, 14, 48), fluorenyl (13); R2 ) H (12),
tBu (10), Ac (13, 14, 46, 48)]. For R1 ) OAlkyl and R2 ) SG, see
Figure 5.
drolysis of Me2-ketal could not be established (cf. Figure
8b). Instead, all decay products identified are known to
be sulfenamide cation descendants. Probably, the sulfe-
namide cation is restored back from the ketal by proto-
nation of the alternative site, i.e., the alkoxy oxygen atom
(Figure 5). Thus, sulfenamide cations appear to be much
more stable than other arylnitrenium ions, which is
explicable by a distinct mesomeric delocalization of the
positive charge to the sulfur atom. This prominent
resonance effect has been clearly demonstrated by kinetic
investigations (2), and was corroborated by molecular
orbital calculations (8).
The additional resonance contributor only possible in
sulfenamide cations enables a unique detoxication reac-
tion, i.e., addition of H2O to the sulfur atom, yielding a
sulfinamide [cf. discussion in (8)]. It might be argued,
therefore, that at limited availability of soft nucleophiles
such as GSH, this reaction with solvent H2O may suffice
for complete detoxication of sulfenamide cations inside
cells, particularly as their pronounced resonance stabi-
lization does not require an instant reaction with the
nearest nucleophile. The reported formation of ring
addition products with weak nucleophilic alcohols and
H2O (8), however, indicates striking similarity of the
In contrast, ipso-adducts derived from sulfenamide
cations apparently are not protonated at the imino
nitrogen atom, since formation of 4,4-dimethoxycyclo-
hexa-2,5-dienone or 1,4-benzoquinone during acid hy-
3
The lack of some product ions (Table 1) may stem from electronic
effects of the strong electronacceptor substituents on CID fragmenta-
tion.
4
The term “sulfenamide cation” will be used throughout in order
to distinguish N-sulfenylnitrenium ions from other arylnitrenium ions.

1432 Chem. Res. Toxicol., Vol. 11, No. 12, 1998
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Ack n ow led gm en t. The financial support of the DFG
(Deutsche Forschungsgemeinschaft; AZ Ga 495/2-1) is
gratefully acknowledged.
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