Additional pathways of S-conjugate formation during interaction of 4- nitrosophenetole with glutathione

Article abstract of DOI:10.1021/tx980087q

The rapid reactions of nitrosoarenes with cellular SH groups have proved to be main metabolic conversions during detoxication. Interactions of the phenacetin metabolite 4-nitrosophenetole with glutathione have been investigated in detail during the last years, revealing a complex pattern of products depending on the stoichiometry of the reactants and reaction conditions. Eight metabolites have been identified hitherto, and the present work extends this medley by six additional products. Three metastable sulfenamides, 4-ethoxy-2,N-bis(glutathion-S-yl)-aniline, N⁴-(glutathion-S- yl)-4-amino-4'-ethoxydiphenylamine, and N-(glutathion-S-yl)-4-aminophenol, as well as the N-sulfenylquinonimine N-(glutathion-S-yl)-l,4-benzoquinonimine were characterized by chemical reactivity, chromatographic behavior, UV/vis absorption, ¹H NMR, and FAB-MS data. The structure of the sulfenamide 2,N⁴- bis(glutathion-S-yl)-4-amino-4'-ethoxydiphenylamine could not be proved unequivocally, but is strongly suggested due to the chemical reactivity, chromatographic behavior, and UV/vis absorption of the compound. Finally, traces of 4-aminophenol were detected. A reaction scheme is presented explaining the formation of all identified metabolites via a central sulfenamide cation. Molecular orbital calculations for this sulfenamide cation have been performed, corroborating the proposed reaction mechanisms on the basis of Klopman's generalized perturbation theory.

Full text of DOI:10.1021/tx980087q

Chem. Res. Toxicol. 1998, 11, 1411-1422
1411
Ad d ition a l P a th w a ys of S-Con ju ga te F or m a tion d u r in g
In ter a ction of 4-Nitr osop h en etole w ith Glu ta th ion e
Dieter Gallemann,* Anke Greif, Peter Eyer, Hans-Ulrich Wagner,†
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
The rapid reactions of nitrosoarenes with cellular SH groups have proved to be main metabolic
conversions during detoxication. Interactions of the phenacetin metabolite 4-nitrosophenetole
with glutathione have been investigated in detail during the last years, revealing a complex
pattern of products depending on the stoichiometry of the reactants and reaction conditions.
Eight metabolites have been identified hitherto, and the present work extends this medley by
six additional products. Three metastable sulfenamides, 4-ethoxy-2,N-bis(glutathion-S-yl)-
aniline, N4-(glutathion-S-yl)-4-amino-4′-ethoxydiphenylamine, and N-(glutathion-S-yl)-4-ami-
nophenol, as well as the N-sulfenylquinonimine N-(glutathion-S-yl)-1,4-benzoquinonimine were
1
characterized by chemical reactivity, chromatographic behavior, UV/vis absorption, H NMR,
and FAB-MS data. The structure of the sulfenamide 2,N4-bis(glutathion-S-yl)-4-amino-4′-
ethoxydiphenylamine could not be proved unequivocally, but is strongly suggested due to the
chemical reactivity, chromatographic behavior, and UV/vis absorption of the compound. Finally,
traces of 4-aminophenol were detected. A reaction scheme is presented explaining the formation
of all identified metabolites via a central sulfenamide cation. Molecular orbital calculations
for this sulfenamide cation have been performed, corroborating the proposed reaction
mechanisms on the basis of Klopman’s generalized perturbation theory.
nitrosoarenes reacting with thiols (Figure 1). A strong
electron acceptor substituent promotes thiolytic cleavage
of the N-S bond of the semimercaptal, giving rise to
N-hydroxyarylamines. Electron-rich semimercaptals,
however, liberate a sulfenamide cation by cleavage of the
N-O bond, since the positive charge can be stabilized
by resonance effects of the aryl substituent [for details,
see (2) and literature cited therein]. First hints for the
intermediate occurrence of such a sulfenamide cation
have been presented by Klehr (4) and on a broader basis
by Kazanis and McClelland (5). Addition of solvent water
to the sulfur atom of the sulfenamide cation easily
explains the formation of sulfinamides (Figure 1, path-
way I), while GSH-mediated reduction leads to sulfen-
amides and arylamines (Figure 1, pathway III) (5). In
physiological systems, low molecular weight nucleophiles
such as GSH and H2O may not be the only sites of attack
by the sulfenamide cation. Addition to nucleophilic sites
of proteins and DNA has been discussed as well (5-8).
However, the reactivity of the sulfenamide cation toward
weak nucleophiles has been hardly studied and awaits a
detailed characterization.
In tr od u ction
Nitrosoarenes are known to be biological reactive
intermediates that are involved in toxic, allergic, mu-
tagenic, and carcinogenic effects [for review, see (2) and
literature cited therein]. This family is formed in vivo
during metabolic reactions of aromatic amines and ni-
troarenes which are inevitably incorporated as constitu-
ents of fried food, cigarette smoke, exhaust fumes, and
drugs. Hence, reactions of nitrosoarenes with cellular
thiols, predominantly GSH, are considered to be impor-
tant for detoxication.
During the interaction of nitrosoaromatics with thiols,
a variety of reaction pathways are followed depending
on the stoichiometry of the reactants, substituent(s) of
the nitrosoarene, pH and polarity of the reaction medium,
and pKa of the thiol (2). During these interactions,
formation of semimercaptals, N-hydroxyarylamines, sul-
finamides, sulfenamides, and arylamines has been de-
scribed. While electron-deficient nitrosoaromatics pre-
dominantly form the aforementioned compounds, a product
shift to sulfenamides and arylamines is observed with
increasing electron-donating character of the substituent
in the nitroso aryl ring. Initially, an intermediate
addition product, so-called semimercaptal, is formed from
The interaction of the phenacetin metabolite 4-nitro-
sophenetole (NOPt1) with GSH was found to yield a
highly complex product pattern (Figure 1). While forma-
tion of N-hydroxy-4-phenetidine could not be established
hitherto (7, 9, 10) and intermediate occurrence of a
semimercaptal could only be proved with 1-thioglycerol
(9), the main metabolites N-(glutathion-S-yl)-4-pheneti-
dine S-oxide (GSO-NHPt) (9, 11), N-(glutathion-S-yl)-4-
phenetidine (GS-NHPt) (9, 11, 12), and 4-phenetidine
* To whom correspondence should be addressed. Telephone: 49 89
5160-7281. Fax: 49 89 5160-7224. e-mail: gallemann@lrz-muenchen.de.
† Present address: Institut fu¨r Organische Chemie der Ludwig-
Maximilians-Universita¨t Mu¨nchen, Karlstrasse 23, D-80333 Mu¨nchen,
Germany.
‡ Present address: Max-Planck-Institut fu¨r Biochemie, Am Klopfer-
spitz 18a, D-82152 Martinsried, Germany.
10.1021/tx980087q CCC: $15.00 © 1998 American Chemical Society
Published on Web 12/03/1998

1412 Chem. Res. Toxicol., Vol. 11, No. 12, 1998
Gallemann et al.
F igu r e 1. Reaction pathways during interaction of NOPt with GSH. Main metabolites identified previously are shown as bold-face
structures; metabolites described in this paper are framed. Glutathione conjugates indicated by italic abbreviations could not be
proved unequivocally. Compound abbreviations were composed as follows: Primary arylamines are labeled by NH2, followed by a
suitable acronym of the aromatic system (Pt, P, EDPA). Sulfen- and sulfinamides, i.e., secondary amines, are accordingly indicated
by NH. Glutathione residues are prefixed in number and type [GS, (GS)2, GSO]. For detailed mechanistic steps of pathways III and
IV, cf. (2, 5, 7).
(NH2Pt) (9-11) have been identified long ago (Figure 1,
pathways I and III). Interestingly, the NOPt/GSH
interaction had shown a poor mass balance (10). In fact,
detailed investigations brought to light some additional
metabolites such as the thioether 4-ethoxy-2-(glutathion-
S-yl)aniline (GS-NH2Pt) (7) (Figure 1, pathway II).
Formation of this thioether was presumed to occur via
the sulfenamide 4-ethoxy-2,N-bis(glutathion-S-yl)aniline
[(GS)2NHPt] which, however, was not proved in those
days. Since (GS)2NHPt is the “missing link” between GS-
NH2Pt and the repeatedly suggested sulfenamide cation,
we tried to isolate and identify this unstable compound
by optimizing the reaction conditions.
In mere chemical systems of NOPt and GSH, the
bicyclic metabolite N-(4-ethoxyphenyl)-N′-(glutathion-S-
yl)-1,4-benzoquinone diimine (GS-EPQDI) and its reduc-
tion products 4-amino-4′-ethoxydiphenylamine (NH2-
EDPA) and 4-amino-4′-ethoxy-2-(glutathion-S-yl)diphen-
ylamine (GS-NH2EDPA) were detected only in traces (7)
(Figure 1, pathway IV). During metabolic conversion of
NOPt in red blood cells, however, the bicyclic arylamine
NH2EDPA was formed as a major metabolite (11). This
finding incited us to study the reaction pathways of
bicyclic metabolites in more depth. To rationalize the
formation of the end products NH2EDPA and GS-NH2-
EDPA, intermediate occurrence of the bicyclic sulfen-
amides N4-(glutathion-S-yl)-4-amino-4′-ethoxydiphenyl-
amine (GS-NHEDPA) and 2,N4-bis(glutathion-S-yl)-4-
amino-4′-ethoxydiphenylamine [(GS)2NHEDPA] has been
presumed (7). Therefore, these bicyclic sulfenamides
1
Abbreviations: AM1, Austin model 1; FAB-MS, fast atom bom-
bardment mass spectrometry; GS-EPQDI, N-(4-ethoxyphenyl)-N′-
(glutathion-S-yl)-1,4-benzoquinone diimine; GS-F5PQDI, N-pentaflu-
orophenyl-N′-(glutathion-S-yl)-1,4-benzoquinone diimine; GS-NH2EDPA,
4-amino-4′-ethoxy-2-(glutathion-S-yl)diphenylamine; GS-NH2Pt, 4-eth-
oxy-2-(glutathion-S-yl)aniline; GS-QI, N-(glutathion-S-yl)-1,4-benzo-
quinonimine; GS-NHEDPA, N4-(glutathion-S-yl)-4-amino-4′-ethoxy-
diphenylamine; (GS)2NHEDPA, 2,N4-bis(glutathion-S-yl)-4-amino-4′-
ethoxydiphenylamine; GS-NHP, N-(glutathion-S-yl)-4-aminophenol;
GS-NHPt, N-(glutathion-S-yl)-4-phenetidine; (GS)2NHPt, 4-ethoxy-2,N-
bis(glutathion-S-yl)aniline; GSO-NHPt, N-(glutathion-S-yl)-4-phen-
etidine S-oxide; LUMO, lowest unoccupied molecular orbital; MNDO,
modified neglect of differential overlap; NH2EDPA, 4-amino-4′-ethoxy-
diphenylamine; NH2Pt, 4-phenetidine; NOEDPA, 4-nitroso-4′-eth-
oxyphenylenediamine; NOF5DPA, 2,3,4,5,6-pentafluoro-4′-nitrosodi-
phenylamine; NOPt, 4-nitrosophenetole; NO2Pt, 4-nitrophenetole;
(2TE)2NHPt, 4-ethoxy-2,N-bis(2-thioethanol-S-yl)aniline; tR, retention
time; VR, retention volume.

4-Nitrosophenetole-Glutathione S-Conjugates
Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1413
Ta ble 1. Qu a n tifica tion of NOP t/GSH Meta bolites F or m ed u n d er Differ en t Stoich iom etr ies a n d Rea ction Con d ition s
1:1b,f
(Figure 2a)
1:5b,f
(Figure 2b)
1:5c,f
(Figure 2c)
1:4d,g
(Figure 3a)
1:4d,e,h
(Figure 3b)
metabolitea
GS-QI
0.8 ( 0.3
9.3 ( 1.1
4.4 ( 0.8
0
1.4 ( 0.5
33 ( 6
1.6 ( 0
ndj
0.7 ( 0.2
4.4 ( 0.2
11 ( 1
0
1.5 ( 0.2
68 ( 1
5.6 ( 0.4
nd
0
0.9 ( 0.1
0
3
1
0
2
10
2i
1
GSO-NHPt
(GS)2NHPt
GS-NH2Pt
4.6 ( 0.4
7.4 ( 0.4
3.9 ( 0.3
0.9 ( 0
4.9 ( 0.9
70 ( 1
nd
12 ( 1
7.5 ( 0.5
0
235/274 nmk
GS-NHPt
4.2 ( 0.5
51 ( 1
NH2Pt
8.3 ( 1.2
(GS)2NHEDPA
GS-NH2EDPA
GS-NHEDPA
GS-EPQDI
NH2EDPA
NOPt/NO2Pt
0
0
nd
nd
nd
9
0.8g
0.5 ( 0.1
0.7 ( 0
0
0
0
2.0 ( 0.3
37
26
1
1.1 ( 0.3
0
0
0
1.5 ( 0.2
1.9 ( 0.1
36 ( 9
2.2 ( 0.1
3.0 ( 0.6
2
Σ
87 ( 11
94 ( 1
94 ( 1
85 ( 2
90
a
Yields of [u-ring-14C]-NOPt metabolites are expressed as percent of the total radioactivity recovered during the HPLC run (HPLC
recovery of the radioactivity injected was 97 ( 3%). GSH added rapidly, reaction time 1 min. c 0.5 h reaction time. GSH added slowly
b
d
within 2 min during vigorous mixing. e GSH added in the presence of 1.0 mM nonradioactive NH2Pt. f xj ( σn-1, n ) 3. xj ( σn-1, n ) 4.
g
h
i
j
k
n ) 1.
[
14C]-NH2Pt. Not detected because of insufficient separation. Unknown metabolite, cf. Results.
retention of hydrophilic compounds such as GS-NHP and
4-aminophenol was the following: 0-6 min, 18-45% MeOH;
6-14 min, 45-90% MeOH; 14-16 min, 90% MeOH; flow, 1.0
mL/min (Figure 2). Chromatography of the bicyclic products
proved to be more successful on Nova-Pak C18 (150 mm × 3.9
mm i.d., 4 µm; Waters) using a sodium phosphate buffer (10
mM, pH 6.5)/MeOH gradient: 0-4 min, 18-36% MeOH; 4-24
min, 36-90% MeOH; 24-26 min, 90% MeOH; flow, 1.0 mL/
min (Figure 3). For semipreparative separations, µ-Bondapak
C18 columns (300 mm × 7.8 mm i.d., 10 µm, Waters) and MeOH/
ammonium hydrogen carbonate buffer (10 mM, pH 6.5) gradi-
ents were used. Peaks were identified by comparing the UV/
vis spectra (220-500 nm) and retention times with those of
authentic compounds.
To determine UV/vis extinction coefficients, the metabolites
were prepared from a radioactive precursor with known specific
activity and separated from byproducts by HPLC. pKa values
were determined by spectroscopic titration. GSH liberation
from purified metabolites was quantified by nonenzymic reac-
tion with 5,5′-dithiobis(2-nitrobenzoic acid) in 2-(N-morpholino)-
ethanesulfonic acid buffer (0.4 M, pH 6) (21). To this end, the
metabolite concentration of an aqueous solution was determined
and applied to the test before and after acidic hydrolysis (pH 1,
10 min at room temperature). GSSG was measured in the same
probes by enzymic reduction (glutathione reductase/NADPH)
in sodium phosphate buffer (0.2 M, pH 7.4) (22).
were searched for in addition. As a result, we succeeded
in isolation and identification of the expected intermedi-
ates, giving further support of the sulfenamide cation
mechanism.
Exp er im en ta l P r oced u r es
Ch em ica ls. NH2Pt was purchased from Aldrich Chemie (D-
89555 Steinheim); ascorbic acid, 2-thioethanol, and 4-nitroso-
phenol were from Fluka AG (CH-9470 Buchs). GSH and
dithiothreitol were obtained from Boehringer (D-68305 Mann-
heim); N-acetyl-[u-ring-14C]-4-phenetidine (specific activity 102
MBq/mmol) was from New England Nuclear (D-63303 Dreieich).
All other reagents were purchased from Merck (D-64283 Darm-
stadt) at the purest grade available.
NOPt (13) (containing 0.7% nitrophenetole), [u-ring-14C]-
NOPt (11) [17 MBq/mmol; containing 2% 4-nitrophenetole (NO2-
Pt), 2% azoxyphenetole, and 0.4% 4-ethoxy-4′-nitrosodiphen-
ylamine], [u-ring-14C]-NH2Pt (14), GS-NH2Pt (7), GS-NHPt (12),
and GS-EPQDI (7) were prepared as described previously.
2-Hydroxy-4-phenetidine was a gift of Dr. K.-G. Eckert (this
institute) (15). Stock solutions of lipophilic reagents were
prepared in methanol.
Ca u tion : Nitrosoarenes bearing π-donating substituents have
tested positive in the Ames mutagenicity assay with Salmonella
typhimurium without activation (16-20).
In str u m en ta tion a n d An a lytica l Meth od s. 1H NMR
spectra were recorded with an AM-400 MHz or an AX-500 MHz
instrument from Bruker (D-76287, Rheinstetten-Forchheim),
0.00 ppm being assigned with external sodium 3-trimethyl-
silylpropanesulfonate in D2O. Electron impact mass spectro-
metry and fast atom bombardment mass spectrometry (FAB-
MS) were performed with a CH-7A instrument from Varian or
a MAT 900 instrument, respectively, each coupled with a data
system SS 200 MS from Finnigan MAT (D-28197 Bremen).
Analytical HPLC was performed with a gradient system con-
sisting of a low-pressure gradient former L 6200 (Merck-Hitachi,
D-64293 Darmstadt), and a UV/vis diode array detector SPDM6A
(Shimadzu, D-47269 Duisburg), coupled with a personal com-
puter. For semipreparative HPLC, a gradient system was used
consisting of an AS-4000A autosampler, a low-pressure gradient
former L 6200A (mixing chamber volume 2 mL), a D-2500
chromato-integrator, an L-7650 fraction collector (Merck-Hita-
chi), and an SPD-6AV UV/vis spectrophotometric detector
(Shimadzu).
For quantification of the products emerging in NOPt/GSH
mixtures (Table 1), [u-ring-14C]-NOPt (about 100 mM in MeOH)
was diluted to 1.0 mM in sodium phosphate buffer (0.2 M, pH
7.4, ambient temperature) and mixed with different volumes of
GSH (100 mM in sodium phosphate buffer, 0.2 M, pH 7.4).
Although NOPt is soluble in sodium phosphate buffer (5 mM,
pH 7.4) up to 2 mM at ambient temperature, the radioactivity
in the reaction mixtures was not totally recovered, probably
because of adsorption effects to the glass vials. This effect was
even more pronounced when using Eppendorf cups. Thus, 0.1
volume of MeOH was added to the incubates directly prior to
sampling whereby recovery was raised to 93 ( 4%. Higher
MeOH proportions were not used in order to maintain chro-
matographic resolution and to prevent reaction of the sulfen-
amide cation with alcohol (8). The profile of products obtained
in the chromatograms is assumed to be representative for the
time of sampling, since GSH separates quickly from the arylic
compounds upon injection into the HPLC system (within less
than 1 min); only the sulfenamides may decay to some extent
during separation. Radioactivity was measured in Bray’s
solution (23) with an LKB Rackbeta 1217 liquid scintillation
counter.
Analytical separations of the monocyclic metabolites were
carried out, if not otherwise stated, on Lichrospher 100 RP-18
(250 mm × 4 mm i.d., 5 µm; Merck) with MeOH/sodium
phosphate buffer (10 mM, pH 6.5) gradients and detection at
230 and 400 nm. A typical gradient which also allowed
Molecular orbital calculations were performed on a COMPAQ
personal computer prolinea 16/66 using the MNDO (modified

1414 Chem. Res. Toxicol., Vol. 11, No. 12, 1998
Gallemann et al.
Ta ble 2. UV/Vis Da ta of Som e N-Su bstitu ted
neglect of differential overlap) (24, 25) or AM1 (Austin model
1) (26, 27) Hamiltonian of the semiempirical molecular orbital
program MOPAC 6.12 (Quantum Chemistry Program Exchange,
Indiana University, Bloomington, IN) (28). Geometries were
optimized for all bond lengths, bond angles, and torsion angles.
All calculations were conducted with PRECISE criteria. Self-
consistent field was achieved for all calculations.
Syn th eses. (A) [14C]-4-Nitr osop h en ol. [u-Ring-14C]-NOPt
(0.15 mL; 86 mM in MeOH) was added to 0.50 mL of NaOH
(2.5 M) and the mixture incubated for 3 days at 37 °C (6). Upon
HPLC chromatography of a neutralized aliquot, the solutions
proved to consist of 4-nitrosophenol containing traces of three
unknown impurities. The alkaline solution was stored at -20
°C and was neutralized prior to use.
Ben zoqu in on (d i)im in es
compound
λ1 (log ꢀ)
λ2 (log ꢀ)
4-nitrosophenola,b (71)
NOEDPAc(11, 29)
NOF5DPAd
265 nm (3.63)
264 nm (3.98)
260 nmf
275 nm (3.69 sh)
264 nm (3.99)
-
397 nm (4.45)
428 nm (4.41)
398 nmf
414 nm (4.34)
418 nm (4.28)
422 nmf
GS-QIe
GS-EPQDIa (7)
GS-F5PQDId
a
b
In phosphate buffer, pH >8. Nitrosoarenes bearing -OH or
-NHR have been shown to undergo nitroso-oxime tautomerism
d
resulting in a quinoid structure (72-74). c In MeOH. In MeOH/
ammonium hydrogen carbonate buffer (10 mM, pH 6.5; 45/55, v/v).
e In phosphate buffer, pH 7.4, 3% MeOH. f ꢀ not determined.
(B) 4-Eth oxy-2,N-bis(glu tath ion -S-yl)an ilin e, (GS)2NHP t.
GSH (50 mM in sodium phosphate buffer, 0.07 M, pH 7.4) was
mixed 1/1 (v/v) with NOPt (10 mM in MeOH) and allowed to
react for about 1 min at 37 °C (yield: 10% of theory as detected
with [u-ring-14C]-NOPt). Since slow decomposition into GS-NH2-
Pt occurred due to excess GSH (12), the product was im-
mediately separated by semipreparative HPLC [isocratic elution
with MeOH/ammonium hydrogen carbonate buffer (10 mM, pH
6.5), 13/87 (v/v), VR ≈ 20 mL]. The collected HPLC cut was
instantly frozen at -70 °C and lyophilized. The corresponding
2-thioethanol derivative was prepared and separated in analogy,
using isocratic elution with MeOH/ammonium hydrogen car-
bonate buffer (10 mM, pH 6.5), 48/52 (v/v), VR ≈ 36 mL.
(C) N4-(Glu t a t h ion -S-yl)-4-a m in o-4′-et h oxyd ip h en yl-
a m in e, GS-NHEDP A. GS-EPQDI (2 mM in H2O, pH
7
adjusted with NH3) was reacted with 2.5 equiv of ascorbate (100
mM in H2O, pH 7 adjusted with NH3) at room temperature. The
solution discolored within a few minutes. To isolate the desired
compound, the reaction mixture was applied to semipreparative
HPLC (MeOH/H2O gradient: 0-10 min, 25-90% MeOH). On
contact with atmospheric oxygen, the isolated sulfenamide
solution turned orange, indicating autoxidation with formation
of the educt. Therefore, the HPLC cuts were collected under
argon and immediately frozen at -78 °C. After lyophilization,
the slightly reddish powder contained small impurities of GS-
EPQDI and NH2EDPA.
(D) N-(Glu ta th ion -S-yl)-1,4-ben zoqu in on im in e, GS-QI.
A saturated solution of 4-nitrosophenol (about 12 mM) in sodium
phosphate buffer (0.2 M, pH 7.4) was rapidly mixed with 1.5
equiv of GSH and the desired product immediately isolated by
HPLC [Lichrospher 100 RP-8, 125 mm × 4 mm i.d.; elution with
MeOH/sodium phosphate buffer (10 mM, pH 7.52), 3/97 (v/v);
4-nitrosophenol
GS-QI
VR
≈ 3.6 mL, VR
≈ 9 mL; yield 7%, as detected
with [14C]-4-nitrosophenol]. To free this compound from sodium
salts disturbing FAB-MS, the product was subjected to a second
HPLC run (PrepNovapak C18, 300 mm × 8 mm i.d., Waters;
GS-QI
isocratic elution with H2O/MeOH, 98/2, v/v; VR
≈ 18 mL).
(E) N-(Glu ta th ion -S-yl)-4-a m in op h en ol, GS-NHP . Isola-
tion of this sulfenamide in substance was not possible because
of an inevitable decay to 4-aminophenol. To obtain the 1H NMR
and FAB-MS spectra of this compound, GS-QI was reduced with
dithiothreitol immediately before recording the spectra. Specif-
ically, 2 µmol of GS-QI (in 0.25 mL of D2O) was mixed with 2.2
µmol of dithiothreitol (in 0.25 mL of D2O, pH 7 adjusted with
ammonia). After complete bleaching, the batch was chromato-
graphed on a Sep Pak C18 cartridge (Waters). GS-NHP was
slowly eluted with 1.5 mL of D2O, rejecting the first 0.5 mL to
remove protons liberated from dithiothreitol. The collected
fraction was investigated by HPLC in order to prove the desired
sulfenamide and stored at -78 °C until recording of the spectra.
(F ) N-P en ta flu or op h en yl-N′-(glu ta th ion -S-yl)-1,4-ben -
zoqu in on e d iim in e, GS-F 5P QDI. Pentafluoroaniline (0.4
mmol in 2 mL of EtOH) was reacted with equimolar amounts
of NOPt (in 2 mL of EtOH) in the presence of hydrochloric acid
F igu r e 2. RP-HPLC chromatograms of various NOPt/GSH
reaction mixtures (in sodium phosphate buffer, 0.2 M, pH 7.4;
ambient temperature). Separation was performed as described
under Experimental Procedures. (a) 1.0 mM NOPt + 1.0 mM
GSH, added rapidly, 1 min reaction time. (b) 1.0 mM NOPt +
5.0 mM GSH, added rapidly, 1 min reaction time. (c) As (b),
but 0.5 h reaction time. Metabolites framed are reported in this
paper.
2
For successful separation of 4-nitrosophenol, the pH of the eluent
4-nitrosophenol
had to be elevated in order to protonate the phenolate (pKa
≈ 6.1).

4-Nitrosophenetole-Glutathione S-Conjugates
Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1415
Ta ble 3. 1H NMR Da ta of GS-QI, GS-EP QDI, a n d GS-F 5P QDI
chemical shifts, ppm, (relative intensity), multiplicities, and coupling constants, Hz
protons
H2/H6
GS-QIa
GS-EPQDIa
GS-F5PQDIb
7.18 (1), dd, 10.0/2.6
7.57 (1), dd, 10.0/2.6
6.61 (1/2), dd, 10.0/1.6
6.76 (1/2), dd, 10.0/1.6
6.93 (1/2), dd, 10.0/1.6
6.95 (1/2), dd, 10.0/1.6
7.03 (1/2), dd, 10.0/1.6
7.10 (1/2), dd, 10.0/1.6
7.23 (1/2), dd, 10.0/1.6
7.34 (1/2), dd, 10.0/1.6
4.61 (1), m
6.8-7.3 (8), m
H3/H5
6.64 (1), dd, 10.0/2.6
6.56 (1), dd, 10.0/2.6
Cys Rc
Cys â1
Cys â2
Gly R
Glu R
Glu â
Glu γ
4.94 (1), dd, 5.0/8.6
3.64 (1), dd, 8.6/14.8
3.87 (1), dd, 5.0/14.8
3.81 (2), m
3.76 (1), m
2.13 (2), m
4.88 (1), dd, 5/9
3.55 (1), dd, 9/15
3.3d
3.86 (4), m
3.5d
3.68 (1), m
1.90 (2), m
2.32 (2), m
2.16 (2), m
2.52 (2), m
2.52 (2), m
a
b
d
In D2O. In DMSO-d6. c Glutathione protons were assigned according to (75). Signal not resolved or overlapped with solvent signals.
Ta ble 4. F AB-MSa F r a gm en ts of (GS)2NHP t a n d Its
(0.4 mmol, 37%) at 80 °C for 1 h to give 2,3,4,5,6-pentafluoro-
4′-nitrosodiphenylamine (NOF5DPA) (29). After removal of the
solvent, the residue was dissolved in 5 mL of CHCl3 and the
product extracted with 10 mL of MeOH/2.5 M NaOH (v/v). The
alkaline phase was neutralized, and the product was reextracted
with diethyl ether and purified by semipreparative HPLC: yield,
1.3% of theory; electron impact mass spectrometry, m/z 288
[M+•]; UV/vis, see Table 2. NOF5DPA [5 µmol in 1 mL of MeOH/
ammonium hydrogen carbonate buffer (0.1 M, pH 10)] was
incubated with equimolar GSH at room temperature overnight.
The desired product, GS-F5PQDI, was purified by semiprepara-
tive HPLC: yield, 75% of theory; FAB-MS (in m-nitrobenzyl
alcohol matrix, acidified with acetic acid), m/z 578 (5%, [M+H]+),
m/z 579/580 (3%, [Mred+H]+) (7), m/z 600 (15%, [M+Na]+), m/z
601/602 (5%, [Mred+Na]+), m/z 622 (6%, [M-H+2Na]+), m/z 623/
624 (2%, [Mred-H+2Na]+), m/z 644 (5%, [M-2H+3Na]+), m/z
645/646 (2%, [Mred-2H+3Na]+). It should be noted that cluster
ions with a difference of 3 × 22 u have been observed repeatedly
for glutathione conjugates, reflecting the additional exchange
2-Th ioeth a n ol Der iva tive (2TE)2NHP t
(GS)2-
NHPtb
(2TE)2-
NHPtc
(2TE)2-
NHPtd
fragment
M ) 747
M ) 289
M ) 289
[M]+
289 (73%) 289 (100%)
290 (38%) 290 (88%)
291 (12%) 291 (22%)
312 (25%) 312 (11%)
382 (9%) 382 (3%)
[M+H]+
[M+2H]+
[M+Na]+
[M+H+glycerol]+
[(M-SR+H)+H]+
[(M+H)-SR]+
443 (5.0%) 214 (22%) 214 (27%)e
213 (73%) 213 (83%)
465 (0.5%)
[(M-SR+H)+Na]+
[(M-SR+H)+H+glycerol]+ 535 (0.5%)
306f
[(M-SR+H)+H-R]+
[(M+H)-SR-R]+
169 (2.5%)
168 (37%) 168 (28%)
a
b
In glycerol matrix. Generation of FAB-MS peaks required
acidification of the target with 1 N HCl. This procedure most
probably resulted in chemical hydrolysis of the N-S conjugated
thiol residue indicated in the fragment row as (M-SR+H).
1
of the two carboxylic protons by sodium (30). For H NMR data,
see Table 3.
d
c Without acidification of the FAB-MS target. FAB-MS target
acidified with 1 N HCl. e After further dilution with glycerol, this
fragment was the main peak in the FAB-MS spectrum, while the
bisthiol peaks disappeared completely. f Only after further dilution
with glycerol.
Resu lts
(GS)2NHP t. This compound was detected in highest
amounts when NOPt was reacted with high excess GSH
at room temperature and the batch immediately applied
to HPLC without any delay [e.g., ether extraction (7)]
(Figure 2b). Upon protracted reaction, the metabolite
decayed slowly with concomitant increase of GS-NH2Pt
(Figure 2c, Table 1). In fact, the isolated compound
slowly liberated the respective arylamine GS-NH2Pt in
aqueous solutions, with acid or alkaline pH (1 and 12.5,
respectively; 10 min at room temperature) accelerating
hydrolysis. Besides GS-NH2Pt, formation of 1/3 equiv of
GSSG was detected. Reaction of isolated (GS)2NHPt with
sodium dithionite resulted in complete formation of GS-
NH2Pt within 3 min as did ascorbate and GSH on
prolonged incubation (1/2 and 2 h, respectively). These
results are in agreement with a sulfenamide structure
which is known to be cleaved under the above-mentioned
reaction conditions (6, 31) liberating the corresponding
amine and glutathione sulfenic acid that is assumed to
decompose into GSO2H and GSSG (2, 4, 32).
The FAB mass spectrum of the glutathione derivative
(Table 4) did not exhibit the expected pseudo molecular
ion [M+H]+ (m/z 748), probably because of insufficient
ionization. In fact, bisglutathione adducts have been
reported to yield particular disappointing results during
FAB-MS (33). However, upon acidification of the FAB-
MS target, a peak was detected at m/z 443 corresponding
to the pseudo molecular ion of GS-NH2Pt, the hydrolysis
product of (GS)2NHPt. Further ions at m/z 465 [(M-
SR+H)+Na]+, m/z 535 [(M-SR+H)+H+glycerol]+, and
m/z 169 [(M-SR+H)+H-(Glu-Ala-Gly)]+ (7) were ob-
served. Because of the lacking molecular ion peak of the
presumed bisglutathione conjugate, the respective 2-thio-
ethanol derivative (2TE)2NHPt was synthesized. (2TE)2-
NHPt exhibited analogous chemical reactivity as the

1416 Chem. Res. Toxicol., Vol. 11, No. 12, 1998
Gallemann et al.
Ta ble 5. 1H NMR Da ta of th e Mon ocyclic Su lfen a m id es (GS)2NHP t, GS-NHP t, a n d GS-NHP a n d of th e Th ioeth er
GS-NH2P t
chemical shifts, ppm,a (relative intensity), multiplicities, and coupling constants, Hz
protons
(GS)2NHPt
GS-NH2Ptb
GS-NHPtc
GS-NHP
CH3-
1.31 (3), t, 7.4
1.47 (3), t, 6.8
1.36 (3), t, 7.0
-CH2O-
H3
4.03(2), q, 7.4
7.06 (1), d, 2.4
6.96 (1), dd, 9.0/2.4
7.36 (1), d, 9.0
4.51 (1), dd, 4.4/9.2
4.32 (1), dd, 4.4/9.0
2.84 (1), dd, 9.2/15.1
3.10 (1), dd, 9.0/14.8
3.17 (1), dd, 4.4/15.1
3.28 (1), dd, 4.4/14.8
4.21 (2), q, 6.8
7.28 (1), d, 2.6
7.07 (1), dd, 9.0/2.6
7.21 (1), d, 9.0
4.09 (2), q, 7.0
H5 (H3)
H6 (H2)
Cys Rd
Cys R′
Cys â1
Cys â1′
Cys â2
Cys â2′
Gly R/R′
Glu R/R′
Glu â/â′
Glu γ/γ′
6.95 (2), d, 9.0
7.09 (2), d, 9.0
4.52 (1), dd, 5.0/8.6
6.80 (4), s
4.59 (1), m
4.62 (1), dd, 4.6/7.6
3.37 (1), dd, 7.6/14.6
2.91 (1), dd, 8.6/14.8
3.18 (1), dd, 5.0/14.8
2.97 (1), m
3.18 (1), m
3.44 (1), dd, 4.6/14.6
3.76 (2), m
3.84 (1), m
2.19 (2), m
2.55 (2), m
3.79 (2), s
3.68 (1), m
2.19 (2), m
2.58 (2), m
3.6 (6), m
3.74 (3), m
1.99 (4), m
2.38 (4), m
2.13 (2), m
2.50 (2), m
a
b
d
Chemical shifts in D2O. From (7). c Mulder et al. have reported similar 1H NMR data determined in CD3OD (12). Glutathione
protons were assigned according to (75).
glutathione derivative except for the alkaline hydrolysis.
The FAB mass spectrum showed a molecular ion cluster
at m/z 289-291 consistent with a bisthiol conjugate.
Further ions emerged at m/z 312 [M+Na]+ and m/z 382
[M+H+glycerol]+, and fragments at m/z 213 [(M+H)-
SR]+ and m/z 168 [(M+H)-SR-R]+, the latter reflecting
one labile bound thiol substituent only. Similarly, the
arylthioether GS-NH2Pt exhibited intense fragmentation
by neutral loss of Glu-Ala-Gly, but not Glu-Cys-Gly (7).
Acidification of the FAB-MS target resulted in increased
ionization yields at first, and in complete hydrolysis to
the mono-conjugate after further dilution with glycerol
(Table 4).
detected in traces only (Figures 2b and 3a), whereas it
was formed as a main product in the presence of equimo-
lar NH2Pt (Figure 3b). As mentioned previously (7), this
trick enlarges the yields of bicyclic metabolites many
times at the expense of monocyclic products, especially
when GSH is added slowly in order to generate but not
to eliminate the sulfenamide cation (cf. Figures 1 and
3a,b; Table 1). Contact of the isolated, colorless (log ꢀ285
) 4.28) GS-NHEDPA with atmospheric oxygen re-
nm
sulted in progressive autoxidation to the orange colored
quinone diimine. Under inert gas atmosphere and room
temperature, the sulfenamide slowly decomposed to yield
mainly NH2EDPA (7, 11). Acid hydrolysis resulted in
complete decomposition to NH2EDPA within 10 min.
Alkaline hydrolysis under the same conditions produced
significant amounts of GS-EPQDI in addition, probably
because of more rapid autoxidation at high pH. During
reaction of the isolated sulfenamide with ascorbate (pH
7), very slow formation of NH2EDPA was observed.
Interaction with GSH proceeded more rapidly, yielding
the arylamine NH2EDPA exclusively. Immediate reduc-
tion to the arylamine was found on reaction with sodium
dithionite.
1
The H NMR spectrum of the glutathione derivative
showed signals belonging to the ethoxy group and two
different glutathionyl substituents, and aromatic signals
of three protons indicating a 1,2,4-substituted aromatic
ring. By comparison of the cysteine â signals with the
thioether GS-NH2Pt and the sulfenamide GS-NHPt, the
glutathione residues could be easily assigned to one
thioether- and one sulfenamide-type substituent (Table
5). The thioether binding site at ring position 2 is
indicated by the above-described decay reactions under
formation of GS-NH2Pt. The UV/vis spectrum of (GS)2-
The 1H NMR spectrum of GS-NHEDPA (Table 6)
showed eight aromatic protons with mutual overlap. The
high field signals were assigned to one ethoxy and one
glutathione residue, the cysteine â signals corroborating
NHPt (log ꢀ242
) 4.04, log ꢀ318
) 3.51; in H2O)
nm
nm
resembles both the sulfenamide GS-NHPt (12) and the
thioether GS-NH2Pt (7). For comparison, the electronic
spectra are shown in Figure 4a. In summary, formation
conditions, chemical reactions, and spectroscopic data of
the compound are consistent with the proposed structure.
GS-EP QDI Descen d a n ts. GS-NHEDPA has been
presumed as an intermediate sulfenamide during reduc-
tion of GS-EPQDI to NH2EDPA (7). In fact, this sulfen-
amide was rapidly and exclusively formed during reaction
of isolated GS-EPQDI with weak reductants such as
ascorbate (cf. Experimental Procedures). In reaction
mixtures of NOPt with GSH, this intermediate could be
1
the presumed sulfenamide structure (cf. H NMR of the
monocyclic sulfenamides in Table 5).
During reaction of NOPt with GSH in the presence of
NH2Pt, not only GS-NHEDPA and NH2EDPA were
formed but also the thioether GS-NH2EDPA (7) and
possibly its sulfenamide precursor (GS)2NHEDPA
emerged. The small HPLC-peak eluting immediately in
front of the thioether GS-NH2EDPA (Figure 3b) was
assumed to reflect the postulated bis-conjugate, since its
UV/vis spectrum resembled both the bicyclic thioether

4-Nitrosophenetole-Glutathione S-Conjugates
Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1417
F igu r e 3. RP-HPLC chromatogram of reaction mixtures of 1.0
mM NOPt and 4.0 mM GSH (injected slowly within 2 min under
vigorous mixing) in the (a) absence and (b) presence of 1.0 mM
NH2Pt (reaction in 0.2 M sodium phosphate buffer, pH 7.4, at
room temperature). Separation of 100 µL aliquots was performed
as described under Experimental Procedures. Metabolites framed
are reported in this paper.
F igu r e 4. Diode array UV/vis spectra of (a) monocyclic and
(b) bicyclic sulfenamides/thioethers in sodium phosphate buffer
(10 mM, pH 6.5)/MeOH. (a) (GS)2NHPt, λmax1 ) 242 nm, λmax2
) 318 nm (s); GS-NH2Pt, λmax1 ) 235 nm, λmax2 ) 312 nm (- - -);
and GS-NHPt, λmax1 ) 245 nm, λmax2 ) 297 nm (‚‚‚). (b) (GS)2-
NHEDPA (presumed), λsh ≈ 255 nm, λmax ) 291 nm (s); GS-
NH2EDPA, λmax1 ) 245 nm, λmax2 ) 287 nm (- - -); and GS-
NHEDPA, λmax ) 285 nm (‚‚‚). Maximum absorptions are
normalized to 80% of the y-coordinate.
Ta ble 6. 1H NMR Da ta of GS-NH2EDP A a n d GS-NHEDP A
based on chemical reactivity as investigated with the
HPLC-isolated peak. During incubation of the HPLC-
cut overnight at room temperature, progressive decay
with liberation of GS-NH2EDPA was observed. Reaction
with ascorbate did not proceed as already observed with
GS-NHEDPA (see above). Reduction with sodium dithio-
nite or GSH, respectively, yielded the expected thioether
GS-NH2EDPA, but amazingly proceeded rather slowly.
Acid hydrolysis liberated the thioether GS-NH2EDPA,
corroborating the presumed sulfenamide structure of the
metabolite. The thioether linkage of (GS)2NHEDPA and
GS-NH2EDPA is probably not affected during the above-
described reduction and hydrolysis reactions, suggesting
a GS-attachment meta to the sulfenamide group of
(GS)2NHEDPA (cf. Figure 1).
GS-QI a n d Its Red u ction P r od u cts. During reac-
tion of NOPt with equimolar amounts of GSH, a very
hydrophilic orange-colored metabolite was observed (Fig-
ure 2a: GS-QI). Despite the low yields of about 1%
(Table 1), our interest in this product was elicited since
it was also formed during reaction of both 4-nitrosoani-
sole [cf. Figure 2 of the following paper (8)] and 4-nitroso-
phenol (cf. synthesis of GS-QI under Experimental
Procedures), indicating loss of the aromatic alkoxy sub-
stituent. Furthermore, its UV/vis spectrum resembled
a quinonimine derivative like the already known GS-
EPQDI (Table 2), and the low amounts of GSH favoring
chemical shifts, ppm,a (relative intensity),
multiplicities, and coupling constants, Hz
protons
GS-NH2EDPAb
GS-NHEDPA
CH3-
-CH2O-
H3
H5
H6
H2′/6′
H3′/5′
Cys Rd
Cys â1
Cys â2
Gly R
Glu R
Glu â
Glu γ
1.46 (3), t, 6.6
4.21 (2), q, 6.6
7.53 (1), s
1.42 (3), t, 7.0
4.17 (2), nrc
7.21 (1), d, 8
7.12 (1), d, 8
7.22 (2), d, 8
7.09 (2), d, 8
4.50 (1), dd, 5/9
3.28 (1), dd, 9/15
3.47 (1), dd, 5/15
3.71 (2), m
7.1 (8), nrc
4.59 (1), dd, 5/9
2.98 (1), dd, 9/15
3.26 (1), dd, 5/15
3.8 (3), m
3.76 (1), m
2.11 (2), m
2.51 (2), m
2.19 (2), m
2.56 (2), m
a
b
d
Chemical shifts in D2O. From (7). c Not resolved. Glu-
tathione protons were assigned according to (75).
GS-NH2EDPA and the sulfenamide GS-NHEDPA (Figure
4b). Because of the low yields, this product was not
amenable to H NMR, and FAB-MS was expected to be
unsuccessful as described for other bisglutathione con-
jugates (33). Thus, indications for its structure are only
1

1418 Chem. Res. Toxicol., Vol. 11, No. 12, 1998
Gallemann et al.
its generation suggested an interesting reaction of the
sulfenamide cation with another nucleophile.
reduction on the target (7)] were consistent with the
assumed structure N-(glutathion-S-yl)-1,4-benzoquinon-
imine. Besides the pseudo molecular ions m/z 413 (3%;
[M+H]+) and 435 (1%, [M+Na]+), a fragment at m/z 107
(28%; [OdC6H4dN+H]+•) was detected which may be a
characteristic fragment of N-sulfenylquinonimines.3
The 1H NMR spectrum of GS-NHP revealed signals
according to one glutathione residue. The chemical shifts
of the cysteine â protons agreed with a sulfenamide
structure rather than with a thioether (cf. Table 5). FAB-
MS of GS-NHP (synthesized instantly before MS as
described under Experimental Procedures) exhibited
pseudo molecular ions at m/z 415 (1.0%) [M+H]+, m/z 437
(1.7%) [M+Na]+, and m/z 459 (0.9%) [M-H+2Na]+
besides m/z 413 (1.4%) probably reflecting residual educt
GS-QI. The poor abundance of the molecular ion is not
unexpected and has been attributed to the low surface
activity of a variety of glutathione conjugates (33).
F u r th er Meta bolites. During reaction of the sulfen-
amide cation with various nucleophiles, addition of
solvent water to the ortho-position may be suggested in
analogy to GSH addition during formation of (GS)2NHPt/
GS-NH2Pt (Figure 1, pathway II). The resulting, eventu-
ally stable product would be 2-hydroxy-4-phenetidine.
This compound could not be detected in the HPLC
chromatograms of different NOPt/GSH incubates (Figure
2: tR ≈ 11.8 min), even not when complete decay of all
metastable sulfenamides had been waited for.
Some new metabolites often observed in NOPt/GSH
reaction mixtures could not be characterized completely
because of low yields and instability. One metabolite
occurring only at low GSH concentrations was eluted
immediately after GS-QI (Figure 2a, tR ) 7.5 min),
exhibiting a characteristic UV/vis spectrum resembling
quinonimines (λmax ) 412 nm, λsh ≈ 390 nm; cf. Table 2).
A second metabolite hidden under the first GSO-NHPt
diasteriomer peak (Figure 2c, shoulder at tR ) 8.5 min)
is formed only at high GSH concentrations and prolonged
reaction times (λmax: 299/318 nm). Interestingly, deriva-
tives with analogous UV/vis spectra were not found when
NOPt was reacted with other thiols. Since this metabo-
lite was formed in higher yields during decomposition of
isolated GS-NHPt, it seemed to be a direct descendant
of this sulfenamide. Another product peak eluting im-
mediately before the sulfenamide GS-NHPt (Figure 3a,
tR ) 7.3 min; λmax: 235/274 nm) is interesting because of
its requirements for formation: This compound emerged
in good yields only at high GSH amounts which had to
be infused slowly into the batch (4%, cf. Table 1). Acid
hydrolysis yielded NH2Pt, excluding this metabolite to
be a sulfenamide of the 2-hydroxy-4-phenetidine men-
tioned above.
GS-QI was stable, both at pH 0.5 and at pH 11.5 for
at least 10 min, but decomposed on prolonged incubation
under formation of unknown compounds. When GS-QI
was reduced with ascorbate (at pH 6.5, ambient temper-
ature), the solution discolored within a few minutes,
yielding only GS-NHP with a UV/vis spectrum resem-
bling monocyclic sulfenamides [λmax1 ) 240 nm, λmax2
)
299 nm, ꢀ1/ꢀ2 ≈ 4; cf. GS-NHPt in Figure 4a and (2)]. GS-
NHP was stable in the presence of ascorbate for at least
1 h, but liberated 4-aminophenol during reduction with
sodium dithionite (rapidly) or GSH (slowly); acid hydroly-
sis (10 min at pH 1) also caused degradation to 4-ami-
nophenol. This chemical behavior suggested GS-NHP to
be the corresponding sulfenamide of 4-aminophenol.
When GS-QI was reduced with sodium dithionite, inter-
mediate GS-NHP was hardly observed but further re-
duced to 4-aminophenol. Reaction of GS-QI with excess
GSH led primarily to GS-NHP followed by transforma-
tion to 4-aminophenol. In addition, one byproduct eluting
in front of GS-NHP was sometimes observed, its UV/vis
spectrum resembling thioethers (λmax ) 317 nm; cf.
Figure 4a). However, this product could not be identified
as the presumed (GS)2NHP or GS-NH2P (Figure 1)
because of poor yields and reproducibility. Direct detec-
tion of GS-NHP and 4-aminophenol in NOPt/GSH reac-
GS-NHP
tion mixtures was unsuccessful (Figure 2: tR
) 3.7
4-aminophenol
min, tR
) 3.9 min).
1
The H NMR spectrum of GS-QI showed four quinoid
protons, each splitting into a double doublet by ortho-
and meta-coupling (Table 3). This pattern reflects an
asymmetric structure of N-substituted 1,4-benzoquinone
monoimines resulting from an sp2 hybridization of the
imino nitrogen atom. In fact, AM1 molecular orbital
calculations for N-(methylthiol-S-yl)-1,4-benzoquinon-
imine revealed a Caryl-N-S bond angle of 126°, a rotation
barrier of 100 kJ /mol for the Caryl-N double bond, and
an inversion barrier of 180 kJ /mol. These barriers are
comparable to those of other sulfenimines determined
experimentally (34), and prevent coalescence (35, 36),
resulting in four distinct signals for the quinoid protons.
An extended splitting pattern has been observed recently
1
in the H NMR spectrum of N-sulfenylquinone diimines
(6). The first isolated derivative of this family, GS-
EPQDI (for structural formula, see Figure 1), did not
allow NMR spectroscopic characterization of the quinoid
protons because of mutual overlap with the aromatic
protons of the 4-ethoxyphenyl ring (7) (Table 3). There-
fore, the pentafluorophenyl derivative GS-F5PQDI (for
structural formula, see Table 3) was synthesized in order
to unmask the quinoid protons. In addition to the above-
mentioned structural effects of quinone monoimines, the
E-Z isomerism of N,N′-disubstituted benzoquinone di-
imines entailed two distinct signals for each of the four
quinoid protons (Table 3).
The high-field signals of GS-QI were totally assigned
to the protons of one glutathione residue. The chemical
shifts of these signals were comparable to those of other
N-(glutathion-S-yl)quinonimine derivatives (cf. Table 3;
the upward shift of 0.2 ppm for the signals of GS-F5PQDI
is caused by the different solvent DMSO-d6). According
to the quinoid character, the cysteine R and â protons
are distinctly shifted downfield compared to other glu-
tathione conjugates (cf. Tables 5 and 6). The FAB-MS
data of GS-QI [recorded in 3-nitrobenzyl alcohol to avoid
Discu ssion
Su lfen a m id e Ca tion Descen d a n ts. During reaction
of NOPt with GSH, formation of the glutathione thioether
GS-NH2Pt was previously reported (7). This finding was
interpreted as additional proof of an intermediate
sulfenamide cation supposed to occur during the interac-
tion of nitrosoarenes with GSH (5). Conceivably, reso-
nance stabilization of this cation should deliver part of
the positive charge to the ortho-position, thereby enabling
nucleophilic attack of good nucleophiles such as alkyl-
3
D. Gallemann, J . Dasenbrock, and E. Wimmer unpublished result.

4-Nitrosophenetole-Glutathione S-Conjugates
Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1419
thiols. The proximate product (GS)2NHPt, however, has
never been observed so far; hence, GS-NH2Pt formation
due to a sulfenamide cation pathway was likely, but still
speculative. In fact, a detailed survey of the pertinent
literature brought to light that o-thioethers may also be
formed from sulfenamides upon rearrangement at low
pH and elevated temperature (37-39). Since the respec-
tive sulfenamide GSNHPt is the main product of the
NOPt/GSH interaction (Table 1), small rates of such a
rearrangement even at neutral pH could not be excluded.
Therefore, the definite proof of (GS)2NHPt formation and
its propensity for decaying into GS-NH2Pt fosters the
sulfenamide cation pathway of thioether formation (Fig-
ure 1, pathway II). Such a mechanism has already been
proposed for 2-amino-5-(glutathion-S-yl)-1-methylimida-
zole formation upon reaction of 1-methyl-2-nitrosoimi-
dazole with a large excess of GSH (40).
Ring-substituted glutathione adducts such as (GS)2-
NHPt and GS-NH2Pt have not been found during me-
tabolism of other nitrosobenzene derivatives investigated
hitherto (2). Since molecular orbital calculations re-
vealed that the sulfenamide cation of nitrosobenzene also
displays substantial positive partial charge at the ortho-
position (2), it remains to be clarified whether thioethers
of other nitrosoarenes have been overlooked due to low
yields and dependence on stoichiometry, reaction time,
and workup.
The benzoquinonimine derivative GS-QI is most prob-
ably also a direct sulfenamide cation descendant. It is
suggesting that solvent water may add to the para-
position of the sulfenamide cation, forming an initial ipso-
adduct. This unstable species may rapidly split off
ethanol by simple proton transfer (eq 1). Similar hy-
drolysis mechanisms have already been suggested for the
structurally homologous N-(thiophenol-S-yl)-4-anisidine
sulfenamide cation (41), and the N-acetyl-4-anisidine and
-phenetidine nitrenium ions (42, 43). Moreover, meta-
stable ipso-adducts such as that depicted in eq 1 were
identified when NOPt was reacted with GSH in solvent
alcohols [cf. the following paper (8)].
F igu r e 5. Molecular orbital calculation results using the
MNDO Hamiltonian for N-(methylthiol-S-yl)-4-phenetidine cat-
ion (heat of formation: 701.7 kJ ). (a) Total charge densities/π-
charge densities at the non-hydrogen atoms (H2, H4, and H5,
+0.11; H3, +0.12). (b) Square of the atomic pπ orbital coefficients
in the LUMO (negative signs of the coefficients are reflected by
shaded circles).
position of the sulfenamide cation.5 Reaction of these
positions with water results in GSO-NHPt and GS-QI,
but both metabolites were formed in distinctly different
yields (cf. Table 1). This divergence can obviously not
be illustrated by only considering charge densities. To
elucidate the reactivity of the sulfenamide cation in more
depth, a theoretical approach was undertaken employing
the generalized perturbation theory of Klopman (44, 45).
To this end, the squares of the atomic pπ-orbital coef-
ficients in the LUMO were calculated (Figure 5b).
Considering these theoretical calculations, reactions of
the sulfenamide cation with various nucleophiles may be
directed by the following factors: Reaction centers dis-
playing “hard electrophilic” properties are the sulfur atom
and the para-position of the aromatic ring, as represented
by high charge densities. Accordingly, only these sites
yield products with the “hard nucleophilic” water, i.e.,
GSO-NHPt and GS-QI. Formation of 2-hydroxy-4-phe-
netidine could not be established in accordance with
distinctly lower charge densities at the ortho-position of
the sulfenamide cation. In parallel, Novak’s group has
repeatedly reported that “arylnitrenium-like” cations
react with water almost exclusively at the para-position
(46-51). The para-position exhibits a high orbital coef-
ficient, too, indicating intermediate hardness/softness of
this reaction center. Thus, the “hard nucleophilic”
solvent water reacting at this site has to compete with
the “soft” nucleophiles GS- and arylamine (44, 52, 53)
[Figure 1, pathways III-V; for detailed mechanistic
steps, cf. (2, 5, 7)]. This may be the reason for the above-
mentioned distinctly lower GS-QI yields compared to
GSO-NHPt.
Thus, solvent water obviously cannot only add to the
sulfur atom of the sulfenamide cation, yielding GSO-
NHPt, but to the para-position as well (Figure 1, path-
ways I and V). This finding was somewhat surprising
since all metabolites identified hitherto indicated the
aromatic ring of the sulfenamide cation only reacting
with (moderately) soft nucleophiles such as GSH or
metabolically formed arylamine. To estimate the reac-
tivity of the three reaction centers in the sulfenamide
cation, i.e., sulfur atom and ortho-/para-position of the
aromatic ring, semiempirical molecular orbital calcula-
tions were performed4 (Figure 5) and the results tenta-
tively related to the experimentally determined product
yields (Table 1).
Comparing the reactions occurring with the soft nu-
cleophile GS-, the soft electrophilic sulfenamide cation
sites have to be taken into account, i.e., ortho- and para-
position.6 As predicted by higher orbital coefficients and
As shown in Figure 5a, similarly high positive partial
charges were calculated for the sulfur atom and the para-
5
The high part of the para-localized resonance contributor (Figure
1) is also reflected by quinoid bond length in the sulfenamide cation:
C1-C2 and C1-C6, 1.47 Å; C2-C3 and C5-C6, 1.37 Å; C4-C3 and
C4-C5, 1.45 Å; C1-N and C4-O, 1.31 Å; N-S, 1.59 Å.
4
To select the most suitable Hamiltonian, MNDO, AM1, and PM3
geometries of [H-N-S-H]+ were compared with ab initio calculations
reported in the literature (1). AM1 delivered deviating structures
(almost linear arrangement of H-N-S), while the MNDO parameter
set produced the best results.
The nitrogen atom with its high orbital coefficient may still not
6
add soft nucleophiles in view of its considerable negative total charge
density (Figure 5a). This prediction is in accordance with all experi-
mental findings reported hitherto (2).

1420 Chem. Res. Toxicol., Vol. 11, No. 12, 1998
Gallemann et al.
higher charge densities in the para-position, products of
pathway III (GSNHPt and NH2Pt) should exceed the
pathway II products [(GS)2NHPt and GS-NH2Pt; Figure
1], which was verified experimentally (Table 1). Accord-
ingly, addition of the “moderately soft” nucleophile NH2-
Pt (44, 52, 53) was only observed at the more reactive
para-position (Figure 1, pathway IV).
acids may efficiently trap this reactive species, thereby
preventing DNA adduct formation.
Red u ction of Qu in on im in e Der iva tives. As shown
with the isolated compound, GS-QI is not an end product
of NOPt metabolism as long as GSH is present. The
quinoid system is slowly reduced with formation of the
metastable sulfenamide GS-NHP which decays to 4-ami-
nophenol during prolonged incubations (Figure 1). The
GS-QI reduction products could not be detected in NOPt/
GSH incubates, although GS-QI disappeared in batches
containing surplus GSH (cf. Figure 2b,c). However, its
products probably remained obscure because of the poor
yields and distinctly lower extinction coefficients. Thio-
ether formation during reaction of GS-QI with GSH was
expected from the sequential reactions of GS-EPQDI
[Figure 1, (7)] and other monocyclic benzoquinon(di)-
imines (68-70). However, (GS)2NHP and GS-NH2P
could not be established unequivocally hitherto. Since
these metabolites are not expected to be detectable in
NOPt/GSH reaction mixtures, as revealed for GS-NHP
and 4-aminophenol, this topic was not investigated
further.
The different yields between GSO-NHPt and sulfen-
amide/arylamine may be explained as well (Figure 1,
pathways I and III; Table 1): While the interaction of
GS- with the para-position is privileged by a high
covalent and a high electrostatic contribution, H2O
addition to the sulfur atom is mainly favored by electro-
static attraction. Correspondingly, GSO-NHPt yield
increased upon lowering GSH availability in the batch7
(Table 1). This theoretical explanation is corroborated
by the results on another nitrosoarene reacting with
GSH: Nitrosobenzene was reported to yield mainly
sulfinamide with low amounts of sulfenamide/arylamine
(2, 10). Molecular orbital calculations for the respective
sulfenamide cation (2, 6) showed a clear increase of the
charge density and the orbital coefficient at the sulfur
atom and a corresponding decrease in the para-position,
compared to the sulfenamide cation dealt with in this
paper.
The reported reactions of the NOPt-derived sulfen-
amide cation (also classified as N-sulfenylnitrenium ion8)
reveal striking similarity to other nitrenium ions. Except
for the kinetic investigations of Kazanis and McClelland
(5), this correspondence was not noticed previously since
the main products of other nitrosoarenes reacting with
GSH were N-hydroxyarylamines, sulfinamides, sulfen-
amides, and arylamines, i.e., metabolites hardly resem-
bling the product pattern of nitrenium ions. As the latter
mainly delocalize the positive charge to the aromatic ring
(1, 5, 54-57), ring addition products are major metabo-
lites. Thus, thioethers have been found during in vitro
and in vivo reactions of nitrenium ion precursors with
GSH (58-61). Trapping by solvent H2O has been shown
to be a major pathway in the absence of better nucleo-
philes (42, 46, 60, 62-64). Finally, addition of aryl-
amines to nitrenium ions has been proved as well (65-
67). In sulfenamide cations, the positive charge is partly
taken over by the sulfur atom, giving rise to high yields
of exocyclic addition products, i.e., sulfinamides (5, 10).
Moreover, acceptor-substituted nitrosoarenes are largely
metabolized in the alternative pathway leading to N-
hydroxyarylamines (Figure 1) (2, 5, 10). In the case of
donor-substituted nitrosoarenes, however, this pathway
is hardly followed (2), but the sulfenamide cation is
formed in the alternative route. This cation obviously
contains sufficient positive partial charge in the aromatic
ring to enable formation of similar ring addition products
as observed with nitrenium ions. In view of this cor-
respondence of the reaction pathways, at least donor-
substituted sulfenamide cations have to be suspected as
ultimate mutagens (5) as has been proved for nitrenium
ions before. Since sulfenamide cations have been shown
to be distinctly more stable than nitrenium ions (1, 5), it
remains to be clarified whether low molecular weight
cellular nucleophiles such as GSH, water, and amino
The bicyclic benzoquinonimine GS-EPQDI is formed
in a similar reaction like its monocyclic counterpart GS-
QI by attack of the sulfenamide cation on already formed
NH2Pt instead of water [Figure 1, (7)]. Identification of
the subsequent sulfenamides GS-NHEDPA and (GS)2-
NHEDPA in reaction mixtures now establishes the
previously suggested pathways which lead to the stable
end products NH2EDPA9 and GS-NH2EDPA, respectively
[for discussion about mechanistic steps of reduction, cf.
Figure 2 of (7)]. As already mentioned above, the yield
in GS-QI and bicyclic metabolites is rather poor, reflect-
ing the distinctly lower nucleophilicity of H2O and NH2-
Pt compared to GS-. Accordingly, high yields of bicyclic
metabolites are obtained when NH2Pt is initially present
and the availability of GSH is restricted (Table 1, last
column). High yields of bicyclic metabolites have also
been observed during metabolism of NOPt in human red
cells (11), indicating similar reaction conditions possibly
occurring in living cells. This scope will be discussed in
detail in a following paper.
F u r th er Meta bolites. The unidentified metabolites
discovered in the HPLC chromatograms (cf. Figures 2a
and 3a) indicate additional reaction pathways running
during interaction of NOPt with GSH. As may be
gathered from the sum of known metabolites calculated
in Table 1, unknown metabolites are formed especially
at limited GSH supply, suggesting reactions of the
sulfenamide cation with alternative nucleophilic sites.
[The extraordinary high standard deviation in metabo-
lites formed during 1:1 stoichiometry (Table 1) conceiv-
ably reflects the reactivity of the sulfenamide cation being
highly dependent on local GSH concentrations during
initial mixing of the batch.] Although these unknown
metabolites are of insignificant quantitative importance,
their structural elucidation could afford deeper insights
in the reactivity of sulfenamide cations. Thus, the recent
discovery of alcohol adducts emerging in alcoholic reac-
tion medium revealed the ability of sulfenamide cations
to react with weak nucleophiles to some extent (8).
7
It should be noted that GSO-NHPt yields are high not only in the
case of low GSH/NOPt stoichiometries but also at high stoichiometries
provided that GSH is added slowly (Table 1).
The term “sulfenamide cation” will be used throughout in order
to distinguish N-sulfenylnitrenium ions from other arylnitrenium ions.
9
A similar pathway has been observed during reduction of the GS-
8
EPQDI analogue N-phenyl-N′-phenylthio-4-benzoquinone diimine with
sodium dithionite (3).

4-Nitrosophenetole-Glutathione S-Conjugates
Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1421
(17) Gupta, R. L., Singh, M., and J uneja, T. R. (1987) Mutagenicity of
certain para substituted nitrosobenzenes-a structure activity
relationship. Indian J . Exp. Biol. 25, 445-449.
(18) Wirth, P. J ., Alewood, P., Calder, I., and Thorgeirsson, S. S. (1982)
Mutagenicity of N-hydroxy-2-acetylaminofluorene and N-hydroxy-
phenacetin and their respective deacetylated metabolites in
nitroreductase-deficient Salmonella TA98FR and TA100FR. Car-
cinogenesis 3, 167-170.
(19) Camus, A. M., Friesen, M., Croisy, A., and Bartsch, H. (1982)
Species-specific activation of phenacetin into bacterial mutagens
by hamster liver enzymes and identification of N-hydroxyphen-
acetin O-glucuronide as a promutagen in the urine. Cancer Res.
42, 3201-3208.
(20) McCoy, E. C., Rosenkranz, H. S., and Bartsch, H. (1986) Mutage-
nicity of the phenacetin metabolites: N-hydroxy-p-phenetidine
and nitrosophenetol in S. typhimurium TA100 and derivatives
deficient in nitroreductase or O-acetylase: probes for testing
intrabacterial metabolic activation. Mutat. Res. 173, 245-250.
(21) Eyer, P., and Podhradsky, D. (1986) Evaluation of the mi-
cromethod for determination of glutathione using enzymatic
cycling and Ellman’s reagent. Anal. Biochem. 153, 57-66.
(22) Bernt, E., and Bergmeyer, H. U. (1970) Glutathion. In Methoden
der enzymatischen Analyse (Bergmeyer, H. U., Ed.) Vol. II, pp
1605-1609, Verlag Chemie, Weinheim.
Con clu sion s. In summary, all NOPt/GSH metabo-
lites elucidated hitherto can be explained starting from
a central sulfenamide cation that enables multiple reac-
tion pathways. The product pattern reveals striking
similarity with other arylnitrenium ions accused as
ultimate carcinogens. Hence, the DNA reactivity of
sulfenamide cations may be of concern as well. Moreover,
formation of quinoid metabolites is expected to contribute
to the toxic potential of nitrosoarenes reacting with thiols.
Ack n ow led gm en t. The financial support of the DFG
(Deutsche Forschungsgemeinschaft; AZ:Ga 495/2-1) is
gratefully acknowledged.
Refer en ces
(1) Glover, S. A., and Scott, A. P. (1989) MNDO properties of
heteroatom and phenyl substituted nitrenium ions. Tetrahedron
45, 1763-1776.
(2) Eyer, P., and Gallemann, D. (1996) Reactions of nitrosoarenes
with SH groups. In The Chemistry of Amino, Nitroso, Nitro and
Related Groups (Patai, S., Ed.) Vol. Suppl. F2, Part 2, pp 999-
1040, J ohn Wiley & Sons, Chichester.
(23) Bray, G. A. (1960) A simple efficient scintillator for counting
aqueous solutions in a liquid scintillation counter. Anal. Biochem.
1, 279-285.
(3) Balboni, C., Benati, L., Montevecchi, P. C., and Spagnolo, P. (1983)
Benzenesulfenanilidyl radicals. Part 2. Substituent and solvent
effects on the reactivity of 4′-substituted benzenesulphenanilidyl
radicals produced by oxidation with lead dioxide of the corre-
sponding benzenesulphenanilides. J . Chem. Soc., Perkin Trans.
1 1983, 2111-2117.
(4) Klehr, H. (1988) Zum Mechanismus der Reaktionen von Ni-
trosoaromaten mit Thiolen. Ph.D. Thesis, Ludwig-Maximilians-
Universita¨t, Mu¨nchen.
(5) Kazanis, S., and McClelland, R. A. (1992) Electrophilic intermedi-
ate in the reaction of glutathione and nitrosoarenes. J . Am. Chem.
Soc. 114, 3052-3059.
(6) Gallemann, D. (1993) Metabolismus und Wirkungen von 4-Ni-
trosophenetol in Humanerythrozyten. Ph.D. Thesis, Ludwig-
Maximilians-Universita¨t, Mu¨nchen.
(24) Dewar, M. J . S., and Thiel, W. (1977) Ground states of molecules.
38. The MNDO method. Approximations and parameters. J . Am.
Chem. Soc. 99, 4899-4907.
(25) Dewar, M. J . S., and Reynolds, C. H. (1986) An improved set of
MNDO parameters for sulfur. J . Comput. Chem. 7, 140-143.
(26) Dewar, M. J . S., Zoebisch, E. G., Healy, E. F., and Stewart, J . J .
P. (1985) AM1: A new general purpose quantum mechanical
molecular model. J . Am. Chem. Soc. 107, 3902-3909.
(27) Dewar, M. J . S., and Yuan, Y. C. (1990) AM1 parameters for
sulfur. Inorg. Chem. 29, 3881-3890.
(28) Stewart, J . J . P. (1990) MOPAC: A semiempirical molecular
orbital program. J . Comput. Aid Mol. Des. 4, 1-105.
(29) Hays, J . T., Young, H. L., and Espy, H. H. (1967) p-Nitrosophenol
chemistry. II. Amination of p-nitrosophenol ethers with primary
aromatic amines. J . Org. Chem. 32, 158-162.
(30) Stanley, J . S., Lay, J . O., J r., Miller, D. W., and DeLuca, D. C.
(1989) Identification of the glutathione conjugate of 4-nitroquino-
line 1-oxide formed in the reaction catalyzed by murine glu-
tathione transferases. Carcinogenesis 10, 587-591.
(31) Klehr, H., Eyer, P., and Scha¨fer, W. (1985) On the mechanism of
reactions of nitrosoarenes with thiols. Formation of a common
intermediate “semimercaptal”. Biol. Chem. Hoppe-Seyler 366,
755-760.
(32) van den Brock, L., Delbressine, L., and Ottenheijm, H. (1990)
Biochemistry and metabolic pathways of sulfenic acids and their
derivatives. In The Chemistry of Sulphenic Acids and their
Derivatives (Patai, S., Ed.) pp 701-721, J ohn Wiley & Sons,
Chichester.
(33) Baillie, T. A., and Davis, M. R. (1993) Mass spectrometry in the
analysis of glutathione conjugates. Biomed. Mass Spectrom. 22,
319-325.
(34) Davis, F. A. (1973) Chemistry of the sulfur-nitrogen bond in
sulfenamides. Int. J . Sulfur Chem. 8, 71-81.
(35) Hesse, M., Meier, H., and Zeeh, B. (1984) Spektroskopische
Methoden in der organischen Chemie, Thieme Verlag, Stuttgart.
(36) Gu¨nther, H. (1983) NMR-Spektroskopie. Eine Einfu¨hrung in die
Protonenresonanz-Spektroskopie und ihre Anwendungen in der
Chemie, Thieme Verlag, Stuttgart.
(37) Capozzi, G., Modena, G., and Pasquato, L. (1990) Chemistry of
sulphenyl halides and sulphenamides. In The Chemistry of
Sulphenic Acids and their Derivatives (Patai, S., Ed.) pp 403-
516, J ohn Wiley & Sons, Chichester.
(7) Gallemann, D., and Eyer, P. (1994) Additional pathways of
S-conjugate formation during the interaction of thiols with
nitrosoarenes bearing π-donating substituents. Environ. Health
Perspect. 102, 137-142.
(8) Gallemann, D., Greif, A., Eyer, P., Dasenbrock, J ., Wimmer, E.,
Sonnenbichler, J ., Sonnenbichler, I., Scha¨fer, W., and Buhrow, I.
(1998) Formation of 4,4-dialkoxycyclohexa-2,5-dienone N-(thiol-
S-yl)imine during reaction of 4-alkoxynitrosobenzenes with thiols
in alcoholic solvents. Chem. Res. Toxicol. 11, 1423-1433.
(9) Klehr, H., and Eyer, P. (1987) Reactions of p-nitrosophenetole with
thiols. Naunyn-Schmiedeberg’s Arch. Exp. Pathol. Pharmacol. 335
Su p p l., R 12.
(10) Diepold, C., Eyer, P., Kampffmeyer, H., and Reinhardt, K. (1982)
Reactions of aromatic nitroso compounds with thiols. In Biological
Reactive Intermediates: 2. Chemical Mechanisms and Biological
Effects (Snyder, R., Parke, V. D., Kocsis, J . J ., J ollow, D. J .,
Gibson, C. G., and Witmer, C. M., Eds.) pp 1173-1181, Plenum
Press, New York.
(11) Klehr, H., Eyer, P., and Scha¨fer, W. (1987) Formation of 4-ethoxy-
4′-nitrosodiphenylamine in the reaction of the phenacetin me-
tabolite 4-nitrosophenetol with glutathione. Biol. Chem. Hoppe-
Seyler 368, 895-902.
(12) Mulder, G. J ., Kadlubar, F. F., Mays, J . B., and Hinson, J . A.
(1984) Reaction of mutagenic phenacetin metabolites with glu-
tathione and DNA. Possible implications for toxicity. Mol. Phar-
macol. 26, 342-347.
(13) Gallemann, D., and Eyer, P. (1993) Effects of the phenacetin
metabolite 4-nitrosophenetol on glycolysis and pentose phosphate
pathway in human red cells. Biol. Chem. Hoppe-Seyler 374, 37-
49.
(38) Braverman, S. (1990) Rearrangements involving sulfenic acids
and their derivatives. In The Chemistry of Sulphenic Acids and
their Derivatives (Patai, S., Ed.) pp 311-360, J ohn Wiley & Sons,
Chichester.
(14) Eyer, P., and Kampffmeyer, H. (1982) Inhibition of glutathione
excretion, bile flow, and alterations of the glutathione status by
4-nitrosophenetol during perfusion of rat liver. Chem.-Biol.
Interact. 42, 209-223.
(15) Eckert, K. G., and Eyer, P. (1983) Differences in the reactions of
isomeric ortho- and para-aminophenols with hemoglobin. Bio-
chem. Pharmacol. 32, 1019-1027.
(39) Craine, L., and Raban, M. (1989) The chemistry of sulfenamides.
Chem. Rev. 89, 689-712.
(40) Farah, S. F. (1991) Aspects of the reduction of 1-methyl-2-
nitroimidazole with glutathione and the reaction of 1-methyl-2-
nitrosoimidazole with glutathione. Ph.D. Thesis, University of
Toronto.
(41) Sayo, H. (1990) Electrochemistry of sulfenic acids and their
derivatives. In The Chemistry of Sulphenic Acids and their
Derivatives (Patai, S., Ed.) pp 571-585, J ohn Wiley & Sons,
Chichester.
(16) Gupta, R. L., Dey, D. K., and J uneja, T. R. (1985) Structure-
mutagenicity relationships within
a series of para-alkoxy-
nitrosobenzenes. Toxicol. Lett. 28, 125-132.

1422 Chem. Res. Toxicol., Vol. 11, No. 12, 1998
Gallemann et al.
(42) Novak, M., Pelecanou, M., and Pollack, L. (1986) Hydrolysis of
the model carcinogen N-(pivaloyloxy)-4-methoxyacetanilide: In-
volvement of N-acetyl-p-benzoquinone imine. J . Am. Chem. Soc.
108, 112-120.
(43) Novak, M., Kahley, M. J ., Lin, J ., Kennedy, S. A., and Swanegan,
L. A. (1994) Reactivity patterns of N-arylnitrenium ions: lack of
correlation with σ+. J . Am. Chem. Soc. 116, 11626-11627.
(44) Klopman, G. (1974) The generalized perturbation theory of
chemical reactivity and its application. In Chemical Reactivity
and Reaction Paths (Klopman, G., Ed.) pp 55-166, J ohn Wiley
& Sons, New York.
(59) van den Goorbergh, J . A., Meerman, J . H., de Wit, H., and Mulder,
G. J . (1985) Reaction of 2-acetylaminofluorene-N-sulfate with
RNA and glutathione: Evidence for the generation of two reactive
intermediates with different reactivities towards RNA and glu-
tathione. Carcinogenesis 6, 1635-1640.
(60) Novak, M., and Lin, J . (1996) Reactions of glutathione with
carcinogenic esters of N-arylhydroxamic acids. J . Am. Chem. Soc.
118, 1302-1308.
(61) Meerman, J . H. N., Beland, F. A., Ketterer, B., Srai, S. K. S.,
Bruins, A. P., and Mulder, G. J . (1982) Identification of glu-
tathione conjugates formed from N-hydroxy-2-acetylaminofluo-
rene in the rat. Chem.-Biol. Interact. 39, 149-168.
(62) Fishbein, J . C., and McClelland, R. A. (1987) Azide ion trapping
of the intermediate in the Bamberger rearrangement. Lifetime
of a free nitrenium ion in aqueous solution. J . Am. Chem. Soc.
109, 2824-2825.
(63) Sukhai, P., and McClelland, R. A. (1996) Direct observation of
4-alkoxyphenylnitrenium ions upon irradiation of 4-alkoxyphenyl
azides in aqueous solution. J . Chem. Soc., Perkin Trans. 2 1996,
1529-1530.
(64) Novak, M., and Kennedy, S. A. (1995) Selective trapping of
N-acetyl-N-(4-biphenylyl)nitrenium and N-acetyl-N-(2-fluor-
enyl)nitrenium ions by 2′-deoxyguanosine in aqueous solution. J .
Am. Chem. Soc. 117, 574-575.
(65) Helmick, J . S., Martin, K. A., Heinrich, J . L., and Novak, M. (1991)
Mechanism of the reaction of carbon and nitrogen nucleophiles
with the model carcinogens O-pivaloyl-N-arylhydroxylamines:
competing SN2 substitution and SN1 solvolysis. J . Am. Chem.
Soc. 113, 3459-3466.
(66) Novak, M., and Rangappa, K. S. (1992) Nucleophilic substitution
on the ultimate hepatocarcinogen N-(sulfonatooxy)-2-(acetylami-
no)fluorene by aromatic amines. J . Org. Chem. 57, 1285-1290.
(67) Novak, M., Rangappa, K. S., and Manitsas, R. K. (1993) Nucleo-
philic aromatic substitution on ester derivatives of carcinogenic
N-arylhydroxamic acids by aniline and N,N-dimethylaniline. J .
Org. Chem. 58, 7813-7821.
(68) Ludwig, E., and Eyer, P. (1994) Structural requirements for the
ferrihemoglobin forming activity of glutathione S-conjugates of
4-dimethylaminophenol. Environ. Health Perspect. 102, 133-136.
(69) Sto¨rle, C., and Eyer, P. (1991) Reactions of the Wurster’s blue
radical cation with thiols, and some properties of the reaction
products. Chem.-Biol. Interact. 78, 333-346.
(70) Sto¨rle, C., and Eyer, P. (1992) Reactions of the Wurster’s red
radical cation with hemoglobin and glutathione during the
cooxidation of N,N-dimethyl-p-phenylenediamine and oxyhemo-
globin in human red cells. Chem.-Biol. Interact. 83, 271-291.
(71) Hays, J . T., de Butts, E. H., and Young, H. L. (1967) p-
Nitrosophenol chemistry. I. Etherification of p-nitrosophenol. J .
Org. Chem. 32, 153-158.
(72) Gowenlock, B. G., and Lu¨ttke, W. (1958) Structure and properties
of C-nitroso-compounds. Quart. Rev. 12, 321-340.
(73) Norris, R. K., and Sternhell, S. (1966) N. M. R. spectra of “p-
nitrosophenol” and its methyl derivatives. Aust. J . Chem. 19,
841-860.
(74) Uffmann, H. (1967) Stereoisomerie und Tautomerie von 1.4-
Benzochinonoximen. Z. Naturforsch. 22B, 491-502.
(75) Rabenstein, D. L., and Keire, D. A. (1989) Nuclear magnetic
resonance spectroscopy of glutathione. In Glutathione. Chemical,
Biochemical, and Medical Aspects (Dolphin, D., Avramovic, O.,
and Poulson, R., Eds.) Vol. A, pp 67-101, J ohn Wiley & Sons,
New York.
(45) Klopman, G. (1968) Chemical reactivity and the concept of charge-
and frontier-controlled reactions. J . Am. Chem. Soc. 90, 223-234.
(46) Novak, M., Kahley, M. J ., Eiger, E., Helmick, J . S., and Peters,
H. E. (1993) Reactivity and selectivity of nitrenium ions derived
from ester derivatives of carcinogenic N-(4-biphenylyl)hydrox-
ylamine and the corresponding hydroxamic acid. J . Am. Chem.
Soc. 115, 9453-9460.
(47) Novak, M., and Lagerman, R. K. (1988) Hydrolysis and Fe2+
-
induced reduction of N-aryl-O-pivaloylhydroxylamines: Aqueous
solution chemistry of model carcinogens. J . Org. Chem. 53, 4762-
4769.
(48) Novak, M., Rovin, L. H., Pelecanou, M., Mulero, J . J ., and
Lagerman, R. K. (1987) Acid- and base-dependent hydrolysis of
N-(sulfonatooxy)-3- bromoacetanilide: involvement of N-(3-bro-
mophenyl)hydroxylamine O-sulfonate. J . Org. Chem. 52, 2002-
2010.
(49) Novak, M., and Roy, A. K. (1985) Hydrolysis of N-(sulfonatooxy)-
p-acetotoluidide: Solution chemistry of models for carcinogenic
metabolites of aromatic amines. J . Org. Chem. 50, 571-580.
(50) Novak, M., Pelecanou, M., Roy, A. K., Andronico, A. F., Plourde,
F. M., Olefirowicz, T. M., and Curtin, T. J . (1984) Solvolysis of
N-sulfonoxyacetanilides in aqueous and alcohol solutions: gen-
eration of electrophilic species. J . Am. Chem. Soc. 106, 5623-
5631.
(51) Davidse, P. A., Kahley, M. J ., McClelland, R. A., and Novak, M.
(1994) Flash photolysis observation and lifetimes of 2-fluorenyl-
and 4-biphenylylacetylnitrenium ions in aqueous solution. J . Am.
Chem. Soc. 116, 4513-4514.
(52) March, J . (1977) Advanced Organic Chemistry: Reactions, Mech-
anisms, and Structure, McGraw-Hill International Book Co.,
Tokyo.
(53) Pearson, R. G., and Songstad, J . (1967) Application of the principle
of hard and soft acids and bases to organic chemistry. J . Am.
Chem. Soc. 89, 1827-1835.
(54) Ford, G. P., and Scribner, J . D. (1981) MNDO molecular orbital
study of nitrenium ions derived from carcinogenic aromatic
amines and amides. J . Am. Chem. Soc. 103, 4281-4291.
(55) Ford, G. P., and Herman, P. S. (1990) Alkyl and acyl substituent
effects on nitrenium ion stabilities: ab initio molecular orbital
calculations. THEOCHEM 204, 121-130.
(56) Ford, G. P., and Herman, P. S. (1991) Comparison of the relative
stabilities of polycyclic aryl nitrenium ions and arylmethyl
cations: ab initio and semiempirical molecular orbital calcula-
tions. THEOCHEM 236, 269-282.
(57) Ford, G. P., and Herman, P. S. (1991) Conformational preferences
and energetics of N-O heterolyses in aryl nitrenium ion precur-
sors: ab initio and semiempirical molecular orbital calculations.
J . Chem. Soc., Perkin Trans. 2 1991, 607-616.
(58) Varghese, A. J ., and Whitmore, G. F. (1985) Properties of
2-hydroxyaminoimidazoles and their implications for the biologi-
cal effects of 2-nitroimidazoles. Chem.-Biol. Interact. 56, 269-
287.
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