Cysteine and glutathione mixed-disulfide conjugates of thiosulfinates: Chemical synthesis and biological activities

Article abstract of DOI:10.1021/jf9029354

The chemical syntheses of cysteine (CYS) and glutathione (GSH) mixed -disulfide conjugates (CySSR, GSSR, respectively) of mercapto residues representing most of the R groups of thiosulfinates (R = methyl, ethyl, propyl, and allyl) are described. Gram-scale conjugates were prepared as >98% pure preparations, with 80% reaction yield for each of the two seminal synthesis steps, with structures confirmed by ¹H NMR and high-resolution MS analyses. These conjugates are derivatives of thiosulfinates that may be evolved in processed foods, in the digestive tract, and through in vivo metabolism. The prepared conjugates were found to be able to induce quinone reductase (QR, a representative phase II enzyme) in murine hepatoma cells (Hepa 1c1c7) and to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated macrophage cells (RAW 264.7), indicating they have potential cancer preventive and anti-inflammatory activities. Among the prepared conjugates, the allyl conjugates of CYS and GSH, S-allylmercaptocysteine (CySSA) and S-allylmercaptoglutathione (GSSA), showed the most potent activity regarding QR induction and NO production inhibition. The conjugates with saturated R groups were also active and conferred biological activity as cystine and oxidized glutathione exhibited no effects in these cellular assays.

Full text of DOI:10.1021/jf9029354

1564 J. Agric. Food Chem. 2010, 58, 1564–1571
DOI:10.1021/jf9029354
Cysteine and Glutathione Mixed-Disulfide Conjugates of
Thiosulfinates: Chemical Synthesis and Biological Activities
†
‡
†,§
,†
G
UODONG
Z
HANG, BIN
L
I
, CHEN-HSIEN
L
EE
,
AND
KIRK L. PARKIN*
†
Department of Food Science, Babcock Hall, University of Wisconsin;Madison, 1605 Linden Drive,
‡
Madison, Wisconsin 53706 and College of Food Science and Technology, South China Agricultural
§
University, Guangzhou, People’s Republic of China. Present address: Department of Food Science and
Biotechnology, 250 Kuo Kuang Road, National Chung Hsing University, Taichung 402, Taiwan.
The chemical syntheses of cysteine (CYS) and glutathione (GSH) mixed -disulfide conjugates
(CySSR, GSSR, respectively) of mercapto residues representing most of the R groups of
thiosulfinates (R = methyl, ethyl, propyl, and allyl) are described. Gram-scale conjugates were
prepared as >98% pure preparations, with 80% reaction yield for each of the two seminal synthesis
1
steps, with structures confirmed by H NMR and high-resolution MS analyses. These conjugates are
derivatives of thiosulfinates that may be evolved in processed foods, in the digestive tract, and
through in vivo metabolism. The prepared conjugates were found to be able to induce quinone
reductase (QR, a representative phase II enzyme) in murine hepatoma cells (Hepa 1c1c7) and to
inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated macrophage cells (RAW
2
64.7), indicating they have potential cancer preventive and anti-inflammatory activities. Among the
prepared conjugates, the allyl conjugates of CYS and GSH, S-allylmercaptocysteine (CySSA) and
S-allylmercaptoglutathione (GSSA), showed the most potent activity regarding QR induction and NO
production inhibition. The conjugates with saturated R groups were also active and conferred
biological activity as cystine and oxidized glutathione exhibited no effects in these cellular assays.
KEYWORDS: Allium; thiosulfinates; mixed-disulfide conjugates; S-allylmercaptocysteine; S-allylmer-
captoglutathione; phase II enzyme induction; anti-inflammatory
INTRODUCTION
with increasing pH toward neutrality (9), and in the gastrointest-
inal tract both cysteine and glutathione are made available by
secretion into the lumen (5). In blood, thiosulfinates have a half-
life of <1 min, and the presumed fate is through reaction with
thiols in plasma or blood cells (3).
The health benefits of Allium vegetables are widely attributed
to or correlated with the enzyme-transformed organosulfur
compounds called thiosulfinates (1) (Figure 1) because they have
exhibited many biological activities (1-3). However, the rapid
metabolism of thiosulfinates in situ and in vivo may indicate that
their metabolic products may contribute to bioactivity or be the
putative bioactive agents. Thiosulfinates can freely permeate cell
membranes and rapidly react with glutathione (GSH) to form
intracellular GSH mixed-disulfide conjugates (GSSR) (4). In
extracellular fluids where cysteine/cystine is the dominant
thiol (5), similar thiol-thiosulfinate chemical conjugations may
occur. GSSR (R = propyl or allyl) species may also be derived
from metabolism of other Allium organosulfur compounds such
as dipropyl disulfide, diallyl disulfide, and diallyl sulfide through
reactions mediated by cytochrome P450 or glutathione S-trans-
ferase (6, 7) (Figure 2). In foods, thiosulfinates are produced in
minced, but unheated, Allium vegetable tissues (3). Inthe presence
of free thiols, including those in proteins, conjugation reactions
can occur in foods containing thiosulfinates. Thiosulfinates are
most stable at acidic pH in foods and of the stomach, while also
persisting for several hours at duodenal pH (1, 3, 8). Thiosulfi-
nates react more favorably in conjugation reactions with thiols
Some thiosulfinate conjugates of cysteine (CYS) have demon-
strable biological activities. For example, the S-allylmercaptocys-
teine (CySSA) derivative is believed to be a major bioactive principle
in aged garlic extract and has antioxidant (10), antiproliferative (11),
apoptosis-inducing (12), and antimetastatic activities (13). Aside
from the studies on CySSA, there is a dearth of information on other
alk(en)yl mixed disulfides of biologically relevant thiols, likely
because they are not commercially available. Anticancer effects
can be mediated through the antioxidant response element to afford
up-regulation of phase II enzymes (14). Phase II enzyme activities,
such as quinone reductase, glutathione-S-transferase, and UDP-
glucuronyltransferase, are responsible for detoxifying carcinogens
and facilitating their excretion from the body.
The current methods to prepare CYS/GSH mixed-disulfide
conjugates of thiosulfinates rely mainly on the reaction of
thiosulfinates and CYS/GSH (4, 9, 15). However, preparation
of some thiosulfinates, particularly the heterologous and S-1-
propenyl-containing species, by chemical synthesis is challen-
ging (3, 16). Enzymatic methods to prepare thiosulfinates have
been described, but appear limited to small-scale prepara-
tions (17, 18). Thiosulfinates are not available commercially,
*
Corresponding author [e-mail klparkin@wisc.edu; telephone
(608) 263-2011; fax (608) 262-6872].
pubs.acs.org/JAFC
Published on Web 01/20/2010
© 2010 American Chemical Society

Article
J. Agric. Food Chem., Vol. 58, No. 3, 2010 1565
˚
likely because they are relatively unstable (8, 19). Thus, to
facilitate studies on CYS/GSH mixed-disulfide conjugates that
represent the range of derivatives that can be formed from Allium
tissue constituents and through in vivo metabolism, it is necessary
to adopt facile chemical methods for large-scale preparation of
these conjugates. In this paper, we describe a simple combination
of steps to synthesize cysteine and glutathione S-alk(en)yl disul-
fide conjugates (CySSR and GSSR, respectively) representative
of many thiosulfinates common to Allium and chemotaxonomi-
cally related species. A dose-dependent assessment of cellular
bioactivities of these conjugates related to cancer chemoprotec-
tion and anti-inflammatory effects is also provided.
Silica gel (60 A, 230-400 mesh ASTM; Fisher Scientific, Fair Lawn,
˚
NJ) and C18 reverse phase silica gel (60 A, 230-400 mesh ASTM; EMD
Chemical Inc., Gibbstown, NJ) were used for flash column chromatogra-
phy. NMR spectra were collected on a Varian Unity-Inova 400 and
00 MHz NMR spectrometer (Analytical Instrumentation Center, School
of Pharmacy, University of Wisconsin (UW);Madison). High-resolution
ESI-MS analyses were conducted on an Agilent ESI-TOF mass spectro-
meter (Mass Spectrometry/Proteomics Facility, Biotechnology Center,
UW;Madison).
Synthesis of Bis(5,5-dimethyl-2-thiono-1,3,2-dioxaphosphorina-
nyl) Disulfide. The synthesis of bis(5,5-dimethyl-2-thiono-1,3,2-diox-
aphosphorinanyl) disulfide (3) was carried out according to the methods
described by Edmundson (20) (Scheme 1). 2,2-Dimethylpropane-1,3-diol
5
(
2 5
41.6 g) was added to a suspension of 44.8 g of P S in 200 mL of toluene
under reflux for 2-3 h. The product mixture was then subjected to solvent
removal by vacuum rotary evaporation and the residue extracted with
diethyl ether to obtain 5,5-dimethyl-2-thiolo-2-thiono-1,3,2-dioxapho-
sphorinane (2), which was used for the next step without further purifica-
MATERIALS AND METHODS
Materials and General Procedures. Chemicals were obtained from
Sigma (St. Louis, MO) or Aldrich (Milwaukee, WI) Chemical Companies.
Solvents used for extraction or chromatography were purchased from
Fisher Scientific (Chicago, IL). Cell culture media and adjuvants were
from Invitrogen (Carlsbad, CA).
1
tion after vacuum rotary evaporation of ether. H NMR of 2 (400 MHz,
CDCl
CH -).
A volume of 20mL of concentrated NH
3
): δ 4.08 (s, 2H, -CH
2
O-), 4.04 (s, 2H, -CH
2
O-), 1.09 (s, 6H,
3
4
OH (28-30%) was thenadded
dropwise to a solution of (2) in 300 mL of toluene with vigorous stirring,
and the mixture was extracted with water (3 ꢀ 300 mL) to recover the
þ
NH
4
salt of 2. Then, 50 g of I
2
dissolved in KI solution (100 g of KI in
4
00 mL water) was added portion-wise to the aqueous phase over 30 min
with vigorous stirring. The aqueous-insoluble product (3) was extracted
with CH Cl
(3 ꢀ 500 mL). The organic layers were combined, washed
with water, dried over anhydrous MgSO , and vacuum rotary evaporated
to afford the disulfide product (3), which was used for the next stepwithout
2
2
4
1
further purification. H NMR of 3(400MHz, CDCl
H CCH O), 4.38 (d, J = 11.0 Hz, 2H, CCH O), 4.04 (d, J = 11.0 Hz, 2H,
CCH O), 3.99 (d, J = 11.0 Hz, 2H, CCH O), 1.30 (s, 6H, C(CH ), 0.96
3
): δ 4.39 (d, J =11.5Hz,
Figure 1. Organosulfur transformation in disrupted Allium tissues. Tran-
sient sulfenic acid (RSOH) can condense to form thiosulfinates
2
2
2
2
2
3 2
)
0
(
nethial-S-oxide (PTSO, a tear-inducing agent, only when R = 1-propenyl).
R-S(O)S-R ) of (dis)similar R groups or rearrange to form propa-
þ
(
s, 6H C(CH
3
)
2
); APCIMS: [M þ H] 395.0 (calcd 394.97).
Synthesis of 5,5-Dimethyl-2-thiono-1,3,2-dioxaphosphorinanyl-
-disulfanylmethane. The synthesis of 5,5-dimethyl-2-thiono-1,3,2-diox-
2
aphosphorinanyl-2-disulfanylmethane (methyl-4) was carried out accord-
ing to modified methods described by Antoniow and Witt (21). Br (4 g)
was added to a solution of 10 g (3) in 100 mL of CH Cl
at -5 °C. The
mixture was stirred at this temperature for 30 min, and 3.55 g of sodium
methanethiolate (CH
SNa) was then added, followed by stirring at 20-22 °C
overnight. The resulting reaction mixture was washed three times with
vol of water. The organic phase was dried over anhydrous MgSO and
subjected to vacuum rotary evaporation to afford the target product
methyl-4). This derivative was directly used for the next step without
further purification. For identification, a >98% pure methyl-4 was
obtained by silica gel column chromatography eluted with hexane/CH Cl
O),
2
2
2
3
1
4
(
2
2
1
(
1:1). H NMR of methyl-4 (500 MHz, CDCl
3
): δ 4.12 (m, 4H, CCH
2
2
.65 (s, 3H, SCH
3
), 1.22 (s, 3H, CCH
3
), 1.05 (s, 3H, CCH ).
3
Synthesis of 5,5-Dimethyl-2-thiono-1,3,2-dioxaphosphorinanyl-
-disulfanylethane (Ethyl-4). The preparation of the ethyl form of 4 was
2
Figure 2. Metabolic transformation of organosulfur components derived
from Allium tissues. CYP, cytochrome P450; GST, glutathione-S-transfer-
ase. Adapted from refs6 and 7.
similar to the above method, except that (i) ethanethiol was used instead of
sodium methanethiolate and (ii) after the addition of ethanethiol, the
1
reaction was stirred for 30 min instead of overnight. H NMR of ethyl-4
a
Scheme 1. Synthesis of CYS and GSH Conjugates of Thiosulfinates (R = Methyl, Ethyl, Propyl, or Allyl)
a
3 3
To prepare CSSM/GSSM, CH SNa was used in reaction step 3 because CH SH is a gas at normal conditions. The chemical synthesis was carried out using methods adapted
from refs 20 and 21.

1566 J. Agric. Food Chem., Vol. 58, No. 3, 2010
Zhang et al.
(
Scheme 1). Similar purities of the GSSR species were obtained as reported
for the CySSR species following purification by RP-18 flash chromatog-
raphy using the conditions described in the preceding paragraph.
Phase II Enzyme Induction Assay. A bioassay based on cultured
murine hepatoma cells (Hepa 1c1c7; ATCC, Rockville, MD) was used to
assess QR induction as a phase II enzyme biomarker essentially as
described in the original method (14). Hepa 1c1c7 cells were grown for
3
2
4 h in 96-well plates (5 ꢀ 10 cells/well) in 200 μL of minimal essential
medium (MEM) supplemented with 10% fetal bovine serum (treated
with activated charcoal to remove any traces of endogenous QR inducers),
1
00 U/mL of penicillin G, and 100 μg/mL of streptomycin at 37 °C in 5%
2
CO in air. The medium was replaced with fresh MEM containing test
compounds (serially diluted 1:2 in MEM), and the cells were incubated for
another 48 h.
After the culture medium was decanted, cells were lysed by adding 50 μLof
saturated digitonin aqueous solution in 2 mM EDTA, pH 7.8, and then
incubated at 37 °C for 30 min with gentle shaking. For the QR assay, a
standard assay cocktail (containing the essential components of FAD,
glucose-6-phosphate, glucose-6-phosphate dehydrogenase, NADP, 3-(4,5-
dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and
menadione) was prepared and added in a final volume of 200 μL to each
well. QR activity was determined by measuring the absorbance of the reduced
tetrazolium dye over a 10 min period using a Spectra Max plus optical
microtiter plate scanner (Molecular Devices, Sunnyvale, CA) set at 490 nm.
A duplicate plate was prepared for cell protein (viability) assays and to
facilitate relative enzyme specific activity calculations. The second plate
was emptied of liquid contents, 100 μL of crystal violet (0.2% in 2%
ethanol) was added to eachwell and incubated for 10 min at 20-22 °C, and
finally the plates were rinsed under cold running water to remove excess
stain. A 150 μL aliquot of 0.5% (w/v) SDS (prepared in 50% aqueous
ethanol) was added to each well, and the plates were incubated for 1 h in a
shaker oven at 37 °C to dissolve the dye. The optical density of each well
was read at 610 nm. The absorbance values of no-cell blanks were
subtracted from those of the controls and treated samples. The degree
of staining (absorbance) with crystal violet was used as a measure of cell
protein (density) and provided the basis for calculating changes in QR
specific activity. QR specific activity was not determined in cases when loss
of viability exceeded 50% as suggested (14).
For a given induced or control sample, QR specific activity was
calculated using the linear portion of the reaction progress curve
(typically 5 min) from the QR assay relative to the absorbance value
(protein) of the crystal violet stain. The degree of QR induction was then
calculated as the ratio of QR specific activity in the treated (induced)
sample relative to the control sample. A “CD value” (concentration
required to double the specific activity of QR) was interpolated from
dose-dependent response plots for each conjugate tested and was used as
an indicator of relative inducer potency. Untreated control cells were
assigned a relative QR specific activity of 1.0.
Figure 3. Chemical structures of prepared GSH and CYS conjugates of
thiosulfinates. GSSM, S-methylmercaptoglutathione; GSSE, S-ethylmer-
captoglutathione; GSSP, S-propylmercaptoglutathione; GSSA, S-allylmer-
captoglutathione; CySSM, S-methylmercaptocysteine; CySSE, S-ethyl-
mercaptocysteine; CySSP, S-propylmercaptocysteine; CySSA, S-allylmer-
captocysteine.
(
500 MHz, CDCl
SCH CH ), 1.37 (tr, J = 7.2 Hz, 3H, SCH
s, 3H, CCH ).
3
): δ 4.12 (m, 4H, CCH
2
O), 3.02 (qr, J = 7.2 Hz, 2H,
CH ), 1.21 (s, 3H, CCH ), 1.05
2
3
2
3
3
(
3
Synthesis of 5,5-Dimethyl-2-thiono-1,3,2-dioxaphosphorinanyl-
-disulfanylpropane (Propyl-4). The propyl form of 4 was synthesized
2
similarly to the method of the ethyl conjugate except propanethiol was
1
used. H NMR of propyl-4 (500 MHz, CDCl
.98 (tr, J = 7.0 Hz, 2H, SCH CH ), 1.75 (sex., J = 7.0 Hz, 2H, CH
CH CH ), 1.20 (s, 3H, CCH ), 1.05 (s, 3H, CCH ), 1.01 (tr, J = 7.0 Hz,
H, CH CH ).
3
): δ 4.12 (m, 4H, -CCH
2
O),
2
-
2
2
2
2
3
3
3
3
2
3
Synthesis of 5,5-Dimethyl-2-thiono-1,3,2-dioxaphosphorinanyl-
-disulfanyl-2-propene (allyl-4). The allyl form of 4 was synthesized
2
similarly to the described method of the ethyl conjugate except 2-propene-
1
1
-thiol was used. H NMR of allyl-4 (500 MHz, CDCl
CH CHdCH ), 5.23 (m, 2H, CH dCH-), 4.11 (m, 4H, CCH
J = 7.6 Hz, 2H, SCH CH), 1.20 (s, 3H, CCH ), 1.05 (s, 3H, CCH
3
3
): δ 5.86 (m, 1H,
O), 3.65 (d,
).
2
2
2
2
2
3
Synthesis of Cysteine (CYS) Alkyl Disulfide Conjugates. The
mixed-disulfide conjugates CySSR (5; Scheme 1) that were prepared are
shown in Figure 3 (7-10). For the propyl disulfide conjugate of CYS
Nitric Oxide (NO) Evolution Bioassay. Mouse macrophage cells
(RAW 264.7, ATCC) were cultured in phenol red-free Dulbecco’s
modified Eagle’s medium (DMEM; Invitrogen) medium supplemented
with 15% fetal bovine serum with antibiotic (a mixture of penicillin and
(
CySSP, 9), triethylamine (5.7 mmol) was combined with
hydrochloride monohydrate (1.42 mmol) and (propyl-4) (2.83 mmol) in
5 mL of ethanol. The reaction mixture was vigorously stirred at 20-22 °C
for 1-2 h. The solvent was removed by vacuum rotary evaporation, and
the residue was washed with CH Cl to remove the excess reactant (propyl-4)
L-cysteine
5
streptomycin) at 37 °C in 5% CO in a 96-well plate (5 ꢀ 10 cells/well) for
2
1
24 h. The medium was then replaced with fresh medium containing test
compounds and 1 μg/mL LPS, and the plate was incubated for another
24 h. A 100 μL sample of culture supernatant from each well was then
combined with 100 μL of freshly prepared Griess reagent (equal volumes
of 2% (w/v) sulfanilamide containing 4% (w/v) H PO and 0.2% (w/v)
2
2
and the major byproduct (2). An alternative workup protocol after reaction
involved filtering the product mixture to recover 5 (largely insoluble in
ethanol) and washing the particulate material with cold ethanol to obtain the
3
4
naphthylethylenediamide) and incubated for 10 min, and then the absor-
bance was measured at 542 nm (22). The concentration of nitrite (evolved
from NO) in the supernatant was calculated from a standard curve
prepared with sodium nitrite dissolved in DMEM.
Cell viability was measured by the MTT assay (23). After a 100 μL
sample had been taken from the cell culture supernatant for the NO assay,
the residual medium was decanted, 100 μL of MTT dye (0.05 mg/mL in
phosphate-buffered saline) was added to each well, and the cells were
incubated for 1 h at 37 °C. Then the solution was decanted, and 200 μL of
DMSO was added to each well and incubated for 10 min under gentle
shaking at 37 °C to dissolve the dye. Relative cell viability was calculated
by measuring the optical density at 550 nm with untreated control cells
assigned a value of 1.0.
final product. In both cases, >80% pure CySSP was obtained (the only
1
impurity shown on H NMR was unreacted
L-cysteine). A >98% pure
sample of 9 was obtained by RP-18 column chromatography eluted with
0% methanol in water. Methyl (CySSM, 7), ethyl (CySSE, 8), and allyl
CySSA, 10) conjugates of CYS were prepared according to the same
5
(
method except that methyl, ethyl, and allyl forms of 4 were used.
Synthesis of Glutathione (GSH) Alkyl Disulfide Conjugates. The
mixed-disulfide conjugates GSSR (6; Scheme 1) that were prepared are
shown in Figure 3. Glutathione conjugates of propyl (GSSP, 13), ethyl
(GSSE, 12), methyl (GSSM, 11), and allyl residues (GSSA, 14) were
prepared as just described for the corresponding cysteine conjugates,
except that GSH (1.40 mmol) replaced CYS in the reaction mixtures

Article
J. Agric. Food Chem., Vol. 58, No. 3, 2010 1567
Statistical Analysis. Results are expressed as mean values ( SD for
used equimolar ratios of organic soluble 4 and thiols as a means to
two or three separate experiments, each with two to three replicates
for each condition assessed. Differences between responses were deter-
mined using a one-tailed t test, with a P value of <0.05 considered to be
significant.
minimize residual byproduct and unreacted materials (21). Sub-
1
sequently, >98% pure (assessed by peak integration by
H
NMR) CYS/GSH mixed-disulfide conjugates (7-14) were ob-
tained by a final RP-18 column chromatography purification
step.
RESULTS AND DISCUSSION
High-resolution ESI-TOF MS profiles data of the CySSR/
GSSR derivatives (7-14; Figure 3) are summarized in Table 1.
(ESI-TOF HR-MS spectra are available in the Supporting
Information.) The molecular formulas of the prepared com-
pounds were confirmed by high-resolution MS analysis. As an
example, the reaction product of cysteine and sodium methyl
Synthesis of CYS/GSH Mixed-Disulfide Conjugates of Thiosul-
finates. The first stage of synthesizing the CYS/GSH disulfide
conjugates of thiosulfinates (5, 6) involved the preparation of the
methyl, ethyl, propyl, and allyl disulfide conjugates of dithiopho-
sphoric acid (4) according to protocols described for preparing
other compounds (20, 21). This stage posed no complications
with typical yieldsof70-80%, and derivatives of4 appearedto be
stable at 20-22 °C for several months. In previous papers, only
hydrophobic thiol substrates were investigated for the prepara-
tion of unsymmetrical disulfides (21). In the present study, some
modifications were necessitated by making use of the water-
soluble thiol substrates CYS and GSH in the reaction. The second
stage of the synthesis involved reaction of CYS or GSH with
excess dithiophosphoric-alk(en)yl disulfides (4) in the presence of
triethylamine as base in ethanol or ethanol-water as solvent.
CYS/GSH conjugates (5, 6) were generated with yields of ∼80%,
likely limited by the partial solubility of 4 in aqueous or polar
organic media. The major byproduct of the reaction (2) was
difficult to remove from the reaction product mixture, and it was
difficult to resolve from the reaction products (5, 6) under the RP-
þ
mercaptoate exhibited the molecular ions m/z [M þ H] at
-
168.0153 and [M - H] at 165.9996, consistent with the molec-
þ
ular formula of CySSM (7): C H NO S (calcd [M þ H] m/z
4
9
2 2
-
168.0153, [M - H] m/z 165.9996). Compared with the starting
material cysteine (exact molecular weight of 121.0198), a
þ45.9877 Da fragment was observed, corresponding to the
addition of the -SCH group to form the disulfide bond.
3
1
H NMR spectral signals of the CySSR and GSSR derivatives
in D O are summarized in Tables 2-4. The H NMR signal
1
2
assignments could be compared only for those species previously
analyzed (24, 25), which include CySSA (10), GSSA (14), and
CYS and GSH, with differences identified in the balance of this
1
section. No reports of H NMR spectra could be found for the
other conjugates (7-9, 11-13). For CySSA (10), the two germ-
inal protons of C-3 are adjacent to the chiral center C-2, and, thus,
the H-3 protons should exhibit different chemical shifts and split
18 flash chromatography conditions used. It was found that
2
evaporation of the ethanolic reaction medium followed by
extracting the product mixture with CH Cl circumvented this
each other with a germinal coupling constant of J = 15.0 Hz. H-
2
2
2 and H-3 protons should exhibit three groups of doublets of
doublets signals at δ 4.10 (H-2, dd, J = 4.0, 8.5 Hz, 1H), 3.10 (H-3,
dd, J = 8.5, 14.5 Hz, 1H), and 3.35 (H-3, dd, J = 4.0, 15.0 Hz,
1H) (Table 3). This contrasts with the previously reported (24)
spectral characteristics of shifts of δ 4.07 (H-2, q) and 3.41 (H-3,
dq). Also, the previously reported δ 3.19 (H-4, dq) appears to be a
transposing error, as the published spectrum reveals a value close
to our δ 3.45 (H-4, br d) signal, consistent with our results. As
with CySSA (10), the two germinal protons of H-7 of GSSA (14)
were interpreted as two doublets of doublets signals at δ 3.01
problem and removed the main contaminants (2 and 4), as well as
other unidentified impurities in the reaction mixture. This af-
forded >80% pure CYS/GSH mixed-disulfide conjugate pre-
parations (7-14), and the only impurity remaining after CH Cl2
2
washing was identified as unreacted CYS or GSH. The utility of
this washing/purification step allowed the use of a molar excess of
4
in the reaction to favor complete thiol conversion into the
intended disulfide conjugates (5, 6), whereas the original method
(
1
H-7, dd, J = 9.5, 14.5 Hz, 1H) and 3.27 (H-7, dd, J = 4.5, 14.5 Hz,
Table 1. High-Resolution ESI-TOF MS of CySSR/GSSR Species
compd molecular formula [M þ H]þ calcd [M þ H]þ [M - H]- calcd [M - H]-
H) (Table 4) as opposed to the reported δ 5.88 (H-7, m) (12). We
1
also interpret each of the H NMR signals of H-3 and H-4 as
having two chemical shifts (δ 2.19, 2.20 for H-3, tr; δ 2.57and 2.58
for H-4, tr) instead of single shifts (δ 2.14 for H-3, q; δ 2.52 for
H-4, q) as reported in ref9 based on the conformational analysis
of the glutathione skeleton (25). In addition, H-9 protons should
exhibit a singlet signal because of the absence of coupling protons,
instead of the doublet signal previously reported (12). The
hydrochloride forms of CySSR were also prepared and charac-
terized by NMR (Table 2), and the only major difference with
CySSM C
CySSE C
CySSP C
CySSA C
4
5
6
6
H
H
H
H
9
NO
2
S
2
168.0153
182.0303
196.0465
194.0300
354.0784
368.0942
382.1097
380.0944
168.0153
182.0309
196.0466
194.0309
354.0794
368.0950
382.1107
380.0950
165.9996
180.0155
194.0315
192.0170
352.0633
366.0790
380.0943
378.0912
165.9996
180.0153
194.0309
192.0153
352.0637
366.0794
380.0950
378.0794
11NO
13NO
11NO
2
S
2
S
2
S
2
2
2
GSSM C11
H
19
H
21
H
23
H
21
N O
3
N O
3
N O
3
N O
3
6
S
S
S
S
2
2
2
2
GSSE
GSSP
GSSA
C
12
C
13
C
13
6
6
6
1
a
Table 2. H NMR of Hydrochloride CYS Conjugates of Thiosulfinates (CySSR-HCl)
position
CySSM
CySSE
CySSP
CySSA
2
4.30 (dd, 4.0, 8.5)
4.37 (dd, 4.0, 8.5)
4.36 (dd, 4.0, 8.0)
4.30 (dd, 4.0, 8.0)
3
4
5
3.15 (dd, 8.0, 15.0)
3.35 (dd, 4.0, 15.0)
3.21 (dd, 8.5, 15.0)
3.38 (dd, 4.0, 15.0)
3.15 (dd, 8.0, 15.0)
3.31 (dd, 4.0, 15.0)
3.14 (dd, 8.0, 15.0)
3.32 (dd, 4.0, 15.0)
2.42 (s)
2.82 (qr, 7.0),
2.81 (qr, 7.0)
2.72 (tr, 7.2)
2.73 (tr, 7.2)
3.39 (br d, 7.5)
1.34 (tr, 7.5)
1.66 (sex, 7.2)
0.92 (tr, 7.2)
5.87 (ddtr, 7.0, 10.0, 17.0)
5.20 (m)
6
a
See Figure 3 for position assignments.

1568 J. Agric. Food Chem., Vol. 58, No. 3, 2010
Zhang et al.
1
Table 3. H NMR of CYS Conjugates of Thiosulfinates (CySSR)
a
position
CySSM
CySSE
CySSP
CySSA
2
4.12 (dd, 4.0, 8.5)
4.11 (dd, 4.0, 9.0)
4.11 (dd, 4.0, 9.0)
4.10 (dd, 4.0, 8.5)
3
4
5
3.13 (dd, 8.5, 15.0)
3.39 (dd, 3.5, 15.0)
3.11 (dd, 9.0, 15.0)
3.35 (dd, 4.0, 15.0)
3.10 (dd, 8.5, 14.5)
3.35 (dd, 4.0, 15.0)
3.10 (dd, 8.5, 14.5)
3.35 (dd, 4.0, 15.0)
2.49 (s)
2.82 (qr, 7.0)
2.81 (qr, 7.0)
2.81 (tr, 7.5)
2.80 (tr, 7.5)
3.45 (br d, 7.5)
1.35 (tr, 7.5)
1.75 (tr, 7.0)
1.00 (tr, 7.5)
5.95 (ddtr, 7.0, 10.0, 17.0)
5.28 (m)
6
a
See Figure 3 for position assignments.
1
Table 4. H NMR of GSH Conjugates of Thiosulfinates (GSSR)
a
position
GSSM
GSSE
GSSP
GSSA
2
3.82 (tr, 6.0)
3.83 (tr, 6.0)
3.82 (tr, 6.0)
3.85 (tr, 6.5)
3
4
2.19 (qr, 7.0)
2.20 (qr, 7.0)
2.19 (qr, 7.0)
2.20 (qr, 7.0)
2.19 (qr, 7.0)
2.20 (qr, 7.0)
2.19 (qr, 7.0)
2.20 (qr, 7.0)
2.58 (tr, 7.0)
.56 (tr, 7.5)
2.56 (tr, 8.0)
2.58 (tr, 8.0)
2.56 (tr, 7.5)
2.58 (tr, 7.5)
2.57 (tr, 7.5)
2.58 (tr, 7.5)
2
6
7
4.76 (dd, 4.0, 9.5)
4.76 (dd, 4.5, 9.5)
4.76 (dd, 5.0, 9.5)
4.75 (dd, 4.5, 9.5)
3.02 (dd, 9.5, 14.5)
3.30 (dd, 4.5, 14.5)
3.00 (dd, 9.5, 14.5)
3.27 (dd, 4.0, 14.5)
3.00 (dd, 9.5, 14.5)
3.27 (dd, 4.5, 14.5)
3.01 (dd, 9.5, 14.5)
3.27 (dd, 4.5, 14.5)
9
3.97 (s)
2.46 (s)
3.98 (s)
3.95 (s)
4.01 (s)
11
12
13
2.77 (qr, 7.5)
1.32 (tr, 7.5)
2.76 (tr, 7.5)
1.72 (sex., 7.5)
0.99 (tr, 7.5)
3.42 (br d, 6.5)
5.93 (ddtr, 7.0, 10.0, 17.0)
5.25 (m)
a
See Figure 3 for position assignments.
spectra and corresponding CySSR species (Table 3) was a
dominate species in garlic (50-94%), and allicin is widely
believed to be responsible for the health benefits of garlic (1, 19).
1-Propenyl thiosulfinates are major species in fresh onion tissue
macerates (19), and the biological effects of the 1-propenyl mixed-
disulfide conjugate derivatives may be key to understanding the
health benefits of onion. However, the method described in
this paper cannot be used directly to prepare the conjugates of
1-propenyl thiosulfinates because the corresponding 1-propene-
1-thiol (RSH, step 3 in Scheme 1) is not available.
QR Induction as a Marker for Phase II Enzyme Up-regulation.
Previous studies have shown that many Allium organosulfur
compounds, such as thiosulfinates and (poly)sulfides, can in-
duce phase II enzymes (27, 28), an important mechanism for
conferring protection against cancer (14). It was of interest to
determine if similar activities are associated with CYS and GSH
mixed-disulfide conjugates CySSR/GSSR, which are the poten-
tial in vivo metabolic products of Allium organosulfur com-
pounds. Representative dose-dependent responses for QR
induction and cell viability for Hepa 1c1c7 cells treated with
CySSE (8) and GSSA (10) are shown in Figure 4. Do-
se-responses for QR induction were considered to be valid
for levels up to >50% retention of cell viability because further
losses in viability may confer intolerable increases in variability
in the analysis (14). These dose-responses allowed for the
determination of relative potency of the mixed-disulfide con-
jugates as the concentration required to double (CD value) QR
∼
0.2 ppm downfield signal of H-2.
The bioavailability (absorption) of thiosulfinates approaches
100%, and they are metabolized rapidly in human blood in vitro,
where the half-life is <1 min (15). Thiosulfinates progressively
decay over a 24 h period in Allium tissue macerates (19). The
cellular membrane permeability and ability to react with
thiols (4, 9, 24) are properties that can account for the short
half-life of thiosulfinates in physiological milieu. GSSA and other
GSSR species may be formed in the intestinal lumen following
consumption of freshly minced Allium vegetables with foods rich
in either GSH or GSH provided by biliary excretion (5). These
observations implicate the formation of mixed-disulfide conju-
gates of thiosulfinates with biological/physiological thiols as
representing major derivatives that form in foods in situ and in
the gut or through metabolic processes in vivo. The relative
importance of these conjugates in the diet is likely proportional to
the relative abundance of thiosulfinates containing the alk(en)yl
groups that form these conjugates. Methyl-containing thiosulfi-
nates are most ubiquitous and found in virtually all species of
Allium vegetables (around 30% in total thiosulfinates of onions,
garlic, leek, and shallot, 86% in Chinese chive) (19). Ethyl-
containing thiosulfinates are found in trace amounts (26),
whereas propyl-containing thiosulfinates are present at levels of
∼
20% in onions and ∼50% in shallot, leek, and scallion while
being virtually absent in garlic. Allyl thiosulfinates are the

Article
J. Agric. Food Chem., Vol. 58, No. 3, 2010 1569
Figure 5. Dose-dependent inhibition of NO production in lipopolysacchar-
ide-stimulated macrophage cells (RAW 264.7) by CySSR conjugates of
G
thiosulfinates. L-NAME (N -nitro-
L-arginine-methyl-ester) was used as a
positive control in the experiment. The results are expressed as mean
percent NO inhibition values ( SD of treated cells compared to control
(
untreated cells).
Figure 4. Quinone reductase (QR) induction in murine Hepa 1c1c7 cells
by CySSE and GSSA. The potency of the QR inducer is indexed by a CD
value, which is the concentration required to double QR specific activity
interpolated from the response curves (indicated by broken lines). Results
are expressed as mean values ( SD for relative QR specific activity.
Table 5. QR-Inducing Potency of CYS and GSH Conjugates of Thiosulfinates
(CySSR/GSSR)
compd
CD value (μg/mL)
(μM)
CySSM
CySSE
CySSP
CySSA
GSSM
GSSE
GSSP
GSSA
88.9 ( 7.6
76.5 ( 20.3
92.0 ( 13.0
25.8 ( 8.9
86.9 ( 5.9
72.4 ( 17.0
72.7 ( 9.3
33.1 ( 9.9
(532)
(423)
(472)
(134)
(246)
(197)
(191)
(87.3)
Figure 6. Dose-dependent inhibition of NO production in lipolysaccharide-
stimulated macrophage cells (RAW 264.7) by GSSR conjugates of
thiosulfinates. The results are expressed as mean percent NO inhibition
values ( SD of treated cells compared to control (untreated cells).
specific activity in Hepa cells. All mixed-disulfide conjugates
behaved qualitatively similarly and induced QR in Hepa 1c1c7
cells relative to noninduced controls (P < 0.05). CD values
derived by interpolation of the dose-response curves are
summarized in Table 5. The CYS and GSH mixed-disulfide
conjugates with saturated R groups (methyl, ethyl, and propyl)
showed similar trends in relative potency of methyl = ethyl =
propyl < allyl based on micromolar CD values (P < 0.05), with
the group of GSSR derivatives about twice as potent as the
CySSR derivatives. Thus, the presence of the unsaturated R
group in these conjugates conferred greatest potency to the
mixed-disulfide conjugates. The mere presence of the R group in
these conjugates also conferred QR-inducing activity because
both cystine (CySSCy) and oxidized glutathione (GSSG) had no
effect on relative QR specific activity and viability of Hepa cells
relative to noninduced controls (data not shown) when tested
over the same dose range as the conjugates.
NO-Based Anti-inflammatory Assay. Allicin and ajoene (an R-
sulfinyl disulfide from garlic) exert the anti-inflammatory effect of
inhibiting inducible nitric oxide synthase (iNOS) expression in
LPS-activated macrophages (30). In the present work, all CySSR/
GSSR mixed-disulfide conjugates inhibited LPS-induced NO
evolution in activated macrophages (Figures 5 and 6). Inhibition
of NO production by the CySSR species exhibited a dose-
G
dependent response qualitatively similar to N -nitro-
L-arginine-
methyl-ester (L-NAME) (Figure 5), a well-known inhibitor of
iNOS activity. CySSA (10) was the most potent inhibitor among
the conjugates (P < 0.05) with the saturated CySSR species (7-9)
having similar potencies. IC50 values estimated from semilog
plots (not shown) revealed values of 40 μM for CySSA, 300 μM
for CySSM, 560 μM for CySSE, and 360 μM for CySSP.
The viability of macrophages treated with all CySSR species
was >94% over the dose range examined, indicating the magni-
tude of inhibition of NO production observed cannot be ac-
counted for by loss of viable cells. Evaluation of the GSSR species
revealed similar responses of macrophages (Figure 6) as observed
for the CySSR species. GSSA (14) was a more potent inhibitor of
NO production than the saturated GSSR species (P < 0.05), and
an IC50 value of 240 μM for GSSA was estimated. As a group, the
The CD values of 100-500 μM exhibited for phase II enzyme
QR) induction by CySSR/GSSR species were several-fold great-
(
er (less potent) than the CD values of 30-100 μM reported for
homologous thiosulfinates with the same R groups and 10-20 μM
for 1-propenyl-containing thiosulfinates (19). A more recent
study revealed allicin at 10-20 μM to up-regulate GSH and
some phase II enzymes in vascular epithelial cells (29).

1570 J. Agric. Food Chem., Vol. 58, No. 3, 2010
Zhang et al.
other GSSR species were less potent at inhibiting NO evolution in
the macrophage bioassay than the CySSR species (P < 0.05).
The viability of macrophages treated with all GSSR species
was >82% over the dose range examined, indicating that loss
of viability could have only a marginal influence on NO produc-
tion at doses where substantial inhibition occurred. CySSA (10)/
GSSA (14) showed more potent NO inhibitory activity than the
positive control L-NAME, a standard iNOS inhibitor (IC50 value
estimated at 430 μM). CySSCy and GSSG had no effect on
NO evolution and viability of macrophages when tested at up to
formed intracellularly after consumption of thiosulfinates (9) or
through metabolic activity (6, 7) (cf. Figure 2).
Further evaluation of the biological effects of CySSR and
GSSR, including in vivo studies, would help to determine if
they could serve as vehicles for delivering Allium-related health
benefits through dietary interventions.
ACKNOWLEDGMENT
We thank Dr. Tomas Stringfellow at the Analytical Instru-
mentation Center at the University of Wisconsin;Madison for
technical assistance with the collection of NMR data and Viriya
Nitteranon for assistance with experiments related with RAW
200 μg/mL relative to LPS-activated controls (data not shown).
As far as we are aware, ours is one of the first studies on in vitro
anti-inflammatory effects of mixed-disulfide conjugates of CYS/
GSH and thiosulfinates. The potency of CySSA (IC50 of 40 μM)
was comparable to the corresponding thiosulfinate allicin (IC50 of
264.7 cells.
Supporting Information Available: High-resolution ESI-
TOF MS of various compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
1
5-20 μM) (30) in terms of NO inhibition in RAW 264.7 cells.
The other CySSR derivativesand GSSA were as much as an order
of magnitude less potent, with IC50 values of 240-560 μM. No
reports on the effects of thiosulfinates other than allicin (29) on
NO evolution in cultured macrophages could be found to afford
further comparisons. An expanded evaluation of other anti-
inflammatory responses, such as modulation of pro-inflamma-
tory cytokines/mediators such as PGE , or inflammation-med-
iating enzymes iNOS and COX-2 evoked by CySSR/GSSR and
the corresponding thiosulfinates is warranted.
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Received for review October 7, 2009. Revised manuscript received
January 4, 2010. Accepted January 05, 2010. This work was
supported by the College of Agricultural and Life Sciences of the
University of Wisconsin (Hatch Grant WIS01184) and the U.S.
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