Isothiocyanates as H2S Donors Triggered by Cysteine: Reaction Mechanism and Structure and Activity Relationship

Article abstract of DOI:10.1021/acs.orglett.9b02117

Isothiocyanates are reported as H₂S donors, yet the mechanisms are not clear. Herein, we reported that isothiocyanates (ITCs, R-NCS) rapidly formed adducts with cysteine. These adducts underwent intramolecular cyclization followed by releasing

Full text of DOI:10.1021/acs.orglett.9b02117

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Isothiocyanates as H2S Donors Triggered by Cysteine: Reaction
Mechanism and Structure and Activity Relationship
Yi Lin,† Xin Yang,† Yuyun Lu,† Dong Liang,‡ and Dejian Huang*,†,§
†Food Science and Technology Programme, Department of Chemistry, National University of Singapore, 3 Science Drive 3,
Singapore 117543, Singapore
‡
̈
̈
Food Chemistry and Molecular Sensory Science, Technische Universitat Munchen, Lise-Meitner-Straße 34, 85354 Freising,
Germany
§National University of Singapore (Suzhou) Research Institute, 377 Lin Quan Street, Suzhou Industrial Park, Jiangsu 215123, China
S
* Supporting Information
ABSTRACT: Isothiocyanates are reported as H2S donors, yet the mechanisms are not clear. Herein, we reported that
isothiocyanates (ITCs, R-NCS) rapidly formed adducts with cysteine. These adducts underwent intramolecular cyclization
followed by releasing organic amine R−NH2 and raphanusamic acid (RA) as major products with formation of H2S and 2-
carbylamino-4,5-dihydrothiazole-4-carboxylic acids as minor products. The ratios of the two groups of products are dependent
on the R groups which have strong impact on H2S releasing rates.
Scheme 1
ydrogen sulfide (H2S) plays pivotal roles in various
Hphysiological and pathological processes of cardiovas-
cular, nervous, and respiratory systems.1−4 In a cardiovascular
system, H2S has been critically involved in the modulation of
blood pressure homeostasis, serving as a potent vasodilator in
endothelial cells, and believed to be responsible for antiaging
effects of calorie restriction.5 As such, exogenous H2S donors,
particularly those slow H2S donors found in foods, attract a
great deal of attention.6,7 Dietary organosulfides are attractive
H2S donors for health promotion. Diallyl disulfide (DADS)
and diallyl trisulfide, the main organosulfides found in garlic,
were shown to H2S donors under physiological conditions by
reacting with gluthathione.8,9 Rich in cruciferous vegetables,
isothiocyanates (ITCs) have been extensively studied for their
cancer chemoprevention activity, yet its mechanism of action is
not clear.10 Recently it was reported that ITCs were H2S
donors.11,12 The H2S releasing activity may be partially
responsible for underlying principles for the cancer chemo-
prevention activity as well as other biological activities.13−16 It
has been widely accepted that the in vivo metabolism pathway
of ITCs involves the formation of glutathione conjugates, 1,
which undergo enzymatic hydrolysis of the peptide bonds to
give cysteine-ITC conjugate intermediates, 2 (Scheme 1). The
latter was acetylated to give NAC-ITC, 3, conjugate that was
found in urine. This pathway does not lead to H2S formation.
To explain the H2S formation mechanism, we hypothesized
that the cysteine-ITC intermediate may undergo intra-
molecular cyclization reaction and lead to releasing of H2S.
To test our hypothesis, we first measured the H2S releasing
capacity of ITCs with different R groups. Mixing of 2 equiv of
cysteine with ITCs resulted in the browning of the lead acetate
paper placed on top of microplate lids, which detected the H2S
in the vapor phase above each well as an indicator of H2S
concentration in the solutions. The amount of H2S was
quantified with Na2S as a reference standard, and the results
were shown in Figure 1 (the dose response curves shown in
Figure S1). p-Nitrophenylisothiocyanate was the most
effective, by at least 4 times more H2S released than other
ITCs, which generated comparable amounts of H2S
independent of the different aryl groups. The aliphatic ITC
(R = phenethyl) generated the least amount of H2S. This result
Received: June 19, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b02117
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Figure 1. H2S releasing capacity of isothiocyanates in the presence of
2 equiv of L-cysteine (PEITC, PhCH2CH2NCS; BITC, PhCH2NCS,
PITC, PhNCS; MeOPITC, p-CH3OC6H4NCS, ClPITC, p-Cl-
C6H4NCS; NO2PITC, p-NO2-C6H4NCS). Reaction conditions:
20% N,N-dimethylformamide (DMF) in PBS (10 mM, pH 7.4) at
37 °C for 24 h.
Figure 2. (a) Full scale reaction time course of H2S formation over
time of different ITCs. (b) Apparent first-order rate constants of H2S
generation reaction between aromatic ITCs with different para
substituents. (c) Hammett plot of the rate of H2S release of p-R-
C6H4NCS (R = H, MeO, Cl, NO2).
suggested that the basicity of the amine and the electron-
withdrawing group in the para position of phenyl group plays
an important role in the H2S releasing capacity of aryl ITCs.
The low yields of H2S formed from the ITCs revealed that
there were other known H2S generation reaction pathways.
From the intermediate 4, H2S (pathway a) and amine
(pathway b) may be eliminated. To fully characterize the
reaction products, we selected p-chlorophenyl isothiocyanate
(ClPITC) as an example and elucidated the reaction products
by LC-MS shown in Figure 3. Similar reaction product
1
Monitored by H NMR spectra and HPLC, we found that
ITCs rapidly form adducts with cysteine, 2-S and 2-N, yet the
H2S releasing rates were much slower. The reaction pathway
leading to H2S release was shown in Scheme 2. The adducts
Scheme 2
underwent intramolecular nucleophilic additions to form
unobserved intermediate 4. Based on this mechanism, the
H2S releasing rate was expected to be first order to the adduct.
We measured the reaction kinetics, and the results are shown
in Figure 2 (and Figure S2).
Figure 3. HPLC chromatogram (UV detector at 254 nm) of the
products of the reaction of p-chlorophenylisothiocyanate (4 mM) and
L-cysteine (8 mM) in 20% N,N-dimethylformamide (DMF) in PBS
(10 mM, pH 7.4) at 37 °C for 24 h.
The H2S formation rates are dependent on the substituent of
the ITCs (Figure 2a). The initial rates have an apparent first-
order relationship with the concentration of the ITCs (Figure
S2). The apparent rate constants are dependent on the R
groups of the ITCs (Figure 2b). The Hammett plot (Figure
2c) resulted in a fairly good linear line with a slope (ρ) of 1.56,
suggesting that the rate-limiting step for H2S releasing from the
ITC-Cys adduct is influenced by the electronic effects of para
substituents on the phenyl. The electron-withdrawing group
facilitated the intramolecular nucleophilic addition of amines.
Our results are in agreement with that by Matson and co-
workers, who reported synthetic S-aroylthiooximes as H2S
donors, triggered by cysteine, exhibiting controllable kinetics
correlated to the electronic withdrawing/donating properties
of para-substituents with a linear Hammett plot (ρ = 1.05).17
distributions were observed for other ITCs such as phenyl
isothiocyanate (PITC), p-nitrophenyl isothiocyanate (NO2-
PITC), p-methoxyphenyl isothiocyanate (MeOPITC), benzyl
isothiocyanate (BITC), and phenethyl isothiocyanate
(PEITC) (data in Supporting Information).
Indeed the LC-MS results revealed raphanusamic acid, 6
(peak 1), and p-chlorophenylamine, 7-ClPh (peak 2), as well
as its adduct with ClPhITC, 8-ClPh (peak 5). This proved that
pathway b is operational. In addition, the pathway a also
happened as 2-(p-chlorophenylamino)-4,5-dihydro-4-thiazole-
carboxylic acid, 5-ClPh (peak 3) was detected. In addition, we
detected adducts of ITC with cystine 9-ClPh (peak 4) and the
oxidative products of the 2-N through the disulfide bond, 10-
ClPh (peak 6). To confirm the structure of 5-ClPh, it has been
B
DOI: 10.1021/acs.orglett.9b02117
Org. Lett. XXXX, XXX, XXX−XXX

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Letter
isolated by semipreparative HPLC and its structure was
1
confirmed by H and 13C NMR as well as high resolution MS
spectra.
ITCs form adducts with cysteine under the physiological pH
buffered conditions, through either the amine or thiol group of
cysteine,18 and the reaction of ITCs with thiol groups is about
1000 times faster than with amine groups.19 Therefore, we
speculated that 2-S is a predominant reaction product in the
first step of the reaction. Both intermediates led to the same
cyclic intermediate 4. The outcome of the reaction was
determined by competitive elimination reaction of H2S or
amine. It was expected that the proportion of β-elimination of
amines shall be influenced by the basicity of the respective
amine. From our HPLC results, we observed that the formed
amines could further react with ITCs to give adducts (such as
compound 8-ClPh in Figure 3). The amine concentration was
indirectly quantified by determining the concentration of
raphanusamic acid 6. While that of H2S was quantified by the
concentration of dihydrothiazole carboxylic acids, 5. The ratio
of [6]/[5] is an indication of which pathway is preferred for a
given amine (Figure 4). Consistent with H2S releasing capacity
Figure 5. HPLC chromatograms at 254 nm. (a) Metabolites of RAW
264.7 cells after incubated with 100 μM fluorescein isothiocyanate
(FITC) at 37 °C for 24 h. (b) Products of the reaction of FITC (4
mM) + L-cysteine (8 mM) in 20% DMF in PBS (10 mM, pH 7.4) at
37 °C for 24 h.
believed that ITCs were metabolized by multistep reactions
starting from forming adducts with gluthathione and ending up
in urine as N-acetylcysteine conjugates (NAC-ITCs) that were
suggested to be the active compounds for their cancer
chemopreventive activity.21 As the precursor of NAC-ITC,
Cys-ITC may undergo intramolecular nucleophilic addition
and result in formation of H2S and dihydrothiazole derivatives.
Alternatively organic amines and raphanusamic acid could be
generated, and they may collectively play different roles for
broad bioactivities of ITCs.22
Figure 4. Ratio of raphanusamic acid (RA), 6, to corresponding
dihydrothiazole carboxylic acid, 5, in the reaction mixture of
isothiocyanate and L-cysteine (1:2) at 37 °C after 24 h (PEITC,
PhCH2CH2NCS; BITC, PhCH2NCS, PITC, PhNCS; MeOPITC, p-
CH3OC6H4NCS, ClPITC, p-Cl-C6H4NCS; NO2PITC, p-O2N-
C6H4NCS).
In summary, we have uncovered new reaction pathways for
ITCs as H2S and amine donors. All the ITCs form adducts
with cysteine rapidly, yet the rate of H2S releasing is dependent
on the R groups. Among them, p-nitrophenylisothiocyanate
generates H2S/amine relatively fast while benzylisothiocyanate
and phenethyl isothiocyanate are slow H2S donors and they
would take more than 1 day at physiological pH conditions to
finish the reaction. Biologically, dihydrothiazoles derived from
ITCs have been observed in insects upon consumption of
glucosinolate rich plant leaves.23 Slugs metabolized (likely as a
detoxification mechanism) isothiocyanate (i.e., 4-methyl-
sulfonylbutyl-ITC) through formation of a glutathione adduct
which was converted to a cysteine conjugate (4MSOB-Cys).
The latter underwent cyclization to give 2-(4-(methylsulfinyl)-
butylamino)-4,5-dihydrothiazole-4-carboxylic acid.24 More re-
cently, this compound was found as an end metabolite of the
cabbage stem flea beetle (Psylliodes chrysocephala).23 The
formation mechanism is likely through the intramolecular
cyclization reaction we proposed herein. Overall, there are only
a few studies on medicinal properties or biological activities of
the dihydrothiazoles derived from ITCs and cysteine except a
patent which claimed that a group of synthetic thiazoles from
ITCs and cysteine are analgesic agents.25 Taken together, our
results warrant further study to understand the underlying
mechanisms of dietary ITCs and their health promotion
benefits as H2S/dihydrothiazole donors as well as organic
amine/raphanusamic acid donors.
results (Figure 1), NO2PITC led to H2S as the major product
and p-nitrophenamine as a minor product. For other ITCs, the
dominant products were respective amines. However, there is
no apparent correlation of the selectivity vs the pKa values of
the respective amines. This suggested that selectivity may be
influenced by factors other than the electron-withdrawing
property of R groups.
To study whether ITCs would undergo similar trans-
formations in vitro under cell culture conditions, we treated
cells (RAW 264.7) with a commonly used fluorescein
isothiocyanate (for sensitive detection of the fluorecent
metabolites) (FITC). FITC metabolites including both a
dihydrothiazole derivative and fluoresceinamine were detected
by HPLC, and the structures were confirmed by LC-MS/MS
and compared with test tube reaction products of FITC and
cysteine (Figure 5). The results suggested that FITC, the
commonly used fluorescent probe for labeling proteins, may
generate H2S.
Our results could shed some light on ITC metabolism
pathways and help to understand their biological activities
through the complex products generated. Although the free
cysteine concentration in human blood is low, glutathione
concentration is very high (∼1.0 mM).20 It has been long
C
DOI: 10.1021/acs.orglett.9b02117
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
(15) Fahey, J. W.; Haristoy, X.; Dolan, P. M.; Kensler, T. W.;
Scholtus, I.; Stephenson, K. K.; Talalay, P.; Lozniewski, A. Proc. Natl.
Acad. Sci. U. S. A. 2002, 99, 7610−7615.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.9b02117.
■
S
(16) Bai, Y.; Wang, X.; Zhao, S.; Ma, C.; Cui, J.; Zheng, Y. Oxid.
Med. Cell. Longevity 2015, 2015, 1.
(17) Foster, J. C.; Chadwick, R.; Powell, C. R.; Radzinski, S. C.;
Matson, J. B. Org. Lett. 2014, 16, 1558−1561.
(18) Zheng, G. Q.; Kenney, P. M.; Lam, L. K. J. Med. Chem. 1992,
35, 185−8.
Experimental procedures and data (PDF)
AUTHOR INFORMATION
(19) Kaschula, C. H.; Hunter, R. Chapter 1 In Studies in Natural
Products Chemistry; Attaur, R., Ed.; Elsevier: 2016; Vol. 50, pp 1−43.
(20) Richie, J. P., Jr.; Skowronski, L.; Abraham, P.; Leutzinger, Y.
Clin Chem. 1996, 42 (1), 64−70.
■
Corresponding Author
*E-mail: chmhdj@nus.edu.sg.
ORCID
(21) (a) Conaway, C. C.; Yang, Y. M.; Chung, F. L. Curr. Drug
Metab. 2002, 3, 233−255. (b) Tang, L.; Zhang, Y. S. Curr. Drug
Metab. 2004, 5, 193−201.
Xin Yang: 0000-0003-1304-7492
Dejian Huang: 0000-0002-2305-3960
Notes
(22) Grundemann, C.; Huber, R. Cancer Lett. 2018, 414, 26−33.
(23) Beran, F.; Sporer, T.; Paetz, C.; Ahn, S.-J.; Betzin, F.; Kunert,
̃
G.; Shekhov, A.; Vassao, D. G.; Bartram, S.; Lorenz, S.; Reichelt, M.
Front. Plant Sci 2018, 9, 1754.
The authors declare no competing financial interest.
̈
(24) Falk, K. L.; Kastner, J.; Bodenhausen, N.; Schramm, K.; Paetz,
C.; Vassao, D. G.; Reichelt, M.; von Knorre, D.; Bergelson, J.; Erb, M.;
ACKNOWLEDGMENTS
■
̃
Authors thank the Singapore Ministry of Education for
financial support (Grant Number: R-143-000-B04-114) and
Scholarships to Y.L. and X.Y. We thank Professor Jinsong Bian
for supplying RAW264.7 cells.
Gershenzon, J.; Meldau, S. Mol. Ecol. 2014, 23, 1188−1203.
(25) Mannion, J. C.; Dax, S. L. Analgesic compounds, compositions,
and uses thereof. WO 2011112602 A1, 2011.
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DOI: 10.1021/acs.orglett.9b02117
Org. Lett. XXXX, XXX, XXX−XXX