Thioglycine and l-thiovaline: Biologically active H2S-donors

Article abstract of DOI:10.1016/j.bmc.2012.02.028

Thioglycine and l-thiovaline are stable under acidic and basic conditions but in the presence of bicarbonate they liberate the gasotransmitter H ₂S. In cells both thioamino acids were proven to enhance cGMP formation and promote vasorelaxation in mouse aortic rings. Given that H ₂S is known to lower arterial hypertension, reduce oxidative stress and exhibit cardioprotective effects in preclinical models, H₂S donors hold promise as novel treatments for cardiovascular diseases.

Full text of DOI:10.1016/j.bmc.2012.02.028

Bioorganic & Medicinal Chemistry 20 (2012) 2675–2678
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Thioglycine and L-thiovaline: Biologically active H2S-donors
Zongmin Zhou a, Margarete von Wantoch Rekowski b, Ciro Coletta c, Csaba Szabo c, Mariarosaria Bucci d,
b,
Giuseppe Cirino d, Stavros Topouzis a, Andreas Papapetropoulos a, , Athanassios Giannis
⇑
⇑
a Department of Pharmacy, Laboratory of Molecular Pharmacology, University of Patras, Greece
b Institut für Organische Chemie, Universität Leipzig, Johannisallee 29, 04103 Leipzig, Germany
c Department of Anaesthesiology, The University of Texas Medical Branch, Galveston, TX, USA
d Department of Experimental Pharmacology, Faculty of Pharmacy, University of Naples-Federico III, Naples, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Thioglycine and L-thiovaline are stable under acidic and basic conditions but in the presence of bicarbon-
Received 23 January 2012
Accepted 8 February 2012
Available online 27 February 2012
ate they liberate the gasotransmitter H2S. In cells both thioamino acids were proven to enhance cGMP
formation and promote vasorelaxation in mouse aortic rings. Given that H2S is known to lower arterial
hypertension, reduce oxidative stress and exhibit cardioprotective effects in preclinical models, H2S
donors hold promise as novel treatments for cardiovascular diseases.
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Gasotransmitters
Hydrogen sulfidel
Thiovalin
Thioglycin
1. Introduction
on its beneficial effects would be desirable. This, theoretically,
could be achieved by delivery of H2S gas, but such an approach
Hydrogen sulfide (H2S) is a gas with a characteristic pungent
smell of rotten eggs, mostly known for its toxic properties in living
organisms.1 More recently, H2S has attracted the attention of bio-
medical researchers, as it was discovered that low amounts of this
gas are endogenously produced in many mammalian tissues.2
Increasing evidence shows that H2S acts as a signaling molecule
in cells and is now considered the third member of the gasotrans-
mitter family along with nitric oxide and carbon monoxide.3 Most
of H2S is produced by two enzymes cystathionine-synthase (CBS)
would have major disadvantages that include practical hurdles
associated with the delivery of gaseous molecules and lack of
targeted delivery. Small molecules that can release H2S in a slow,
regulated fashion, mimicking the endogenous release of H2S,
would be preferable.
The majority of researchers use NaHS or Na2S as a source for
H2S in their experiments.7 Under physiological conditions (pH
7.4), ca. 28% of hydrogen sulfide (pKa = 7) is undissociated and
present in biological fluids as H2S. It should, however, be noted that
it is unclear whether H2S, HSꢀ or both are the biologically relevant
species. Exposure of biological systems to NaHS causes a burst of
H2S that does not recapitulate the low level continuous production
of H2S that occurs in vivo. In addition, NaHS is rapidly oxidized by
O2. Another approach used to increase H2S production in cells and
and cystathionine c-lyase (CSE) that use L-cysteine as a substrate
and pyridoxal phosphate as a co-factor.4 CBS is highly expressed
in the central nervous system, while CSE is abundantly present in
the heart, liver, kidney and to a smaller extent in the blood ves-
sels.2b,5 A third enzymatic system that contributes to the endoge-
nous production of H2S is 3MST/CAT.4a,5
tissues, is to provide L-cysteine that is enzymatically converted to
H2S exhibits neuroprotective and cardioprotective actions, re-
duces vascular tone stimulates the growth of new blood vessels
(angiogenesis).2b,4b,5,6 The role of H2S in inflammation is less clear
as both anti-inflammatory and pro-inflammatory actions of this
gasotransmitter have been reported.2a Several disease states,
including diabetes, hypertension, stroke and Alzheimer’s disease,
are associated with decreased H2S production.2 Therefore, admin-
istration of H2S to compensate for decreased levels or to capitalize
H2S through the action of CSE and CBS. This would, however, not
yield the expected results in conditions where the levels and/or
activity of these enzymes are down-regulated by disease pro-
cesses; in addition cysteine has biological effects that are not re-
lated to H2S production. On the other hand, only a handful of
H2S-donors have been reported in the literature. These include
GYY4137,8 a synthetic derivative of the Lawesson’s reagent, N-ben-
zoylthiobenzamides,9 the garlic constituents diallyl disulfide and
trisulfide (DADS and DATS),10 modified forms of clinically used
agents that carry a H2S-releasing moiety (such as sildenafil, dic-
⇑
Corresponding authors.
E-mail addresses: apapapet@upatras.gr (A. Papapetropoulos), giannis@uni-
lofenac, etc),7 as well as
L
-cysteine analogues.11 Obvious disadvan-
tages exist for most of the above. For example, the biological
leipzig.de (A. Giannis).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.028

2676
Z. Zhou et al. / Bioorg. Med. Chem. 20 (2012) 2675–2678
O
properties/potential toxicity of the backbone of GYY4137 that re-
mains after H2S release are unknown, while agents like S-diclofe-
nac have biological activities unrelated to H2S release that
complicates the interpretation of experimental data. Moreover,
N-benzoylthiobenzamides that release H2S in the presence of cys-
teine, will not only do so when added to cells, but in addition they
will also acylate biological molecules carrying nucleophilic groups.
The aim of the present study was to design, synthesize and
pharmacologically characterize new safe, water-soluble, slow-
releasing H2S donors. We thought, thioaminoacids could satisfy
the above mentioned criteria and could, thus, be ideal H2S donors.
In fact such amino acid derivatives have been reported in the liter-
ature and used as starting materials for the synthesis of peptides.12
Interestingly, thioaminoacids are stable under acidic and basic con-
ditions. However, in the presence of bicarbonate they are sponta-
neously transformed at mild alkaline pH values to the
O
O
a, b
R
R
R
c
SH
OH
SH
NH2
1a,b
NHBoc
NHBoc
5a,b
4a,b
Figure 2. Synthesis of thioglycine 1a and thiovaline 1b. Reaction conditions: (a)
1,10-carbonyldiimidazole, CH2Cl2; (b) H2S-gas; (c) TFA, CH2Cl2 (1:1), 52% over two
steps; Boc, tert-butoxy-carbonyl; H2S, hydrogen sulfide; TFA, trifluoroacetic acid; a
series R = H, b series R = iso-propyl.
corresponding
a-amino acid N-carboxyanhydrides by simulta-
neous liberation of H2S. This property is described as the bicarbon-
ate effect (Fig. 1).12
Taking into consideration that the bicarbonate concentration in
blood at physiological pH (7.4) is about 27 mmol/l13 we hypothe-
sized that thioaminoacids could be optimal H2S donors. In order
to prove our hypothesis we synthesized thioglycine as outlined
in Figure 2. A similar strategy was used to synthesize thiovaline.
Starting from commercially available Boc-protected glycine 4a
(Fig. 2) the amino acid was activated as imidazolide and subse-
quently, without prior isolation, H2S gas was bubbled through
the solution according to known protocols.14,15 Treatment of the
obtained derivatives 5a,b with trifluoroacetic acid (TFA) afforded
the known free thioglycine 1a. In an analogous way we synthesized
thiovaline 1b. Both thioaminoacids showed identical properties as
described by Wieland.16
Thioglycine gave a positive result in the sodium nitroprusside
assay17 indicating its ability to liberate H2S. This was confirmed
additionally using a specific H2S electrode (Fig. 3).
In addition, 1H NMR-spectroscopy studies (see Supplementary
data Fig. S1) were carried out, measuring the time- and tempera-
ture-dependent decomposition of thioglycine in the presence of
NaHCO3 to give its N-carboxyanhydride 318 and HSꢀ. These exper-
iments revealed stability of thioglycine in a 40 mM bicarbonate
solution at 25 °C. However, raising the temperature at 40 °C
showed a slow decomposition of thioglycine until an equilibrium
was reached after 72 h. At this time the mixture consisted of 65%
of thioglycine and 35% of N-carboxyanhydride 3. It should be kept
in mind that under these conditions (alkaline pH, closed system)
complete transformation of thioglycine to N-carboxyanhydride 3
and HSꢀ is not possible because HSꢀ remains in the reaction
mixture.
To compare the release of H2S from thioglycine and thiovaline
to the release of H2S from known donors, we used the methylene
blue method,19 a standard method for H2S determination. In this
assay, both thioaminoacids seemed to release only minor amounts
of H2S (Fig. S2). The inability to detect larger amounts of H2S com-
ing from thioaminoacids relates to the fact that they are stable in
the acid conditions utilized during of the methylene blue assay.
Therefore we developed a new assay based on the fluoresence
emitted by thiobimane in order to detect H2S release from donor
Figure 3. Thioglycine releases H2S. H2S released by thioglycine was detected using
the H2S specific amperometric sensor. (A) Concentration-dependent release of H2S
by thioglycine. (B) Time dependency of H2S release by thioglycine (100
Supplementary data for details).
lM). (see
compounds that could also be used in the future to detect H2S in
biological samples. We hypothesized that dibromobimane 6 could
be used as a specific reagent to detect H2S as only 6 after reaction
with H2S/HSꢀ would yield fluorescent20 thiobimane 7 (Fig. 4).
Dibromobimane 6 does not show significant emission under the
same conditions (excitation at 360 nm and emission at 465 nm,
see also Supplementary data Fig. S3–S5). Although free thiols and
aminoacids like cysteine are known to react21 with dibromobi-
mane the corresponding products are only weakly fluorescent un-
der the detection conditions (Fig. S6A). These physicochemical
properties make dibromobimane an ideal reagent for specific sen-
sitive detection of H2S. In fact we developed a sensitive fluores-
cence assay, which detects H2S (HSꢀ, S2ꢀ) at concentrations as
O
O
O
O
N
N
N
N
HS-
-
O
O
O
HCO3
H2N
HN
HN
O
+
HS
SH
SH
O
Br
Br
S
7
O
OH
2
1
3
6
Figure 1. Proposed mechanism for the formation of glycine N-carboxy- anhydride 3
Figure 4. Transformation of dibromobimane 6 to thiobimane 7 in the presence of
and release of H2S (HSꢀ) in the presence of bicarbonate.
H2S/HSꢀ.

Z. Zhou et al. / Bioorg. Med. Chem. 20 (2012) 2675–2678
2677
low as 5
l
M (Fig. S6 B). When this fluorescent method was used, it
Increased levels of cGMP in smooth muscle are known to trigger
relaxation through multiple pathways that lower intracellular cal-
cium, cause myosin light chain dephosphorylation and activate
potassium channels.24 To further characterize the biological activ-
became evident that thioglycine and thiovaline released ample
amounts of H2S (Fig. 6S C)22 Using the thiobimane method we ob-
served that thioglycine released more H2S than L-thiovaline; re-
lease of the gasotransmitter from the thioaminoacids was
gradual, reaching a plateau after 60 min, in contrast to the rapid
rate of H2S generation observed with NaHS and Na2S (Fig. S7).
ities of thioglycine and L-thiovaline and to test their potential to be
used as smooth muscle relaxing agents, we evaluated the ability of
the thioaminoacids to cause dilatation. Mouse aortic rings were
precontracted with phenylephrine and then exposed to various
Thioglycine and L-thiovaline liberated more H2S than GYY4137.
Interestingly, thioamino acid addition in Hanks Buffered Salt Solu-
tion (HBSS) which contains calcium- and magnesium-ions pro-
moted the formation of glycine N-carboxyanhydride and showed
faster decomposition at 37 °C (data not shown).
H2S donors (Fig. 6A). In these experiments, we observed that
ovaline was more potent, but elicited the same maximal response
with thioglycine. Both -thiovaline and thioglycine were more effi-
cacious and potent than GYY4137. The observation that -thiova-
L-thi-
L
L
In order to pharmacologically characterize the thioaminoacids,
we initially determined their ability to increase intracellular cGMP
levels. We have recently shown that NaHS increases this cyclic
nucleotide in smooth muscle cells by inhibiting the activity of
phosphodiesterase (PDE), the enzymes responsible for breakdown
of this cyclic nucleotide.23 Exposure of cultured rat aortic cultured
smooth muscle to thioglycine or thiovaline lead to a concentration-
dependent increase in cGMP levels (Fig. 5).
Glycine and valine failed to increase the levels of cGMP (Fig. S8).
It is noteworthy that cGMP in response to NaHS reached a plateau
at 50 lM yielding a twofold increase, while exposure to thioglycine
was much more robust (approximately 10-fold increase) (Fig. 2)
and increased further up to 3 mM (data not shown). Similarly,
line is more potent than thioglycine in eliciting vasorelaxation,
although might be perceived to be in apparent contradiction to
the lower release of H2S by thiovaline in buffered solutions can
be explained by a more extensive uptake of this thioaminoacid into
cells compared to thioglycine or by faster liberation of H2S from
thiovaline promoted by biomolecules present in tissues.
To evaluate the ability of thioglycine to promote H2S release
in vivo, this aminoacid was administered to rats and plasma sam-
ples were removed at the indicated times points (Fig. 6B). Hydro-
gen sulfide levels increased after intraperitoneal (ip) injection of
5 mg/kg in a time-dependent manner. H2S plasma levels first be-
came significantly increased after 30 min and remained elevated
for 8 h.
exposure to
compared to NaHS.
L
-thiovaline increased cGMP levels to a greater extent
In conclusion, we have synthesized two new H2S donors that
use endogenous molecules (proteinogenic amino acids) as carrier
scaffolds of the active moiety (H2S) and are, thus, non-toxic. In con-
trast to the commonly used prototype H2S donor NaHS, thioamino-
acids liberate H2S at
a slow rate mimicking the sustained
endogenous H2S production. Thioglycine and thiovaline were pro-
ven to enhance cGMP formation and promote vasorelaxation. Gi-
ven that H2S is known to lower arterial hypertension, reduce
oxidative stress and exhibit cardioprotective effects in preclinical
Figure 6. Biological activity of thioglycine (A) Aortic rings were denuded of
endothelium and contracted with phenylephrine PE (1 lM); once the plateau was
Figure 5. Thioamino acids increase cGMP levels in smooth muscle cells. Rat aortic
smooth muscle cells were exposed to the indicated concentration of NaHS (A),
reached, cumulative-concentration response curves to GYY4137, NaHS, thioglycine
or thiovaline were performed. n = 6, (B): Blood samples were obtained 15 min,
30 min, 1 h, 2 h, 4 h, 6 h, or 12 h after intraperitoneal injection of thioglycine (5 mg/
kg) or saline.
thioglycine (B) or
L-thiovaline (C) for 5 min; cellular cGMP was then extracted and
measured by EIA (see supp. info for details); n = 4;⁄P <0.05 form control.

2678
Z. Zhou et al. / Bioorg. Med. Chem. 20 (2012) 2675–2678
5. Kimura, H. Antioxid. Redox Signal. 2010, 12, 1111.
models, H2S donors hold promise as novel treatments for cardio-
vascular diseases.
6. a Papapetropoulos, A.; Pyriochou, A.; Altaany, Z.; Yang, G.; Marazioti, A.; Zhou,
Z.; Jeschke, M.; Branski, L.; Herndon, D.; Wang, R.; Szabó, C. Proc. Natl. Acad. Sci.
USA 2009, 106, 21972; b Whiteman, M.; Moore, P. J. Cell. Mol. Med. 2008, 13,
488; c Lavu, M.; Bhushan, S.; Lefer, D. Clin. Sci. 2011, 120, 219.
7. Caliendo, G.; Cirino, G.; Santagada, V.; Wallace, J. J. Med. Chem. 2010, 53, 6275.
8. Li, L.; Whiteman, M.; Guan, Y.; Neo, K.; Cheng, Y.; Lee, S.; Zhao, Y.; Baskar, R.;
Tan, C.; Moore, P. Circulation 2008, 117, 2351.
Acknowledgments
In memoriam of Theo Angelopoulos (1936–2012).
9. Zhao, Y.; Wang, H.; Xian, M. J. Am. Chem. Soc. 2011, 133, 15.
10. Benavides, G.; Squadrito, G.; Mills, R.; Patel, H.; Isbell, T.; Patel, R.; Darley-
Usmar, V.; Doeller, J.; Kraus, D. Proc. Natl. Acad. Sci. USA 2007, 104, 17977.
11. Wang, Q.; Wang, X.; Liu, H.; Rose, P.; Zhu, Y. Antioxid. Redox Signal. 2010, 12,
1155.
12. Wieland, T.; Lambert, R.; Lang, H. U.; Schramm, G. Liebigs Ann. Chem. 1955, 597,
181.
13. Tietz Fundamentals of Clinical Chemistry; Burtis, C. A., Ashwood, E. R., Bruns, D.
E., Eds., 6th ed.; Saunders: St. Louis, USA, 2007.
14. Kolb, J.; Beck, B.; Almstetter, M.; Heck, S.; Herdtweck, E.; Dömling, A. Mol. Diver.
2003, 6, 297.
15. Le, H.-T.; Gallard, J.-F.; Mayer, M.; Guittet, E.; Michelot, R. Bioorg. Med. Chem.
1996, 4, 2201.
This work has been co-financed by the European Union (Euro-
pean Social Fund –ESF) and Greek national funds through the oper-
ational Program ‘Education and Lifelong Learning’ of the National
Strategic Reference Framework (NSRF) — Research Funding Pro-
gram: Thalis, Investing in knowledge society through the European
Social Fund to AP, by a grant from the Shriners’ Burns Hospitals
(#8661) to CS and by the COST Action BM1005 (ENOG: European
network on gasotransmitters). We thank also the University of
Leipzig for financial support.
16. Wieland, T.; Sieber, D.; Bartmann, W. Chem. Ber. 1954, 8, 1093.
17. Rock, P. A.; Swinehart, J. H. Inorg. Chem. 1966, 5, 1078.
18. Lamy, C.; Lemoine, J.; Bouchu, D.; Goekjian, P.; Strazewski, P. ChemBioChem
2008, 9, 710.
19. Stipanuk, M.; Beck, P. Biochem. J. 1982, 206, 267.
20. Kosower, E. M.; Pazhenchevsky, B.; Dodiuk, H.; Ben-Shoshan, M.; Kanety, H. J.
Org. Chem. 1981, 46, 1673.
Supplementary data
Supplementary data (experimental procedures, spectral data for
all unknown compounds) associated with this article can be found,
in the online version, at doi:10.1016/j.bmc.2012.02.028.
21. Krishnan, B.; Szymanska, A.; Gierasch, L. M. Chem. Biol. Drug Des. 2007, 69, 31.
22. Recently three methods for hydrogen sulphide detection were published: (a)
Peng, H.; Cheng, Y.; Dai, C.; King, A. L.; Predmore, B. L.; Lefer, D. J.; Wang, B.
Angew. Chem., Int. Ed. 2011, 50, 9672; (b) Liu, C.; Pan, J.; Li, S.; Zhao, Y.; Wu, L.
Y.; Berkman, C. E.; Whorton, A. R.; Xian, M. Angew. Chem., Int. Ed. 2011, 50,
10327; (c) Shen, X.; Pattillo, C. B.; Pardue, S.; Bir, S. C.; Wang, R.; Kevil, C. G. Free
Radic. Biol. Med. 1021, 2011, 50.
23. Bucci, M.; Papapetropoulos, A.; Vellecco, V.; Zhou, Z.; Pyriochou, A.; Roussos,
C.; Roviezzo, F.; Brancaleone, V.; Cirino, G. Arterioscler. Thromb. Vasc. Biol. 1998,
2010, 30.
References and notes
1. Reiffenstein, R.; Hulbert, W.; Roth, S. Annu. Rev. Pharmacol. Toxicol. 1992, 32,
109.
2. a Li, L.; Moore, P. Trends Pharmacol. Sci. 2008, 29, 84; b Szabó, C. Nat. Rev. Drug
Discov. 2007, 6, 917; c Dodge, M. E.; Lum, L. Annu. Rev. Pharmacol. Toxicol. 2011,
51, 289.
3. Szabo, C. Sci. Transl. Med. 2010, 2, 59.
4. a Elsey, D.; Fowkes, R.; Baxter, G. Cell. Biochem. Funct. 2010, 28, 95; b Wang, R.
Kidney Int. 2009, 76, 700.
24. Hofman, F. J. Biol. Chem. 2005, 280, 1.