Tetrasulfanes as selective modulators of the cellular thiolstat

Article abstract of DOI:10.1080/10426507.2012.746691

Naturally occurring polysulfanes often exhibit a wide spectrum of biological activity, which results from their ability to react with, and hence modify cysteine residues in key proteins and enzymes of the cellular thiolstat. Such interactions frequently proceed via S-thiolation of cysteine residues and subsequent formation of Reactive Oxygen Species. Polysulfanes are highly effective, yet also surprisingly selective for cysteine residues and enable the control of numerous cellular processes ranging from an activation of antioxidant defenses to the induction of programed cell death. Unfortunately, the arsenal of natural tri-and tetrasulfanes is limited. Here, we showcase the synthesis of asymmetric tetrasulfanes, which exhibit an interesting nematocidal activity and represent a new generation of tailor-made polysulfanes with potential applications in the field of agriculture and medicine. Supplemental materials are available for this article. Go to the publisher's online edition of Phosphorus Sulfur and Silicon and the Related Elements.

Full text of DOI:10.1080/10426507.2012.746691

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Tetrasulfanes as Selective Modulators of
the Cellular Thiolstat
Brigitte Czepukojc a , Uma M. Viswanathan a , Ahsan Raza a , Sher Ali
a , Torsten Burkholz a & Claus Jacob a
a Division of Bioorganic Chemistry, School of Pharmacy , Saarland
University , D-66123 , Saarbruecken , Germany
Accepted author version posted online: 02 Jan 2013.Published
online: 29 May 2013.
To cite this article: Brigitte Czepukojc , Uma M. Viswanathan , Ahsan Raza , Sher Ali ,
Torsten Burkholz & Claus Jacob (2013) Tetrasulfanes as Selective Modulators of the Cellular
Thiolstat, Phosphorus, Sulfur, and Silicon and the Related Elements, 188:4, 446-453, DOI:
10.1080/10426507.2012.746691
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Phosphorus, Sulfur, and Silicon, 188:446–453, 2013
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2012.746691
TETRASULFANES AS SELECTIVE MODULATORS
OF THE CELLULAR THIOLSTAT
Brigitte Czepukojc∗, Uma M. Viswanathan∗, Ahsan Raza,
Sher Ali, Torsten Burkholz, and Claus Jacob
Division of Bioorganic Chemistry, School of Pharmacy, Saarland University,
D-66123 Saarbruecken, Germany
GRAPHICAL ABSTRACT
Abstract Naturally occurring polysulfanes often exhibit a wide spectrum of biological activity,
which results from their ability to react with, and hence modify cysteine residues in key proteins
and enzymes of the cellular thiolstat. Such interactions frequently proceed via S-thiolation
of cysteine residues and subsequent formation of Reactive Oxygen Species. Polysulfanes are
highly effective, yet also surprisingly selective for cysteine residues and enable the control of
numerous cellular processes ranging from an activation of antioxidant defenses to the induction
of programed cell death. Unfortunately, the arsenal of natural tri- and tetrasulfanes is limited.
Here, we showcase the synthesis of asymmetric tetrasulfanes, which exhibit an interesting
nematocidal activity and represent a new generation of tailor-made polysulfanes with potential
applications in the field of agriculture and medicine.
Supplemental materials are available for this article. Go to the publisher’s online edition of
Phosphorus Sulfur and Silicon and the Related Elements.
Keywords Cysteine; cellular thiolstat; nematodes; polysulfane; S-thiolation
Received 5 October 2012; accepted 31 October 2012.
The authors are grateful for financial support from Saarland University, the Government of Saarland (LFFP
T/1-14.2.1.1-LFFP 12/23), the Interreg IVa Programme (Corena-Network; 35GR11051), and the European Com-
munity’s Seventh Framework Programme (FP7/2007-2013) under grant agreement 215009 RedCat. Ahsan Raza
and Sher Ali were in part funded by the DAAD programme for German–Pakistani Research Cooperation (ID-
54374219). The authors would further like to thank Dr. Joseph Zapp for the NMR measurements and Thomas
Hoffmann for the HRMS measurements.
∗Both authors contributed equally.
Address correspondence to Prof. Dr. Claus Jacob or Dr. Torsten Burkholz, Division of Bioorganic Chemistry,
School of Pharmacy, Saarland University, Campus B 2.1, PO Box 151150, D-66041 Saarbruecken, Germany.
E-mail: c.jacob@mx.uni-saarland.de; t.burkholz@mx.uni-saarland.de
446

THIOLSTAT-MODULATING TETRASULFANES
447
INTRODUCTION
Organic sulfur compounds (OSCs) form a class of natural compounds associated
with an impressive chemical diversity, reactivity and—above all—wide range of biological
activities, which include antioxidant, chemopreventive, antimicrobial, anti-inflammatory,
and even anticancer activity.1–11 At the same time, some of these OSCs occur in edible
plants, such as garlic, onions, Shiitake mushrooms, asparagus, and mustard, and therefore
have formed part of the human diet for centuries without any major adverse effects on
human health.12 Not surprisingly, certain OSCs, such as diallyltrisulfane (DATS) and dial-
lyltetrasulfane (DATTS) from garlic, have recently attracted considerable interest as these
compounds seem to possess a high, yet selective activity against bacteria, fungi, and certain
cancer cells.7,10,11,13,14–17,18–21
This surprising combination of high activity and selectivity found in such chemically
rather simple molecules can be rationalized using the model of the cellular thiolstat.8,9,19,22,23
The latter represents a major intracellular regulatory network composed of redox sensitive
cysteine proteins and enzymes, whose respective function and activity is in part controlled
by the oxidation state of key cysteine residues. Oxidation of such proteins, which include
β-tubulin, by compounds such as DATS or DATTS, usually occurs in form of S-thiolation,
and may trigger major cellular response pathways, which can ultimately also lead to cell
death via apoptosis.24,25 During the last couple of years, a range of polysulfanes have
therefore been synthesized and tested for biological activity, among them various tri- and
tetrasulfanes, which usually are more reactive than the corresponding disulfanes, yet also
chemically more stable than the higher (penta-, hexa- and hepta-) polysulfanes.1,26 At the
same time, the biological activity of nanoscopic sulfur (S8)—which is easy to obtain and
to handle and also does not smell—has been studied rather extensively, underlining the
special (re-)activity of substances containing longer sulfur–sulfur chains.10,11
Here, we report our latest attempts to expand the scope of tetrasulfanes for biological
applications by synthesizing asymmetric polysulfanes for possible agricultural and medical
uses. These studies represent only the first step toward a wider range of new compounds
and their biological activities, and hence are of a preliminary nature.
RESULTS AND DISCUSSION
As natural polysulfanes such as DATS and DATTS are associated with undesired
properties (e.g., low solubility in aqueous media and an intense, arguably unpleasant smell),
it is necessary to vary the side chains in order to “fine tune” the physical, chemical, and
pharmacological properties of such compounds before they can be applied in practice. In
the case of the tetrasulfanes, the synthetic method reported by Derbesy and Harpp in 1994
enables the rapid and effective synthesis based on the reaction of sulfur monochloride
(S2Cl2) with a given thiol.27 Whilst this method is fairly simple to employ, it traditionally
results in symmetric tetrasulfanes, which are not always useful from a more biological
perspective. We have therefore decided to expand the original method by Derbesy and
Harpp to synthesize asymmetric tetrasulfanes (AS4) in a one-pot synthesis employing
S2Cl2 and sequential addition of equimolar amounts of two different thiols (Figure 1).27
Our results obtained for compounds AS4-1 and AS4-2 demonstrate that this synthetic
approach is fruitful as it results in the desired, i.e., asymmetric products, which can then be
separated from the corresponding symmetric tetrasulfanes due to differences in the polarity
of the three products formed.

448
B. CZEPUKOJC ET AL.
Figure 1 General scheme for the synthesis of asymmetric tetrasulfanes based on the method initially reported by
Derbesy and Harpp in 1994. The insert shows the chemical structures of the lead compounds DATS and DATTS
and the propyl analogues DPTS and DPTTS, which are found in garlic and onions, respectively.
To date, several biologically interesting AS4 have already been synthesized employing
this method, of which AS4-1 (yield 19%) and AS4-2 (yield 75%) are of particular interest
and will be presented as part of this report: These compounds combine an acceptable
lipophilicity with good solubility and stability in aqueous media, have virtually no smell,
can be handled easily, and hence may be of interest for practical applications.
As redox activity is deemed to be key to biological activity, the redox behavior of
AS4-1 and AS4-2 was studied using Cyclic Voltammetry in conjunction with a dropping
mercury working electrode. As expected, AS4-1 and AS4-2 are both redox active. Their
cathodic reduction potentials Epc were determined as −787 mV and −779 mV versus
the Ag/AgCl reference electrode, respectively, which is comparable to the Epc of DATTS
(−680 mV). Anodic oxidation potentials Epa were −560 mV for AS4-1, −554 mV for
AS4-2, and −603 mV for DATTS, respectively. The midpoint E1/2 potentials (often casually
referred to as “redox potentials”) were −674 mV, −667 mV, and −642 mV for AS4-1,
AS4-2, and DATTS, respectively. Whilst such electrochemical studies using the dropping
mercury electrode are not devoid of complications, they confirm that compounds such as
AS4-1 and AS4-2 are redox active and possess redox properties comparable to the ones of
DATTS.
We have then investigated the biological activity of AS4-1 and AS4-2 against the
nematode Steinernema feltiae, which serves as a readily available, robust, and reproducible
model of an agricultural pest. As Figure 2 indicates, both AS4-1 and AS4-2 possess a
reasonable activity against S. feltiae. After 24 h, the viability of the nematode is decreased
dramatically in the presence of both polysulfanes when compared to the DMSO control,
with LD50 values for AS4-1 and AS4-2 in the range of 40 μM and 200 μM, respectively. The
toxic effects observed for both compounds are statistically highly significant (compared
to the DMSO control), and compare well with the one of DATTS (LD50 value around
150 μM).

THIOLSTAT-MODULATING TETRASULFANES
449
Figure 2 Activity of AS4-1 and AS4-2 against the nematode S. feltiae at 30 min (ꢀ) and after 24 h (ꢁ). Whilst
most nematodes are still alive after 24 h in the DMSO control, there is a statistically highly significant reduction
of the number of viable nematodes in the presence of AS4-1 (Panel A) and AS4-2 (Panel B), especially at
concentrations above 50 μM. The difference between measurements were examined by using Student’s t-test
versus DMSO control, a value p < 0.05 was considered statistically significant. Symbols: n.s. = not significant;
∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Data are depicted as mean SD (n = 9).

450
B. CZEPUKOJC ET AL.
Figure 3 Selected aspects of the biochemical mode(s) of action of polysulfanes based on the established bio-
chemical behavior of DATTS and related tetrasulfanes. In this model, the polysulfane reacts with key cellular
proteins, modifying their (essential, regulatory) cysteine residues and hence changing their function or activity.
Such modifications subsequently initiate cellular signaling and control cascades, which may result in a range of
cellular responses. As a polysulfane-specific caveat, S-thiolation of proteins may not only occur by the direct
reaction of the polysulfane with a cysteine thiol but may also proceed via initial formation of a mixed disulfide
(RSSG) or GSSG, both of which can also S-thiolate proteins. Cysteine oxidation and S-thiolation may be enhanced
further by the formation of superoxide radical anions (O2•−) from O2.
Our results show that it is possible to produce asymmetric tetrasulfanes with desired
properties using a simple method based on S2Cl2 and mixtures of specifically selected
thiols. Whilst this method necessarily also results in the formation of symmetric products
(which may also be useful), the latter can be separated from the asymmetric compounds
with comparable ease. In fact, it is possible to enhance the formation of the asymmetric
product by adding the two thiol-containing educts sequentially, as the intermediate RS3Cl,
which is formed initially from RSH and S2Cl2, can be reacted with R’SH to the final product
RS4R’. This approach has been used quite successfully in the case of AS4-2 (75% yield),
and yields for the AS4 compounds are generally quite acceptable, i.e., in the range of yields
also encountered for most polysulfanes.
Whilst AS4-1 and AS4-2 are the first asymmetric tetrasulfanes we have explored, a
wide range of asymmetric as well as symmetric tetrasulfanes can now be designed and
synthesized, including multifunctional tetrasulfanes and amphiphilic structures. Asymmet-
ric trisulfanes may also be considered, as the method of synthesis published in 1961 by
Milligan et al. involves a two-step procedure, which provides scope for the employment of
two different side-chain bearing educts.28
Indeed, from the perspective of pharmaceutical chemistry, asymmetric polysulfanes
have several advantages: First, it is easier to fine-tune lipophilicity of the tetrasulfanes as

THIOLSTAT-MODULATING TETRASULFANES
451
the two side chains differ and individually can include specific lipophilic and hydrophilic
groups. Whilst lipohilicity can also be controlled in symmetric tetrasulfanes, the fact that
each function is added necessarily twice implies that control is more problematic—and
as both chains are identical, such symmetric tetrasulfanes are often either too lipophilic
or too hydrophilic. Symmetric derivatives of DATTS (calculated clogP = 2.68), such as
1,4-dibenzyltetrasulfane (DBTTS) and 3,3ꢀ-dipropanoic acid tetrasulfane (DPSTTS), for
instance, possess clogP values of 4.64 and 0.71, respectively, which are both suboptimal
from the perspective of Lipinski’s rules. The asymmetric analogue AS4-2, in contrast, pos-
sesses more amenable clogP value of 2.76. Similarly, asymmetric polysulfanes enable the
incorporation of different functional groups into each side chain. The modified method of
Derbesy and Harpp also compares favorably to alternative synthetic methods for tetrasul-
fanes, such as the reaction of (symmetric or asymmetric) disulfanes with elemental sulfur.
Whilst such “sulfur enrichment” of disulfanes also results in the corresponding tri- and
tetrasulfanes, it is complicated by the appearance of a wide range of other (poly-)sulfane
contaminants, which are difficult to separate.26 And finally, S2Cl2 and thiols are readily
available and larger scale syntheses of asymmetric tetrasulfanes for practical applications
using this method can be considered.
CONCLUSIONS
Whilst considerably more tests in diverse biological systems—and with a much wider
range of compounds—are required, initial results indicate that the DATTS derivatives such
as AS4-1 and AS4-2 retain redox activity and also aspects of the biological activity of
DATTS (Fig. 3), yet lack some of the “undesired” properties associated with this natural
product, such as its distinct smell or oily consistency. AS4-2, for instance, is a non-smelling,
yellow solid which, like DATTS, is active against S. feltiae, yet is considerably easier to
handle. Ultimately, such compounds may combine sufficient chemical stability, solubility,
and bioavailability with otherwise amenable physicochemical properties, and hence may
also be superior to DATTS when it comes to agricultural or medical applications. Future
studies will need to consider the spectrum of biological activity associated with such tailor-
made polysulfanes in more detail, and also investigate their mode(s) of biological action.
EXPERIMENTAL
Synthesis of Polysulfanes (AS4-1 and AS4-2): General Procedure
To a solution of S2Cl2 (2.70 g, 20 mmol) in diethyl ether (50 mL), an equimolar
amount of the respective mercaptane (20 mmol) and pyridine (1.58 g, 20 mmol) in diethyl
ether (25 mL) was added dropwise for 1 h at −78 ◦C. The solution was kept at −78 ◦C for
45 min. Then, another equimolar solution of 3-mercaptopropionic acid (2.12 g, 20 mmol)
and pyridine (1.58 g, 20 mmol) in diethyl ether (25 mL) was added dropwise for 1 h at
◦
◦
−78 C. The reaction mixture was stirred for another 1 h at −78 C and then allowed to
warm up to room temperature. Subsequently, the organic layers were washed with distilled
water (three times) and dried over MgSO4. After evaporating the solvent, the product was
purified by column chromatography. AS4-1 was purified by column chromatography using
CH2Cl2/methanol 9:1 v/v as eluent (Rf = 0.36). In case of AS4-2, the compound was
purified by column chromatography using CH2Cl2 as eluent (Rf = 0.38).

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B. CZEPUKOJC ET AL.
Nematode Assay
Steinernema feltiae was purchased from Sautter & Stepper GmbH (Ammerbruch,
Germany) in the form of a soft cake and was used as a model organism for agricultural
◦
nematodes. Nematode suspensions were prepared in 250 mL of distilled water at 27 C,
compounds were dissolved in DMSO, and added to the nematode suspension, which was
incubated at 27 ◦C. Viability readings were taken after 30 min and 24 h using a microscope
(TR 200, VWR International, Belgium) at four-fold magnification. Each experiment was
conducted in triplicate and on three separate occasions. In essence, the examination of each
concentration of each compound was therefore carried out nine times in total.
ABBREVIATIONS
AS4-1
AS4-2
DATS
3-(propyl tetrasulfanyl)-propanoic acid;
3-(phenyl tetrasulfanyl)-propanoic acid;
diallyl trisulfane;
DATTS
DBTTS
diallyl tetrasulfane;
1,4-dibenzyltetrasulfane;
DPSTTS 3,3ꢀ-dipropanoic acid tetrasulfane;
DMSO
GSSG
OSC
dimethyl sulfoxide;
glutathione disulfide;
Organic Sulfur Compound;
S. feltiae Steinernema feltiae
For the origin of the materials, the analytical methods used—including details of the
nematode assay employed—and the analytical data for each compound synthesized (please
consult the Supplementary Materials, available online).
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