Applications of Synthetic Organic Tetrasulfides as H2S Donors

Article abstract of DOI:10.1021/acs.orglett.7b00858

In an effort to expand the availability of simple polysulfides for H₂S donation, we report here the synthesis and H₂S release profiles of bis(aryl) and bis(alkyl) tetrasulfides. The tetrasulfide donors release H₂S in a first-order dependence on reduced glutathione (GSH) and release more H₂S than the commonly used trisulfide DATS.

Full text of DOI:10.1021/acs.orglett.7b00858

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Letter
pubs.acs.org/OrgLett
Applications of Synthetic Organic Tetrasulfides as H2S Donors
Matthew M. Cerda,† Matthew D. Hammers,† Mary S. Earp, Lev N. Zakharov, and Michael D. Pluth*
Department of Chemistry and Biochemistry, Materials Science Institute, Institute of Molecular Biology, University of Oregon,
Eugene, Oregon 97403, United States
S
* Supporting Information
ABSTRACT: In an effort to expand the availability of simple
polysulfides for H2S donation, we report here the synthesis and
H2S release profiles of bis(aryl) and bis(alkyl) tetrasulfides. The
tetrasulfide donors release H2S in a first-order dependence on
reduced glutathione (GSH) and release more H2S than the
commonly used trisulfide DATS.
itric oxide (NO), carbon monoxide (CO), and hydrogen
sulfide (H2S) are small gaseous molecules, often called
N
gasotransmitters, which are produced endogenously, freely
permeate cell membranes, and act on molecular or cellular
targets to exert physiological effects.1 Over the past decade, the
accepted roles of H2S in biology have expanded and now
include important roles in neurotransmission,2 vasodilation,3
apoptosis,4 and other physiological processes.5 Aligned with
these physiological roles, new chemical tools that enable H2S
delivery are needed for both research and potential therapeutic
applications.6,7 Because direct administration of H2S gas is not
practical in most biological contexts, many researchers employ
inorganic sulfide salts, such as sodium hydrosulfide (NaSH) or
sodium sulfide (Na2S), as a primary sulfide source. By
comparison to enzymatic H2S production profiles, these sulfide
salts release H2S instantaneously upon dissolution, often
resulting in unwanted volatilization, oxidation, and/or reduced
effective residence times.8 To address this issue, small molecule
H2S donors that release H2S at controlled rates comparable to
enzymatic H2S production have been prepared and inves-
tigated.9 Current common motifs found in H2S donation
include aryl isothiocyanates,10 phosphinodithioates,11 thio-
acids12/amides,13 dithiolethiones,14 thiolysis of trisulfides,15
and protected persulfides16 and stimulated release of carbonyl
Figure 1. (a) Mechanism of H2S release from DATS in the presence
of GSH. (b) Representative examples of natural products containing
polysulfides.
sulfide (COS) from thiocarbamates, which is subsequently
converted to H2S by carbonic anhydrase.17 Of these methods,
one of the simplest and most commonly used for H2S donation
is the allium-derived diallyl trisulfide (DATS), which releases
H2S upon reaction with thiols (Figure 1a). The mechanism of
this reaction has been investigated in detail,18 and the sulfide
from DATS has been attributed to the beneficial cardiovascular
effects of garlic.19 Despite this widespread utility, DATS is the
only polysulfide that has been used broadly as an H2S donor
despite other accessible polysulfides, many of which have higher
sulfane sulfur content and therefore higher H2S releasing
capacity.
1b), have also been isolated from natural sources indicative of
their abundance and biological importance. Independent of
H2S releasing capacities, polysulfides are also involved directly
in a variety of physiological processes, including inhibition of
cholesterol synthesis, anti-inflammation, and antioxidant
activity.21 Inspired by the prevalence of naturally occurring
polysulfides and the widespread utility of DATS, we envisioned
accessing synthetic polysulfides for GSH-triggered H2S
donation and expanding the toolbox of polysulfide-based H2S
Polysulfides are a class of naturally occurring molecules and
are in part responsible for the beneficial cardiovascular effects
associated with consumption of alliums such as garlic.15,20 More
exotic polysulfides, including varacin and chaetocin C (Figure
Received: March 22, 2017
© XXXX American Chemical Society
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donors. Herein, we report the synthesis and evaluation of
bis(aryl)- and bis(alkyl) tetrasulfides which release H2S in the
presence of excess GSH.
With the goal of expanding the toolbox of polysulfide-based
H2S donors, we evaluated previous methods for simple
polysulfide synthesis. Both trisulfides and tetrasulfides are
synthetically accessible by treating a desired thiol with sulfur
dichloride (SCl2) or sulfur monochloride (S2Cl2), respec-
tively.22 We chose to focus on tetrasulfides due to their higher
sulfane sulfur content, thus higher H2S releasing potential with
respect to trisulfides. Treatment of the parent thiol with S2Cl2
and pyridine in anhydrous solvent at −78 °C afforded the
desired tetrasulfide in good to excellent yield with no required
purification (Table 1).23 In addition to facile tuning of the
Table 1. Synthesis of Bis(aryl) and Bis(alkyl) Tetrasulfides
Figure 2. (a) Mass spectrum (EI-MS) of 3. (b) ORTEP
representation (50% ellipsoids) of the molecular structure of 4.
(282.0), as well as daughter fragmentation peaks corresponding
to extrusion of one and two sulfane sulfur atoms to generate
Ph2S3 (250.0) and Ph2S2 (218.0), respectively. In addition, the
most intense ion peak corresponds to homolysis of the weak26
internal S−S bond to generate PhSS• (141.0). Additionally,
HPLC analysis of 3 further supports the high purity of the
synthesized tetrasulfide (Figure S20). Taken together, the
spectroscopic and spectrometric data support clean formation
of the desired tetrasulfide product. In addition, single crystals of
4 suitable for X-ray diffraction were obtained by slow
evaporation from n-pentane, further confirming the molecular
structure (Figure 2b). Tetrasulfide 4 crystallized in P21/c with a
central S−S dihedral angle of 82.8° and nearly perpendicular
89.9° angle between the two phenyl rings, which were oriented
in a slip stacked orientation with a 3.50 Å interplanar spacing.
Additional stabilizing forces in the lattice include C−H
hydrogen bonds from the methyl (3.7 Å) and meta aryl (3.9
Å) hydrogens, consistent with recent work demonstrating that
sulfur is a C−H hydrogen bond acceptor.27
With tetrasulfides in hand, we next investigated the release of
H2S upon addition of GSH. We chose to use GSH as the
trigger for H2S release due to higher intracellular levels (∼1−10
mM) relative to other biological thiols, although other
biological reductants, such as NADH, can also trigger H2S
release from polysulfides.28 We hypothesized that H2S release
from tetrasulfides is likely to proceed through an analogous
mechanism to that of trisulfides (Figure 1a), although the
addition of a second sulfane sulfur in tetrasulfides increases the
mechanistic complexity because GSH can attack at either the α-
or β-sulfur (Figure 3). For example, nucleophilic attack by GSH
at the β-sulfur can either lead to a nonproductive pathway by
formation of the parent thiol and a mixed glutathione
tetrasulfide or to a productive pathway via formation of a
persulfide and mixed glutathione trisulfide (Figure 3a). These
reactive intermediates would further react with GSH leading to
the generation of 2 equiv of H2S and terminating in the
formation of a mixed glutathione disulfide. Alternatively,
electron-donating or -withdrawing ability of the bis(aryl)
tetrasulfides (1−6), this route also enabled access to bis(alkyl)
tetrasulfides, including N-acetylcysteine-derived tetrasulfides (7
and 8). These two compounds provide a promising scaffold for
combining the physiological effects of H2S with those of N-
acetylcysteine (NAC), which is used as an antidote for
acetaminophen overdose,24 CO poisoning,25 and other
conditions. Notably, the use of S2Cl2 to access tetrasulfides is
tolerant of varying thiol nucleophilicity and could prove useful
in the synthesis of other organic tetrasulfides.
1
The prepared tetrasulfides were characterized by H and
13C{1H} NMR spectroscopy to assess the purity of each
product and EI-MS to confirm selective tetrasulfide formation.
A representative mass spectrum of 3 is shown in Figure 2a,
which clearly shows the parent ion peak corresponding to Ph2S4
B
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problem, we used initial rate analysis and construction of a log−
log plot of rate as a function of [GSH], which demonstrated a
first-order rate dependence (Figure 4b).
Given the ease of functionalization for the bis(aryl)
tetrasulfides, we also investigated the effects of electronic
substitution on H2S release. We anticipated that electronic
modulation of the bis(aryl) tetrasulfides would favor a specific
pathway due to change in electrophilicity at the α- or β-sulfur.
Alternatively, it is possible that although the balance of the
competing pathways could shift, it would not do so sufficiently
to result in different H2S release profiles. Similar to our
observation for 3, GSH-triggered H2S release from the
remaining bis(aryl) tetrasulfides was observed with initial
rates ranging from 0.0032 to 0.054 nA·s−1 (Table S2). The
Hammett plot constructed from the initial rate data did not
provide a linear relationship, suggesting that factors other than
electronic effects may influence H2S release rates from the
bis(aryl) tetrasulfides (Figure S1). We next compared the rates
of release of alkyl tetrasulfides 7 and 8, both of which released
H2S more slowly than the aryl tetrasulfides (Figure 5). Notably,
Figure 3. Proposed mechanisms of H2S release from tetrasulfides,
including initial nucleophilic attack by GSH at the (a) β-sulfur or (b)
α-sulfur.
nucleophilic attack by GSH at the α-sulfur results in the
formation of a hydropolysulfide and a mixed glutathione
disulfide (Figure 3b). This hydropolysulfide would further react
with another equivalent of GSH to generate H2S and a mixed
glutathione trisulfide. Repetition of this process generates
oxidized GSH (GSSG) and a persulfide, which should rapidly
react with GSH to generate another equivalent of H2S and
terminate in a mixed glutathione disulfide. Overall, a single
tetrasulfide molecule should release 2 equiv of H2S following
reduction by GSH and terminate in a mixed glutathione
disulfide.
To gain preliminary mechanistic insights into H2S release
from the prepared tetrasulfides, we measured the GSH
dependence of H2S release from 5 μM 3 (10 mM PBS, pH
7.4) using a sulfide-selective electrode. As expected, no H2S
release was observed in the absence of GSH, demonstrating
that the tetrasulfides are stable in solution in the absence of
thiols. Increasing the GSH concentration resulted in an
increased rate of H2S production (Figure 4a). The acquired
H2S release data did not fit cleanly to pseudo-first-order
kinetics, suggesting that the complexity of the H2S-releasing
pathway and possibility for competing reactions may
complicate the observed rate profiles. To circumvent this
Figure 5. GSH-triggered (250 equiv) H2S release from 5 μM
tetrasulfides 1−8 and 5 μM DATS (pH 7.4, room temperature).
all of the prepared aryl and alkyl tetrasulfides resulted in greater
H2S donation than the well-established trisulfide donor DATS
under the reported conditions, thus highlighting the efficacy of
tetrasulfides as viable H2S donors. The differences in H2S
releasing profiles of the reported donors are likely due to
varying electronic contributions between the different com-
pounds. Clearly further mechanistic investigations are required
to fully elucidate the mechanism of GSH-mediated release of
H2S from tetrasulfides.
In summary, a diverse array of bis(alkyl) and bis(aryl)
tetrasulfides are readily accessible from simple synthetic
procedures. The prepared tetrasulfides readily release H2S
upon treatment with GSH and release more H2S per molecule
than commonly used DATS. Overall, we expect that these and
other simple polysulfides will provide researchers interested in
H2S donating molecules with a broader palette of available
polysulfide donor motifs with different release profiles and
biological activities.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.7b00858.
Figure 4. (a) GSH-dependent H2S release from 3. (b) log(k) vs
log[GSH] plot demonstrating a 1st-order dependence in [GSH] on
H2S release from 3.
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Experimental procedures, characterization data (PDF)
Letter
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(20) Pluth, M. D.; Bailey, T. S.; Hammers, M. D.; Hartle, M. D.;
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Crystallographic data (CIF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: pluth@uoregon.edu.
ORCID
(23) Zysman-Colman, E.; Harpp, D. N. J. Org. Chem. 2003, 68,
2487−2489.
(24) Smilkstein, M. J.; Knapp, G. L.; Kulig, K. W.; Rumack, B. H. N.
Engl. J. Med. 1988, 319, 1557−1562.
(25) Flanagan, R. J.; Meredith, T. J. Am. J. Med. 1991, 91, S131−
Michael D. Pluth: 0000-0003-3604-653X
Author Contributions
†M.M.C. and M.D.H. contributed equally.
S139.
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Notes
The authors declare no competing financial interest.
(27) Hartle, M. D.; Hansen, R. J.; Tresca, B. W.; Prakel, S. S.;
Zakharov, L. N.; Haley, M. M.; Pluth, M. D.; Johnson, D. W. Angew.
Chem., Int. Ed. 2016, 55, 11480−11484.
ACKNOWLEDGMENTS
■
(28) Peng, B.; Liu, C.; Li, Z.; Day, J. J.; Lu, Y.; Lefer, D. J.; Xian, M.
Bioorg. Med. Chem. Lett. 2017, 27, 542−545.
Research reported in this publication was supported by the
Sloan Foundation (M.D.P.) and the Dreyfus Foundation
(M.D.P.). Basic research on synthetic polysulfide chemistry
was supported by the NSF (CHE-1454747). Applications of
polysulfide donors, and HPLC analysis was supported by the
NIH (R01GM113030). NMR spectroscopy capabilities in the
UO CAMCOR facility are supported by the NSF (CHE-
1427987). We would like to thank Hillary A. Henthorn (UO)
for her assistance in kinetic analysis.
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