Understanding hydrogen sulfide storage: Probing conditions for sulfide release from hydrodisulfides

Article abstract of DOI:10.1021/ja505371z

Hydrogen sulfide (H₂S) is an important biological signaling agent that exerts action on numerous (patho)physiological processes. Once generated, H₂S can be oxidized to generate reductant-labile sulfane sulfur pools, which include hydrodisulfides/persulfides. Despite the importance of hydrodisulfides in H₂S storage and signaling, little is known about the physical properties or chemical reactivity of these compounds. We report here the synthesis, isolation, and characterization (NMR, IR, Raman, HRMS, X-ray) of a small-molecule hydrodisulfide and highlight its reactivity with reductants, nucleophiles, electrophiles, acids, and bases. Our experimental results establish that hydrodisulfides release H₂S upon reduction and that deprotonation results in disproportionation to the parent thiol and S⁰, thus providing a mechanism for transsulfuration in the sulfane sulfur pool.

Full text of DOI:10.1021/ja505371z

Communication
pubs.acs.org/JACS
Open Access on 07/10/2015
Understanding Hydrogen Sulfide Storage: Probing Conditions for
Sulfide Release from Hydrodisulfides
T. Spencer Bailey, Lev N. Zakharov, and Michael D. Pluth*
Department of Chemistry and Biochemistry, Institute of Molecular Biology, Material Science Institute, 1253 University of Oregon,
Eugene, Oregon 97403, United States
S
* Supporting Information
problems, often leading to variable results from different
ABSTRACT: Hydrogen sulfide (H2S) is an important
biological signaling agent that exerts action on numerous
(patho)physiological processes. Once generated, H2S can
be oxidized to generate reductant-labile sulfane sulfur
pools, which include hydrodisulfides/persulfides. Despite
the importance of hydrodisulfides in H2S storage and
signaling, little is known about the physical properties or
chemical reactivity of these compounds. We report here
the synthesis, isolation, and characterization (NMR, IR,
Raman, HRMS, X-ray) of a small-molecule hydrodisulfide
and highlight its reactivity with reductants, nucleophiles,
electrophiles, acids, and bases. Our experimental results
establish that hydrodisulfides release H2S upon reduction
and that deprotonation results in disproportionation to the
parent thiol and S0, thus providing a mechanism for
transsulfuration in the sulfane sulfur pool.
labeling strategies. A better functional understanding of the
chemical reactivity of hydrodisulfides would not only contribute
to the quickly evolving strategies for detection, but would also
clarify the potential roles of hydrodisulfides in H2S chemistry
and signaling. Based on this current need, we viewed that
isolation of a discrete small-molecule hydrodisulfide model
would allow for the reactivity of this important functional group
to be established under controlled conditions. Motivated by
these challenges, we report here the preparation, isolation, and
full characterization of tritylhydrodisulfide (TrtSSH) and
confirm that under hydrophobic conditions hydrodisulfides
release H2S upon reduction, but not upon protonation or by
nucleophilic attack.
To establish the spectroscopic and reactivity properties of
hydrodisulfides, we chose to prepare tritylhydrodisulfide due to
stability of the S-nitrosothiol analogue, TrtSNO, and the
crystallinity of the trityl group. Although there are various
reports of hydrodisulfides in the literature, to the best of our
knowledge, none of these have been characterized completely.9
We viewed that isolation and characterization of the −SSH
functional group would allow for other researchers to better
identify and characterize similar compounds in the future.
Additionally, studies focused on hydrodisulfide reactivity with
different electrophiles have used hydrodisulfides generated in
situ rather than isolated hydrodisulfides.10 Although the trityl
group precludes water solubility, the investigation of hydro-
disulfide reactivity in organic solution may provide insights into
the chemistry of hydrodisulfides in hydrophobic environments
in protein architectures, which are proposed to be involved in
the activation of various enzymes. To prepare TrtSSH, we first
treated TrtSH with AcSCl (3) in Et2O to generate TrtSSAc
(4). Removal of the acetyl protecting group by acidic
ethanolysis affords the desired hydrodisulfide product, TrtSSH
(Scheme 1).
ydrogen sulfide (H2S) is now accepted as an important
Hsignaling molecule that exerts physiological functions in
the cardiovascular, neuronal, and immune systems.1 Produced
endogenously from cystathionine-β-synthase (CBS), cystathio-
nine-γ-lyase (CSE), and cysteine amino transferase (CAT) in
combination with 3-mercaptosulfurtransferase (3MST), H2S
exists primarily as HS− under normal physiological conditions.1
In addition to this freely diffusible form, H2S oxidation can
generate reductant-labile sulfane sulfur pools containing
hydrodisulfides/persulfides (RSSH), hydropolysulfides (RS-
(Sn)SH), and polysulfides (RS(Sn)SR), all of which may be
present in micromolar concentrations.2 In addition to their
possible roles in H2S storage, the S-sulfhydration (or S-
persulfidation) of protein Cys residues is postulated to be an
important oxidative post-translational modification that con-
tributes to H2S signaling and action.3 In addition to reductant-
labile sulfane sulfur, sulfide can also be stored in acid-labile
sulfur pools, such as iron sulfur cluster-containing proteins.4
Although, Kevil et al. recently reported methods to separate the
free, acid-labile, and reductant-labile sulfide pools,5 there
remains controversy surrounding the basic reactivity of sulfane
sulfur, specifically persulfides. For example, although some
reports suggest that reductant-labile sulfane sulfur is also acid-
labile,6 it is more likely that acid-labile and reductant-labile
sulfur form two chemically distinct pools.7
Scheme 1. Synthesis of TrtSSH
Because the chemical reactivity of hydrodisulfides is poorly
established, the chemical underpinnings for detecting3a,8 and
differentiating −SSH from −SH groups have been fraught with
Received: May 28, 2014
Published: July 10, 2014
© 2014 American Chemical Society
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Communication
With TrtSSH in hand, we fully characterized the compound
and compared its chemical properties to those of TrtSH. Both
Although reports have suggested that H2S can be released
from hydrodisulfides upon protonation, reduction, and
nucleophilic attack, we expected that only reduction would
liberate sulfide because the sulfur oxidation state is higher in
hydrodisulfides (either two S1− or one S0 and S2−) than in H2S
(S2−). As expected, treatment of TrtSSH with reductants, such
as PPh3 or [NBu4 ][BH4 ] (Figures S6, S7) resulted in clean
conversion of TrtSSH to TrtSH with concurrent liberation of
sulfide.11 These results confirm that hydrodisulfides are a
source of reductant-labile sulfane sulfur (Table 1, entries 1−2).
1
the H and 13C{1H} NMR spectra of TrtSSH are similar to
1
those of TrtSH, but the H NMR resonance of the sulfhydryl
SSH is shifted upfield to 2.7 ppm, by comparison to 3.1 ppm in
TrtSH (Figures S4, S5). Much of the infrared and Raman
spectra of TrtSSH and TrtSH are also similar due to their
related structures; however, the S−H stretch shifts to a lower
wavenumber by 65 cm−1 for TrtSSH when compared to TrtSH
(Figure 1b). Comparison of the Raman spectrum of TrtSSH
+
−
Table 1. Reactivity of TrtSSH with Different Reactants
Including Reductants, Nucleophiles, Electrophiles, Bases,
a
and Acids
entry
reactant
completion
products
1
PPh3
<2 min
<2 min
n.r.
Ph3PS, TrtSH
TrtSH, BH3, SH−
−
+
−
2
[NBu4 ][BH4
]
3
DTP
4
TrtSH
BnSH
H2S
n.r.
−
−
−
5
n.r.
6
n.r.
7
AcOH
n.r.
−
8
AcOH + TrtSH
[Na+][TrtS−]
n.r.
−
9
<2 min
<2 min
1 h
TrtSH, S8
TrtSH, S8
TrtSH, S8
TrtSH, S8
TrtSH, S8
TrtSH, S8
TrtSH, S8
TrtSH, S8
TrtSH, S8
TrtSH, S8
TrtSH, S8
TrtSH, S8
TrtSS-NEM
[NBu4 ][CN−]
+
10
11
12
13
14
15
16
17
18
19
20
21
[NBu4 ][Cl−]
+
[NBu4 ][Br−]
24 h
+
[NBu4 ][I−]
>48 h
<2 min
30 min
<2 min
<2 min
<2 min
<2 min
>48 h
24 h
+
[NBu4 ][OAc−]
+
[NBu4 ][TFA−]
+
NEt3
10% NEt3
DMEDA
DMAP
Pyridine
b
NEM
a
Experiments were performed in CD2Cl2 at room temperature with 18
mM TrtSSH and reactant. All products were confirmed by 1H or
31P{1H} NMR spectroscopy. BH3 formation was identified by trapping
with THF and characterization by 11B{1H} NMR spectroscopy. S8
formation was confirmed and quantified by titration with PPh3 at the
Figure 1. (a) X-ray crystal structure of TrtSSH. Thermal ellipsoids are
drawn at the 50% probability level. Hydrogen atoms on the phenyl
rings are omitted for clarity. Full crystallographic details are available in
the Supporting Information. (b) Infrared (black) and Raman (blue)
spectra of TrtSH (top) and TrtSSH (bottom). The Raman spectrum
of S8 (red) is shown for comparison.
b
end of the reaction. Reaction was incomplete due to slower reactivity
of protonated TrtSSH, however addition of NEt3 increased the
reaction rate. Abbreviations: DTP: dithiopropane; DMEDA: N,N′-
dimethylethylenediamine; DMAP: dimethylaminopyridine; NEM: N-
ethyl-maleimide.
with that of S8 showed similarities in the 200−500 cm−1 region
corresponding sulfur−sulfur bond formation. HRMS analysis of
TrtSSH confirmed its atomic composition, affording an m/z
value of 307.0631 amu, which agrees well with the calculated
value of 307.0621 amu for TrtSS−. To further confirm the
molecular structure of TrtSSH, single crystals suitable for X-ray
diffraction were grown from toluene/Et2O. TrtSSH crystallizes
in P212121 with an S−S bond length of 2.0396(12) Å and a
CSSH dihedral angle of 82.2°, both of which are within the
ranges common for disulfides (Figure 1a). The terminal −SSH
hydrogen was located from the residual density map, and
inspection of the packing diagram shows that it does not form
hydrogen bonds with other atoms in the solid state (Figure
S28).
When TrtSSH was treated with sulfhydryl-containing species,
such as TrtSH, BnSH, dithiopropane (DTP), or H2S, however,
sulfide release did not occur, suggesting that the thiols were
either insufficiently reducing or that proton transfer is a
mechanistically important step (Table 1, entries 3−6).
Treatment of TrtSSH with acid, either alone or in the presence
of a thiol as a potential electron source, also failed to liberate
sulfide (Table 1, entries 7−8). These results confirm that
hydrodisulfides are a source of reductant-labile but not acid-
labile sulfide.
To further understand the reactivity of thiols toward TrtSSH,
1
we treated TrtSSH with TrtS−. Monitoring the reaction by H
Having confirmed the molecular identity of TrtSSH and
established its spectroscopic properties, we next investigated
the reactivity of TrtSSH to determine under which conditions
sulfide or other sulfur-containing products were released.
NMR spectroscopy revealed clean conversion to TrtSH and the
formation of S0 (vide infra) (Table 1, entry 9). To clarify this
reactivity, we investigated whether the TrtS− was acting as a
nucleophile or as a base by treating TrtSSH with different
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Communication
nucleophiles (CN−, I−, Br−, Cl−, TFA−, and AcO−) and bases
(NEt3, DMEDA, pyridine, and DMAP). Monitoring these
reactions by H NMR spectroscopy (Table 1, entries 10−20)
deprotonation initially generates TrtSS−, which subsequently
disproportionates into TrtS− and S0, thus providing a
mechanism for transsulfuration in the sulfane sulfur pool
without disruption of redox homeostasis.
In summary, we have prepared, isolated, and fully
characterized the stable hydrodisulfide TrtSSH and studied its
reactivity toward reductants, nucleophiles, acids, and bases. The
chemical reactivity data obtained from TrtSSH demonstrate
that hydrodisulfides do not react with nucleophiles or acids in
organic solution, but rather release sulfide upon reduction and
disproportionate to release S0, likely as S8 under physiological
conditions, upon deprotonation (Figure 3). These results
1
revealed that the rate of conversion of TrtSSH to TrtSH and S0
correlated with the basicity (CN− > AcO− > TFA− > Cl− > Br−
> I−) rather than with anion nucleophilicity (Figures S15−
S20). The same trends for reaction rate were maintained with
amine bases: NEt3 ∼ DMEDA > DMAP > pyridine (Figures
S21, S23−S25). Introduction of catalytic NEt3 (entry 17, Figure
S22) resulted in the same reactivity, thus supporting a base-
mediated disproportionation mechanism. We also wanted to
verify the multitude of reports highlighting the nucleophilicity
of hydrodisulfides. To verify the nucleophilicity of TrtSSH, we
treated TrtSSH with N-ethylmaleimide (NEM), a common
thiol labeling agent, which resulted in slow conversion to the
NEM-labeled disulfide (Figure S26). The slow reaction rate is
consistent with proton transfer being important in the observed
reactivity. Adding a small amount of NEt3 to the reaction
drastically increases the reaction rate, mimicking the observed
reactivity of hydrodisulfides with electrophiles in water.
Having determined that deprotonation results in dispropor-
tionation of TrtSSH to TrtSH and S0, we wanted to further
investigate the mechanistic details involved in this reaction.
Monitoring the formation of TrtSH from the reaction between
1
TrtSSH and different nucleophiles by H NMR spectroscopy
resulted in clean conversion to the TrtSH product without the
formation of any NMR-active intermediates (Figure 2a). After
Figure 3. Reactivity of TrtSSH with acids, electrophiles (E+),
reductants ([red]), bases, or nucleophiles (nuc).
clarify the innate reactivity of hydrodisulfides and confirm that
hydrodisulfides are viable sources of reductant-labile sulfur.
Additionally, deprotonation of hydrodisulfides may provide a
mechanism for interconversion of different species within the
reductant-labile sulfur pool, thereby allowing for facile
conversion back to the parent thiol without disruption of
thiol or redox homeostasis. We envision that these studies as
well as future work using isolated hydrodisulfides will be
invaluable in furthering our understanding of biological H2S
signaling and storage by providing clear insight into the
reactivity of hydrodisulfides under different reactive conditions.
ASSOCIATED CONTENT
* Supporting Information
Experimental details, NMR spectra, and UV−vis and crystallo-
graphic data. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
1
Figure 2. (a) H NMR spectra of the conversion of 18 mM TrtSSH
+
(blue circle) to TrtSH (red circle) with 1 equiv of [NBu4 ][TFA−]
recorded every 60 s. (b) Addition of PPh3 (yellow circle) in 0.5 equiv.
increments at the end of the reaction shows the clean formation of
Ph3PS (green circle).
AUTHOR INFORMATION
Corresponding Author
pluth@uoregon.edu
■
Notes
The authors declare no competing financial interest.
the completion of each reaction, titration with PPh3 and
monitoring the formation of Ph3PS, which is generated by
reaction of PPh3 with S0, by 31P{1H} NMR spectroscopy
established that 1 equiv of S0 was formed for each equiv of
TrtSSH (Figure 2b). Following the reaction of TrtSSH with
ACKNOWLEDGMENTS
■
We thank Milton Jackson, Jr. for assistance with Raman
spectroscopy. This work was supported by the Oregon Medical
Research Foundation and the NIGMS (R00GM092970). The
NMR facilities at the UO are supported by NSF/ARRA (CHE-
0923589). The Biomolecular Mass Spectrometry Core of the
Environmental Health Sciences Core Center at Oregon State
University is supported, in part, by the NIEHS (P30ES000210)
and the NIH.
+
[NBu4 ][CN−] by UV−vis spectroscopy, however, showed the
formation of a transient intermediate with λmax = 340 nm
(Figure S27), which we attribute to TrtSS−. Disappearance of
this band is accompanied by the appearance of a broad
absorption band centered at 300 nm consistent with S8
formation. Taken together, these data suggest that TrtSSH
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(11) PPh3 reduces S0 to S2− which is trapped as SPPh3. This sulfide
can be liberated by hydrolysis in water. Direct reduction of the
hydrodisulfide by PPh3 is more likely than deprotonation followed by
reaction with S8 because compounds of similar or higher basicity than
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