An Esterase-Sensitive Prodrug Approach for Controllable Delivery of Persulfide Species

Article abstract of DOI:10.1002/anie.201704117

A strategy to deliver a well-defined persulfide species in a biological medium is described. Under near physiological conditions, the persulfide prodrug can be activated by an esterase to generate a “hydroxymethyl persulfide” intermediate, which rapidly collapses to form a defined persulfide. Such persulfide prodrugs can be used either as chemical tools to study persulfide chemistry and biology or for future development as H₂S-based therapeutic reagents. Using the persulfide prodrugs developed in this study, the reactivity between S-methyl methanethiosulfonate (MMTS) with persulfide was unambiguously demonstrated. Furthermore, a representative prodrug exhibited potent cardioprotective effects in a murine model of myocardial ischemia-reperfusion (MI/R) injury with a bell shape therapeutic profile.

Full text of DOI:10.1002/anie.201704117

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Accepted Article
Title: A Novel Esterase-sensitive Prodrug Approach for Controllable
Delivery of Persulfide Species
Authors: Yueqin Zheng, Bingchen Yu, Zhen Li, Zhengnan Yuan, David
J. Lefer, Binghe Wang, Siming Wang, Chelsea L. Organ, and
Rishi K. Trivedi
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201704117
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10.1002/anie.201704117
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COMMUNICATION
A Novel Esterase-sensitive Prodrug Approach for Controllable
Delivery of Persulfide Species
Yueqin Zheng,#[a] Bingchen Yu,#[a] Zhen Li,# [b] Zhengnan Yuan,[a] Chelsea L. Organ,[b] Rishi K.
Trivedi,[b] Siming Wang,[a] David J. Lefer,[b] and Binghe Wang*[a]
Abstract: A new strategy to deliver a well-defined persulfide species
in a biological medium is described herein. Under near physiological
conditions, the persulfide prodrug can be activated by an esterase to
generate a “hydroxyl methyl persulfide” intermediate, which rapidly
collapses to form a defined persulfide. Such persulfide prodrugs can
be used either as chemical tools to study persulfide chemistry and
biology or for future development as H2S-based therapeutic reagents.
Using the persulfide prodrugs developed in this study, the reactivity
between S-methyl methanethiosulfonate (MMTS) with persulfide was
unambiguously demonstrated. In addition, a representative prodrug
exhibited potent cardioprotective effects in a murine model of
myocardial ischemia-reperfusion (MI/R) injury with a bell shape
therapeutic profile.
it is hard to understand how a persulfide species can be stronger
reducing agents than sulfide. Such findings suggest that more
work is needed at the molecular level to elucidate the mechanism
of action. Other examples include the findings that reactive sulfur
species (RSS) such as sulfane sulfurs or polysulfides are more
effective in S-sulfhydration than H2S.[1g, 9] There is a clear need
for investigation of persulfide chemistry and chemical biology in
this field.[7a] However, there are two major challenges that face the
chemistry field. The first one is a lack of good persulfide
precursors/prodrugs that allow for easy and reliable access to
persulfides as research tools; and the second one is the limited
availability of ‘easy to use’ detection methods for protein S-
sulfhydration.[7a] There has been reported work to address the
second issue.[1g, 6, 10] We herein focus on developing novel and
easy to handle persulfide prodrugs to address the first issue.
The difficulty in developing persulfide prodrugs comes from
the unstable nature of persulfide species, which can rapidly
decompose to disulfides, polysulfides, elemental sulfur and
H2S.[11] This is especially true if there is an exposed free sulfhydryl
group (-SH). To achieve controlled delivery of persulfide in
biological studies, the free sulfhydryl group has to be protected,
and then regenerated when needed. One class of existing
persulfide precursors contains the acyl disulfide group.[3a, 10c] They
were cleverly designed to release the persulfides through
nucleophilic attack by a thiol group. However, the release of
persulfide relies on the addition of an excess amount of thiol in
solution.[3a, 10d] Therefore, there is a mix of various sulfur species
present at any given time. An approach to directly achieve “pure”
persulfide species under physiological conditions without using
other sulfide or thiol species is needed.
Enzyme-sensitive prodrugs have been widely used in drug
delivery.[12] We are interested in designing esterase-sensitive
persulfide prodrugs, which are stable under physiological
conditions and can efficiently generate persulfide in the presence
of an enzymatic trigger. Specifically, similar to the idea of using
“hemiacetal” as an unstable intermediate in prodrug design,[13] we
were interested in exploiting the “hydroxyl methyl disulfide”
(HOCHRSSR) analog as a key intermediate in our design. In this
design, an ester group is introduced to mask the hydroxyl group,
leading to a stable precursor. Activation of this prodrug thus relies
on the cleavage of this ester bond, resulting in an unstable
HOCHRSSR intermediate, which would collapse to give an
aldehyde and a persulfide (Scheme 1).
The physiologic and potentially therapeutic roles of hydrogen
sulfide (H2S) and related sulfur species are well accepted.[1]
However, the detailed mechanism(s) of action for these sulfur
species is far from clear. Further, even the question of the “active”
sulfur species is not always clear for a given biological response.
To complicate this further, different donors of sulfur species
without defined chemistry are often used in various studies,
leading to difficulty in result interpretation and comparison. For
example, polysulfides and garlic-derived sulfur species are often
used in biological studies.[2] The chemistry of such sulfur donors
is not well defined; consequently, it is not always clear what the
“active” species is and the relative ratio of the various species. To
advance the field of sulfur biology, it is important to devise
chemical strategies that allow for the precise production of various
specific sulfur species for mechanistic studies at the molecular
level and for understanding the biology. Several labs have made
significant contributions in developing prodrugs of hydrogen
sulfide[3] and COS[4] as well as sulfur donors at various oxidation
states.[5] Along this line, one molecular pathway is known to play
an important role in sulfur-mediated signaling, S-sulfhydration.[6]
Clearly, this is not the kind of chemistry that can be achieved with
hydrogen sulfide per se. It would require sulfur species at the
oxidation state of a persulfide or polysulfide. Indeed, perthiol
species have drawn growing attention owing to its potentially
dominant roles in H2S-related signaling pathways.[1g, 7] There are
already many studies that explore persulfide chemistry in biology.
For example, it was reported that persulfide species such as
glutathione persulfide (GSSH) have much stronger “reducing”
ability for ferric-cytochrome c than H2S and GSH.[8] Such results
are puzzling in terms of the redox chemistry because chemically
[a] Yueqin Zheng, Bingchen Yu, Zhengnan Yuan, Siming Wang and
Prof. Binghe Wang
Department of Chemistry
Georgia State University
Atlanta, Georgia 30303-3083
E-mail: wang@gsu.edu
[b] Zhen Li, Chelsea L. Organ, Rishi K. Trivedi and Prof. David J. Lefer
Cardiovascular Center of Excellence,
Louisiana State University Health Science Center,
New Orleans, Louisiana 70112, United States
Scheme 1. General design of persulfide prodrugs, and their release mechanism.
To test our design, persulfide prodrug 1 (P1, BW-HP-
201,Scheme 2 ) was synthesized by a one-step reaction between
1,2-dibenzyldisulfane and propionic acid using KMnO4 as the
#
These authors contributed equally.
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10.1002/anie.201704117
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COMMUNICATION
oxidant by following a similar literature procedure.[14] P1 is a
colorless oil without the characteristic smell of sulfide such as
benzyl mercaptan and is stable at room temperature for 5 days
and at -20 °C for 3 months. P2~6 (BW-HP-202-206) were also
synthesized using the same procedures.
than 5% benzaldehyde was detected in the solution with 500 µM
compound 3 without the addition of PLE. Such results indicated
that the prodrugs were most effectively activated by an esterase,
which controlled the rate-determining step.
Figure 1. P1 hydrolysis at 37 °C in PBS (2% DMSO). □:100 μM P1; :100
μM P1 + 500 μM Compound 3;
:100 μM P1 + PLE; :100 μM P1 + 500 μM
Compound 3 + PLE;
100 μM P1 + 3 mM Compound 3 + PLE. a) H2S release
detection by MB method; b) Benzaldehyde formation detection by HPLC.
Scheme 2. Structures of persulfide prodrugs.
For small molecule bioregulators (SMBs), release rate is a
very important factor in determining the sustained effective
concentration and thus biological effect. Donors at the same
concentration could lead to different biological effects when the
release rates are different. Our earlier work on H2S prodrugs
demonstrated that varying the acyl moiety allows for tuning the
esterase-catalyzed hydrolysis rates.[15] Thus we reasoned that
similarly designed persulfide prodrugs should also show different
release profiles. Therefore, we next examined release kinetics.
Specifically, we treated all these precursors (100 μM) with 0.5
u/ml PLE in PBS containing 500 μM compound 3 at 37 °C.
Aldehyde formation was monitored by HPLC (Table 1), and H2S
release was tested by the MB method (Figure S5).
We first studied the ability for P1 to undergo the intended
reaction by monitoring the formation of both benzyl persulfide and
benzaldehyde by HPLC. It turned out that the benzyl persulfide
(compound 2) (Scheme 3a) was not sufficiently stable for
detection on an HPLC time scale. This further affirms the rationale
for designing these prodrugs, i.e., without the protection of the
terminal sulfhydryl group, the persulfide species would not be
stable enough for long-term storage. For detection purpose, we
therefore, trapped benzyl persulfide using N-acetyl-L-cysteine
(compound 3) to give compound 4, which is stable for HPLC
detection. Specifically, 100 μM P1 was treated with porcine liver
esterase (0.5 u/ml, PLE) for 10 min at 37 °C in the phosphate
buffered saline (PBS, pH= 7.4) containing 500 μM compound 3
(Scheme 3a). The results show that the amount of benzaldehyde
1 detected was the equivalent of 96% conversion. However, only
55% recovery of disulfide 4 were detected under such conditions,
clearly indicating that the trapping reaction was not fast enough
on the time scale of the study. By increasing the concentration of
3 to 3 mM, we were able to detect about 95% recovery of disulfide
4 (Scheme 3a). Such results clearly indicated that the release
reaction occurred as designed.
Table 1. Total percentage of aldehyde formation (A%) and 50% aldehyde
formation time (t1/2
)
Compo
und
P1
P2
P3
P4
P5
P6
A%
96±3
25±5
∆
91±4
85±9
97±3
17±6
97±2
29±6
89±6
145±12
t1/2 (s)
12±6*
∆: not detectable because of low boiling point of acetaldehyde;*: 50% P2
remaining at the time of sampling. n= 3 p=0.95.
The results demonstrated that all the prodrugs were sensitive
to PLE with hydrolysis half-lives ranging from 12 to 145 s. As a
consequence of their respective release rates, the peak
concentrations and sustained concentrations are also different.
For example, H2S level released from P1 reached a peak
concentration of 33 µM within 30 s and then decreased slowly.
On the other hand, the slowest prodrug P6 showed a gradual
increase in H2S concentration, reaching a maximum of 15 µM
after 3 min (Figure S5).
The above studies clearly demonstrated the chemical
feasibility of the prodrug activation and allow for studies of
reaction kinetics. However, such studies used benzaldehyde and
hydrogen sulfide as surrogates for product detection. Next, it was
important to demonstrate persulfide formation without the added
the thiol species, 3. In the field of designing detection methods
for protein S-sulfhydration, a key step is developing trapping
reagents, which could efficiently trap the persulfide under
physiological conditions.[7a] For this study, we used
dinitrofluorobenzene (DNFB, Compound 5), which was known to
trap persulfides (Scheme 3b).[16] Specifically, we incubated 100
μM of the prodrugs with 1u/ml PLE at 37 °C for 10 s and then
added 4 mM of DNFB to the mixture. The resulting solution was
further incubated for another 30 min. Then the formation of
disulfide compound 6 was analyzed by HPLC (Table 2). We were
able to trap 70%-82% of the persulfide released from the prodrugs.
There could be several reasons for the incomplete “conversion”
to 6. First, it is possible that the reaction rate between the released
persulfide and 5 is not high enough for 100% conversion.
Scheme 3a. Benzyl persulfide was release from P1 and trapped by 3
Besides using HPLC to detect the formation of 1 and 4, we
also used the standard methylene blue (MB) method to detect the
H2S release from P1 (Figure 1) in the presence of 3. The results
showed no obvious H2S generation in the absence of PLE, and
only less than 5 µM H2S (5%) were formed in the presence of 500
µM compound 3, suggesting the possibility of a small percentage
of the prodrug to undergo sulfur exchange. However, with the
addition of 0.5 u/ml PLE at 37 °C in PBS P1 generated about 23
µM, 33 µM and 45 µM H2S in the presence of 0 µM, 500 µM and
3 mM compound 3, respectively. Compared with H2S release, the
formation of benzaldehyde was not affected by compound 3
(Figure 1). More than 90% benzaldehyde was detected in 2 min
in PLE-containing solution with or without compound 3, and less
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However, the fact that all prodrugs gave similar percentages of 6
(Table 2) suggests that this is not controlled by reaction kinetics
at the trapping step because the various prodrugs gave
substantially different peak concentrations, and trapping yields
are not correlated with release rates. The second possible reason
is the competition between trapping and disproportionation
reaction of the persulfide released. We tend to think that the latter
was the reason for the less than 100% conversion to 6. To
confirm this, we incubate 100 μM of the prodrug with 1u/ml PLE
and 4 mM DNFB at r.t., and found that the trapping yields
increased to 82%-93%. Clearly, at lower temperature (room
temperature vs. 37 °C), the disproportion reaction was slower,
allowing for the improved trapping efficiency. The results
demonstrated that the prodrugs could efficiently afford persulfide
for further studies.
released from P5. Specifically, 100 μM of the prodrug was
incubated with 1u/ml PLE and 4 mM MMTS at r.t. for 30 min
(Scheme 4d). Then LC-MS was applied to analyze product
formation. For comparison, we also incubated 4 mM of MMTS
with 1 mM dibenzyl disulfide BnSSBn and 1 mM of Na2S (Scheme
4c). The results are shown in Figure 2a and 2b. The reaction in
4c led to BnSSMe as the major product, and only a small amount
of BnSSSMe was formed. Such results are quite similar to the
product formation pattern in the reported reaction between MMTS
with GSSG and Na2S.[10d] However in the reaction of MMTS with
benzyl persulfide released from P5 (Scheme 4d, Figure 2b),
BnSSSMe clearly formed as the dominant product, and other
possible sulfide products were only observed in minute quantities.
The results here clearly demonstrated that MMTS could react with
persulfide. By taking advantage of the unique property of our
persulfide prodrug system, we reconfirmed that MMTS can
efficiently react with persulfide.
Scheme 3b. Persulfide released from precursors and trapped by DNFB
Table 2. Persulfide released from precursors and trapped by DNFB (A: trapping
yield at 37 °C, B: trapping yield at r.t. n= 3, p=0.95)
Prodrugs
A (%)
P1
P2
P3
P4
P5
P6
79±5
70±4
71±5
80±4
82±5
78±4
B (%)
87±5
80±6
81±5
88±6
93±5
86±5
As a research tool, the system can provide relatively “pure”
persulfide, which is very unique and important in the field of
protein S-sulfhydration. In 2009, sulfide signaling through S-
sulfhydration was first demonstrated.[6] A modified biotin switch
technique, using S-methyl methanethiosulfonate (MMTS) as an
alkylating reagent, was used to identify a large number of proteins
that may undergo S-sulfhydration. The underlying chemical
mechanism was based on the assumption that MMTS would
selectivity react with thiol groups (RSH), but not persulfide group
(RSSH) (Scheme 4a).
Figure 2. a) HPLC trace of BnSSBn reacting with Na2S and MMTS. b) HPLC
trace of esterase mediate P5 hydrolysis and persulfide trapping by MMTS.
Besides using the prodrugs as chemical tools to study
persulfide chemistry under near physiological conditions, we also
examined their biological effects. We first assessed the
cytotoxicity of these prodrugs using the H9c2 cell line. All
prodrugs showed no obvious toxicity at up to 100 µM after 24 h of
incubation (Figure S2 and S3). Various sulfur species have been
shown to exhibit protective effects in heart myocardial infarction
reperfusion (MI/R) injury studies.[1f, 3a, 17] As a test of the biological
relevance of the prodrugs designed, we selected P2, which has a
relative good water solubility and low toxicity of side product
(acetaldehyde), as a representative for examination of its
protective effect in a murine model of MI/R injury. The compounds
were tested in a mouse heart ischemia reperfusion injury model.
Mice were subjected to 45 min of ischemia induced by left
coronary artery (LCA) occlusion followed by 24 h of reperfusion of
P2 (0, 12.5, 50, 100, 500 μg/kg) or vehicle, administered by
intracardiac injection at the time of reperfusion. The LCA was re-
occluded at 24h of reperfusion at the same position. Then Evens
Blue dye was injected through right common carotid artery to
delineate the area-at-risk (AAR). Due to the occlusion of LCA,
AAR did not receive blood flow nor the Evens Blue dye, while the
remainder of the heart was stained blue. Therefore, AAR were the
non-blue regions in the mid-ventricular slice (Figure 3a). The
infarct size (INF) was stained white by triphenyltetrazolium
chloride solution. Thus, the white regions in the mid-ventricular
slice represent INF (Figure 3a). Left ventricle (LV) is the sum of
blue and non-blue regions. All of the animal groups displayed
Scheme 4. Persulfides react with MMTS
However, later studies suggested that this assumption on the
lack of reactivity between MMTS with RSSH was questionable
(Scheme 4a).[10d] One reported work in studying the reactivity
between MMTS with RSSH used a mixture of GSSG and sodium
sulfide (Scheme 4b). In doing so, a small peak of GSSSMe was
indeed detected. However, the origin of this “trisulfide” peak could
be interpreted in more than one way due to complexity of the
mixture formed in the process of preparing GSSH (Scheme 4b).
To further probe the reactivity between MMTS with RSSH, we
conducted the reaction between MMTS and the persulfide
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In summary, we have developed a series of persulfide prodrugs
with controllable release rates. These persulfide prodrugs release
persulfide through an esterase-mediated hydrolysis mechanism.
In the presence of the PLE, the prodrugs efficiently released
persulfides under near physiological conditions. Using the
prodrug, we reaffirmed the reactivity between persulfide and
MMTS. The protective effects of P2 in a murine model of MI/R
injury have also been demonstrated. All the studies above
demonstrate that this novel type of persulfide prodrugs not only
can be used as research tools, but also are possible therapeutic
agents.
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Partial financial support from the GSU Brains and Behaviors
Fellowship Program (BY) is gratefully acknowledged. D.J.L is
supported by grants from the National Heart, Lung, and Blood Institute
(R01HL092141, R01HL116571, R43HL131356).
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Keywords: Persulfide prodrugs • H2S • Persulfide reactivity •
bell shape therapeutic profile
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