Development of S-Substituted Thioisothioureas as Efficient Hydropersulfide Precursors

Article abstract of DOI:10.1021/jacs.8b08469

Because of their inherent instability, hydropersulfides (RSSH) must be generated in situ using precursors, but very few physiologically useful RSSH precursors have been developed to date. In this work, we report the design, synthesis, and evaluation of novel S-substituted thiosiothioureas as RSSH precursors. These water-soluble precursors show efficient and controllable release of RSSH under physiological conditions.

Full text of DOI:10.1021/jacs.8b08469

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Communication
Development of S-Substituted Thioisothioureas
as Efficient Hydropersulfide Precursors
Vinayak S. Khodade, and John P. Toscano
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b08469 • Publication Date (Web): 03 Dec 2018
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Development of S-Substituted Thioisothioureas as Efficient
Hydropersulfide Precursors
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Vinayak S. Khodade and John P. Toscano*
Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA
Supporting Information Placeholder
A general strategy for RSSH generation is to substitute the
terminal sulfhydryl group with a suitable protecting group. The
reaction of alkyl halides with thiourea is a commonly used
ABSTRACT: Due to their inherent instability, hydropersulfides
RSSH) must be generated in situ using precursors, but very few
(
physiologically useful RSSH precursors have been developed to
date. In this work, we report the design, synthesis, and evaluation
of novel S-substituted thiosiothioureas as RSSH precursors. These
water-soluble precursors show efficient and controllable release of
RSSH under physiological conditions.
2
7
method for thiol synthesis (Scheme 2, eq. 1).
This multistep
one-pot process proceeds via the intermediacy of an isothiourea,
which is then hydrolyzed under basic condition to produce the
2
8-29
desired thiol and urea as a byproduct.
Based on this reaction,
we thought to protect the terminal sulfhydryl group of RSSH in
the form of an S-alkylthioisothiourea (Scheme 2, eq. 2), which can
then potentially be deprotected under physiological condition to
produce RSSH. We expected that the more acidic nature of
Hydrogen sulfide (H
2
S) is a biologically important signaling
molecule that exerts diverse roles in a myriad of physiological
1
-3
processes.
However, recent reports indicate that much of H
2
S
7
RSSH vs. RSH should facilitate RSSH release. In this design,
biological activity may be attributed to hydropersulfides (RSSH)
thiols are used to construct the RSSH precursors in a convenient
one-pot process. We also anticipated that different thiol and
thiourea substituents could potentially affect the rates of
deprotection, thereby offering tunability of RSSH release.
4-11
and/or polysulfides (RSS
n
R).
RSSH have been detected at
significant levels in tissues, cells, and plasma and play a
6
, 12-13
regulatory role in redox biology.
RSSH are also excellent
1
4-15
hydrogen transfer agents towards a variety of radicals.
Despite increasing evidence of the role of RSSH in redox
signaling, the fundamental chemistry and biological roles of
RSSH are still poorly understood. This deficiency is partly due to
Scheme 2. Design of S-Substituted-Thioisothioureas as
Hydropersulfide Precursors
the unstable nature of RSSH. Although sterically hindered
16-17
hydropersulfides are isolable in organic solvents,
they rapidly
convert to a variety of products including trisulfides, tetrasulfides,
1
7-19
elemental sulfur, and H
2
S in aqueous solution.
Because of
this inherent reactivity, RSSH are typically generated in situ.
Previously reported generation strategies are summarized in
1
8, 20-26
Scheme 1.
To advance our understanding of RSSH roles in
biology, the development of new precursors that can cleanly and
reliably produce RSSH under biological conditions is critical.
To test this hypothesis, we synthesized precursor 1 (Scheme 2, eq.
3
0-31
Scheme 1. Methods for Hydropersulfide Generation
2),
and examined RSSH generation using N-ethylmaleimide
3
2
(
NEM) as a trap; see Supporting Information (SI) for synthetic
and analytical details. The RSS-NEM adduct was monitored by
ultra-performance liquid chromatography–mass spectrometry
(
°
UPLC–MS). Incubation of 1 with NEM in pH 7.4 buffer at 37
C for 30 min led to complete disappearance of 1, with no
evidence of persulfide-NEM adduct formation. Instead, formation
of dialkyltrisulfide 2 was observed (SI, Figure S1). Precursor 1
does indeed produce RSSH; however, due to the more
electrophilic nature of the precursor itself compared with NEM,
RSSH reacts preferentially with 1 to produce trisulfide 2 (Scheme
3
, Path A). In addition, we also observe thiosulfinate RS(O)-SR 3
as a major product (SI, Figure S1), suggesting an intramolecular
cyclization mechanism and subsequent production of a sulfenic
acid (RSOH) intermediate that is also trapped by precursor 1
(
Scheme 3, Path B).
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Scheme 3.
Proposed Mechanism of Trisulfide
2
and
evidence of RS(O)-NEM 6b formation, RSS-NEM adduct 5b is
1
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9
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Thiosulfinate 3 Formation from Precursor 1
formed in less than 50% yield with other products also observed
(
SI, Figure S23). These additional non-RSSH producing reaction
pathways for 4c are under further investigation.
Next, we examined precursor 4d in which the thiourea is
substituted with a phenyl group. Analysis of RSSH generation
from 4d in the presence of NEM shows clean conversion of 4d to
the RSS-NEM adduct 5a with no evidence of the sulfenic acid-
derived product 6a. Based on the mechanism of isothiourea
hydrolysis which produces alkyl thiol and urea (Scheme 2, eq.
28-29
1
),
we expected N-phenylurea to be formed in high yield.
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However, UPLC-MS analysis showed no evidence for N-
phenylurea, indicating that hydrolysis is not the operative
mechanism for RSSH generation. Instead, phenyl cyanamide is
observed in 93% yield (SI, Figures S27-30), indicating an
3
1, 35
elimination mechanism for RSSH generation (Scheme 5).
To gain additional insight into the mechanism of RSSH
formation, 4d was analyzed by UPLC-MS in acetonitrile. Here,
we observe a peak at 0.66 min (Figure 2a, bottom trace).
Immediately following introduction of 4d to pH 7.4 buffer this
peak disappears with concomitant appearance of a new peak at 4.2
To reduce reaction with the precursor, we examined precursor 4a,
equipped with an inhibiting dimethyl substituent alpha to the
disulfide (Figure 1). UPLC-MS analysis shows formation of
RSS-NEM 5a (SI, Figure S12), confirming RSSH generation. In
+
min, which is assigned to the neutral form of 4d (4d-H ; see SI for
experimental details, Figures S32-34). This result suggests that
precursor 4d rapidly undergoes neutralization at pH 7.4 to form an
addition, we also observe RS(O)-NEM 6a formation (SI, Figure
+
intermediate 4d-H , which then decomposes to release RSSH and
33-34
S12), analogous to previous obervations,
indicating that the
phenyl cyanamide. The ability of 4d to release RSSH in the
presence of thiols (likely to be present under physiological
conditions) was also examined (SI). We observe formation of the
RSSH-derived trisulfide as the major product. The anticipated
disulfide product formed from reaction of 4d with thiol is also
detected, but only in minor amounts, confirming the ability of 4d
to release RSSH even in the presence of excess thiol (SI, Figures
S67-73). RSSH generation from 4d was also confirmed using
RSOH-producing reaction (Scheme 4, Path B) remains
competitive with the desired production of RSSH (Path A).
Figure 1. Hydropersulfide Precursors 4a-h with Synthetic
Yields
3
2, 36
iodoacetamide (IAM),
another commonly used RSSH trap
(
SI, Figures S35-37). Together, these results confirm the
improved ability of 4d to produce RSSH.
Next, precursor 4b, equipped with methyl group on the nitrogen
of the thiourea moiety, was synthesized to examine the effect of
N-alkyl substituents on RSSH generation. The RSSH yield was
reduced compared with that from 4a (SI, Figure S18), suggesting
that an alkyl substituent on nitrogen disfavors RSSH generation
and RSOH generation remains the dominant reaction path.
Scheme 4.
Proposed Mechanism of Precursors 4a-c
Decomposition in the Presence of NEM
Figure 2. (a) UPLC-MS chromatograms of RSSH generation from 4d
(200 µM) in the presence of NEM (4 mM) incubated in pH 7.4
ammonium bicarbonate (100 mM) with the metal chelator DTPA (100
µM) at 37 ˚C. A peak at 3.7 min corresponding to N-ethylmaleamic
acid (NEMA), derived from NEM hydrolysis, was observed under
these conditions (see SI); (b) MS spectrum (ESI+ mode) of the
product eluting at 5.06 min corresponding to 5a with m/z 349.0885
(expected m/z 349.0886).
1
Next, we used H NMR spectroscopy to monitor the kinetics of
2
RSSH generation from 4d by trapping with NEM in 10% D O,
pH 7.4 buffer containing DMSO as an internal standard at 37 °C.
An increase in the peak intensity attributed to one of the
diasterotopic protons of the succinimide ring of the two product
To prevent sulfenic acid generation from a carboxylic acid-
mediated intramolecular cyclization, we examined precursor 4c in
which the carboxylic acid was masked as methyl ester. Although
decomposition of 4c in the presence of NEM produced no
diastereomers (H , δ 2.99-2.91 ppm) was observed (Figure 3a).
b
The other diasterotopic proton (H , δ 3.30-3.21 ppm) overlaps
c
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with N-ethylmaleamic acid (NEMA), a product derived from
electron withdrawing substituents maintain efficient RSSH
3
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NEM hydrolysis (SI, Figure S56).
Similarly, the intensity of
generation, with a small effect on the kinetics of RSSH release.
For example, 2-Cl substituted precursor 4f produces RSS-NEM in
95% yield with a 17.3 min half-life, and 4-Cl substituted
precursor 4g produces the NEM adduct in 91% with a 9.3 min
half-life. Similarly, 4-CF substituted precursor 4h produces 95%
3
of RSS-NEM with a half-life of 6.8 min.
two methyl group protons of precursor 4d (δ 1.44 and 1.41 ppm)
decreased as four methyl group protons of the two diastereomers
a
of 5a (H , δ 1.40, 1.37 ppm and 1.40, 1.34 ppm) increased,
1
supporting the clean conversion of 4d to 5a. The H NMR peak
assignments for 5a are consistent with the RSS-NEM adduct
formed during S-methoxycarbonyl penicillamine disulfide
Based on the clean formation of hydropersulfide and aryl
cyanamide during decomposition of 4d-h, we propose the general
mechanism shown in Scheme 5. Neutralization of S-alkyl-
thioisothiourea 7 (or its resonance partner 7a) occurs at pH 7.4 to
produce a neutral species 8 and/or 8a, which can further undergo
(
MCPD) decomposition (Scheme 1, eq. 3) in the presence of
NEM (SI, Figure S57). The half-life of precursor 4d at 37 °C is
5.6 min with a 96% yield of RSS-NEM adduct 5a (Figure 3b).
1
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1, 35
an elimination reaction to produce RSSH and aryl cyanamide.
The elimination reaction can be initiated by either donation of the
imine-nitrogen lone pair of 8 to form aryl cyanamide and RSSH
(
Scheme 5, eq. 2) or deprotonation of the arylamino-nitrogen
proton to form RSSH and an aryl carbodiimide, which can further
undergo tautomerization to afford the aryl cyanamide (Scheme 5,
eq. 3). Alternately, an elimination reaction can be initiated by
donation of the nitrogen lone pair of intermediate 8a to produce
3
8
RSSH and aryl carbodiimide (Scheme 5, eq. 1).
We have
preliminarily examined the decomposition of precursor 4d at pH 6
and pH 8.5 (SI, Figures S64-S66). Decomposition is substantially
slowed at lower pH and increased at higher pH, consistent with
the mechanism proposed in Scheme 5. The reduced RSSH yield
from methoxy-substituted 4e suggests that an electron donating
group on the phenyl ring disfavors neutralization to form 8/8a,
and as a result carboxylate anion attack of the disulfide to produce
sulfenic acid can contribute. Slower RSSH generation from 4f–h
suggests that an electron withdrawing group on the phenyl ring
might cause the nitrogen lone pair (e.g., in 8a) to be less available
for the elimination reaction. In addition, the thermodynamic
stability of the aryl cyanamide produced could contribute to the
observed rate of RSSH release.
1
Figure 3. (a) Representative stacked H NMR spectra showing the
decomposition of precursor 4d (1 mM) with NEM (20 mM) in 10%
D O, phosphate buffer (PB), pH 7.4 at 37°C to produce RSS-NEM
2
adduct 5a. Precursor 4d was introduced to a solution of NEM in PB at
t=0 min and following equilibration and shimming, the first H NMR
1
spectrum was obtained after 6 min; (b) The kinetics of decomposition
of 4d (circles represent the -CH
formation of 5a (squares represent the diasterotopic protons (H
the succinimide ring of the two product diastereomers). The curves
3
protons (H
a
) of the precursor) and
b
) of
Scheme 5.
Proposed Mechanism of Hydropersulfide
−
1
are calculated best fits to a single-exponential function (k = 0.12 min
and t1/2 = 5.6 min for each fit).
Generation from S-substituted Thioisothioureas
Table 1.
Hydropersulfide yields and half-lives for
Precursors 4d-h
1
2
Hydropersulfide
Yield (%)
t
1/2
(min)
Precursor
4d
R
R
a
b
c
H
H
Ph
96 ± 1
5.6 ± 0.3
5.0 ± 0.5
4
4
e
f
4-MeOPh 75 ± 2
1
0
7.3
.4
±
H
2-ClPh
4-ClPh
95 ± 2
4g
H
H
91 ± 1
95 ± 2
9.3 ± 0.6
6.8 ± 0.2
d
d
4
h
3
4-CF Ph
To demonstrate the generality of S-substituted thioisothioureas as
hydropersulfide precursors, we also examined tert-butylthiol-
based precursor 9 (Figure 4), which produces a near quantitative
yield (97%) of RSS-NEM adduct 10 with a half-life of 17.3 min
a
RSSH yield was calculated based upon % precursor consumed.
b
RSSHꢀprecursorsꢀ(1ꢀmM)ꢀwereꢀincubatedꢀinꢀtheꢀpresenceꢀofꢀNEMꢀ
20ꢀmM)ꢀinꢀpH 7.4 PB containing 10% D O at 37 °C. Reported data
(
2
c
represent averages ± SD (n = 3). The half-life of 4d is 4.3 min in pH
7.4 buffer with 20% MeOD. Reduced aqueous solubility required
this experiment to be carried out in pH 7.4 buffer with 20% MeOD.
(
SI, Figure S62). Slower RSSH generation observed from 9
d
compared with 4d suggests additional influence of the S-alkyl
substituent on RSSH generation.
Precursor 4e, equipped with a 4-OMe substituent, was synthesized
to study the effect of an electron donating group on the phenyl
ring. The half-life of 4e was found to be 5.0 min (Table 1),
suggesting no significant effect on the kinetics of RSSH
generation. However, the RSSH yield (75%) was reduced and
sulfenic acid-derived product 6a is now observed (SI, Figures S38
and S58), suggesting that an electron donating group on the
phenyl ring disfavors RSSH generation. In contrast, we find that
Figure 4. Precursor 9 and RSS-NEM adduct 10
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In summary, we have designed, synthesized, and evaluated S-
substituted-thioisothioureas as RSSH precursors. These
precursors show efficient and controllable release of RSSH under
physiological conditions. We also demonstrated that RSSH
generation can be tuned by structural modifications. Importantly,
these precursors are found to be stable for several months on the
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bench-top in the solid state and for a few weeks in D O at room
temperature (SI, Figure S63). Given the significance of RSSH in
redox biology, we hope that these S-substituted thioisothioureas
will find use as research tools to advance our understanding of
RSSH chemical biology.
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ASSOCIATED CONTENT
(
16) Bailey, T. S.; Zakharov, L. N.; Pluth, M. D., J. Am. Chem.
Supporting Information
Soc. 2014, 136, 10573-10576.
(17) Bailey, T. S.; Pluth, M. D., Free Radic. Biol. Med. 2015, 89,
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The Supporting Information is available free of charge on the
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(
18) Isabelle, A.; Erwan, G., ChemBioChem 2014, 15, 2361-
364.
19) Yadav, P. K.; Martinov, M.; Vitvitsky, V.; Seravalli, J.;
Experimental details and supplementary figures, S1−S84 (PDF)
AUTHOR INFORMATION
Corresponding Author
jtoscano@jhu.edu
Wedmann, R.; Filipovic, M. R.; Banerjee, R., J. Am. Chem. Soc.
2016, 138, 289-299.
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Ferrer-Sueta, G.; Filipovic, M. R.; Alvarez, B., J. Biol. Chem.
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Notes
The authors declare no competing financial interests.
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ACKNOWLEDGMENT
(
We gratefully acknowledge the National Science Foundation
K.; Wang, S.; Lefer, D. J.; Wang, B., Angew. Chem., Int. Ed.
2017, 56, 11749-11753.
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Day, J. J.; Xian, M., Angew. Chem., Int. Ed. 2018, 130, 5995-
(
CHE-1566065) for generous support for this research. We also
thank Dr. Ananya Mujumdar for assistance with NMR kinetics
experiments and Dr. I. Phil Mortimer for assistance with
compound identification and characterization by mass
spectrometry.
5
(
999.
25) Powell, C. R.; Dillon, K. M.; Wang, Y.; Carrazzone, R. J.;
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