Development of Acid-Mediated H2S/COS Donors That Respond to a Specific pH Window

Article abstract of DOI:10.1021/acs.joc.9b01873

Hydrogen sulfide (H₂S) is a biologically relevant molecule, and recent efforts have focused on developing small molecular donors that deliver H₂S on demand. Acid-activated donors have garnered significant interest due to the potential application of such systems in myocardial ischemia injury or for suppressing tumor growth. In this work, we report a new strategy for tuning H₂S delivery to a specific pH window. Specifically, we utilize self-immolative thiocarbamates with an imine-derived triggering group. After imine hydrolysis, the self-immolative decomposition releases carbonyl sulfide (COS), which is quickly hydrolyzed to H₂S by carbonic anhydrase. Although acid-mediated hydrolysis results in imine cleavage, environments that are too acidic result in protonation of the aniline intermediate and results in inhibition of COS/H₂S release. Taken together, this mechanism enables access to donor motifs that are only activated within specific pH windows. Here, we demonstrate the design, preparation, and pH evaluation of a series of imine-based COS/H₂S donor motifs, which we anticipate that will have utility in investigating H₂S in acidic microenvironments.

Full text of DOI:10.1021/acs.joc.9b01873

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Article
Development of Acid-Mediated H2S/COS
Donors that Respond to a Specific pH Window
Annie K. Gilbert, Yu Zhao, Claire E Otteson, and Michael D Pluth
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b01873 • Publication Date (Web): 03 Sep 2019
Downloaded from pubs.acs.org on September 3, 2019
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Development of Acid-Mediated H2S/COS Donors that Respond to a Specific pH Window
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Annie K. Gilbert, Yu Zhao, Claire E. Otteson, and Michael D. Pluth*
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Department of Chemistry and Biochemistry, Materials Science Institute, Institute of Molecular
Biology, University of Oregon, Eugene, Oregon, 97403, USA. E-mail: pluth@uoregon.edu
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Abstract
Hydrogen sulfide (H2S) is a biologically-relevant molecule, and recent efforts have focused on
developing small molecular donors that deliver H2S on demand. Acid-activated donors have
garnered significant interest due to the potential application of such systems in myocardial
ischemia injury or for suppressing tumor growth. In this work, we report a new strategy for tuning
H2S delivery to a specific pH window. Specifically, we utilize self-immolative thiocarbamates
with an imine-derived triggering group. After imine hydrolysis, the self-immolative decomposition
releases carbonyl sulfide (COS), which is quickly hydrolyzed to H2S by carbonic anhydrase.
Although acid-mediated hydrolysis results in imine cleavage, environments that are too acidic
result in protonation of the aniline intermediate and results in inhibition of COS/H2S release. Taken
together, this mechanism enables access to donor motifs that are only activated within specific pH
windows. Here we demonstrate the design, preparation, and pH evaluation of a series of imine-
based COS/H2S donor motifs, which we anticipate will have utility in investigating H2S in acidic
microenvironments.
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Introduction
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Hydrogen sulfide (H2S) is an important biological signaling molecule and the most recent
addition to the gasotransmitter family alongside nitric oxide and carbon monoxide.1 Endogenous
H2S production is primarily attributed to four main enzymes including cystathionine β-synthase
(CBS), cystathionine γ-lyase (CSE), 3-mercaptopyruvate sulfur transferase (3-MST), and cysteine
aminotransferase (CAT) through cysteine and homocysteine catabolism. H2S can also be generated
through non-enzymatic pathways such as the thiol-mediated release of H2S from allium- and
garlic-derived polysulfides.2 Once generated, H2S is involved in different physiological processes,
including KATP channel activation3 and antioxidant and antiapoptotic signaling.4 In efforts to
modulate H2S levels in different systems, most studies have used inorganic sulfide salts, such as
sodium sulfide (Na2S) and sodium hydrosulfide (NaSH), as exogenous H2S sources. Although
these sulfide sources increase endogenous H2S levels, the large and immediate dose can result in
unwanted toxicity and sulfide oxidation.5 To better understand and leverage H2S levels in various
biological systems, a variety of chemical tools have been developed in the last decade and include
CSE and CBS inhibitors, H2S donors, and activity-based H2S probes for H2S detection.6,7,8
Of the wide array of available H2S donors, one common strategy to engineer H2S release is to
leverage water- or acid-mediated hydrolysis. For example, GYY4137 is one of the most widely
used synthetic H2S donors and releases H2S slowly upon hydrolysis in water at physiological pH
(Figure 1a).9 Similarly, deprotection of certain thioacetals10-11 generates an unstable gem-dithiol
intermediate that is hydrolyzed through an acid-mediated mechanism to release H2S. In more
recent examples, JK donors, which are based on the phosphorothioate core of GYY4137, were
developed and designed to undergo an intramolecular cyclization upon protonation of the P-S
moiety.12 Highlighting potential applications of pH-activated donors, the JK family of donors
showed cytoprotective effects in cell models of oxidative damage and cardioprotective effects in
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an in vivo mouse model of myocardial ischemia-reperfusion injury. More broadly, mildly-acidic
pH environments are found during ischemia injury, within the extracellular environment of
cancerous cells, and in certain subcellular compartments, such as the lysosome. These acidic
environments, when taken in combination with prior work showing the beneficial effects of H2S
in myocardial ischemia-reperfusion injury13 and the ability to induce cell cycle arrest to suppress
tumor growth14 suggest potential application for acid-activated H2S donors. Aligned with these
potential opportunities, a key need remains developing chemistry that enables access to donors
that can be activated in specific pH ranges rather than just at increasing rates at more acidic pH.
Such a strategy could be useful in developing design strategies for oral administration of
hydrolysis-based H2S donor motifs.
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a)
S
R2
S
R1
S
O
P
S
P
N
MeO
S
Ph
NH
R1
O
O
H2N
O
NO2
N
O2
O
GYY4137
Caged gem-dithiols
JK donors
O
b)
Carbonic
Anhydrase
Ph
pH 4.6 - 6.0
N
S
H
2S
COS
H
(CA)
R
N
Figure 1. (a) Representative examples of current acid-labile H2S donors. (b) Design of pH-
dependent COS/H2S release from caged thiocarbamate scaffolds.
To address this challenge, we viewed that triggerable caged thiocarbamates could be modified
to develop donor motifs activated within a specific pH range. Caged thiocarbamates have recently
emerged as a highly tunable class of donors that undergo a triggered, self-immolative elimination
to release carbonyl sulfide (COS), which is rapidly hydrolyzed to H2S by the ubiquitous enzyme
carbonic anhydrase (CA).15,16-17 Examples of triggers employed within this scaffold include
reactive oxygen species,18-20 esterases,21-23 light,24-26 and cysteine.27 We envisioned that using a pH
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sensitive group, such as an imine, as the trigger could be used to develop acid-triggered COS/H2S
donors (Figure 1b). Here we demonstrate that use of an imine trigger, when coupled to the
mechanism of 1,6-elimination required in the self-immolative thiocarbamates, provides donors
that respond within a specific pH window. This new class of donors improves on the pH activation
specificity of current acid-labile donors by providing H2S delivery within a specific pH range. We
expect this pH activation specificity will be useful in different applications requiring compound
stability in strongly acidic or basic environments prior to H2S release.
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Results and Discussion
Donor design. To develop donor motifs that are activated within a specific pH range, we chose
to use an imine as the acid-sensitive trigger because imines are readily hydrolyzed under acidic
conditions and have been used previously to initiate the 1,6-benzyl elimination of carbamate-
containing prodrugs.28 In our designed system, imine cleavage would generate a p-
aminobenzylcarbamothioate intermediate, which would undergo a subsequent 1,6-elimination to
release COS (Scheme 1). Although imine hydrolysis is acid-mediated, if the solution is too acidic,
then the resultant aniline intermediate will be protonated, which will inhibit the 1,6-elimination
and should decrease the rate of COS release. Similarly, the imine should be stable under basic
conditions and not release COS. Combining these parameters, we expected that the efficiency of
the imine-based donor would peak at pH values between the pKa of the iminium (pKa ~5-7) and
anilinium ion (pKa ~4.6). In contrast to other available acid- or hydrolysis-based donors, this would
result in a pH window for activation rather than a direct dependence of release rate with pH.
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O
O
pH < 7
N
N
S
S
H
H
pH > 7
Ph
N
Ph
N
-pHTCM
S
H
Imine
Hydrolysis
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O
O
pH < 4.6
pH > 4.6
N
S
N
H
S
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H
H3N
H2N
CA
H
2S
COS
COS
Scheme 1. Proposed pH-dependent COS/H2S-Release pathway: Basic conditions decrease the rate
of imine hydrolysis. Strongly acidic conditions protonate the aniline intermediate and prevent COS
release.
Synthesis. To test our hypothesis that the acid-triggered thiocarbamate donors undergo a pH-
dependent release, we prepared a pH sensitive imine-containing thiocarbamate donor. Treatment
of 4-aminobenzyl alcohol with benzaldehyde in the presence of acetic acid formed 4-
(benzylideneamino)benzyl alcohol, which was coupled with phenyl isothiocyanate in the presence
of sodium hydride to obtain the O-alkyl thiocarbamate isomer (Scheme 2a). Although analogous
O-alkyl thiocarbamate donors have been prepared and are stable under ambient conditions, we
found that the O-alkyl thiocarbamate with the imine trigger isomerized to the S-alkyl pH-sensitive
thiocarbamate (S-pHTCM) isomer both in solution and also in the solid state (Figure S1). Thione-
thiol isomerization is well known to occur in thiocarbamates via the Newman-Kwart
rearrangement,29,30 but typically requires high temperatures or catalysts.31,32 Similarly, benzylic
Newman-Kwart rearrangements have recently been reported to occur at elevated temperatures.33
We hypothesize the electron-donating imine substituent aids in the stabilization of the benzylic
carbocation intermediate formed in the thiocarbamate rearrangement and thus may facilitate this
isomerization under more mild conditions.
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(a)
OH
OH
O
PhCHO, AcOH
THF
65 °C, 3 hr
68%
H2N
Ph
N
Ph
Ph
O-alkyl to S-alkyl
isomerization
PhNCS, NaH
N
S
THF
0° to r.t., 2 hr
74%
H
Ph
N
S
-pHTCM
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O
(b)
OH
SH
PhNCO, TEA
N
O
THF
65 °C, 2 hr
44%
H
Ph
N
Ph
N
pHCM
O
PhNCO, TEA
CH2Cl2
0 °C to r.t., 1 hr
92%
(c)
Ph
S
N
H
-TCM
S
Scheme 2. (a) Synthetic scheme for acid-triggered thiocarbamate COS/H2S donor (S-pHTCM),
(b) carbamate control compound (pHCM), and (c) triggerless control compound (S-TCM).
In addition to S-pHTCM, the corresponding pH-sensitive carbamate (pHCM) and triggerless
S-alkyl thiocarbamate (S-TCM) were prepared as control compounds to confirm that the COS/H2S
release is triggered by imine hydrolysis of the thiocarbamate. In contrast to the model
thiocarbamate, pHCM should undergo the same imine hydrolysis to release CO2 instead of COS
while generating the same byproducts as S-pHTCM. In the absence of the imine trigger, S-TCM
is not expected to decompose and release COS.
Measurement of H2S-release. To evaluate COS/H2S release from this series of compounds, we
used the colorimetric methylene blue (MB) assay.34 We measured COS/H2S release from S-
pHTCM (50 µM) across a range of pH values (4.0-8.0) in the presence of CA (25 µg/mL) at 37
°C. We confirmed CA activity at pH 5.5 using a p-nitrophenyl acetate assay (see Figure S2). To
span this range of pH, we used sodium citrate buffered and phosphate buffered saline (PBS)
solutions. We observed no buffer dependence when evaluating H2S release from S-pHTCM in
citrate and PBS buffer at pH 6.0 as shown in Figure S3. Consistent with our expectations, we
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observed more efficient COS/H2S release in weakly-acidic conditions from pH 4.6 to 6.5 than from
pH values outside of this range (Figure 2). The rates of H2S release have a similar pH dependence,
but because the rate limiting step of the reaction may change as a function of pH and not all the
H2S release curves peaked at the same levels, we chose to measure H2S concentration at 5 hours
for these investigations (Figure S4). Further supporting our hypothesis, we found that the inflection
points of the pH response curve (4.3 and 7.3) matched the expected pKa values of the iminium and
the anilinium ions (Figure S5). Taken together, these data support the mechanism of COS/H2S
release outlined in Scheme 1.
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Figure 2. (a) COS/H2S release from S-pHTCM (50 µM) at pH 4.0 - 8.0 containing CA (25
µg/mL). (b) pH curve of H2S concentration at 5 h. Experiments were performed in quadruplicate
with results expressed as mean ± S.D. (n = 4).
To confirm that H2S release from S-pHTCM requires both imine hydrolysis and the
thiocarbamate moiety, we measured the H2S release from the pHCM and S-TCM control
compounds at pH 5.5. Under identical conditions as those for S-pHTCM we failed to observe H2S
generation from either pHCM or S-TCM (Figure 3). In the absence of CA, S-pHTCM showed a
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significantly reduced rate of H2S release (Figure S6). To further support the proposed release
mechanism, we monitored the reaction by HPLC analysis (Figure S7 and S8). Consistent with the
proposed 1,6-elimination mechanism, we observed benzaldehyde, aniline, and 4-aminobenzyl
alcohol during the course of the reaction (Scheme 3).
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Figure 3. H2S release from S-pHTCM and triggerless (S-TCM) and carbamate (pHCM) control
compounds (50 µM) in citrate buffer (10 mM, pH 5.5) containing CA (25 µg/mL) at 37 °C.
Experiments were performed in quadruplicate with results expressed as mean ± S.D. (n = 4)
O
Imine
O
Hydrolysis
N
N
S
S
H
H
O
H
Ph
N
H2N
Ph
Ph-NH2
CA
+H+
OH
H
2O
-H+
COS
H2S
H2N
HN
Scheme 3. Proposed mechanism of acid-triggered COS/H2S release from caged-thiocarbamate
donors with byproducts of self-immolation.
We next investigated the tolerance of the acid-mediated cleavage and self-immolation to the
presence of different biological nucleophiles by measuring H2S release from S-pHTCM at pH 5.5
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in the presence of different analytes. We did not observe a significant change in donor efficiency
in the presence of 250 µM of GSH, Cys, Lys, Ser, Hcy, Gly, or GSSG, which demonstrates the
compatibility of our approach with common nucleophiles and reactive species (Figure 4). In the
presence of higher levels of GSH (5 mM), however, we did observe modest inhibition. We do not
view this as a significant problem, however, because about 90% of GSH is localized in the cytosol,
about 10% is localized in the mitochondria and the endoplasmic reticulum, leaving negligible GSH
levels in acidic cellular compartments.35 To further investigate whether this observed inhibition
was observed at physiological pH values, we repeated the measurement at pH 7.4 in the presence
of 5 mM GSH and did not observe a significant difference in efficiency in the absence or presence
of GSH (Figure S9). These data suggest that high concentrations of GSH should not interfere with
application of this triggering motif in normal cellular environments.
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Figure 4. COS/H2S release at 5 h from S-pHTCM (50 µM) in citrate (pH 5.5) in the presence of
CA (25 µg/mL) and various analytes (250 µM unless otherwise noted): no analyte, GSH (5 mM),
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GSH, Cys, Lys, Ser, Hcy, Gly, and GSSG. Experiments were performed in triplicate with results
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expressed as mean ± S.D. (n = 3).
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Although our primary goal was to develop chemistry that enabled donor response within a
specific pH range, we also wanted to demonstrate the feasibility of appending the developed motif
to commonly-used subcellular targeting groups that direct compounds to acidic subcellular
compartments. To demonstrate this compatibility, we prepared a thiocarbamate donor (Lyso-
pHTCM), as well as the associated control compounds (Lyso-pHCM and Lyso-TCM), with an
aminoethyl-morpholine group, which has been used previously to direct compounds to the
lysosome. We measured the H2S release efficiency from the compounds and found that Lyso-
pHTCM showed a 33% H2S release efficiency over 5 h and that the control compounds did not
release H2S (Figure 5). Although beyond the scope of the present investigations, we anticipate that
these compounds may be of use in investigating the role of lysosomal H2S delivery in various
contexts.
Figure 5. (a) H2S release comparison of Lyso-pHTCM and S-pHTCM. (b) H2S release from
Lyso-pHTCM and related control compounds. All experiments performed in quadruplicate in
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citrate buffer (pH 5.5, 10 mM) containing CA (25 µg/mL) at 37 °C. Results expressed as a mean
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Conclusions
Based on the broad utility of hydrolysis- and acid-sensitive H2S donors, we developed a new
strategy that enables for the efficiency of H2S release to be tuned to specific pH ranges. This
response profile is in contrast to currently-available acid-mediated H2S donor motifs. By including
an acid-sensitive imine group on a caged thiocarbamate, we demonstrated that the acid-mediated
imine hydrolysis in combination with inhibitory protonation of aniline product after hydrolysis
resulted in an operative pH response range of 4.6 – 6.5. The imine-based donor also shows good
tolerance to a wide array of biological nucleophiles at physiologically-relevant concentrations. We
also demonstrated the modularity of our approach by attaching a common lysosomal targeting
group to the donor motif and demonstrated that H2S release is still efficient in this targeted
construct. Overall, we anticipate that this approach will provide new opportunities to tune donor
motifs to respond to specific pH windows associated with subcellular organelles and that the
developed chemistry will enable specific pH windows to be targeted to other acidic
microenvironments.
Experimental Section
Materials and methods. Reagents were purchased from Sigma-Aldrich, Tokyo Chemical
Industry (TCI), Fisher Scientific, and VWR. Column chromatography was performed with 230-
400 mesh silica gel. Deuterated solvents were purchased from Cambridge Isotope Laboratories.
13
1H and C{1H} NMR spectra were recorded on a Bruker 500 MHz or Varian Inova 500 MHz
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instrument. Chemical shifts are reported relative to residual protic solvent resonances. Air-free
experimental procedures were performed in an Innovative Atmospheres N2-filled glove box or
using Schlenk technique. UV-Vis spectra were acquired on an Agilent Cary 60 UV-Vis
spectrometer. Mass spectrometric data was acquired by the University of Illinois, Urbana
Champaign MS Facility, or on a Xevo Waters ESI LC/MS instrument.
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General Procedure for H2S Detection. In an N2-filled glovebox, scintillation vials with septa
caps were filled with 20 mL of degassed buffer (citrate/PBS, pH 4.0-8.0, 10 mM). All stock
solutions were prepared in the glovebox with degassed solvents. The 10 mM donor stock solution
was prepared in DMSO, and a 10 mg/mL carbonic anhydrase (CA) stock solution was prepared in
Millipore H2O. Each scintillation was charged with 50 µL of the CA stock solution to achieve a
final concentration of 25 µg/mL. The solutions were thermally equilibrated to 37 °C while stirring
for 20 min. During this time, methylene blue cocktail solutions (0.3 mL) were prepared in 1.5 mL
disposable cuvettes. The methylene blue cocktail solutions contained 60 µL of 1% (w/v)
Zn(OAc)2, 120 µL of 30 mM FeCl3 in 1.2 M HCl, and 120 µL of 20 mM N,N-dimethyl-p-
phenylene diamine in 7.2 M HCl. To each solution, 100 µL of the 10 mM donor stock solution
was added to reach a final concentration of 50 µM. Immediately after donor addition at t = 0 min,
a 0.3 mL reaction aliquot was removed and added to a methylene blue cocktail solution. This
process was repeated at 5, 10, 15, 30, 60, 90, 120, 180, 240, and 300 min time points. The reaction
aliquots added to the methylene blue cocktail solutions were mixed thoroughly and incubated for
1 h at room temperature in the dark, after which the absorbance values at 670 nm were measured.
MB Assay Calibration Curve. The methylene blue cocktail solution (0.5 mL) and PBS pH 7.4
buffer (0.5 mL) was added to 1.5 mL disposable cuvettes. A NaSH stock solution (100 mM) was
prepared in degassed Millipore H2O under an inert atmosphere and diluted to 1 mM. Immediately
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after dilution, aliquots of the NaSH stock solution were added to the methylene blue solutions to
reach final concentrations of 10, 20, 30, 40, and 50 µM. The solutions were mixed and incubated
at room temperature for 1 h, after which the absorbance values at 670 nm were measured.
Syntheses.
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4-(Benzylideneamino)benzyl alcohol. (Modified from previous report.)28 4-Aminobenzyl
alcohol (0.50 g, 4.1 mmol) and benzaldehyde (0.82 mL, 8.2 mmol) were added to anhydrous THF
(15 mL) in the presence of MgSO4 (~1g). Glacial AcOH was then added dropwise (~4 drops) and
the resultant reaction mixture was refluxed for 2 h. The reaction was then quenched with a solution
of saturated NaHCO3 (30 mL) and extracted with EtOAc (3 x 15mL). The organic layers were
combined, washed with deionized H2O (30 mL) and brine (30 mL), and dried over MgSO4. The
drying agent was removed by filtration, and the organic solvent was removed under reduced
pressure. The product was isolated and purified by column chromatography using EtOAc/Hexanes
(10-100% gradient; 5% NEt3) to yield a yellow solid (343 mg, 68% yield). 1H NMR (500 MHz,
DMSO-d6) δ: 8.63 (s, 1H), 8.02 – 7.87 (m, 2H), 7.59 – 7.48 (m, 3H), 7.36 (d, J = 8.2 Hz, 2H), 7.24
(d, J = 8.2 Hz, 2H), 5.18 (t, J = 5.7 Hz, 1H), 4.51 (d, J = 5.7 Hz, 2H).13C{1H} NMR (126 MHz,
DMSO-d6) δ: 160.1, 149.9, 140.5, 136.1, 131.4, 128.8, 128.6, 127.3, 120.8, 62.6. HRMS (ASAP
TOF) (m/z): [M + H]+ cacld for C14H14NO, 212.1075; found, 212.1090.
S-4-(Benzylideneamino)benzyl
phenylcarbamothioate
(S-pHTCM).
4-
(Benzylideneamino)-benzyl alcohol (60.0 mg, 0.284 mmol) and phenyl isothiocyanate (41 µL,
0.34 mmol) were combined in anhydrous THF (15 mL). The reaction mixture was cooled to 0 °C
before adding NaH (18 mg, 0.75 mmol). The reaction mixture was then stirred at room temperature
for 2 h. The solvent was removed under reduced pressure, and the crude mixture was purified by
column chromatography with EtOAc/Hexanes (4-34% gradient; 5% NEt3). The product was
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isolated as a yellow solid, which isomerizes from the O-alkyl to S-alkyl isomer over 3 days in the
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solid state (73 mg, 74% yield). H NMR (500 MHz, DMSO-d6) δ: 10.35 (s, 1H), 8.62 (s, 1H),
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8.02–7.82 (m, 2H), 7.59–7.44 (m, 5H), 7.41 (d, J = 8.2 Hz, 2H), 7.31 (dd, J = 8.4, 7.2 Hz, 2H),
7.23 (d, J = 8.2 Hz, 2H), 7.05 (t, J = 7.4 Hz, 1H), 4.19 (s, 2H). 13C{1H} NMR (126 MHz, DMSO-
d6) δ: 164.3, 160.6, 150.3, 138.9, 136.6, 136.0, 131.5, 129.6, 128.9, 128.8, 128.6, 123.4, 121.1,
119.0, 32.6. HRMS (ES+ TOF) (m/z): [M + H]+ cacld for C21H19N2OS, 347.1218; found,
347.1212.
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4-(Benzylideneamino)benzyl phenylcarbamate (pHCM). 4-(Benzylideneamino)benzyl
alcohol (61 mg, 0.28 mmol) and phenyl isocyanate (31 µL, 0.28 mmol) were added to anhydrous
THF (15 mL). The reaction mixture was cooled to 0 °C before addition of NEt3 (0.50 mL, 3.6
mmol), after which the reaction mixture was refluxed for 2 h. The reaction was quenched with
brine (30 mL) and extracted with EtOAc (3 x 15 mL). The organic layers were combined, washed
with deionized H2O (30 mL) and brine (30 mL), and dried over MgSO4. The drying agent was
removed by filtration and the solvent was removed under reduced pressure. The crude mixture was
purified by column chromatography with EtOAc/Hexanes (4-34% gradient; 5% NEt3) to afford
the product as a white solid (41 mg, 44%). 1H NMR (500 MHz, DMSO-d6) δ: 9.76 (s, 1H), 8.63
(s, 1H), 7.94 (dd, J = 7.3, 2.2 Hz, 2H), 7.63–7.37 (m, 7H), 7.37–7.20 (m, 4H), 6.99 (t, J = 7.4 Hz,
1H), 5.17 (s, 2H).13C{1H} NMR (126 MHz, DMSO-d6) δ: 161.0, 153.4, 151.3, 139.1, 136.0, 134.3,
131.6, 129.3, 128.8, 128.8, 128.7, 122.4, 121.1, 118.2, 65.5. HRMS (ES+ TOF) (m/z): [M + H]+
cacld for C21H19N2O2, 331.1447; found, 331.1435.
S-Benzyl phenylcarbamothioate (S-TCM). Benzyl mercaptan (61 µL, 0.52 mmol) and phenyl
isocyanate (58 µL, 0.51 mmol) were combined in anhydrous THF (15 mL). The reaction mixture
was cooled to 0 °C before adding NEt3 (0.1 mL), after which the reaction was stirred at room
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temperature for 1 h. The reaction was then quenched with brine (30 mL) and extracted with EtOAc
(3x15mL). The organic layers were combined and dried over MgSO4. The organic layer was
filtered, and the solvent was removed under reduced pressure. The product was isolated by column
chromatography using EtOAc/Hexanes (4-34% gradient; 5% NEt3) to afford the product as a white
solid (120 mg, 92% yield). 1H NMR (500 MHz, DMSO-d6) δ: 10.33 (s, 1H), 7.55–7.46 (m, 2H),
7.39 – 7.20 (m, 7H), 7.05 (t, J = 7.4 Hz, 1H), 4.15 (s, 2H). 13C{1H} NMR (126 MHz, DMSO-d6)
δ: 164.3, 138.9, 138.7, 128.9, 128.7, 128.4, 127.0, 123.4, 119.0, 32.9. HRMS (ASAP TOF) (m/z):
[M + H]+ cacld for C14H14NOS, 244.0796; found, 244.0791.
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4-(2-Aminoethyl)morpholine phenyl isothiocyanate. 4-(2-Morpholinoethyl) aniline (61 mg,
0.30 mmol) was added to anhydrous CH2Cl2 (20 mL). The reaction mixture was cooled to °C, then
a solution of 1,1′-thiocarbonyldiimidazole (53 mg, 0.30 mmol) in anhydrous CH2Cl2 (5 mL) was
added dropwise. The reaction was stirred at room temperature for 2 h, after which the solvent was
removed under reduced pressure. The crude mixture was purified by column chromatography with
MeOH/CH2Cl2 (2-20% gradient) and isolated as a yellow oil (180 mg, 86% yield). 1H NMR (500
MHz, CDCl3) δ 7.22 – 7.11 (m, 4H), 3.73 (t, J = 4.6 Hz, 4H), 2.79 (dd, J = 9.7, 6.3 Hz, 2H), 2.57
(dd, J = 9.6, 6.4 Hz, 2H), 2.50 (t, J = 4.6 Hz, 4H). 13C{1H} NMR (126 MHz, CDCl3) δ 140.0,
135.0, 130.0, 129.3, 125.9, 67.1, 60.5, 53.8, 33.1. HRMS (ASAP TOF) (m/z): [M + H]+ cacld for
C13H17N2OS, 249.1062; found, 249.1078.
S-4-(Benzylideneamino)benzyl(4-(2-morpholinoethyl)phenyl) carbamothioate (Lyso-
pHTCM). 4-(Benzylideneamino)benzyl alcohol (100 mg, 0.473 mmol) and morpholine
isothiocyanate (118 mg, 0.473 mmol) were added to anhydrous THF (20 mL). The reaction
mixture was cooled to 0 °C before addition of NaH (18 mg, 0.75 mmol) and then stirred for 10 h
at room temperature. The solvent was removed under reduced pressure. The crude mixture was
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purified by column chromatography with EtOAc/Hexanes (8-66% gradient; 5% NEt3) to afford
the product as a white solid that isomerizes from the O-alkyl to S-alkyl isomer over 4 days (77 mg,
36%).1H NMR (500 MHz, DMSO-d6) δ: 10.26 (s, 1H), 8.61 (s, 1H), 7.95–7.89 (m, 2H), 7.56–7.48
(m, 3H), 7.43–7.37 (m, 4H), 7.22 (d, 2H), 7.15 (d, 2H), 4.17 (s, 2H), 3.56 (d, J = 5.0 Hz, 4H), 2.67
(t, J = 7.7 Hz, 2H), 2.46 (t, 1H), 2.40 (s, 4H). 13C{1H} NMR (126 MHz, DMSO-d6) δ: 164.6, 161.0,
150.7, 137.3, 137.1, 136.5, 136.0, 132.0, 130.1, 129.5, 129.3, 129.1, 121.6, 119.6, 66.7, 60.5, 53.7,
33.1, 32.3. HRMS (ES+ TOF) (m/z): [M + H]+ cacld for C27H30N3O2S, 460.2059; found, 460.2076.
S-4-(Benzylideneamino)benzyl(4-(2-morpholinoethyl)phenyl) carbamate (Lyso-pHCM).
4-(2-Morpholinoethyl) aniline (61 mg, 0.30 mmol) and triphosgene (41 mg, 0.36 mmol) were
combined in anhydrous CH2Cl2 (15 mL). The reaction was cooled to 0 °C and NEt3 (0.2 mL, 5
equiv.) was added. After stirring at 0 °C for 2 h, the reaction mixture was purged with N2 before
adding a solution of phenyl imine benzyl alcohol (74 mg, 0.35 mmol) in anhydrous CH2Cl2 (5 mL)
dropwise. The reaction was stirred at room temperature for 2 h, after which the solvent was
removed under reduced pressure. The crude mixture was purified by column chromatography with
EtOAc/Hexanes (12-100% gradient; 5% NEt3) to yield the product as a white solid (70.1 mg, 54%
yield). 1H NMR (500 MHz, DMSO-d6) δ: 9.66 (s, 1H), 8.63 (s, 1H), 7.98–7.90 (m, 2H), 7.57–7.50
(m, 3H), 7.50–7.44 (m, 2H), 7.37 (d, J = 8.1 Hz, 2H), 7.33–7.26 (m, 2H), 7.17–7.09 (m, 2H), 5.15
(s, 2H), 3.56 (t, J = 4.7 Hz, 4H), 2.66 (dd, J = 9.3, 6.4 Hz, 2H), 2.46 (dd, J = 9.2, 6.5 Hz, 2H), 2.40
(s, 4H). 13C{1H} NMR (126 MHz, DMSO-d6) δ: 160.9, 153.4, 151.2, 136.9, 135.9, 134.4, 134.3,
131.6, 129.2, 128.9, 128.8, 128.7, 121.0, 118.2, 66.2, 65.4, 60.2, 53.3, 31.8. HRMS (ASAP_TOF)
(m/z): [M + H]+ cacld for C27H30N3O3, 444.2287; found, 444.2282.
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S-Benzyl(4-(2-morpholinoethyl)phenyl)
carbamothioate
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4-(2-
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in anhydrous CH2Cl2 (15 mL). The reaction mixture was cooled to 0 °C and NEt3 (0.20 mL, 1.5
mmol) was added. The reaction mixture was warmed from 0 °C to room temperature and stirred
overnight, after which it was purged with N2 before adding benzyl mercaptan (41 µL, 0.35 mmol)
dropwise. The reaction mixture was stirred at room temperature for 10 h. The solvent was removed
under reduced pressure, and the product was purified by column chromatography with
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MeOH/CH2Cl2 2-20% gradient to afford the product as a yellow solid (41 mg, 39% yield). H
NMR (500 MHz, DMSO-d6) δ 10.25 (s, 1H), 7.43–7.28 (m, 6H), 7.27–7.21 (m, 1H), 7.18–7.12
(m, 2H), 4.14 (s, 2H), 3.56 (t, J = 4.7 Hz, 4H), 2.67 (t, 2H), 2.47 (t, J = 8.0 Hz, 1H), 2.40 (s, 4H).
13C: NMR (126 MHz, DMSO-d6) δ: 164.6, 139.3, 137.3, 129.5, 129.2, 128.9, 127.4, 119.5, 66.7,
60.5, 55.4, 53.7, 40.5, 33.4, 32.3. HRMS (ES+ TOF) (m/z): [M + H]+ cacld for C20H25N2O2S,
357.1637; found, 357.1641.
ASSOCIATED CONTENT
Supporting Information
H2S release data, HPLC data, and NMR Spectra.
AUTHOR INFORMATION
Corresponding Author
pluth@uoregon.edu
ACKNOWLEDGMENT
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This work was supported by the NIH (MDP; R01GM113030), NSF/GRFP (AKG; DGE-1842486),
and the Dreyfus Foundation. The NMR and MS facilities in CAMCOR are supported through NSF
awards CHE-0923589 and CHE-1625529.
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References
5
6
1. Wang, R. Physiological Implications of Hydrogen Sulfide: A Whiff Exploration that
7
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Blossomed. Physiol. Rev. 2012, 92, 791-896.
9
2. Benavides, G. A.; Squadrito, G. L.; Mills, R. W.; Patel, H. D.; Isbell, T. S.; Patel, R. P.;
Darley-Usmar, V. M.; Doeller, J. E.; Kraus, D. W. Hydrogen sulfide mediates the vasoactivity of
garlic. Proc. Natl. Acad. Sci. USA 2007, 104, 17977-17982.
10
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12
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48
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52
53
54
55
56
57
58
59
60
3. Zhao, W.; Zhang, J.; Lu, Y.; Wang, R. The vasorelaxant effect of H2S as a novel endogenous
gaseous KATP channel opener. EMBO J. 2001, 20, 6008-6016.
4. Calvert, J. W.; Jha, S.; Gundewar, S.; Elrod, J. W.; Ramachandran, A.; Pattillo, C. B.; Kevil,
C. G.; Lefer, D. J. Hydrogen sulfide mediates cardioprotection through Nrf2 signaling.
Circulation Res. 2009, 105, 365-374.
5. Whiteman, M.; Li, L.; Rose, P.; Tan, C.-H.; Parkinson, D. B.; Moore, P. K. The effect of
hydrogen sulfide donors on lipopolysaccharide-induced formation of inflammatory mediators in
macrophages. Antioxid. Redox Signal. 2010, 12, 1147-1154.
6. Zhao, Y.; Biggs, T. D.; Xian, M. Hydrogen sulfide (H2S) releasing agents: chemistry and
biological applications. Chem. Commun. 2014, 50, 11788-11805.
7. Hartle, M. D.; Pluth, M. D. A practical guide to working with H2S at the interface of
chemistry and biology. Chem. Soc. Rev. 2016, 45, 6108-6117.
8. Powell, C. R.; Dillon, K. M.; Matson, J. B. A review of hydrogen sulfide (H2S) donors:
Chemistry and potential therapeutic applications. Biochem. Pharmacol. 2018, 149, 110-123.
9. Li, L.; Whiteman, M.; Guan, Y. Y.; Neo, K. L.; Cheng, Y.; Lee, S. W.; Zhao, Y.; Baskar, R.;
Tan, C. H.; Moore, P. K. Characterization of a novel, water-soluble hydrogen sulfide - Releasing
molecule (GYY4137): New insights into the biology of hydrogen sulfide. Circulation 2008, 117,
2351-2360.
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10. Devarie-Baez, N. O.; Bagdon, P. E.; Peng, B.; Zhao, Y.; Park, C.-M.; Xian, M. Light-
induced hydrogen sulfide release from “caged” gem-dithiols. Org. Lett. 2013, 15, 2786-2789.
11. Shukla, P.; Khodade, V. S.; SharathChandra, M.; Chauhan, P.; Mishra, S.; Siddaramappa,
S.; Pradeep, B. E.; Singh, A.; Chakrapani, H. "On demand" redox buffering by H2S contributes
to antibiotic resistance revealed by a bacteria-specific H2S donor. Chem. Sci. 2017, 8, 4967-
4972.
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12. Kang, J. M.; Li, Z.; Organ, C. L.; Park, C. M.; Yang, C. T.; Pacheco, A.; Wang, D. F.;
Lefer, D. J.; Xian, M. pH-Controlled Hydrogen Sulfide Release for Myocardial Ischemia-
Reperfusion Injury. J. Am. Chem. Soc. 2016, 138, 6336-6339.
13. Elrod, J. W.; Calvert, J. W.; Morrison, J.; Doeller, J. E.; Kraus, D. W.; Tao, L.; Jiao, X. Y.;
Scalia, R.; Kiss, L.; Szabo, C.; Kimura, H.; Chow, C. W.; Lefer, D. J. Hydrogen sulfide
attenuates myocardial ischemia-reperfusion injury by preservation of mitochondrial function.
Proc. Natl. Acad. Sci. USA 2007, 104, 15560-15565.
14. Wu, D.; Si, W.; Wang, M.; Lv, S.; Ji, A.; Li, Y. Hydrogen sulfide in cancer: friend or foe?
Nitric Oxide 2015, 50, 38-45.
15. Steiger, A. K.; Pardue, S.; Kevil, C. G.; Pluth, M. D. Self-Immolative Thiocarbamates
Provide Access to Triggered H2S Donors and Analyte Replacement Fluorescent Probes. J. Am.
Chem. Soc. 2016, 138, 7256-7259.
16. Powell, C. R.; Foster, J. C.; Okyere, B.; Theus, M. H.; Matson, J. B. Therapeutic delivery
of H2S via COS: small molecule and polymeric donors with benign byproducts. J. Am. Chem.
Soc. 2016, 138, 13477-13480.
17. Steiger, A. K.; Zhao, Y.; Pluth, M. D. Emerging Roles of Carbonyl Sulfide in Chemical
Biology: Sulfide Transporter or Gasotransmitter? Antioxid. Redox Signal. 2018, 28, 1516-1532.
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18. Zhao, Y.; Pluth, M. D. Hydrogen Sulfide Donors Activated by Reactive Oxygen Species.
Angew. Chem. Int. Ed. 2016, 55, 14638-14642.
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19. Zhao, Y.; Henthorn, H. A.; Pluth, M. D. Kinetic insights into hydrogen sulfide delivery
from caged-carbonyl sulfide isomeric donor platforms. J. Am. Chem. Soc. 2017, 139, 16365-
16376.
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20. Chauhan, P.; Jos, S.; Chakrapani, H. Reactive Oxygen Species-Triggered Tunable
Hydrogen Sulfide Release. Org. Lett. 2018, 20, 3766-3770.
21. Chauhan, P.; Bora, P.; Ravikumar, G.; Jos, S.; Chakrapani, H. Esterase Activated Carbonyl
Sulfide/Hydrogen Sulfide (H2S) Donors. Org. Lett. 2017, 19, 62-65.
22. Steiger, A. K.; Marcatti, M.; Szabo, C.; Szczesny, B.; Pluth, M. D. Inhibition of
Mitochondrial Bioenergetics by Esterase-Triggered COS/H2S Donors. ACS Chem. Biol. 2017,
12, 2117-2123.
23. Levinn, C. M.; Steiger, A. K.; Pluth, M. D. Esterase-Triggered Self-Immolative
Thiocarbamates Provide Insights into COS Cytotoxicity. ACS Chem. Biol. 2019, 14, 170-175.
24. Zhao, Y.; Bolton, S. G.; Pluth, M. D. Light-Activated COS/H2S Donation from
Photocaged Thiocarbamates. Org. Lett. 2017, 19, 2278-2281.
25. Sharma, A. K.; Nair, M.; Chauhan, P.; Gupta, K.; Saini, D. K.; Chakrapani, H. Visible-
Light-Triggered Uncaging of Carbonyl Sulfide for Hydrogen Sulfide (H2S) Release. Org. Lett.
2017, 19, 4822-4825.
̌
26. Stacko, P.; Muchova, L.; Vıtek, L.; Klan, P. Visible to NIR light photoactivation of
́
́
́
hydrogen sulfide for biological targeting. Org. Lett. 2018, 20, 4907-4911.
27. Zhao, Y.; Steiger, A. K.; Pluth, M. D. Cysteine-activated hydrogen sulfide (H2S) delivery
through caged carbonyl sulfide (COS) donor motifs. Chem. Commun. 2018, 54, 4951-4954.
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Page 22 of 22
1
2
3
4
5
6
7
8
9
28. Müller, I. A.; Kratz, F.; Jung, M.; Warnecke, A. Schiff bases derived from p-aminobenzyl
alcohol as trigger groups for pH-dependent prodrug activation. Tetrahedron Lett. 2010, 51, 4371-
4374.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
29. Lloyd-Jones, G. C.; Moseley, J. D.; Renny, J. S. Mechanism and application of the
Newman-Kwart O→ S rearrangement of O-aryl thiocarbamates. Synthesis 2008, 2008, 661-689.
30. Zonta, C.; De Lucchi, O.; Volpicelli, R.; Cotarca, L., Thione–Thiol Rearrangement:
Miyazaki–Newman–Kwart Rearrangement and Others. In Sulfur-Mediated Rearrangements II,
Springer: 2006; pp 131-161.
31. Perkowski, A. J.; Cruz, C. L.; Nicewicz, D. A. Ambient-Temperature Newman-Kwart
Rearrangement Mediated by Organic Photoredox Catalysis. J. Am. Chem. Soc. 2015, 137,
15684-15687.
32. Pedersen, S. K.; Ulfkjaer, A.; Newman, M. N.; Yogarasa, S.; Petersen, A. U.; Solling, T. I.;
Pittelkow, M. Inverting the Selectivity of the Newman-Kwart Rearrangement via One Electron
Oxidation at Room Temperature. J. Org. Chem. 2018, 83, 12000-12006.
33. Eriksen, K.; Ulfkjaer, A.; Solling, T. I.; Pittelkow, M. Benzylic Thio and Seleno Newman-
Kwart Rearrangements. J. Org. Chem. 2018, 83, 10786-10797.
34. The MB assay is generally not suitable for use with acid-labile H2S donors because of the
strongly acidic conditions required during the assay, but we expected that such strongly acidic
conditions would protonate the aniline intermediate and prevent release of COS/H2S.
35. Lu, S. C. Regulation of hepatic glutathione synthesis: current concepts and controversies.
FASEB J. 1999, 13, 1169-1183.
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