Inhibition of Mitochondrial Bioenergetics by Esterase-Triggered COS/H2S Donors

Article abstract of DOI:10.1021/acschembio.7b00279

Hydrogen sulfide (H₂S) is an important biological mediator, and synthetic H₂S donating molecules provide an important class of investigative tools for H₂S research. Here, we report esterase-activated H₂S donors that function by first releasing carbonyl sulfide (COS), which is rapidly converted to H₂S by the ubiquitous enzyme carbonic anhydrase (CA). We report the synthesis, self-immolative decomposition, and H₂S release profiles of the developed scaffolds. In addition, the developed esterase-triggered COS/H₂S donors exhibit higher levels of cytotoxicity than equivalent levels of Na₂S or the common H₂S donors GYY4137 and AP39. Using cellular bioenergetics measurements, we establish that the developed donors reduce cellular respiration and ATP synthesis in BEAS 2B human lung epithelial cells, which is consistent with COS/H₂S inhibition of cytochrome c oxidase in the mitochondrial respiratory chain although not observed with common H₂S donors at the same concentrations. Taken together, these results may suggest that COS functions differently than H₂S in certain biological contexts or that the developed donors are more efficient at delivering H₂S than other common H₂S-releasing motifs.

Full text of DOI:10.1021/acschembio.7b00279

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Article
Inhibition of Mitochondrial Bioenergetics
by Esterase-Triggered COS/H2S Donors
Andrea K Steiger, Michela Marcatti, Csaba Szabo, Bartosz Szczesny, and Michael D Pluth
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00279 • Publication Date (Web): 14 Jun 2017
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Inhibition of Mitochondrial Bioenergetics by Esterase-Triggered COS/H2S Donors
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Andrea K. Steiger,a Michela Marcatti,b Csaba Szabo,b Bartosz Szczesny,b Michael D. Plutha,*
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Department of Chemistry and Biochemistry, Materials Science Institute, Institute of Molecular
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Biology. University of Oregon, Eugene, OR 97403, USA
b Department of Anaesthesiology, University of Texas Medical Branch, Galveston, TX 77555, USA.
Abstract
Hydrogen sulfide (H2S) is an important biological mediator, and synthetic H2S donating molecules
provide an important class of investigative tools for H2S research. Here we report esterase-activated
H2S donors that function by first releasing carbonyl sulfide (COS), which is rapidly converted to H2S
by the ubiquitous enzyme carbonic anhydrase (CA). We report the synthesis, self-immolative
decomposition, and H2S release profiles of the developed scaffolds. In addition, the developed
esterase-triggered COS/H2S donors exhibit higher levels of cytotoxicity than equivalent levels of Na2S
or the common H2S donors GYY4137 and AP39. Using cellular bioenergetics measurements, we
establish that the developed donors reduce cellular respiration and ATP synthesis in BEAS 2B human
lung epithelial cells, which is consistent with COS/H2S inhibition of cytochrome c oxidase in the
mitochondrial respiratory chain although not observed with common H2S donors at the same
concentrations. Taken together, these results may suggest that COS functions differently than H2S in
certain biological contexts or that the developed donors are more efficient at delivering H2S than other
common H2S-releasing motifs.
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Introduction
Hydrogen sulfide (H2S) is an endogenously-produced signaling molecule that plays critical roles in
mammalian biology. Physiological sulfide levels are tightly regulated, and enzymatic production
derives primarily from cysteine and homocysteine metabolism by cystathionine--synthase (CBS),
cystathionine--lyase (CSE), and 3-mercaptopyruvate transferase (3-MST).1 Continually broadening in
scope, H2S plays important roles in cardioprotection,2 inflammation,3 vasodilation,4 as well as other
processes. Because of this diversity, there is significant interest in developing both research and
therapeutic strategies for regulating sulfide levels in different biological contexts.5 At the biological
level, inhibition, knockout, and/or overexpression of H2S-producing enzymes can be used to modulate
endogenous H2S levels. Alternatively, chemical approaches using exogenous H2S donation often
provide a more convenient and broader approach. For example, inorganic sulfide salts, such as NaSH
and Na2S provide a convenient source of sulfide, however, the large and instantaneous bolus of
sulfide released from such salts is often rapidly oxidized and fails to mimic the continuous H2S release
associated with enzymatic synthesis, thus limiting the utility of these exogenous H2S sources.6
Because of these limitations, developing small molecules that undergo specific reactions to release
H2S in a controlled manner to more closely mimic well-regulated enzymatic production remains an
important goal.7, 8 Aligned with these needs, naturally-occurring polysulfides such as diallyl trisulfide
(DATS), which releases sulfide upon reaction with thiols,9, 10 hydrolysis-based H2S donors such as
GYY4137 and ADT-OH,11, 12 which slowly produce H2S in water, and a palette of thiol-activated
donors, have all been developed.7, 13 Although such donors have been used in applications ranging
from probe development to in vivo studies, key challenges include low H2S donation efficiencies from
hydrolysis-based donors and thiol consumption and redox perturbation from polysulfides and thiol-
activated donors.
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As a step toward addressing these challenges, our group recently developed a new H2S donation
strategy based on the intermediate release of carbonyl sulfide (COS), which is rapidly hydrolyzed to
H2S by the ubiquitous enzyme carbonic anhydrase (CA).14 By leveraging well-established work on the
triggered decomposition of benzylic carbamates commonly used in pro-drug and fluorophore release
strategies,15-17 we demonstrated that self-immolative thiocarbamates can be readily engineered to
respond to different stimuli and release COS/H2S. Specifically, cleavage of an analyte-specific
protecting group unmasks a phenol, which then undergoes a self-immolative decomposition to release
COS. Importantly, this strategy enables significant control over H2S donation depending on the trigger
and provides access to important carbamate control compounds, which release CO2/H2O rather than
COS/H2S. Furthermore, although COS hydrolysis by CA is rapid, some evidence suggests that COS
itself may have unique roles in chemical biology, as evidenced by COS detection in the headspace
above ex vivo porcine coronary arteries and by increased COS levels in exhaled breath from patients
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with cystic fibrosis, organ rejection, or liver disease.18-21 Therefore, although self-immolative
thiocarbamates have recently been shown to be useful as responsive H2S donors, they may also
provide a platform for future studies of COS chemical biology.22 Following our initial report of caged
COS/H2S release, we have expanded this approach to include COS/H2S donors activated by reactive
oxygen species (ROS),23 bio-orthogonal “click-and-release” donors based on trans-cyclooctene /
tetrazine click chemistry,24 and light-activated COS/H2S donors.25 Related COS-based donors based
on nucleophilic addition to small molecule and polymeric cyclic N-thiocarboxyanhydrides resulting in
the release of COS have also emerged recently.26 Although the nucleophile-based donors provide
slow H2S release akin to enzymatic synthesis, the required consumption of cellular nucleophiles, such
as thiols, to release COS/H2S is similar to other thiol-activated donors.
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To address these limitations, we viewed that installation of an ester as the triggering group to
thiocarbamate-based platforms would provide access to slow-release COS/H2S donors upon ester
cleavage by intracellular esterases that do not require consumption of cellular nucleophiles for
activation (Scheme 1). The strategy of using intracellular esterases to cleave esterified moieties on
small molecules is a well-established method used extensively to impart cellular trappability, improve
membrane permeability, and in the activation of caged pro-drugs and other biological payloads.27-30
Additionally, activation by intracellular esterases eliminates the consumption of cellular nucleophiles
for activation. Consistent with this design strategy, the Wang group recently reported esterase-
triggered H2S donors utilizing a trimethyl lock unmasking of caged thioacids and demonstrated their
anti-inflammatory effects.31 Similarly, during the preparation of this manuscript an esterase-activated
S-alkyl thiocarbamate COS/H2S donor was reported, but detailed biological applications were not
investigated.32 Here, we report the design, evaluation, and application of esterase-activated COS/H2S
donors and provide the first insights into the influence of COS donors on cellular toxicology and
mitochondrial bioenergetics.
Scheme 1. Design of esterase-triggered self-immolative COS/H2S donors and proposed COS/H2S
release mechanism.
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Results and Discussion
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Triggered COS/H2S donors benefit from a high degree of modularity and facile introduction of
different triggering functionalities. To access an esterase-functionalized thiocarbamate, we first
prepared 4-pivaloyl benzyl alcohol in one step,33 which was then treated with p-tolylisothiocyanate in
the presence of DBU to afford donor 1 in moderate yield (Scheme 2). The choice of pivalic acid to
construct the donor was due to the increased hydrolytic stability of pivalic ester over other ester
protecting groups, and 4-methyl aniline was used as the payload of the donor to provide a simple
spectroscopic handle. The analogous carbamate control 2 was prepared in good yield by treating 4-
pivaloyl benzyl alcohol with p-tolylisocyanate and DBU. Carbamate 2 provides an important control
compound that undergoes the same self-immolative decomposition as 1, but releases CO2 instead of
COS, thus enabling separation of the action of COS/H2S from that of the organic byproducts formed
after donor activation. Additionally, we also prepared triggerless thiocarbamate 3 as a control
compound to test the thiocarbamate stability toward esterases.
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Scheme 2. (a) Synthesis of H2S donor 1, CO2-releasing analogue 2, and triggerless thiocarbamate 3.
The mechanism of the cascade decomposition of similar benzyl carbamates has established
previously to release CO2, the amine payload, and a quinone methide intermediate.15-17, 34 The final
fate of the electrophilic quinone methide is dependent on which nucleophiles are present in the
system. For example, it is often scavenged by water to form 4-hydroxy benzylalcohol, or can be
scavenged by biological nucleophiles to afford thiol or amine-derived conjugates.35, 36 To confirm
similar reactivity in the present system, we first demonstrated that the addition of porcine liver
esterase (PLE) initiated a self-immolative decomposition reaction as anticipated. After stirring 1 (14
mM) with PLE (28 U/mL) in PBS (pH 7.4) with 10% DMSO for 48 hours, the organic layer was
extracted and analyzed by NMR spectroscopy and mass spectrometry (Figure 1). Loss of the benzylic
1
and thiocarbamate N-H protons at ~5.5 ppm and 11.1 ppm, respectively, in the H NMR spectrum of
the reaction mixture confirmed self-immolation (Figure 1a). As further evidence of the triggered
cascade decomposition, the broad NMR resonances characteristic of O-alkyl thiocarbamates, which is
due to the slow rotation around the thiocarbamate moiety on the NMR time scale, sharpen
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significantly upon ester cleavage with PLE. Additionally, the 13C{1H} NMR spectrum after treatment
with PLE (Figure 1b), clearly showed the loss of the C=S and benzylic carbon resonances at 185 and
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70 ppm, respectively. Both the H and 13C{1H} NMR spectra also show the formation of new aromatic
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species corresponding to several products, which were further characterized using mass
spectrometry. Because the generated p-quinone methide is electrophilic, we expect that it would be
scavenged by biological nucleophiles, such as thiols, amines, or water, under physiological
conditions. Given that similar scaffolds have been reported previously for drug and fluorophore
delivery, no significant biological interference is anticipated from these side-reactions of the
byproducts alone. The mass spectrum acquired of the reaction mixture after treatment with PLE
clearly showed formation of p-toluidine (m/z: 107.0) and the product corresponding to p-toluidine
trapping of the o-quinone methide intermediate (m/z: 214.1) as expected (See Figure S1 for MS data
and formation details). Taken together, the NMR spectroscopy and mass spectrometry studies
confirm self-immolation of the scaffold as described in Scheme 1.
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Figure 1. (a) H NMR spectrum of 1 before (top) and after (bottom) treatment with PLE. (b) 13C{1H}
NMR spectrum of 1 before (top) and after (bottom) stirring with PLE.
With esterase-triggered donors in hand, we first confirmed that 1 was stable in aqueous solution in
the absence of esterase. We next verified that addition of 1 to pH 7.4 PBS buffer containing
physiologically relevant levels of CA (25 µg/mL) did not result in H2S generation when monitored
using an H2S-selective electrode, confirming that the esterase does not cleave thiocarbamates directly
(Figure 2a). Further control experiments using the parent benzyl thiocarbamate 3, which lacks the
ester trigger, confirmed that the benzyl thiocarbamate moiety is not cleaved directly by PLE. Having
confirmed the stability of the donor platform prior to activation, we next treated 1 with 1 U/mL PLE in
the presence of CA and observed immediate H2S release (Figure 2a). As expected, increasing the
PLE concentration to 20 U/mL under otherwise identical conditions resulted in significantly faster H2S
release. Additionally, treatment of 1 with acetazolamide (AAA), a known CA inhibitor, significantly
reduced the rate of H2S production, confirming that CA is necessary for COS conversion to H2S under
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the reaction conditions. Supplementing the H2S electrode measurements, we also confirmed H2S
release from 1 using an H2S-responsive fluorescent probe (Figure 2b). Consistent with the electrode
data, incubation of 50 M 1 with 5 M MeRho-Az37 in the presence of CA and 1 U/mL PLE resulted in
a fluorescence turn-on consistent with H2S release. We also attempted to obtain fluorescent live cell
images by incubating 1 in BEAS 2B cells with MeRho-Az, but the high cytotoxicity of 1 and limited
permeability of MeRho-Az in BEAS 2B cells limited our ability to obtain high quality images. As a
whole, these data demonstrate that the thiocarbamate donors are stable until activated by esterases
and release H2S in a COS-dependent manner.
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Figure 2. (a) H2S release from 1 in PBS (pH 7.4) in the presence of CA (25 µg/mL) with 1 U/mL
(green trace) or 20 U/mL (blue trace) PLE. Addition of CA inhibitor AAA (2.5 µM) significantly reduces
H2S release (black trace). No H2S release was observed from 1 in the absence of esterase (grey
trace) or from thiocarbamate 3 lacking an ester trigger (brown trace). (b) Detection of H2S released
from 1 with the H2S-responsive probe MeRho-Az. Conditions: 50 µM 1, 5 µM MeRho-Az, 25 µg/mL
CA, 1 unit/mL PLE, 3 mL PBS (pH 7.4). 37 C, ex= 476 nm, em= 480-650 nm.
We next investigated the cytotoxicity of 1-3 on BEAS 2B, human lung epithelial cells, by
measuring the reduction of a tetrazolium compound (MTT) to formazan by metabolically active cells,
and by measuring the release of lactate dehydrogenase (LDH) due to permeability of the plasma
membrane, which is a sign of necrotic cell death. BEAS 2B cells exhibit low expression of all three
canonical H2S-producing enzymes, CBS, CSE, and 3-MST.38 To provide suitable comparisons with
commonly-used synthetic donors, we first obtained comparable cytotoxicity data for known H2S
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donors GYY4137, which does not localize in the mitochondria, and also with mitochondrially-targeted
AP39 (Figure 3a,b).39 When compared to the DMSO vehicle, GYY4137 showed no significant
cytotoxicity up to 10 µM using either the MTT or LDH assay. Similarly, AP39 did not exhibit
cytotoxicity at 10 µM nor at lower concentrations, indicating that neither of these H2S donors are
significantly cytotoxic. Additionally, no cytotoxicity was observed when BEAS 2B cells were treated
with up to 10 µM of Na2S, a direct source of sulfide (See Figure S2 for MTT and LDH assays with
Na2S). By contrast, 10 µM of 1 resulted a significant decrease cell viability and increase in LDH levels,
which was not observed for control compounds 2 or 3 (Figure 3c,d). The lack of cytotoxicity of control
compound 2 suggests that the mechanism of cytotoxicity does not result from the formation of the
electrophilic p-quinone methide intermediate40 because this species is formed upon activation of both
donor 1 and control compound 2. Similarly, compound 3 does not reduce cell viability, confirming that
the observed cytotoxicity of 1 relies on triggering by cellular esterases and is not a result of the
thiocarbamate scaffold itself. We note that although the extensive cytotoxicity data reported is only in
BEAS 2B cells, preliminary cytotoxicity evaluations in HeLa cells additionally showed increased
cytotoxicity of donor 1 below 100 µM with no cytotoxicity observed for control compound 2 (See
Figure S3 for viability evaluation in HeLa cells). Importantly, the esterase-triggered COS/H2S donor 1
provides a significantly different toxicological profile from other commonly-used H2S donors.
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Figure 3. Cell viability studies of AP39, GYY4137, and 1-3 in BEAS 2B using the (a,c) MTT and (b,d)
LDH cell viability assays. The chemical structures of AP39 and GYY4137 are shown in the upper right
panel.
To investigate the underpinnings of the increased cytotoxicity of 1, and because H2S is a well-
known inhibitor of mitochondrial cytochrome c oxidase,41 we analyzed the mitochondrial respiration of
BEAS 2B cells treated with 1-3 using an Extracellular Flux Analyzer.42 We chose this assay because
H2S is well known to impact mitochondrial respiration, even if the H2S is not delivered directly to the
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mitochondria, and because COS has been suggested to inhibit the mitochondrial respiratory chain. An
Extracellular Flux Analyzer measures the cellular oxygen consumption rate (OCR) as an indicator of
mitochondrial respiration in real time. Using this technique, we were able to probe three key
parameters of mitochondrial function upon exposure to compounds 1-3 as well as AP39 and
GYY4137 through sequential addition of oligomycin (an ATP synthase inhibitor), carbonyl cyanide 4-
(trifluoromethoxy)phenylhydrazone (a mitochondrial uncoupler), and a combination of rotenone and
antimycin A (complex I and complex III inhibitors, respectively).43 As expected, incubation of BEAS 2B
cells with increasing concentrations of 1 for 24 hours negatively affected all major bioenergetics
parameters, namely oxygen consumption linked with basal respiration, maximal respiration, and ATP
synthesis (Figure 4a-f). These reductions are consistent with known inhibitory effects of H2S on
cellular bioenergetics, primarily through inhibition of mitochondrial cytochrome c oxidase (Complex
IV). By contrast, control compound 2 did not negatively impact the measured bioenergetics
parameters, but rather resulted in increases in basal respiration and ATP synthesis. Compound 3 had
no effect on basal respiration or ATP synthesis, and only slightly decreased maximal respiration at 10
µM. To better compare these results with known H2S donors, we also analyzed the mitochondrial
respiration of BEAS 2B cells incubated with AP39 and GYY4137 under the same conditions (Figure
4g-i). We failed to observe significant inhibition of cellular bioenergetics at the observed
concentrations, raising the possibility that the inhibitory effects of 1 may not be from H2S release
alone, but could also be due to direct COS inhibition of cytochrome c oxidase,44, 45 which has been
reported previously but received less scrutiny than direct H2S inhibition. Our previous investigations
with ROS-activated23 and “click-and-release”24 COS/H2S donors did not reveal donor cytotoxicity
below 100 M, suggesting that the observed cytotoxicity from 1 may be either specific to lung
epithelial cells or may be amplified by preferential release in certain cellular locales that enable
efficient interaction with the mitochondrial respiratory chain.
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Figure 4. Cellular bioenergetics analysis including (a) basal respiration, (b) maximal respiration, and
(c) ATP synthesis in BEAS 2B cells upon addition of 1-10 µM of 1-3 as well as full bioenergetics data
at (d) 1 µM, (e) 3 µM, and (f) 10 µM. For comparison, data was additionally collected for (g) basal
respiration, (h) maximal respiration, and (i) ATP synthesis of BEAS 2B cells incubated with AP39 and
GYY4137.
Abbreviations:
oligo:
oligomycin,
FCCP:
carbonyl
cyanide
4-
(trifluoromethoxy)phenylhydrazone, AA/Rot: antimycin A and rotenone. Values represent the means ±
SEMs from three independent biological experiments each with five technical replicates. The values
that are significantly different by Student's t test are indicated by asterisks as follows: **, P < 0.01; *, P
< 0.05
In conclusion, we have designed an easy-to-access, esterase-triggered COS/H2S donor and
shown that it is rapidly activated by esterases to generate H2S in vitro using isolated PLE. Using
toxicity assays and bioenergetics measurements, we demonstrated that the increased cytotoxicity of 1
is due inhibition of mitochondrial respiration, whereas carbamate control compound 2 and triggerless
thiocarbamate 3 failed to negatively perturb normal bioenergetics. Using mitochondrial-targeted AP39
and the non-targeted donor GYY4137, we confirmed that the disruption of cellular bioenergetics
observed from 1 is significantly different than is seen with other H2S donors, potentially suggesting
that either the efficiency of H2S release, specific localization of the esterase-triggered scaffold, or
direct inhibition of mitochondrial respiration by COS itself is responsible for the observed cytotoxicity.
As a whole, the reported esterase-cleaved donor provides a slow-release method of COS/H2S release
in cellular environments. Further work investigating the impacts of H2S/COS donors on cellular
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bioenergetics and applications toward harnessing the selective cytotoxicity of these donor motifs is
ongoing in our labs and will be reported in due course.
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Methods
Materials and Methods. Reagents were purchased from Sigma-Aldrich or Tokyo Chemical
Industry (TCI) and used as received. 4-Pivaloyl benzyl alcohol was synthesized as previously
reported.33 Spectroscopic grade, inhibitor-free THF was deoxygenated by sparging with argon
followed by passage through a Pure Process Technologies solvent purification system to remove
water. Deuterated solvents were purchased from Cambridge Isotope Laboratories and used as
received. Silica gel (SiliaFlash F60, Silicycle, 230-400 mesh) was used for column chromatography.
Column chromatography was performed using a Biotage Isolera One automatic flash chromatography
purification system. 1H and 13C{1H} spectra were recorded on a Bruker 600 MHz instrument. Chemical
shifts are reported in ppm relative to residual protic solvent resonances. H2S electrode data were
acquired with a World Precision Instruments (WPI) ISO-H2S-2 sensor connected to a TBR4100 Free
Radical Analyzer. All air-free manipulations were performed under an inert atmosphere using
standard Schlenk techniques or an Innovative Atmospheres N2-filled glove box.
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H2S Electrode Materials and Methods. Phosphate buffered saline (PBS) tablets (1X, CalBioChem)
was used to make buffered solutions (PBS, 140 mM NaCl, 3 mM KCl, 10 mM phosphate, pH 7.4) in
Millipore water. Buffer solutions were sparged with N2 to remove dissolved oxygen and stored in an
N2-filled glovebox. Carbonic anhydrase (CA) from bovine erythrocytes (3,500 W/A units/mg) was
obtained from Sigma Aldrich and a 10 mg/ mL CA stock solution was prepared in deoxygenated buffer
(PBS, 140 mM NaCl, 3 mM KCl, 10 mM phosphate, pH 7.4) in a glovebox. The stock solution was
stored under nitrogen at 4 C and warmed to room temperature immediately before use. Stock
solutions of porcine liver esterase (PLE) and acetazolamide (AAA) were prepared under N2 with
degassed buffer (PBS, pH 7.4) immediately prior to use. Thiocarbamate and carbamate stock
solutions were prepared in an N2-filled glovebox in DMSO and stored at -25 C until immediately
before use.
General Procedure for H2S Electrode Experiments. Scintillation vials containing 20.00 mL of
phosphate buffer (140 mM NaCl, 3 mM KCl, 10 mM phosphate, pH 7.4) were prepared in an N2-filled
glovebox. A split-top septum cap was placed on the vial after probe insertion. The WPI electrode was
then inserted into the vial and the measured current was allowed to equilibrate before starting the
experiment. With moderate stirring, the CA stock solution (50 µL, 25 µg/mL) was injected, followed by
subsequent injections of thiocarbamate donor, carbamate control, PLE, or AAA.
MTT assay. The MTT assay was performed as reported.1 Briefly, BEAS 2B cells were incubated
with vehicle (DMSO), 1-3 at the desired concentration for 24 hours, then 3-(4,5-dimethyl-2-thiazolyl)-
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2,5-diphenyl-2H-tetrazolium bromide (MTT) was added to the cells at a final concentration of 0.5
mg/mL and cells were cultured at 37 ºC for 1 hour. The cells were washed with PBS and the formazan
dye was dissolved in isopropyl alcohol. The amount of converted formazan dye was measured at 570
nm with background measurement at 690 nm on a Powerwave reader (Biotek).
LDH assay. Lactate dehydrogenase (LDH) release was determined as a cytotoxicity assay, a
measurement of necrotic cell death, as described previously.46 Briefly, 30 μL of supernatant was
saved before addition of MTT and mixed with 100 μL of freshly prepared LDH assay reagent to reach
final concentrations of 85 mM lactic acid, 1040 mM nicotinamide adenine dinucleotide (NAD+), 224
mM N-methylphenazonium methyl sulfate (PMS), 528 mM 2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-
phenyl-2H-tetrazolium chloride (INT) and 200 mM Tris (pH 8.2). The changes in absorbance were
read kinetically at 492 nm for 15 min (kinetic LDH assay) on a monochromator-based reader
(Powerwave HT, Biotek) at 37 ºC. LDH activity values are shown as Vmax for the kinetic assays in
mOD/min.
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Bioenergetic analysis in cultured cells. The XF24 Extracellular Flux Analyzer (Agilent
Technologies, Santa Clara, CA, USA) was used to measure bioenergetic function as described
previously.46 Briefly, BEAS 2B cells were incubated with vehicle (DMSO), 1-3 (1, 3, 10 M) for 24h.
Oxygen consumption rate (OCR) after oligomycin (1.5 µg/mL) was used to assess ATP production
rate and OCR after FCCP (0.5 µM) to assess maximal mitochondrial respiratory capacity. Antimycin A
(2 µg/mL) and rotenone (2 µM) were used to inhibit the flux of electrons through complex III and I, to
detect residual non-mitochondrial OCR, which is considered to be due to cytosolic oxidase enzymes.
Synthesis
O-(4-pivaloylbenzyl)-N-(p-tolyl)thiocarbamate (1). 4-Pivaloylbenzyl alcohol (131 mg, 0.668 mmol)
and p-tolylisothiocyanate (107 mg, 0.717 mmol) were combined in anhydrous THF (3 mL) and DBU
(125 µL, 0.835 mmol) was added dropwise. The reaction mixture was stirred under nitrogen at room
temperature for 4 hours. The solvent was removed under reduced pressure and the crude mixture
was purified using column chromatography (hexanes:EtOAc gradient) to yield the pure product as a
white solid (123 mg, 52%). Note: The resonances of thiocarbamates are doubled as a result of slow
rotation and the peaks associated with these rotamers are denoted with “major” and “minor” when
delineation is possible. 1H NMR (600 MHz, DMSO-d6)  (ppm): 11.13 (s, 1H), 7.54-7.42 (m, 3H), 7.21-
7.07 (m, 5H), 5.57 (minor); 5.48 (major) (s, 2H), 2.28 (major); 2.23 (minor) (s, 3H), 1.30 (s, 9H).
13C{1H} NMR (150 MHz, DMSO-d6)  (ppm): 187.8, 187.2, 176.9, 151.1, 136.5, 135.5, 134.9, 134.6,
134.0, 133.6, 130.2, 129.9, 129.7, 129.4, 123.4, 122.5, 122.3, 71.9, 70.3, 39.0, 27.2, 20.9. FTIR (ATR,
cm-1): 3207, 2972, 1750, 1704, 1541, 1423, 1107, 1043, 810. HRMS (m/z): [M + Na]+ calcd for
[C20H23NO3SNa]+ 380.1296, found 380.1299.
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O-(4-pivaloylbenzyl)-N-(p-tolyl)carbamate (2). 4-Pivaloyl benzyl alcohol (100 mg, 0.480 mmol) was
dissolved in anhydrous THF (4 mL) and DBU (89.6 µL, 0.600 mmol) was added. p-Tolylisocyanate
(64.3 mg, 0.480 mmol) was added dropwise in anhydrous THF (1 mL). The reaction mixture was
stirred under nitrogen at room temperature for 18 hours. The solvent was removed under reduced
pressure and the crude mixture was purified using column chromatography (hexanes:EtOAc gradient)
to yield the pure product as a clear oil (119 mg, 73%). 1H NMR (600 MHz, DMSO-d6)  (ppm): 9.65 (s,
1H), 7.47 (d, J=8.2 Hz, 2H), 7.32 (d, J=8.8 Hz, 2H), 7.07 (d, J=8.1 Hz, 2H), 6.85 (d, J=8.7 Hz, 2H),
5.05 (s, 2H), 2.23 (s, 3H), 1.30 (s, 9H). 13C{1H} NMR (150 MHz, DMSO-d6)  (ppm): 176.9, 153.8,
150.9, 137.1, 134.8, 131.9, 129.9, 129.6, 122.2, 118.6, 65.5, 39.0, 27.3, 20.8. FTIR (ATR, cm-1): 3364,
2974, 1725, 1595, 1524, 1508, 1192, 1111, 1015, 812, 785, 766. HRMS (m/z): [M + Na]+ calcd for
[C20H23NO4Na]+ 364.1525, found 364.1525.
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Supporting Information
The Supporting Information is available free of charge on the ACS Publications website. NMR
spectra, mass spectrometry data, additional cytotoxicity data.
Acknowledgements
Research reported in this publication was supported by the Sloan Foundation (M.D.P.), Dreyfus
Foundation (M.D.P.), and the NSF/GRFP (A.K.S.; DGE-1309047). Applications of H2S sensing were
supported by the NIH (R01GM113030 to M.D.P.). NMR spectroscopy capabilities in the UO CAMCOR
facility are supported by the NSF (CHE-1427987). Cell toxicity and cellular bioenergetics studies were
supported by the NIH/NCI (R01GM10784604 to C.S.) and NIH/NIEHS (R21ES024648 to B.S.). The
Q-Tof Ultima mass spectrometer at UIUC was purchased in part with a grant from the NSF (DBI-
0100085).
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