Esterase-Triggered Self-Immolative Thiocarbamates Provide Insights into COS Cytotoxicity

Article abstract of DOI:10.1021/acschembio.8b00981

Hydrogen sulfide (H₂S) is an important gasotransmitter and biomolecule, and many synthetic small-molecule H₂S donors have been developed for H₂S-related research. One important class of triggerable H₂S donors is

Full text of DOI:10.1021/acschembio.8b00981

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Letter
Tunable Esterase-Triggered Self-Immolative
Thiocarbamates Provide Insights into COS Cytotoxicity
Carolyn M Levinn, Andrea K Steiger, and Michael D Pluth
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00981 • Publication Date (Web): 14 Jan 2019
Downloaded from http://pubs.acs.org on January 17, 2019
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Esterase-Triggered Self-Immolative Thiocarbamates Provide Insights into COS
Cytotoxicity
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Carolyn M. Levinn†, Andrea K. Steiger†, and Michael D. Pluth*
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ǂDepartment of Chemistry and Biochemistry, Materials Science Institute, Institute of Molecular
Biology, University of Oregon, Eugene, Oregon 97403, United States. pluth@uoregon.edu
†These authors contributed equally to this work.
ABSTRACT: Hydrogen sulfide (H2S) is an important gasotransmitter and biomolecule, and many
synthetic small-molecule H2S donors have been developed for H2S-related research. One
important class of triggerable H2S donors are self-immolative thiocarbamates, which function by
releasing carbonyl sulfide (COS), which is rapidly converted to H2S by the ubiquitous enzyme
carbonic anhydrase (CA). Prior studies of esterase-triggered thiocarbamate donors reported
significant inhibition of mitochondrial bioenergetics and toxicity when compared to direct sulfide
donors, suggesting that COS may function differently than H2S. Here, we report a suite of modular
esterase-triggered self-immolative COS donors and include the synthesis, H2S release profiles,
and cytotoxicity of the developed donors. We demonstrate that the rate of ester hydrolysis
correlates directly with the observed cytotoxicity in cell culture, which further supports the
hypothesis that COS functions as more than a simple H2S shuttle in certain biological systems.
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INTRODUCTION
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Hydrogen sulfide (H2S), the most recent addition to the gasotransmitter family,1 plays
important physiological roles in the cardiovascular,2 respiratory, as well as other organ systems.3
Significant interest in both research and therapeutic approaches for H2S delivery has led to the
development of a library of synthetic small molecules that release H2S (H2S donors) by using
different strategies.4-8 In one recently-developed approach, our group, as well as others, has
reported H2S donors based on the triggerable, self-immolative decomposition of thiocarbamates
to release carbonyl sulfide (COS), which is rapidly hydrolyzed to H2S by the ubiquitous
mammalian enzyme carbonic anhydrase (CA).9 This COS-dependent H2S-releasing strategy is
highly tunable and allows for triggering of H2S release by a variety of stimuli, including ROS,10, 11
nucleophiles,12 cysteine,13 and light.14-16
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In addition to functioning as a precursor for CA-mediated H2S release, COS is the most
prevalent sulfur-containing gas in Earth’s atmosphere and plays important roles in the global
sulfur cycle. Despite this significance, few studies have investigated the physiological properties
of COS directly.17 Currently, there are no established mechanisms of eukaryotic COS
biosynthesis, although it has been shown that acetylcholine stimulation of porcine coronary artery
(PCA) leads to an observed increase in COS, indicating that muscarinic acetylcholine receptors
could play a role in regulating COS synthesis.18 Additionally, it has been detected in the
headspace of PCA and cardiac muscle,18 suggesting potential endogenous production. Although
simple methods for the direct detection of COS in aqueous solutions are not currently available,
COS can be detected through GC-MS analysis or by other spectroscopic methods. Moreover,
COS has also been recognized as a potential exhaled breath biomarker for a variety of diseases,
including cystic fibrosis19 as well as liver disease and rejection,20, 21 which suggests a possible
role in disease physiology. The consumption of COS by CA is well established and COS toxicity
closely resembles that of H2S, which is likely due to CA-mediated hydrolysis within mucous
membranes upon exposure. The rapid conversion of COS to H2S, with an associated rate
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constant of 2.2x104 M-1s-1 (for bovine CA II), makes COS a convenient source of sulfide, but also
makes disentangling the chemical biology of COS from H2S inherently challenging.22
We recently reported an esterase-triggered COS-mediated H2S donor,23 wherein ester
cleavage reveals an intermediate phenol that undergoes a 1,4-self-immolation cascade to release
COS, followed by rapid hydrolysis to H2S. Contrary to previous reports of similar donors, however,
these compounds exhibit significant cytotoxicity and fully inhibited major mitochondrial
bioenergetic pathways in bronchial epithelium BEAS2B cells. Similar cytotoxicity profiles were not
observed for other H2S donors, including NaSH, GYY4137, or AP39, at similar concentrations.
Furthermore, the analogous CO2-releasing carbamate control compound was non-cytotoxic,
confirming that the observed cytotoxicity or bioenergetics impacts were not due to organic
byproducts of donor activation. Taken together, these results led to the hypothesis that the
observed effects could be due to a buildup of COS. Supporting this hypothesis, the rate of small
ester cleavage by mammalian esterases is likely faster (~5.1x104 – 5.8x105 M-1s-1) than the rate
of CA-mediated COS hydrolysis to H2S,22, 24 which would result in a buildup of intracellular COS.
Here we extend this hypothesis by preparing a library of esterase-cleaved COS-releasing donors
in which the steric bulk of the ester and the electronic properties of the aniline payload are
modified. We demonstrate that the differential cytotoxicity of these donors maps to the COS
release rates, thus furthering the hypothesis that COS may exert different biological effects than
H2S alone (Figure 1).
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Figure 1. Esterase-triggered thiocarbamate-based H2S donors exhibit increased cytotoxicity,
potentially due to the buildup of intracellular COS.
RESULTS AND DISCUSSION
To further investigate whether the cytotoxicity of these esterase-activated COS/H2S
donors could be related to COS directly, we chose to probe the relationship between COS release
rates and the corresponding cytotoxicity. We hypothesized that if COS buildup was responsible
for the observed cytotoxicity, then esters cleaved more quickly should result in increased cell
death, whereas esters cleaved more slowly should have a diminished effect. In the esterase-
activated donors, the rate of COS release depends not only on the rate of ester cleavage
(“triggering”), but also on the rate of self-immolative decomposition. There have been a number
of reports demonstrating that rate of esterase activity varies directly with the steric bulk of the
ester being cleaved,25, 26 providing a rational strategy for manipulating the rate of triggering by
intracellular esterases. Similarly, recent work has demonstrated that the electronics of the amine
payload can affect the rate of thiocarbamate self-immolation.27, 28
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Figure 2. (a) Synthetic scheme for the development of a library of esterase-activated
thiocarbamate COS/H2S donors (TCM1-14) and (b) table showing all compounds used in this
study (TCM1-14 and CM1-14) with yields.
To probe the effects of steric bulk on the rate of COS release and cytotoxicity of esterase-
triggered COS donors, we prepared a library of thiocarbamates functionalized with different
esters. To prepare the donors, we first treated 4-hydroxy benzyl alcohol with different alkyl and
aryl carbonyl chlorides to afford the corresponding esters. Reaction with p-tolyl isothiocyanate
furnished the desired thiocarbamates (TCM1 to TCM9) in 14-90% yield (Figure 2a). In parallel,
we also prepared the carbamate control compounds, which release CO2 rather than COS, by
treatment of the carbonyl chloride intermediates with p-tolyl isocyanate (Figure 2b). To investigate
the role of electronic modulation of the aniline payload on the rate of self-immolation and
cytotoxicity of these compounds, a similar synthetic sequence was followed to access esterase-
triggered COS donors with electron-rich and electron-deficient amine payloads, (TCM10 to
TCM14).
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With the library of esterase-activated COS/H2S donors in hand, we next measured the H2S
release from these compounds in the presence of CA (Isozyme II from bovine erythrocytes) and
porcine liver esterase (PLE). Direct detection of H2S using a sulfide-selective electrode is simple
and fast, but analogous methods do not exist for rapid COS detection directly in solution. For this
reason, we added excess CA to these experiments to ensure no buildup of COS and used the
detection of H2S as an indirect measurement of COS release. We treated compounds TCM1 –
TCM9 (Figure 2b, left) and TCM10 – TCM14 (Figure 2b, right) with 5 U/mL PLE in the presence
of CA (25 µg/mL) in PBS buffer (pH 7.4) and observed H2S release from each of compounds
using a H2S-sensitive electrode. These data confirm that physiologically relevant amounts of CA
and PLE are sufficient to result in H2S release from each of these donors. Consistent with our
expectation that steric changes to the esters would results in different cleavage rates, we
observed significantly different H2S release rates and efficiencies from the donor compounds
containing a variety of different ester groups (Figure 3a). For example, donors with bulkier esters
(cyclohexyl (TCM7), adamantyl (TCM8), or naphthyl (TCM9), yellow, orange, and light blue
traces, respectively) generated H2S more slowly than those with smaller esters (methyl (TCM1),
t-butyl (TCM5), or methyl cyclopropyl (TCM4), dark green, grey, and magenta traces,
respectively). This qualitatively confirms that donors containing larger ester groups produce
COS/H2S more slowly, consistent with slower hydrolysis by PLE.
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Figure 3: H2S release curves for compounds (a) TCM1 – TCM9 and (b) TCM5, TCM10-TCM14)
in the presence of PLE (5 U/mL) and CA (25 µg/mL) at pH 7.4. (c and d) Cytotoxicity of
compounds in HeLa cells. Data for donors (TCM1 – TCM14) is shown for 10 – 100 µM and
compared to the cytotoxicity of the carbamate control compounds (CM1 – CM14) at 100 µM. (c)
Cytotoxicity data for donors containing varying ester groups (TCM1 – TCM9), with steric bulk of
the ester group decreasing from left to right. (d) Cytotoxicity data for donors containing varying
amine payloads (TCM5, TCM10 – TCM14), with electron donating-ability of the payload
decreasing from left to right. Results are expressed as mean  SD (n=6). The values that are
significantly different by Student's t test are indicated by asterisks as follows: ***, p < 0.001 **, p
< 0.01; *, p < 0.05. (e) Dual-axis comparison of cytotoxicity of various ester-containing donors at
100 µM and the rate of H2S release from these compounds in the presence of PLE and CA. (f)
Table of the rates of H2S release and percent cell viability of various ester-containing donors at
100 µM.
H2S release kinetics were also compared for a library of t-butyl ester functionalized donors
containing a variety of electronically modulated amine payloads. We hoped to systematically
decrease the rate of COS release through the introduction of electron-donating groups, although
acidification of the N-H proton of the thiocarbamate has been reported to decrease the rate of
COS release from similar donors functionalized with electron-withdrawing groups as well.27
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Consistent with this hypothesis, the introduction of either strongly electron-withdrawing (NO2
(TCM14), CF3 (TCM13), pink and purple traces, respectively) or electron-donating (Ph (TCM10),
black trace) groups decreased the rate and efficiency of the donors, indicating that both electron-
withdrawing and electron-donating groups slow down the rate of self-immolation following
esterase hydrolysis. The donors containing weakly electronically modified amine payloads TCM5,
TCM11, and TCM12) appear to have very similar initial rates (Figure 3b).
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We next sought to determine whether the observed differences in COS/H2S release rates
translated to differences in cytotoxicities of compounds TCM1 – TCM14. To probe these effects,
we incubated HeLa cells with the COS donor compounds at 10, 25, 50, and 100 µM for 90 minutes
and measured the resultant cell viability against the vehicle using the formazan dye-based CCK-
8 cytotoxicity assay. We found that the cytotoxicity of the donors increased as the size of the ester
decreased (Figure 3c), with the smallest ester (Me, TCM1) resulting in about 70% cell death at
100 µM. No significant cell death was observed, however, when cells were incubated with 100
µM of TCM9, which requires hydrolysis of a much larger naphthyl ester and has a much slower
rate of COS/H2S release. To confirm that the observed cytotoxicity was not due to the organic
byproducts of the donor constructs after activation, we also investigated the cytotoxicity of the
corresponding carbamate control compounds (CM1 – CM14) using the same conditions. Overall,
we found significantly less cytotoxicity of all of the carbamates up to 100 µM. No significant trend
was observed in the cytotoxicity of the donors as the electronics of the payload were changed.
While many of these donors (TCM5, TCM11 – TCM14) were cytotoxic, even as low as 25 µM, we
did not find any correlation between cytotoxicity and the electronics of the amine payload (Figure
3d). Since the mechanism of decomposition of these donors may change due to acidification of
the N-H proton, the cytotoxicity likely does not correspond to the rate of COS production.
This work provides evidence that cellular accumulation of COS is cytotoxic. Importantly,
the cytotoxicity observed from many of these COS donors was completely eliminated when HeLa
cells were incubated with the analogous, CO2-releasing carbamates, which control for all other
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byproducts, suggesting that COS may be directly responsible for the observed effects. Cell death
is dose-dependent for all of the cytotoxic COS-releasing compounds, and in general, the most
cytotoxic donors were also found the have the most rapid kinetics of H2S release in the presence
of CA (Figure 3e and 3f). Overall, the hypothesis that the inclusion of a larger ester on these
donors would decrease the rate of hydrolysis and prevent the build-up of COS was found to hold
true. We were not able to systematically decrease the rate of COS release through electronic
modulation of the amine payloads, but did find that both strongly electron-withdrawing and
electron-donating groups diminished COS/H2S release, consistent with two previously suggested
effects: that electron-donating groups decrease the rate of self-immolation, and electron-
withdrawing groups can result in a change in the mechanism of H2S release due to acidification
of the N-H proton on the thiocarbamate.27 Due to a potential change in mechanism supported by
a decrease in H2S production from donors containing electron-withdrawing payloads, it is
impossible to correlate the cytotoxicity of these particular donors with their rate of COS/H2S
release.
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In addition to providing new insights into the differential impacts of COS and H2S, this work
also increases the tools available for increasing basal H2S concentrations without the need for
external triggering mechanisms or consumption of cellular nucleophiles. To confirm that these
donors release COS/H2S in a cellular environment, we incubated 100 µM Cy-TCM (TCM7) with
SF7-AM in HeLa cells and observed an increase in fluorescence corresponding to H2S donation
from the scaffold (Figure 4).29 This confirms the basic cellular viability of this compound as an H2S
donor. Although the faster-releasing donors are too cytotoxic for use as efficacious H2S donors,
the slower-releasing donors provide a library of enzyme-activated COS/H2S donors viable for use
in cell-based experiments.
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Figure 4. (a) Live-cell imaging of H2S release from TCM7 in HeLa cells. HeLa cells were treated
with SF7-AM (5 µM) and Hoechst (5 µg/mL) for 30 min, washed, and incubated with FBS-free
DMEM containing TCM7 (100 µM, bottom) or DMSO (0.5%, top) for one hour. Cells were then
washed and imaged in PBS. Scale bar = 100 µm. (b) Relative integrated fluorescence intensity
of cells treated with TCM7 versus vehicle treated cells.
In conclusion, this work supports the hypothesis that rapid accumulation of COS likely
results in cytotoxicity.23 Conclusively disentangling the effects of COS delivery from the
physiological effects of H2S will require a systematic study of COS, the various CA isoforms, and
the potential for subcellular localization of COS delivery from various donors. The work reported
here suggests the likely role of COS in the cytotoxicity of many of these compounds and provides
an important piece of early evidence that COS delivery may produce a cellular response that is
different than that observed from H2S alone.
METHODS
General Materials and Methods. Reagents were purchased from Sigma-Aldrich or Tokyo
Chemical Industry (TCI) and used as received. SF7-AM was synthesized as previously reported.30
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.
1
Silica gel (SiliaFlash F60, Silicycle, 230-400 mesh) was used for column chromatography. H,
13C{1H}, and 19F NMR spectra were recorded on a Bruker 500 or 600 MHz instrument (as
indicated). Chemical shifts are reported in ppm relative to residual protic solvent resonances.
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Mass spectrometric measurements were performed on a Xevo Waters ESI LC/MS instrument or
by the University of Illinois, Urbana Champaign MS facility. H2S electrode data were acquired with
a Unisense H2S Microsensor Sulf-100 connected to a Unisense Microsensor Multimeter. All air-
free manipulations were performed under an inert atmosphere using standard Schlenk techniques
or an Innovative Atmospheres N2-filled glove box. HeLa cells were purchased from ATCC
(Manassas, Virginia, USA). Cell imaging experiments were performed on a Leica DMi8
fluorescence microscope, equipped with an Andor Zyla 4.2+ sCMOS detector. Fluorescence
intensity measurements were calculated using Fiji (ImageJ).31 Fluorescence intensities were
measured in Fiji, with images scaled to 32-bit, and the error is reported as the standard mean
error.
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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. The Unisense
electrode was inserted into the vial and the vial was capped with a split-top septum to minimize
oxidation. The current was allowed to equilibrate prior to starting the experiment. With moderate
stirring, the CA stock solution (50 µL, CAII from Bovine Erythrocytes) was injected, followed by
subsequent injections of TCM stock solution (50 µL) and PLE stock solution (100 µL). H2S release
was monitored until leveling off.
CCK-8 Cell Viability Experiments. HeLa cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin/streptomycin at 37 C under 5% CO2. 96-well plates were seeded with 15,000 cells/well
overnight then washed, incubated in FBS-free DMEM containing vehicle (0.5% DMSO), TCA (10-
100 µM), or carbamate (10100 µM) for 90 minutes. Cells were then washed with PBS and CCK-
8 solution (1:10 in FBS-free DMEM) was added to each well, and cells were incubated for 1-2
hours at 37 C under 5% CO2. The absorbance at 450 nm was measured using a microplate
reader and the cell viability was measured and normalized to the vehicle group. Results are
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expressed as mean  SD (n=6). P values were calculated using a Student’s T-test in Excel
compared to DMSO alone.
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Cell Imaging. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 C under
5% CO2. Imaging dishes were seeded with HeLa cells overnight and then washed and incubated
with SF7-AM (5 µM) and Hoechst 33342 (5 µg/mL) in FBS-free DMEM for 30 min. Cells were then
washed with PBS and incubated with either Cy-TCM (100 µM) or vehicle (DMSO, 0.5%) in FBS-
free DMEM for 60 minutes prior to being washed with PBS and imaged. Imaging was performed
once, and the fluorescence intensities were calculated from the images shown, with 77 cells in
the TCM images and 116 cells in the control.
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Synthesis
General procedure for the synthesis of phenol esters. 4-Hydroxy benzyl alcohol (1.0
equiv.) was dissolved in anhydrous THF (0.1 M solution), under and atmosphere of N2. The
solution was cooled to 0 °C, followed by addition of Et3N. The reaction mixture was let stir for 5
minutes, after which the carbonyl chloride was added dropwise over 20 minutes. The resultant
mixture was stirred at 0 °C until the completion of the reaction indicated by TLC. The reaction was
quenched by adding brine (30 mL), and the aqueous solution was extracted with ethyl acetate (3
x 20 mL). The organic layers were combined, dried over anhydrous MgSO4, and concentrated
under reduced pressure. The crude product was purified by silica column chromatography. Full
spectroscopic data for each compound is reported in the SI. The preparation of MeCp-OH is also
reported in the SI.
General procedure for preparation of thiocarbamates. The functionalized benzyl alcohol
(1.0 equiv.) was dissolved in anhydrous THF (0.2 M solution) under an atmosphere of N2. Aryl
isothiocyanate (1.1 equiv.) was added, followed by DBU (1.25 equiv.) at 0 °C. The resultant
mixture was warmed to rt and stirred monitored by TLC. The reaction was quenched upon
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observation of by-product formation by TLC by addition of brine (20 mL), and extracted with EtOAc
(3 x 20 mL). The combined organic layers were dried over anhydrous MgSO4 or Na2SO4,
concentrated under reduced pressure, and purified by silica column chromatography. Full
spectroscopic data for each compound is reported in the SI.
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General procedure for preparation of carbamate controls. Functionalized benzyl alcohol
(1.0 equiv.) was dissolved in anhydrous THF (0.1 M solution) under an atmosphere of N2. Aryl
isocyanate (0.90 equiv.) was added, followed by DBU (1.25 equiv.) at 0 °C. The resultant mixture
was warmed to rt and stirred monitored by TLC. The reaction was quenched upon observation of
by-product formation by TLC by addition of brine (20 mL), and extracted with EtOAc (3 x 20 mL).
The combined organic layers were dried over anhydrous MgSO4, concentrated under reduced
pressure, and purified by silica column chromatography. Full spectroscopic data for each
compound is reported in the SI.
ASSOCIATED CONTENT
The Supporting Information is available free of charge on the ACS Publications website at
http://pubs.acs.org. NMR spectra, mass spectrometry data (PDF).
ACKNOWLEDGEMENTS.
The research reported here was supported by the Dreyfus Foundation, the NIH (R01GM113030),
and the NSF/GRFP (DGE-1309047 to CML, AKS). NMR, microscopy, and MS capabilities in the
UO CAMCOR facility are supported by the NSF (CHE-1427987, CHE-1531198, CHE-1625529).
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