Use of Dithiasuccinoyl-Caged Amines Enables COS/H2S Release Lacking Electrophilic Byproducts

Article abstract of DOI:10.1002/chem.201905577

The enzymatic conversion of carbonyl sulfide (COS) to hydrogen sulfide (H₂S) by carbonic anhydrase has been used to develop self-immolating thiocarbamates as COS-based H₂S donors to further elucidate the impact of reactive sulfur species in biology. The high modularity of this approach has provided a library of COS-based H₂S donors that can be activated by specific stimuli. A common limitation, however, is that many such donors result in the formation of an electrophilic quinone methide byproduct during donor activation. As a mild alternative, we demonstrate here that dithiasuccinoyl groups can function as COS/H₂S donor motifs, and that these groups release two equivalents of COS/H₂S and uncage an amine payload under physiologically relevant conditions. Additionally, we demonstrate that COS/H₂S release from this donor motif can be altered by electronic modulation and alkyl substitution. These insights are further supported by DFT investigations, which reveal that aryl and alkyl thiocarbamates release COS with significantly different activation energies.

Full text of DOI:10.1002/chem.201905577

A Journal of
Accepted Article
Title: Dithiasuccinoyl-Caged Amines Enables COS/H2S Release
Lacking Electrophilic Byproducts
Authors: Matthew m Cerda, Jenna L Mancuso, Emma J Mullen,
Christopher H Hendon, and Michael Dwight Pluth
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201905577
Link to VoR: http://dx.doi.org/10.1002/chem.201905577
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Dithiasuccinoyl-Caged Amines Enables COS/H2S Release
Lacking Electrophilic Byproducts
Matthew M. Cerda, Jenna L. Mancuso, Emma J. Mullen, Christopher H. Hendon, and Michael D.
Pluth*[a]
[a]
M. M. Cerda, J. L. Mancuso, E. J. Mullen, Dr. C. H. Hendon, Dr. M. D. Pluth
Department of Chemistry and Biochemistry, Materials Science Institute, Knight Campus for Accelerating Scientific Impact, Institute of Molecular Biology
University of Oregon
Eugene, Oregon, 97403, USA
E-mail: pluth@uoregon.edu
Supporting information for this article is given via a link at the end of the document.
Abstract: The enzymatic conversion of carbonyl sulfide (COS) to
hydrogen sulfide (H2S) by carbonic anhydrase has been used to
develop self-immolating thiocarbamates as COS-based H2S donors
to further elucidate the impact of reactive sulfur species in biology.
The high modularity of this approach has provided a library of COS-
based H2S donors that can be activated by specific stimuli. A common
limitation, however, is that many such donors result in the
intermediate formation of an electrophilic quinone methide byproduct
during donor activation. As a mild alternative, we demonstrate here
that dithiasuccinoyl groups can function as COS/H2S donor motifs and
that these groups release two equivalents of COS/H2S and uncage an
amine payload under physiologically relevant conditions. Additionally,
we demonstrate that COS/H2S release from this donor motif can be
altered by electronic modulation and alkyl substitution. These insights
are further supported by DFT investigations, which reveal that aryl and
alkyl thiocarbamates release COS with significantly different
activation energies.
dioxide (CO2) in the presence of bovine carbonic anhydrase II
(kcat/Km = 2.2 x 104 M-1 s-1).[16] In our initial approach, we were
inspired by self-immolative carbamates, which release CO2 as a
byproduct upon activation,[17] and developed analogous self-
immolative thiocarbamates that function as tunable COS-based
H2S donors (Figure 1a).[18]
Introduction
Despite being a malodorous gas,[1] hydrogen sulfide (H2S)
is an important biological signaling molecule often referred to as
a gasotransmitter alongside carbon monoxide and nitric oxide.[2]
H2S-mediated signaling is important in several physiological
processes including vasodilation,[3] neurotransmission,[4] and
inflammation.[5] These findings have led researchers to propose
the use of H2S as a potential therapeutic agent for a variety of
conditions and pathologies.[6] Toward this goal, researchers have
relied heavily on the use of NaSH and Na2S as sources of H2S
due to ease of handling and commercial availability; however, H2S
release from these salts is considerably different relative to
enzymatic H2S generation.[7] To better mimic endogenous H2S
production, methods of generating H2S at controlled rates under
physiologically-relevant conditions are needed,[8] and the
development of small molecule “H2S donors” is an active research
area aimed at addressing this need.[9] Such compounds generate
H2S by passive hydrolysis[10] or activation in the presence of
specific stimuli including light,[11] biological thiols,[12] and cellular
enzymes including esterases.[13]
Figure 1. (a) Generalized reaction mechanism for COS release from self-
immolative thiocarbamates and subsequent hydrolysis of COS to H2S by
carbonic anhydrase. (b) Overall reaction scheme of COS release from
dithiasuccinoyl groups in the presence of thiols.
The high modularity of this scaffold has enabled the rapid
expansion of this approach by our group[9] as well as others to
prepare COS-based H2S donors activated by various stimuli
including acidic pH,[19] esterases,[20] reactive oxygen species,[21]
and cysteine.[22] This approach has also been extended to provide
oligomeric COS-based H2S donors.[23] A critical, yet often
overlooked component of this approach is the formation of a
quinone methide byproduct, which is a potent electrophile and
known Michael acceptor in biological systems.[24] Although we
have not observed cytotoxicity from this byproduct in our studies,
chronic exposure from therapeutic administration of these
compounds is likely to induce electrophilic stress leading to long-
term cytotoxicity.[25] As an alternative approach, Matson and co-
workers have reported both small molecule and polymeric N-
thiocarboxyanhydrides (NTAs) as COS/H2S donors, which only
result in small peptide byproducts.[26] These donor compounds,
however, exhibit a relatively low H2S-releasing efficiency and lack
significant reactivity studies for COS release in response to
Recently, an alternative approach to H2S generation has
utilized the hydrolysis of carbonyl sulfide (COS) by carbonic
anhydrase (CA), a ubiquitous metalloenzyme.[14] Existing in
Nature as the most abundant sulfur-containing gas in the
atmosphere,[15] COS is rapidly converted to H2S and carbon
1
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various analytes. To further develop COS-based H2S donors as
both research tools and potential pharmacological agents,
alternative donor motifs are needed that lack electrophilic
byproducts, maximize COS/H2S release, and allow for simple
tuning of release rates.
To assess the viability of these compounds as COS/H2S
donors, we examined the release of H2S from PhDTS (25 µM) in
the presence of biologically-relevant nucleophiles (500 µM, 20
equiv.) and CA (25 µg/mL) using the methylene blue assay to
measure H2S generation (Figure 2).[29] Our expectation was that
this donor functional group would be activated broadly by different
nucleophiles rather than one specific biological nucleophile, thus
broadening the scope of potential activation pathways.
To address these needs, we focused on the reactivity of the
dithiasuccinoyl (DTS) group, which has been used previously as
a protecting group for amines in peptide synthesis.[27] The DTS
group is cleaved by thiols, which results in reduction to a
symmetric disulfide, two equivalents of COS, and an amine
(Figure 1b). We note that previous studies on thiol-mediated
reduction of DTS groups examined this reactivity in organic
solvents using β-mercaptoethanol as the reductant and quantified
reaction kinetics through the use of an amino acid analyzer
without direct observation of COS.[28] We envisioned that this
reactivity could be harnessed to prepare a library of COS-based
H2S donors that do not release electrophilic byproducts, but that
readily release COS/H2S under physiologically relevant
conditions in the presence of CA. Herein, we demonstrate that
DTS-caged amines function as versatile COS/H2S donors
activated by biological nucleophiles, including cysteine and
reduced glutathione (GSH). Additionally, we use a combination of
experimental and computational investigations to demonstrate
that the rate of COS/H2S release can be readily tuned by
electronic modulation and subsequent stabilization of the COS-
releasing thiocarbamic acid intermediate.
Figure 2. Activation profiles of H2S release from PhDTS (25 µM) in the presence
of different potential nucleophiles (500 µM, 20 equiv.) and carbonic anhydrase
(25 µg/mL). Data were acquired at 1, 5, 10, 15, 30, 45, and 60 min. Methylene
blue absorbance values are relative to the maximum absorbance value obtained
from H2S release in the presence of cysteine (3). Analytes: H2O with no carbonic
anhydrase (1), cysteine with no carbonic anhydrase (2), N-acetyl-L-cysteine (4),
L-cysteine methyl ester (5), N-acetyl-L-cysteine methyl ester (6), homocysteine
(7), reduced glutathione (8), serine (9), lysine (10), only carbonic anhydrase (11),
S-methyl cysteine (12).
Results and Discussion
To prepare a small library of COS-based H2S donors with
tunable release rates, we treated alkyl and aryl isothiocyanates
with N,N-dimethylethanolamine in the presence of sodium hydride
to generate the desired thiocarbamate intermediate. Subsequent
treatment with chlorocarbonylsulfenyl chloride afforded the
desired DTS-caged compounds (Scheme 1). The amines chosen
for this library included aryl amines with different electron
donating/withdrawing properties, as well as alkyl amines with
increasing steric bulk.
In the absence of CA, we did not observe hydrolysis-
mediated H2S release from PhDTS. The addition of cysteine to
PhDTS in the absence of CA resulted in slow, yet gradual H2S
release. We attribute this observation to the hydrolysis of COS at
physiological pH, which has been reported previously to be
slow.[30] Treatment of PhDTS with cysteine in the presence of CA
resulted in significant H2S generation. Using a calibration curve
generated with known concentrations of NaSH, we measured 40
µM H2S generation from 25 µM PhDTS in the presence of 500 µM
cysteine, which corresponds to an H2S releasing efficiency of 80%
(Figure S45). This observation not only highlights the high
efficiency of H2S release from DTS-caged compounds, but also
supports that two equivalents of COS are released per DTS group.
In addition to COS/H2S release, we also observed the
formation of aniline following treatment of PhDTS with cysteine by
HPLC, which further supports the proposed releasing mechanism
(Figure S48). Protection of the amine and/or carboxylate groups
on cysteine did not significantly impact the rate or quantity of
COS/H2S release from PhDTS, which supports a thiol-mediated
releasing pathway. Interestingly, the use of S-methyl cysteine
resulted in slow, yet considerable H2S release suggesting a less
favorable, thiol-independent reaction pathway. In previous studies,
the direct reaction of amines at the carbonyl position of DTS
groups has been observed and proposed to result in the
Scheme 1. Synthesis of DTS-based COS/H2S donors.
2
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generation of COS and sulfane sulfur.[28] We observed a similar
rate of H2S release in the presence of lysine, which further
supports a minor, amine-mediated mechanism of H2S release.
We note an induction phase in the rate of amine activation and
find that this reaction is slower relative to the thiol-mediated
reduction of DTS groups (Figure S49). Together with the
decreased nucleophilicity of amines due to protonation at
physiological pH and lower biological concentrations relative to
thiols, we expect this mechanism of activation to be negligible in
a biological context. In the presence of homocysteine and GSH,
we observed lower quantities of COS/H2S released, which we
attribute to the lower nucleophilicity of these thiols as a function
of thiolate/thiol speciation at physiological pH.[31] We failed to
observe COS/H2S release in the presence of serine implying that
alcohol-mediated mechanisms of activation are not present.
In the presence of CA, but absence of any added
nucleophiles, we did observe slight H2S production. We
hypothesized that this release could be due to coordination of
PhDTS to the Zn2+ center in CA, which would facilitate hydrolysis
by carbonyl activation. To probe this reactivity, we pre-incubated
CA with the CA inhibitor acetazolamide (5 µM) and measured H2S
release from PhDTS (Figure S46). Under these conditions, we
failed to observe H2S generation, which supports the hypothesis
of a minor CA/Zn2+-mediated hydrolysis mechanism. Alternatively,
this could also be due to minor background DTS hydrolysis
followed by COS conversion to H2S by CA. Further experiments
using a catalytic amount of Zn(OAc)2 (5 µM) did not result in
COS/H2S release from PhDTS thus highlighting the need for the
protein microenvironment present in CA for activation of PhDTS
(Figure S47).[32] Similar to the reactivity with amines, the rate of
CA/Zn2+-mediated hydrolysis is slower than the thiol-mediated
reduction. Taken together, these results demonstrate the validity
of PhDTS and related compounds to serve as COS/H2S donors
under physiologically-relevant conditions in the presence of thiols
and CA.
merits future investigation. By contrast, we observed the caging
of alkyl amines resulted in significantly slower rates of H2S release
relative to DTS-caged anilines with tBuDTS and EtDTS displaying
the fastest and slowest rates of H2S release, respectively (Figure
3b). We reasoned that inductively donating alkyl amines likely
stabilize the COS-releasing thiocarbamic intermediates leading to
a decrease in H2S-releasing kinetics.
With a small library of DTS-based donors in hand, we next
examined the effect of the amine payload on COS/H2S release
using each donor (25 µM) in the presence of cysteine (500 µM,
20 equiv.) and CA (25 µg/mL) at physiological pH (Figure 3). We
hypothesized that DTS-caging of functionalized anilines would
allow COS/H2S release rates to be tuned based on prior work
aimed at solvent-dependent linear free energy relationship
investigations into the phosphine-mediated sulfur extrusion from
DTS.[33] Additionally, we expected that the caging of alkyl amines
would lead to stabilization of the COS-releasing thiocarbamic acid
intermediate and subsequently decrease the rate of H2S release
relative to that observed for DTS-caged anilines. In the presence
of cysteine and CA, we observed varying rates and quantities of
H2S release from the reported aryl-based DTS compounds with
3-MePhDTS and 4-tBuPhDTS displaying the fastest and slowest
rates of H2S release, respectively (Figure 3a). The releasing
curves from the DTS-caged anilines did not fit cleanly to first-order
exponential decay, which we attribute to competing COS-
releasing and DTS consumption pathways, such as direct thiol
activation versus CA-mediated activation. Additionally, previous
work has reported the formation of isothiocyanates from
sufficiently acidic thiocarbamates, which likely further complicates
the rates of release from DTS-caged anilines containing electron-
withdrawing groups.[34] Overall, the functionalization of caged
anilines directly alters rates of COS/H2S release from DTS-based
donors, and the ability to control tuning of these releasing kinetics
Figure 3. (a) H2S release from aryl-based DTS compounds. (b) H2S release
from alkyl-based DTS compounds.
To further investigate the differences between aryl and alkyl
amine substitution, we used density functional theory (DFT) to
examine the potential energy surface for COS release from
PhDTS and AlkylDTS compounds. In these systems, we used
methyl thiol (MeSH) to simplify possible protonation states of non-
participating functional groups during the reaction. Calculations
3
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were performed using Gaussian 09 at the B3LYP/6-311++G(d,p)
level of theory applying the IEF-PCM water solvation model
(Figure 4).
Figure 4. Potential energy surface for COS release from PhDTS and AlkylDTS compounds. Calculations were performed using Gaussian 09 at the B3LYP/6-
311++G(d,p) level of theory applying the IEF-PCM water solvation model. MeSH was used as the thiol nucleophile to simplify accessible protonation states of
non-participating functional groups on the thiol nucleophile.
For PhDTS, we found that the initial nucleophilic attack by
the thiolate was the highest barrier (21.7 kcal/mol) on the reaction
coordinate and dethiocarboxylation of the thiocarbamic acid
intermediate was only moderately endothermic (+3.2 kcal/mol)
with respect to the starting materials. By contrast, although the
AlkylDTS compounds showed similar activation barriers for the
initial attack by thiolate (23.5 – 26.8 kcal/mol), the activation
Conclusion
barrier for the final dethiocarboxylation varied significantly as a
function of the alkyl group. The highest activation barrier for
COS/H2S donors in the presence of thiols without the formation of
dethiocarboxylation was found for the EtDTS compound (+29.8
kcal/mol), but this barrier decreased with the increasing donating
model compound were used to investigate COS/H2S release as a
ability of iPrDTS (+27.5 kcal/mol), and tBuDTS (+20.9 kcal/mol);
all of which were competitive with the activation barriers for initial
thiol attack on the DTS motif. These relative energetic barriers are
the rate of COS/H2S release from DTS-based donors can be
consistent with the observed rates of COS/H2S release from the
AlkylDTS compounds. Moreover, these results suggest that the
inductive contributions, rather than the steric bulk differences of
the alkyl substituents, have a larger impact on the release of COS
guide future work on this reactive intermediate. Specifically, this
from thiocarbamic acid intermediates. Taken together, the
combination of experimental and computational data
between aryl and alkyl thiocarbamates as COS-releasing motifs,
demonstrates the ability to tune H2S/COS release from this
We demonstrated the use of DTS-based compounds to serve as
electrophilic byproducts. Reactivity studies using PhDTS as a
function of biological nucleophiles and thiol identity. By modifying
the structure of the amine payloads, we also demonstrated that
modified by simple structural modifications. The results from DFT
calculations has shed light on the impact of amine identity on COS
release from thiocarbamic acids and provides a foundation to
work directly elaborates on the observed reactivity differences
which provides fundamental information upon which to expand
scaffold by simple structural modifications. Moreover, these
the utility of these donors. The simple synthetic conditions and
results provide guidance for controlling the COS/H2S release rate
from any donor motifs that proceed through a thiocarbamic acid
incorporation amine-based payloads including fluorophores[35]
intermediate prior to COS extrusion.
unique reactivity of this donor scaffold would readily allow for the
and known therapeutics[36] to provide COS-based H2S fluorescent
donors and prodrugs.
4
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FeCl3 in 1.2 M HCl, 200 μL of 20 mM N,N-dimethyl-p-phenylene diamine
in 7.2 M HCl, and 100 μL of 1% (w/v) Zn(OAc)2. To begin an experiment,
20 μL of 25 mM donor stock solution was added for a final concentration
of 25 μM. At set time points after the addition of donor, 500 μL reaction
aliquots were added to the methylene blue cocktail solutions and incubated
for 1 h at room temperature shielded from light. Absorbance values at 670
nm were measured 1 h after addition of reaction aliquot. Each experiment
was performed in quadruplicate unless stated otherwise. UV-Vis spectra
were acquired on an Agilent Cary 60 UV-Vis spectrophotometer equipped
with a Quantum Northwest TC-1 temperature controller set at 25 ± 0.05 °C.
Experimental Section
Synthesis Materials and Methods
Reagents were purchased from Sigma-Aldrich, Tokyo Chemical Industry,
or VWR and used directly as received. Deuterated solvents were
purchased from Cambridge Isotope Laboratories and used as received. 1H,
13C{1H}, and 19F NMR spectra were recorded on a Bruker 500 MHz
instrument. Chemical shifts were reported relative to residual protic solvent
resonances. MS data was collected on a Xevo G2-XS QTof (Waters)
instrument. Silica gel (SiliaFlash F60, Silicycle, 230-500 mesh) was used
for column chromatography. All air-free manipulations were performed
under an inert atmosphere using standard Schlenk technique.
Computational Methods
All structures were initially constructed, and optimized using the UFF force
field as implemented, in Avogadro.[37] The resultant structures were further
optimized using the unrestricted hybrid GGA functional, B3LYP, as
implemented in Gaussian 09,[38] with a triple zeta basis set that includes
Synthesis
General Procedure for the Synthesis of Thiocarbamates: This
procedure has been modified from a previous report.[33] In a flame-dried
round bottom flask under nitrogen, sodium hydride (1.25 equiv.) and N,N-
dimethylethanolamine were added to anhydrous toluene (20 mL). After
stirring briefly until gas evolution ceased, the desired isothiocyanate (1.0
equiv.) was added dropwise (if liquid) or in a single portion (if solid). The
reaction was stirred at room temperature for 3 h under nitrogen. The
reaction was quenched with deionized H2O (30 mL) and extracted with
ethyl acetate (3 x 15 mL). The combined organic extractions were washed
with brine (1 x 20 mL), dried over MgSO4, and concentrated under reduced
pressure. The desired product was obtained following purification by
column chromatography. All NMR data for these compounds was obtained
at 60 °C due to hindered rotation of thiocarbamates at room temperature.
We note the alkyl thiocarbamates displayed hindered rotation at 60 °C
giving rise to two sets of peaks corresponding to the E and Z isomers.
diffuse and polarization functions on heavy atoms, 6-311+G*.
A
pseudosolvent polarizable continuum model for water was used to account
for solvation effects. Attacking thiols were modeled as methyl thiol to
reduce computational expense.
Transition state searches were carried out at the same level of theory as
ground state structures. First, a potential energy surface scan of the active
reaction coordinate was used to obtain a good starting point for the
ultimate transition state search algorithm. Vibrational analysis confirmed a
single imaginary frequency corresponding to the direction of bond
formation or breaking for each activated complex. No transition state was
found for thiol disulfide exchange. This could indicate a barrierless
transition, or a shallow potential energy surface with a loose transition state.
Biologically relevant thiols, such as cysteine, whose attack may be
sterically hindered unlike the methyl thiol employed in this model, may
experience a more defined transition state and activation barrier.
General Procedure for the Synthesis of Dithiasuccinoyls: This
procedure has been modified from a previous report.[33] To a flame-dried
round bottom flask under N2, chlorocarbonylsulfenyl chloride (1.0 equiv.)
was added to anhydrous DCM (20 mL). In a separate vial, the desired
thiocarbamate (1.0 equiv.) was dissolved in anhydrous DCM (1 mL) and
added dropwise to the reaction. The reaction mixture was stirred at room
temperature for 1 h. The reaction was quenched with 1 M HCl (15 mL) and
the organic layer separated. The organic layer was washed with deionized
water (2 x 20 mL), brine (1 x 20 mL), dried over MgSO4, and concentrated
under reduced pressure. The desired product obtained by purification via
preparative thin layer chromatography.
Acknowledgements
Research reported in this publication was supported by the NIH
(MDP; R01GM113030) and the Dreyfus Foundation. NMR and
MS instrumentation in the UO CAMCOR facility is supported by
the NSF (CHE-1427987 and CHE-1625529). This work used the
Extreme Science and Engineering Discovery Environment
(XSEDE), which is supported by National Science Foundation
grant number ACI-1548562.
H2S Detection Materials and Methods
Phosphate buffered saline (PBS) tablets (1X, CalBioChem) were used to
prepare buffered solutions (140 mM NaCl, 3 mM KCl, 10 mM phosphate,
pH 7.4) in deionized water. Buffer solutions were sparged with nitrogen to
remove dissolved oxygen and stored in an Innovative Atmosphere
nitrogen-filled glovebox. Donor stock solutions (in acetonitrile) were
prepared inside a nitrogen-filled glovebox immediately before use. Trigger
stock solutions (in PBS) were freshly prepared in an N2-filled glovebox
immediately before use. CA stock solutions (in PBS) were freshly prepared
in a nitrogen-filled glovebox immediately before use.
Keywords: hydrogen sulfide • carbonyl sulfide • reactive sulfur
species
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10.1002/chem.201905577
Chemistry - A European Journal
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No more electrophilic byproducts! The use of dithiasuccinoyl (DTS)-caged amines provides access to COS-based H2S donors that
can be activated in the presence of biological thiols including cysteine and reduced glutathione to generate two equivalents of COS/H2S
and release an amine-based payload. These donor compounds provide researchers with chemical tools to further probe the role of
reactive sulfur species in biology.
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