Amino acid-based H2S donors:N-thiocarboxyanhydrides that release H2S with innocuous byproducts

Article abstract of DOI:10.1039/d1cc01309b

A library ofN-thiocarboxyanhydrides (NTAs) derived from natural amino acids with benign byproducts and controlled H₂S-release kinetics is reported. Minimal acutein vitrotoxicity was observed in multiple cell lines, while longer-term toxicity in cancer cells was observed, with slow-releasing donors exhibiting the greatest cytotoxic effects.

Full text of DOI:10.1039/d1cc01309b

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Amino acid-based H2S donors:
N-thiocarboxyanhydrides that release H2S
with innocuous byproducts†
Cite this: Chem. Commun., 2021,
57, 5522
Received 10th March 2021,
Accepted 22nd April 2021
ab
ac
a
a
Kuljeet Kaur,
Patrick Enders,
Yumeng Zhu,
Abigail F. Bratton,
Chadwick R. Powell,a Khosrow Kashfi
and John B. Matson
*
d
a
DOI: 10.1039/d1cc01309b
rsc.li/chemcomm
A library of N-thiocarboxyanhydrides (NTAs) derived from natural endogenous H2S production to better understand H2S physiol-
amino acids with benign byproducts and controlled H2S-release ogy and explore its therapeutic potential.10,11
kinetics is reported. Minimal acute in vitro toxicity was observed in
Controlled delivery of H2S is challenging due to its gaseous
multiple cell lines, while longer-term toxicity in cancer cells was nature, toxicity at high concentrations, and short half-life in a
observed, with slow-releasing donors exhibiting the greatest biological setting.12 To address these challenges, researchers
cytotoxic effects.
have developed several small and macromolecular synthetic
H2S donors with appreciable control over dosage and release
Three endogenously produced signaling gases, nitric oxide rates.13–17 One way to deliver H2S that has gained interest
(NO), hydrogen sulfide (H2S), and carbon monoxide (CO), recently is via the delivery of carbonyl sulfide (COS),18,19 which
recognized as gasotransmitters, are enzymatically generated, is effectively converted into H2S in eukaryotic organisms by the
can penetrate cell membranes, and have specific cellular tar- action of the enzyme carbonic anhydrase (CA).20 Several groups
gets and physiological functions.1 The most recently recognized have developed and studied thiocarbamate-based COS donors
gasotransmitter, H2S, directly interacts with or initiates major that decompose to release COS in response to specific stimuli,
signaling pathways linked to various disease states, including such as reactive oxygen species (ROS), enzyme activity, and light
cardiovascular disorders, neurological disorders, and other (Fig. 1A).21–25 For example, Pluth and coworkers have developed
conditions including diabetes, chronic kidney disease, and enzyme-triggered, COS-releasing small molecule thiocarba-
cancer.2–9 In some disease indications, endogenous H2S pro- mates, and our group recently described a poly(thiourethane)
duction is disrupted, and administration of exogenous H2S that releases on average 7 equivalents of COS after triggering,
in animal models can alleviate symptoms and improve out- also relying on a thiocarbamate linkage.26,27
comes. Exogenous H2S administration is also needed for
in vitro and in vivo studies focused on better understanding
the physiological roles of this important gas. To conduct these
studies, H2S-releasing molecules (often called H2S donors) are
employed, with
a goal of mimicking or supplementing
a Department of Chemistry, Virginia Tech Center for Drug Discovery, and
Macromolecules Innovation Institute, Virginia Tech, Blacksburg, VA, 24061, USA.
E-mail: jbmatson@vt.edu
b
´
´
Institut des Materiaux and Institut des Sciences et Ingenierie Chimiques,
´
`
´ ´
Laboratoire des Polymeres, Ecole Polytechnique Federale de Lausanne (EPFL),
Lausanne CH-1015, Switzerland
c Institute of Chemistry, Rostock University, Albert-Einstein-Str. 3a, Rostock 18059,
Germany
Fig. 1 Classes of COS donors. (A) Thiocarbamate-based donors (and
related donors such as thionoesters and thioureas) are structurally tunable
but release electrophilic byproducts. (B) N-functionalized NTAs release
COS and an N-alkyl amino acid upon hydrolysis with little control over
release rate. (C) C-functionalized NTAs, described here, release amino acid
byproducts, and the R group can influence COS release rate.
d Department of Molecular, Cellular, and Biomedical Sciences, City University of
New York School of Medicine, New York, NY, 10031, USA
† Electronic supplementary information (ESI) available: H2S release studies, cell
studies, synthetic procedures and NMR spectra. See DOI: 10.1039/d1cc01309b
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These triggerable, self-immolative, thiocarbamate-based
H2S donors present highly tunable COS/H2S release. However,
the byproducts (e.g., (aza)quinone methides) associated with
the activation of a 1,6-elimination reaction are electrophilic,
potentially toxic, and complicate experiments when studying
the chemical biology of COS/H2S.28 For example, an esterase-
triggered thiocarbamate donor reduced viability at much
higher levels than commonly used H2S donors (NaSH and
GYY4137) in human bronchial epithelium (BEAS 2B) cells
and in HeLa cells by inhibiting mitochondrial respiration
pathways.29 The observed toxicity in thiocarbamates was
believed to be the result of rapid COS/H2S release from these
donors rather than the byproduct, leading to a buildup of gas in
toxic concentrations. A detailed follow-up study on similar
thiocarbamates with varying steric bulk of the ester group
suggested that toxicity depended on the rate of ester cleavage,
with compounds generating COS most quickly exhibiting the
highest toxicity; however, by product toxicity could not be fully
ruled out.30 This complication has led our group and others to
identify COS donors that do not generate electrophilic
byproducts,31–33 with the goal of disentangling the direct
chemical biology of COS/H2S from associated byproducts.
One class of COS-mediated H2S donors with benign bypro-
ducts are the N-thiocarboxyanhydrides (NTAs). Our first report
was on an NTA derived from sarcosine, an N-alkyl amino acid
that releases COS along with a sarcosine-containing byproduct
(Fig. 1B).31 More recently, we also described a series of
N-substituted NTAs with bio-orthogonal handles for easy access
to polymeric and peptidic COS/H2S-releasing materials.26 How-
ever, lack of tunability of the rate of H2S release from these
N-functionalized NTAs led us to design a series of C-substituted
NTAs based solely on natural amino acids (Fig. 1C). We envi-
sioned that hydrolysis of the peptide-derived NTAs would
produce COS while regenerating the original amino acid as
the only byproduct. We also hypothesized that the steric bulk of
the substituent on the a-carbon could influence the rate of
hydrolytic ring-opening and subsequent COS release. There-
fore, this series of NTAs could (1) enable the ability to tune the
rate of COS/H2S release from NTAs, and (2) facilitate investiga-
tions into how COS release rate influences cytotoxicity because
COS is the only potentially toxic product.
Fig. 2 Schematic representation of nucleophilic ring-opening of (A) N-
substituted and (B) C-substituted NTAs.
adjacent C-4 carbon atom (Fig. 2B). We hypothesized that bulky
substituents at the C-4 carbon atom (the a carbon in the
original amino acid) would impede the approach of the nucleo-
phile, slowing down COS release. Further, we were also inter-
ested in studying the effects of ring size on the rate of COS
release, and therefore we attempted to synthesize rings with
n 4 5, specifically 6 and 7-membered rings.
In order to test our hypothesis, we synthesized nine different
NTAs from natural L-amino acids in a two-step process (Fig. 3A).
Among these, seven NTAs were derived from canonical amino
acids (Fig. 3B): Gly-NTA, Ala-NTA, Val-NTA, Leu-NTA, Ile-NTA,
Phe-NTA, and Pro-NTA. We also prepared two additional
NTAs: one with two methyl groups on C-4 derived from
2-aminoisobutyric acid (Aib-NTA), and one that included a
6-membered NTA ring derived from b-alanine (b-Ala-NTA). We
also attempted to synthesize a 7-membered NTA derived from
4-aminobutanoic acid, but the 5-membered heterocycle O-ethyl
2-oxopyrrolidine-1-carbothioate formed instead (for details see
ESI†). This might be the result of a higher energy penalty
associated with the formation of a 7-membered cyclic NTA
compared to 5- or 6-membered rings. As a result, we did not
attempt to make NTAs with larger ring sizes.
With nine NTAs in hand, next we measured H2S release from
the NTAs using an H2S sensitive electrochemical probe. To
test our hypothesis, we chose Gly-NTA, Val-NTA, and Ile-NTA
and studied their hydrolytic ring-opening in 5% DMSO-PBS
An interesting similarity among all small molecule N-
substituted NTAs is that their H2S release peaking times, as
measured by an H2S-sensitive electrochemical probe, were all
measured at around 40 min.26 We speculate that this similarity
is related to the mechanism of the nucleophilic ring-opening
reaction. The ring-opening of an NTA likely involves nucleo-
philic addition to the C-5 carbonyl carbon to form a tetrahedral
intermediate, followed by reformation of the carbonyl in the
ring-opening step, and finally release of COS from the resulting
thiocarbamate (Fig. 2A). Substituents present on the N atom of
the ring are therefore likely too far from the C-5 carbonyl
carbon to affect the rate of nucleophilic addition. For this
work, we hypothesized that it would be possible to control
the rate of nucleophilic addition at the C-5 carbonyl carbon,
and subsequently the rate of COS release, by substituting the
Fig. 3 (A) General scheme for synthesis of N-substituted NTAs used in
this work; conditions: (i) NaOH, methanol/H2O (ii) PBr3, EtOAc. (B)
Chemical structures of nine NTAs used in this work.
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(10 mM) in the presence of 300 nM CA (Fig. S1, ESI†). For sake shortest half-lives, ranging from 1–2 h, consists of Gly-NTA,
of simplicity, we only evaluated hydrolysis here and did not b-Ala-NTA, and Ala-NTA; the second group is Leu-NTA, Phe-
include any additional nucleophiles. The H2S peaking times NTA, and Pro-NTA with half-lives ranging from 4–7 h. Finally,
showed a clear trend: the smaller Gly-NTA showed a sharp the third group consists of Aib-NTA, Leu-NTA, and Val-NTA
profile with a peaking time of 51 min, while the NTAs with with half-lives ranging from 10–20 h.
larger substituents at C-4 (i.e., the a-carbon atom) exhibited
The H2S release half-lives of these three groups can be
more gradual release profiles with peaking times of 96 min rationalized by considering the steric bulk near the reactive
(Val-NTA) and 104 min (Ile-NTA) (Table S1, ESI†). While the C-5 carbonyl in each set. Gly-NTA and b-Ala-NTA have no ring
electrochemical probe provides a real-time analysis of H2S substitutions and therefore, little steric bulk to limit nucleo-
release, it is difficult to use this method to determine a rate philic addition to the C-5 carbonyl. The fact that there is little
constant or compare donors with variable release profiles difference in the H2S release half-lives between these two NTAs
because the released H2S is constantly oxidizing and volatiliz- (1.1 h for b-Ala-NTA and 1.7 h for Gly-NTA) indicates that the
ing. To measure and compare pseudo-first-order H2S release difference in ring size did not play a substantial role in
half-lives among all 9 NTAs, we used the methylene blue assay. controlling the rate of COS generation. Therefore, their H2S
In our experiments, water was used as the nucleophile, and we release half-lives are the fastest of all, about 2-fold faster than
included CA in the reaction vials to quickly convert COS Ala-NTA (half-life of 2.2 h), which has a methyl group on C-4.
generated during the NTA ring-opening reaction into H2S. The third group with longest half-lives consists of NTAs with
Fitting the data to a pseudo-first-order kinetics model, as we dialkyl groups present either on C-4 itself (Aib-NTA) or on the
have done previously using the methylene blue assay,34 allowed carbon next to C-4 (Ile-NTA and Val-NTA), which is the b-carbon
us to determine half-lives of H2S release (Fig. 4A).
in relation to the reactive C-5 carbonyl. Aib-NTA with gem-
Half-life values determined by the methylene blue method dimethyl groups at the a-carbon atom had a half-life of 10.2 h,
for Gly-NTA, Val-NTA, and Ile-NTA were 1.7, 18, and 20 h, whereas Ile-NTA and Val-NTA with dialkyl substituents on the
respectively (Fig. 4B and Table S2, ESI†). The methylene blue b-carbon showed twofold longer half-lives (18 and 20 h, respec-
kinetics data revealed a B10-fold difference in release rate tively), indicating that dialkyl groups on the b-carbon may be
between Gly-NTA and NTAs with bulky substituents, highlight- more effective in impeding nucleophilic approach at the C-5
ing the strength of this method compared to the electrochemi- carbonyl than dialkyl groups directly on C-4. The middle group
cal probe method, where only a 2-fold difference in peaking with half-lives in between those of first and third groups
time was observed despite drastically different release profiles. consists of NTAs without dialkyl substitution on C-4 or the
Across all nine NTAs, H2S release half-lives varied from a b-carbon. For example, Leu-NTA with a dimethyl group at
shortest half-life of 1.1 h for b-Ala-NTA to 20 h for Val-NTA. the g-carbon atom had a B4-fold shorter half-life of 4.5 h
Looking more closely, the H2S release half-lives could be compared to Ile-NTA and Val-NTA, further suggesting that a
divided into three separate groups. The first group with the disubstituted b-C atom slows down the ring-opening reaction.
Similarly, Phe-NTA (half-life of 4.6 h), with an aromatic ring at
g-carbon atom, has a similar half-life to Leu-NTA. Finally, Pro-
NTA has a unique structure with a 5-membered ring fused with
the NTA ring. This bicyclic structure appears to make accessi-
bility of a nucleophile to the C-5 carbonyl group in between that
of a C-4 dimethyl (Aib-NTA) and a phenyl group (Phe-NTA),
exhibiting an intermediate half-life of 6.9 h.
Overall, these observations support our hypothesis that side
chain substitution on the C-4 carbon plays a role in slowing
down nucleophilic addition at the C-5 carbonyl. The data
further suggest that dialkyl groups on the side chain b-carbon
atom are more effective in slowing down this reaction than
fused rings or mono- or di-substituents present at other posi-
tions. These results may inform design of other COS/H2S-
releasing NTAs.
We hypothesized that the fast-releasing NTAs would exhibit
greater cytotoxicity compared to slow-releasing NTAs based on
reports detailing acute cytotoxicity within 1.5 h due to rapid
COS/H2S accumulation in esterase-triggered thiocarbamates.30
To test acute toxicity, we treated estrogen-positive human
breast cancer MCF-7 cells with 100 mM NTA for a period of
1.5 h, and viability was measured against untreated cells using
the CCK-8 cytotoxicity assay. In this cell line, none of the NTAs
were cytotoxic, and increases in the CCK-8 signal compared
Fig. 4 (A) H2S release from NTAs (100 mM) measured via the methylene
blue assay in 10 mM PBS solution with 5% DMSO and 300 nM CA. Data
points are solid circles, and dotted lines show pseudo-first-order kinetics
fits; (B) H2S release half-lives for NTAs determined from the pseudo-first-
order kinetics fits. (C) Cell viability data for MCF-7 breast cancer cells
treated with 100 mM NTA solution with 0.25% DMSO in PBS (pH 7.4) for
1.5 h. (D) IC50 data for selected NTAs and GYY4137 after 72 h incubation
time in MCF-7 and HT-29 cancer cells.
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with controls were observed in all treatment groups, likely due instrumentation, Dr. Mehdi Ashraf Khorassani for assistance with
to reported effects of H2S-induced cell proliferation (Fig. 4C).35 mass analysis, and Prof. Yin Wang for assistance with cell studies.
Additionally, b-Ala-NTA, the fastest H2S donor of the group,
showed no cytotoxicity under these conditions. Next, we tested
the two fastest H2S-releasing NTAs, Gly-NTA and b-Ala-NTA, on
Conflicts of interest
a different cell line, H9C2 cardiomyocytes. Again, we observed
no acute cytotoxicity after 1.5 h up to a concentration of 300 mM
(Fig. S3, ESI†). These results are in contrast to fast esterase-
triggered thiocarbamates, which had a similar release profile to
these two fastest NTA-based donors but were cytotoxic to HeLa
cells at concentrations as low as 25 mM upon incubation for the
same time period.30 Therefore, this work suggests that COS/H2S
is not acutely toxic, at least in these cell lines.
There are no conflicts of interest to declare.
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