Thionoesters: A Native Chemical Ligation-Inspired Approach to Cysteine-Triggered H2S Donors

Article abstract of DOI:10.1021/jacs.8b07268

Native chemical ligation (NCL) is a simple, widely used, and powerful synthetic tool to ligate N-terminal cysteine residues and C-terminal α-thioesters via a thermodynamically stable amide bond. Building on this well-established reactivity, as well as adv

Full text of DOI:10.1021/jacs.8b07268

Article
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
pubs.acs.org/JACS
Thionoesters: A Native Chemical Ligation-Inspired Approach to
Cysteine-Triggered H S Donors
2
Matthew M. Cerda, Yu Zhao, and Michael D. Pluth*
Department of Chemistry and Biochemistry, Materials Science Institute, Institute of Molecular Biology, University of Oregon,
Eugene, Oregon 97403, United States
*
S Supporting Information
ABSTRACT: Native chemical ligation (NCL) is a simple,
widely used, and powerful synthetic tool to ligate N-terminal
cysteine residues and C-terminal α-thioesters via a thermody-
namically stable amide bond. Building on this well-established
reactivity, as well as advancing our interests in the chemical
biology of reactive sulfur species including hydrogen sulfide
(
H S), we hypothesized that thionoesters, which are constitu-
2
tional isomers of thioesters, would undergo a similar NCL reaction in the presence of cysteine to release H S under
2
physiological conditions. Herein, we report mechanistic and kinetic investigations into cysteine-mediated H S release from
2
thionoesters. We found that this reaction proceeds with high H S-releasing efficiency (∼80%) and with a rate constant (9.1 ±
2
−
1 −1
0
.3 M s ) comparable to that for copper-catalyzed azide−alkyne cycloadditions (CuAAC). Additionally, we found that the
final product of the reaction of cysteine with thionoesters results in the formation of a stable dihydrothiazole, which is an iron-
binding motif commonly found in siderophores produced by bacteria during periods of nutrient deprivation.
INTRODUCTION
■
Hydrogen sulfide (H S) is now recognized as an important
2
1
biological signaling molecule that is produced endogenously,
cell membrane permeable, and reactive toward cellular and/or
2
molecular targets. The endogenous production of H S stems
2
primarily from catabolism of cysteine and homocysteine by
cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE),
3
and 3-mercaptopyruvate sulfurtransferase (3-MST). Recently,
increasing interest has focused on harnessing H S as a potential
2
4
5
therapeutic agent based on its role in vasodilation, neuro-
6
7
transmission, and angiogenesis. Although the majority of
prior reports have used sodium hydrosulfide (NaSH) or
sodium sulfide (Na S) as sources of H S, the addition of these
2
2
salts to a buffer leads to an almost instantaneous increase in
H S concentration, which is in stark contrast to the slow,
2
8
gradual endogenous production of H S. In efforts to provide
2
more physiologically relevant rates of H S release, researchers
2
have developed different types of H S-releasing molecules
2
9
−11
(
Figure 1a).
For example, Lawesson’s Reagent and related
have been used as hydrolysis-activated H2S
1
2,13
derivatives
donors that function at physiological pH, and dithiolethiones,
such as ADT−OH, have been conjugated to nonsteroidal anti-
Figure 1. (a) Representative examples of common synthetic, small-
inflammatory drugs (NSAIDs) to access H S prodrug
2
1
4
molecule H S donors; (b) selected small-molecule, thiol-triggered
2
conjugates. More recently, “triggered-release” scaffolds have
also been reported, including those activated by light and
enzymatic activation. In addition, recent work has demon-
15
H S donors.
2
16
been developed that are activated by thiols, such as cysteine
and reduced glutathione (GSH) (Figure 1b). Polysulfides, such
strated that carbonyl sulfide (COS)-releasing scaffolds can also
function as H S donors via the rapid conversion of the released
2
17
COS to H S by carbonic anhydrase.
2
Drawing parallels to the enzymatic conversion of cysteine or
homocysteine to H S, a number of H S donor motifs have
Received: July 17, 2018
2
2
©
XXXX American Chemical Society
A
DOI: 10.1021/jacs.8b07268
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
as the commonly used diallyl trisulfide (DATS) or more
20
Article
1
8
H S. Herein, we present a mechanistic and kinetic inves-
tigation of thionoesters with cysteine and related species and
also demonstrate that thionoesters function as cysteine-
2
19
recently reported synthetic trisulfides and tetrasulfides,
release H S in the presence of thiols via an intermediate
2
persulfide. Building in complexity, Xian and co-workers have
selective H S donors that proceed through a native chemical
ligation-type mechanism.
2
reported thiol-triggered H S donors based on protected N-
2
2
1
22
mercaptan or persulfide platforms. Similarly, Matson and
co-workers reported S-aroylthiooxime compounds, which
23
RESULTS AND DISCUSSION
To prepare a model thionoester system, we treated phenyl
■
generate a thiol-reactive intermediate thiooxime. Thiol-
2
4
mediated H S release from arylthioamides and aryl
2
chlorothionoformate with phenylmagnesium bromide at −78
2
5
isothiocyanates has also been reported, although the
°
C in anhydrous THF to yield O-phenyl benzothioate
34 35
mechanisms of H S release remains uninvestigated and low
2
(DPTE). Despite previous reports, we found that treat-
ment of phenyl benzoate with Lawesson’s reagent required
extended reaction times and afforded undesirable yields, which
is consistent with the predicted decrease in reactivity of esters
releasing efficiencies (∼2% and 3%, respectively) are observed.
To the best of our knowledge, the only reported cysteine-
selective H S donor utilizes the established reactivity of
2
26
acrylate Michael acceptors toward cysteine, to subsequently
trigger the generation of COS, which is quickly converted to
H S by carbonic anhydrase.
36
toward Lawesson’s reagent. The structure and purity of
DPTE were confirmed by NMR spectroscopy and HPLC (see
Supporting Information). To determine whether thionoesters
27
2
To further the development of thiol-triggered H S donors,
2
are a viable platform for H S release, we added 25 μM DPTE
2
we were inspired by the well-established chemistry of native
chemical ligation due to the high biological compatibility and
presence of a sulfur atom. Native chemical ligation is the
chemoselective reaction between a thioester and an N-terminal
to buffered aqueous solutions (10 mM PBS, pH 7.4)
containing varying concentrations of cysteine (25−500 μM)
and monitored H S generation using the spectrophotometric
2
37
methylene blue assay (Figure 3a). Consistent with our design
28
cysteine residue to generate a new amide bond. This reaction
has been applied extensively in the field of protein synthesis,
including in the semisynthesis of a potassium channel
2
9
protein. The mechanism of this important ligation reaction
begins by the nucleophilic addition of a cysteine sulfhydryl
group to form an intermediate thioester, which then undergoes
a rapid S to N acyl transfer to generate the more
3
0
thermodynamically stable amide product (Figure 2). Despite
Figure 2. Generalized reaction scheme for native chemical ligation
and release of H S upon addition of cysteine to a bis(phenyl)
2
thionoester.
the broad use of thioesters as activated coupling partners for
native chemical ligation, to the best of our knowledge there
have not been investigations into similar reactions with
thionoesters, which are a constitutional isomer of thioesters.
Building from our interest in the chemistry of reactive sulfur
3
1−33
species,
we hypothesized that thionoesters would undergo
a similar reaction pathway in the presence of cysteine, but
would also generate H S during the S to N acyl transfer step of
2
the reaction. Such reactivity would not only provide access to
new H S-releasing motifs but also provide insights into new
2
Figure 3. (a) Release of H S from DPTE in the presence of
increasing cysteine concentrations (25, 125, 250, and 500 μM) in 10
2
mechanisms of chemical ligation that could be accessed by
simple interchange of oxygen and sulfur atoms in a reactive
electrophile. Additionally, such platforms are also attractive
because they mimic the enzymatic conversion of cysteine to
mM PBS, pH 7.4 at 25 °C. (b) Lack of H S release from structurally
2
related compounds (25 μM) in the presence of cysteine (500 μM, 20
equiv).
B
DOI: 10.1021/jacs.8b07268
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Journal of the American Chemical Society
Article
hypothesis, we observed an increase in H S release from DPTE
at higher cysteine concentrations, suggesting that thionoesters
homocysteine also resulted in H S release, although at a slower
2
2
rate than from treatment with cysteine. This observation is
consistent with a larger, less favorable transition state required
for an intramolecular S to N acyl transfer in the homocysteine
system in comparison with the cysteine system. Alternatively,
the reduced rate may be reflective of the significant pKa
are a viable platform for cysteine-triggered H S donation.
2
To assess the H S-releasing efficiency from thionoesters, we
2
used a methylene blue calibration curve to quantify the H2S
release (Figure S4). We measured that 20 μM of H S was
2
released from a 25 μM solution of DPTE in the presence of
difference between cysteine (pK ≈ 8.5) and homocysteine
a
41
5
00 μM cysteine (20 equiv), which corresponds to a releasing
(pK ≈ 10), meaning that, under physiological conditions,
a
efficiency of 80%. In addition to the thionoester system, we
the effective concentration of cysteine thiolate is much greater
than homocysteine thiolate (∼10% vs ∼0.03%). Surprisingly,
also investigated H S release from structurally related diphenyl
2
ester (2) and diphenyl thioester (3) compounds under our
conditions (Figure 3b). As expected, neither of these
treatment of DPTE with penicillamine did not result in H S
2
release. We anticipated that geminal methyl groups would help
to preorganize the intermediate dithioester generated after
compounds released H S when treated with excess cysteine.
2
42
Similarly, a representative secondary thioamide (4) failed to
nucleophilic attack and would result in faster H S release.
2
release H S in the presence of cysteine, suggesting the release
2
However, the geminal methyl groups also likely significantly
reduce the nucleophilicity of the thiol moiety due to steric
congestion, which would subsequently disfavor the initial
nucleophilic attack on the thionoester.
of H S occurs exclusively from the thionoester moiety in the
2
presence of cysteine.
To further investigate the selectivity of H S release from
2
thionoesters, we treated DPTE with other biologically relevant
We also investigated whether different cysteine derivatives
38
nucleophiles (Figure 4). In the absence of any added
could generate H S release from DPTE to further understand
2
the requirements for H S release from thionoesters. Treatment
2
of DPTE with cysteine methyl ester did not affect H2S
production, suggesting that the carboxylic acid is not required
for H S generation. By contrast, treatment of DPTE with N-
2
acetylcysteine, N-acetylcysteine methyl ester, or S-methylcys-
teine completely abolished H S release, highlighting the
2
requirement of a 2-aminoethanethiol moiety for productive
H S release. Consistent with these results, treatment of DPTE
2
with GSH, the most abundant biological thiol, did not generate
H S, which is consistent with the requirement of a pendant
2
amine to generate H S release. Despite the lack of H S release,
2
2
we anticipated that GSH would still attack DPTE to form an
intermediate dithioester, which should still be sufficiently
electrophilic to react with cysteine to generate H S. To test this
2
hypothesis, we treated DPTE (25 μM) with GSH (1 mM) and
cysteine (500 μM) and observed a reduced rate of H S release.
2
These results suggest that the competitive, nonproductive,
addition of GSH to the thionoester is reversible, and that the
thionoester moiety can still react with Cys in the presence of
Figure 4. Selectivity of H S release from DPTE in the presence of
different analytes. Data were acquired at 1, 5, 10, 15, 30, 45, and 60
min. Methylene blue absorbance values are relative to the maximum
GSH to release H S. Adding to the selectivity investigations,
treatment of DPTE with porcine liver esterase (PLE) failed to
2
2
generate H S; however, we cannot rule out consumption of the
2
absorbance value obtained from H S release in the presence of
2
thionoester moiety by PLE or other native enzymes. Taken
together, these results demonstrate the high selectivity of the
thionoester moiety toward cysteine and homocysteine for H2S
release.
Building from the selectivity studies, as well as from the
established mechanism of native chemical ligation, we
cysteine (1). Analytes: H O/PBS buffer (2), serine (3), lysine (4), L-
2
homocysteine (5), DL-penicillamine (6), L-cysteine methyl ester
hydrochloride (7), N-acetyl-L-cysteine (8), N-acetyl-L-cysteine methyl
ester (9), S-methyl-L-cysteine (10), GSH (11), cysteine + GSH (12),
cysteine + lysine (13), PLE (1.0 U/mL) (14).
proposed a mechanism for cysteine-mediated H S release
from thionoesters (Scheme 1). Initial nucleophilic addition by
2
nucleophiles, no hydrolysis-mediated H S release was observed
from DPTE at physiological pH, although we note prior
2
reports show that thionoesters are hydrolyzed under basic
conditions to afford the corresponding thioacid and alcohol.
Treatment of DPTE with serine or lysine, chosen as
Scheme 1. Proposed Mechanism of H S Release from DPTE
in the Presence of Cysteine
39
2
representative alcohol- and amine-based nucleophiles respec-
tively, did not result in H S release, although prior reports
2
suggest that amines can react with thionoesters to yield
40
thioamides via displacement of the corresponding alcohols.
To investigate this potential side reactivity, cysteine-triggered
500 μM) H S release from DPTE (25 μM) was measured in
(
2
the presence of lysine (500 μM) and we observed no change in
H S-releasing efficiency. We also investigated the reactivity of
2
DPTE with thiol-based nucleophiles. Treatment of DPTE with
C
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J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
cysteine on 1 generates tetrahedral intermediate 5, which
collapses to form dithioester intermediate 6 and extrude 1
equiv of phenol. Similar to native chemical ligation, subsequent
nucleophilic attack by the pendant amine on the thiocarbonyl
nucleophilic attack of the sulfhydryl group on the thioester is
thought to be rate-limiting due to the rapid and irreversible
subsequent S to N acyl transfer to form the more
thermodynamically stable amide bond. In the thionoester
system, the initial attack by a thiol on DPTE results in
extrusion of phenol, which is a much weaker nucleophile than a
thiol and should not attack the generated dithioester
intermediate. If other thiols are present in solution, then it is
likely that they could attack the dithioester intermediate in a
transdithioesterification reaction. This thiol exchange is
30
leads to the formation of substituted thiazolidine 7. Loss of
−
H S, by either direct extrusion of HS or solvent-assisted
2
extrusion of H S, results in formation of dihydrothiazole 8,
2
which could be further hydrolyzed to form N-benzoyl-cysteine
(
9).
As a first step toward investigating our proposed mechanism,
we determined the reaction order in cysteine by treating
DPTE (25 μM) with varying concentrations of cysteine under
pseudo-first-order conditions at 25 °C and measuring H2S
release using the methylene blue assay (Figure 5). As expected,
supported by the observed reduced rate of H S generation
2
from DPTE in the presence of competing thiols, suggesting
that the initial nucleophilic attack on dithioesters is reversible.
Using similar pseudo-first-order conditions as those used for
the cysteine order dependence investigations (25 μM DPTE,
5
00 μM cysteine), we performed an Eyring analysis to
determine the activation parameters for the reaction in an
effort to further understand the amount of disorder in the rate-
limiting transition state for the reaction (Figure 6). Our
Figure 6. (a) Effect of temperature on rate of H S release from DPTE
2
(25 μM) in the presence of cysteine (500 μM, 20 equiv). (b) Eyring
analysis of H S release from DPTE.
2
expectation was that if initial thiol addition is the rate-limiting
step, then we would observe a negative entropy of activation
(ΔS ) of approximately −20 eu, which is typical for a
Figure 5. (a) H S release by DPTE in the presence of increasing
cysteine concentrations (250, 500, 1000, and 1250 μM). (b) Plot of
log(kobs) vs log([Cys]) for DPTE. (c) Plot of kobs vs [Cys].
‡
2
bimolecular reaction. In contrast, if the intramolecular S to N
thioacyl transfer to form the substituted thiazolidine is the rate‡-
limiting step, then we would expect a larger, more negative ΔS
due to the highly ordered structure required for the
intramolecular cyclization. Under our experimental conditions,
we observed that increased cysteine concentrations led to
increased rates of H S production. The resultant releasing
2
‡
curves were fit to obtain pseudo-first-order rate constants
we observed ΔS = −38 ± 3 eu, which is most consistent with
(
k ), and plotting log[Cys] versus log[k ] confirmed a first-
intramolecular cyclization being the rate-determining step of
the reaction.
obs
obs
order dependence in cysteine, which is consistent with our
proposed mechanism. Additionally, the obtained kobs values
were plotted against Cys concentrations to obtain a second-
As a final step of characterizing the proposed mechanism, we
performed a preparative scale reaction and isolated the reaction
products. In addition to recovered starting material, we isolated
a cysteine-derived dihydrothiazole (CysDHT) rather than N-
benzoyl-L-cysteine as the major product of the reaction (Figure
7). These results suggest that the dihydrothiazole is stable
under aqueous conditions and is not further hydrolyzed to N-
benzoyl-L-cysteine. To further confirm the formation of
CysDHT from DPTE, we synthesized an authentic sample
of CysDHT and used HPLC to monitor the reaction progress.
We treated a 100 μM solution of DTPE with 20 equiv of L-
cysteine methyl ester and observed nearly complete conversion
to phenol and CysDHT within 1 h. Using known
concentrations of phenol and CysDHT to construct an
HPLC calibration curve, we measured that the concentrations
order rate constant of 9.1 ± 0.3 M−
1
s
−1
for the reaction. In
comparison to other known reactivities, the rate of cysteine-
triggered H S release from DPTE is comparable to the rate
2
−
1
−1
(
10−100 M
s ) of copper(I)-catalyzed azide−alkyne
cycloadditions (CuAAC), a classic example of a “click
reaction.”
4
3
To further evaluate our proposed mechanism, we sought to
identify the rate-determining step in cysteine-triggered release
of H S from thionoesters. In native chemical ligation, the initial
2
nucleophilic attack by thiols to form intermediate thioesters is
reversible and has been utilized to enhance the reactivity of
alkyl thioesters for native chemical ligation. However, in the
presence of cysteine, the transthioesterification resulting from
D
DOI: 10.1021/jacs.8b07268
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Journal of the American Chemical Society
Article
exploration into the thionoester functional group for cysteine-
selective reactive probes.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.8b07268.
■
*
S
Experimental procedures, NMR spectra, HPLC data
(PDF)
AUTHOR INFORMATION
Corresponding Author
pluth@uoregon.edu
■
*
ORCID
Yu Zhao: 0000-0003-1250-9480
Michael D. Pluth: 0000-0003-3604-653X
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Research reported in this publication was supported by the
Dreyfus Foundation and the NIH (R01GM113030). NMR
and MS instrumentation in the UO CAMCOR facility are
supported by the NSF (CHE-1427987, CHE-1625529).
Figure 7. (a) Reaction conditions; (b) 100 μM DPTE in PBS (10
mM, pH 7.4) with 10% THF; (c) 100 μM PhOH in PBS (10 mM,
pH 7.4) with 10% THF; (d) 100 μM CysDHT in PBS (10 mM, pH
7
.4) with 10% THF; (e) reaction aliquot after 1 h.
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DOI: 10.1021/jacs.8b07268
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX