Esterase activated carbonyl sulfide/hydrogen sulfide (H2S) donors

Article abstract of DOI:10.1021/acs.orglett.6b03336

Hydrogen sulfide (H₂S) is a mediator of a number of cellular processes, and modulating cellular levels of this gas has emerged as an important therapeutic area. Localized generation of H₂S is thus very useful but highly challenging. Here, we report pivaloyloxymethyl-based carbonothioates and carbamothioates that are activated by the enzyme, esterase, to generate carbonyl sulfide (COS), which is hydrolyzed to H₂S.

Full text of DOI:10.1021/acs.orglett.6b03336

Letter
pubs.acs.org/OrgLett
Esterase Activated Carbonyl Sulfide/Hydrogen Sulfide (H S) Donors
2
Preeti Chauhan, Prerona Bora, Govindan Ravikumar, Swetha Jos, and Harinath Chakrapani*
Department of Chemistry, Indian Institute of Science Education and Research Pune, Dr. Homi Bhabha Road, Pashan Pune 411 008,
Maharashtra, India
*
S Supporting Information
ABSTRACT: Hydrogen sulfide (H S) is a mediator of a
2
number of cellular processes, and modulating cellular levels of
this gas has emerged as an important therapeutic area.
Localized generation of H S is thus very useful but highly
2
challenging. Here, we report pivaloyloxymethyl-based carbon-
othioates and carbamothioates that are activated by the
enzyme, esterase, to generate carbonyl sulfide (COS), which is hydrolyzed to H S.
2
1
aseous entities such as nitric oxide (NO), carbon
2
,3
4−6
G
monoxide (CO), sulfur dioxide (SO2)
and hydro-
7
gen sulfide (H S) have emerged as major mediators of cellular
2
3
processes and have therapeutic applications. For example, H S
2
is involved in regulating the homeostasis of various
physiological systems which include−cardiovascular, neuronal,
8
−11
gastrointestinal, renal, liver and reproductive systems.
Modulating levels of H S within cells has tremendous impact
2
1
0,12,13
on disease biology.
generation as well as dissipation assume importance. Due to
Thus, methodologies for controlled
1
4
its gaseous nature, site-specific delivery of H S is challenging. A
2
common strategy to deliver gaseous species is to mask them as
organic or inorganic compounds that dissociate to generate the
1
5
gas. These compounds, known as H S donors, are widely
2
used to generate this gas within cells and in animal
1
0,15,16
models.
Due to the ubiquitous targets of H S, localized
2
delivery of H S is highly desirable but remains a major hurdle.
2
Site-directed delivery requires installation of a structural trigger
in the H S donor which is cleaved by a metabolic stimulus to
2
1
7−24
generate H S.
2
Figure 1. (a) Analyte homeostasis during H S detection is possible
using azide-based thiocarbamates. (b) Nucleophile-activated COS/
2
Carbonyl sulfide (COS) is a naturally occurring gas that
hydrolyzes to H S. This reaction is accelerated by the widely
prevalent enzyme, carbonic anhydrase (CA). Thus, generat-
ing COS within cells is a newly emerging and an attractive
2
H S donors and their corresponding polymeric analogue. (c) Proposed
2
25
enzyme-activated carbonothioate (1) and carbamothioate (2) COS/
H S donors.
2
strategy to localize H S within cells. Recently, Pluth and co-
2
workers reported a new class of H S generating small molecules
2
26
that are triggered by H S. These thiocarbamates generate
2
3
0
COS, which undergoes hydrolysis to produce H S (Figure
presents distinct advantages. We considered carbonothioates
Figure 1c, 1; X = O) as well as carbamothioates (Figure 1c, 2;
2
2
7
(
1
a). Thiocarbamates were proposed as analytical tools for
X = NH) as enzyme activated COS/H S donors. The
detection of H S and may aid in analyte homeostasis. Matson
2
2
significantly lower bond dissociation energy of a typical C−S
bond (shown in green, Figure 1c) when compared with a C−O
bond (shown in blue, Figure 1a) might facilitate the process of
self-immolation. It is anticipated that, upon activation, a
carbonothioate (X = O) will dissociate rapidly in pH 7.4 buffer
to produce COS and an alcohol (Figure 1c).
and co-workers, more recently, reported N-thiocarboxyanhy-
dride (NTA) as a nucleophile activated COS/H S donor
2
2
5
(
Figure 1b). Here, a nucleophile, such as an amine reacts with
31
NTA to generate COS/H S. However, due to the ubiquitous
2
nature of nucleophiles in various biological media, selectivity of
this donor may be compromised. Our laboratory considered
the possibility of using enzyme-activated donors of COS/H S.
2
The use of enzymes to deliver drugs, latent fluorophores (for
Received: November 7, 2016
28,29
imaging), or biological species
has been widely used and
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.6b03336
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
In order to test if the nature of the leaving group X affects the
rate of decomposition, carbamothioates (X = NH) were also
considered (Figure 1c). Together, these compounds will offer
an enzymatic trigger for H S generation and a better
2
understanding of the nature of COS/H S donors. The enzyme
2
that we chose was esterase (ES). Due to its wide occurrence in
nearly all cells, this metabolic trigger will find broad use for the
study of H S biology. The choice of a pivaloyloxymethyl group
2
as the protective group will ensure rapid activation of the donor
3
2
and help with localization of the bioactive molecule.
In order to test this hypothesis, the thiol 4 was synthesized
from 3 (Scheme 1). Reaction of 4 independently with 5a, 5b,
Scheme 1. Synthesis of Thiol 4
and 5c gave the desired compounds 1a, 1b, and 1c (Table 1).
Next, carbamothioates 2a, 2b, and 2c were synthesized from
the corresponding 4-nitrophenolates 6a, 6b, and 6c, respec-
33
tively by reaction with the thiol 4 (Table 1).
Table 1. Synthesis of Carbonothioates and Carbamothioates
Figure 2. (a) H S yields were measured using a Dn-N -based assay.
2
3
Compounds were incubated with ES and CA in pH 7.4 buffer for 60
min followed by addition of Dn-N . Fluorescence intensity at 535 nm
3
(excitation 340 nm) was measured. (b) HPLC analysis of a reaction
mixture consisting of 1c, ES, CA, and Dn-N . Ctrl is Dn-N .
3
3
Fluorescence detector was used: excitation 340 nm; emission 535 nm.
Positive control: Na S (see SI, Figure S2). (c) Formation of methylene
blue as determined by spectrophotometry during incubation of 2b in
a
2
X
Y
R
reactant
prod
yield, %
O
Cl
4-NO Ph
5a
5b
5c
6a
6b
6c
1a
1b
1c
2a
2b
2c
77
64
16
11
44
37
2
the presence of ES and CA. Na S was used as the positive control in
2
O
Cl
Ph
this experiment. (d) Representative trace for H S detection using a
2
O
Cl
CH Ph
H S-sensitive electrode: 1a was incubated in pH 7.4 buffer in the
2
2
b
b
b
NH
NH
NH
OAr
OAr
OAr
Ph
presence of CA. After 5 min, ES was added to the reaction mixture. (e)
Representative images of MCF-7 cells. The H S-sensitive fluorogenic
4-OMePh
2
probe NBD-fluorescein (10 μM) was cotreated with (i) DMSO; (ii)
CH Ph
2
2
b, 50 μM; (iii) 2b, 100 μM, for 40 min followed by imaging using a
a
b
Unoptimized isolated yields. Ar = 4-NO Ph.
2
fluorescence microscope. Scale bar is 200 μm.
(
4-((4-Nitrophenoxy)methyl)phenoxy)methyl pivalate 7 was
2
a). All other compounds 1−2 were similarly found to generate
2
prepared and used as a negative control. This compound
should be cleaved by esterases but will not produce COS. The
formation of 4-nitrophenol (as in the case of 1a) will serve as a
colorimetric indicator for mechanistic investigations.
H S under these conditions (Figure 2a). Compound 7, which is
expected to dissociate in the presence of ES but does not
generate H S, did not show a significant signal attributable to
Dn-NH in this assay (Figure 2a). The formation of the Dn-
2
2
NH fluorophore (see Supporting Information (SI), Figures S1,
2
S2) was also independently confirmed by HPLC analysis of a
reaction mixture containing 1c, ES, CA, and Dn-N (Figure
3
2
b). The formation of H S during decomposition was also
2
35
inferred by a lead acetate assay. Here, incubation of 1c with
ES and CA followed by addition of an aliquot to lead acetate
paper showed a distinct dark coloration that is indicative of the
formation of lead sulfide (see SI, Figure S3).
Using Dansyl-Azide (Dn-N ), a H S-sensitive fluorogenic
3
2
dye, the formation of H S was assessed. This probe is known to
2
−
react with HS to produce dansylamine (Dn-NH ), which has a
Independent verification of H S release from this class of
2
2
distinct fluorescence signal at 535 nm (excitation 340 nm).
compounds was carried out by a colorimetric assay where
When authentic H S was treated with Dn-N , we find a
methylene blue formation is monitored (Figure 2c; see SI,
2
3
25
fluorescence signal at 535 nm (excitation 340 nm) for Dn-NH
Figure S5). An absorbance profile that correlated well with
2
3
4
(Figure 2a). Compound 1a was incubated in the presence of
authentic Na S was observed (see SI, Figure S4). When this
2
ES and CA for 1 h, followed by addition of Dn-N . A
fluorescence signal at 535 nm was indicative of the capability of
experiment was carried out with acetazolamide, a known
inhibitor of CA, we find nearly complete abrogation of the
absorbance for methylene blue formation (see SI, Figure
3
this compound to generate H S under these conditions (Figure
2
B
DOI: 10.1021/acs.orglett.6b03336
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
2
4,26
S6).
Lastly, carbonothioate 1a was incubated with CA and a
carbamothioates 2a, 2b, and 2c in pH 7.4 buffer in the presence
H S-selective electrode was used for detection of H S (Figure
of ES and CA were recorded by using a similar Dn-N assay.
2
2
3
2d). The formation of COS was verified by a reported mass
The relative rates for Dn-NH formation during incubation of
2
spectrometry methodology, and a profile that is consistent with
COS was recorded (see SI, Figure S7).
2a and 2b were found to be comparable with those of 1a−1c
36
(see SI, Table S1, entries 4−5); it is thus likely that a similar
The H S donors prepared herein were evaluated for their
mechanism is operational. The rate of H S release from 2b was
2
2
ability to inhibit proliferation of human breast cancer MCF-7
cells using a standard cell viability assay. We find no significant
monitored by a methylene blue formation assay (see SI, Figure
S5), and we found a similar rate when compared with the
39
cytotoxicity at 25 μM (see SI, Figure S13). The H S generating
dansyl azide method. HPLC analysis of the decomposition of
2b (see SI, Figure S10) showed the formation of an
intermediate (possibly I, Scheme 2) that decomposed to
produce p-anisidine. Thus, rapid cleavage of the pivaloylox-
ymethyl group followed by self-immolation produces a short-
lived intermediate I, which dissociates to produce COS.
2
capability of 2b within cells was evaluated using a fluorogenic
37
probe NBD-Fluorescein for H S (see SI, Figure S14). Upon
2
incubation of MCF-7 cells with this probe and subsequent
treatment of cells with 2b, we find a distinct fluorescence signal
suggesting increased intracellular H S (Figure 2e). Together,
2
these data support the formation of COS/H S under the assay
2
Hydrolysis of COS in the presence of CA produces H S.
2
conditions from donors prepared in this study and their
compatibility for use in cellular studies.
When H S release from 2c was monitored by the Dn-N
2
3
assay as well as methylene blue formation, we found a slower
rate of release when compared with other compounds tested
Next, the kinetics of H S release from these compounds was
2
39
3
studied. Using the Dn-N method, the time course of Dn-NH2
(see SI, Table S1 and Figure S5). Decomposition of 2c in the
presence of ES was carried out: HPLC analysis revealed nearly
complete disappearance of 2c within 15 min suggesting the
triggerable nature of this donor in the presence of the stimulus,
esterase (see SI, Figure S11). An intermediate (possibly I,
Scheme 2) was observed which gradually disappeared during
the course of the reaction (see SI, Figure S12). We were unable
to characterize this intermediate, but it appears that the rate of
COS release may, in part, depend on the decomposition of this
intermediate. Hence, the use of carbamothioates such as the
ones developed in this work may offer distinct advantages in
formation, which is a proxy for H S release, was monitored. A
2
representative curve for H S release from 1a is shown (see SI,
2
Figure S1). The rate constant for H S release was found by
2
fitting the initial rate data to first-order kinetics (see SI, Figure
S1). The rate constants for H S release from 1a, 1b, and 1c
2
were found to be similar in magnitude suggesting no major
dependence on the nature of the leaving group (see SI, Table
S1, entries 1−3). In order to better understand the mechanism
of decomposition (Scheme 2), the decomposition of 1a was
monitored by HPLC (see SI, Figure S8).
modulating H S release through stereoelectronic effects on the
nitrogen. Although these offer mechanistic insight, the
differences among the release profiles are small, and in cellular
experiments, the concentrations of these enzymes will, in part,
2
Scheme 2. Proposed Mechanism for Decomposition of
Carbonothioates and Carbamothioates in the Presence of ES
and CA
40
determine the kinetics of H S release. Wang and co-workers
2
reported an esterase triggered small molecule with tunable H S
2
21
generating capability. Here, the nature of the ester was
modified to attain differences in rates of H S generation. The
2
compounds prepared in our study can perhaps similarly be
modified to achieve tunability. Furthermore, this scaffold is also
amenable to incorporation of other triggers of interest that
facilitate localized delivery of H S: Pluth and co-workers have
2
reported a hydrogen peroxide inducible H S donor based on
2
40
the thiocarbamate scaffold. However, carbonothioates and
carbamothioates prepared in this study, to our knowledge, have
41
thus far not been reported as H S donors.
2
Upon addition of esterase, nearly complete disappearance of
In summary, we show that carbonothioates as well as
carbamothioates can be suitably modified to be triggered to
generate COS under physiologically relevant conditions. The
importance of gaseous species in mediating cellular processes as
well as close association with pathophysiological conditions has
been widely studied. In order to better understand the precise
roles of these gases, new and improved tools are necessary.
While these tools are being developed, they are also being
evaluated for their therapeutic potential. Numerous inves-
1
a within 15 min was recorded suggesting that cleavage of the
32
pivaloyloxymethyl group is fast. During this study, concom-
itant formation of 4-nitrophenol was observed, which was
confirmed by using an authentic sample of this compound (see
SI, Figure S8). The time course of 4-nitrophenol formation
during decomposition of 1a was next independently recorded
by spectrophotometry by monitoring the absorbance of 4-
nitrophenol at 405 nm (see SI, Figure S9). The rate constant
−1
was found as 0.14 min . When a similar experiment was
conducted with 7 where 4-nitrophenol release does not involve
the formation of COS, a comparable rate constant of 0.15
tigations have shown that H S has enormous potential but is
2
limited by its toxicity. Therefore, being able to trigger H S
2
−1
min was recorded. Together, these data suggest the short-
generation intracellularly in a site of interest is of fundamental
importance. Our data reveal that the cleavage of these
compounds by esterase is rapid followed by generation of
lived nature of intermediate I in the case of 1a. Although the
rate of 4-nitrophenol formation is faster than H S release as
2
determined by Dn-NH2 formation, these relatively small
COS/H S. Although esterases are widely present in numerous
2
differences do not support significant accumulation of
cell types, this study lays the foundation for site-directed
38
intermediate I (Scheme 2). Next, H S release profiles of
delivery of COS/H S.
2
2
C
DOI: 10.1021/acs.orglett.6b03336
Org. Lett. XXXX, XXX, XXX−XXX

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AUTHOR INFORMATION
Corresponding Author
E-mail: harinath@iiserpune.ac.in.
■
*
2
(
ORCID
(
6
(
Harinath Chakrapani: 0000-0002-7267-0906
Notes
The authors declare no competing financial interest.
(
ACKNOWLEDGMENTS
■
(
(
The authors thank the Department of Science and Technology
DST, Grant No. EMR/2015/000668) and Council for
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Scientific and Industrial Research (CSIR) for financial support.
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DOI: 10.1021/acs.orglett.6b03336
Org. Lett. XXXX, XXX, XXX−XXX