Visible-Light-Triggered Uncaging of Carbonyl Sulfide for Hydrogen Sulfide (H2S) Release

Article abstract of DOI:10.1021/acs.orglett.7b02259

Generation of hydrogen sulfide (H₂S) is challenging and few methods are capable of localized delivery of this gas. Here, a boron dipyrromethene-based carbamothioate (BDP-H₂S) that is uncaged by visible light of 470 nm to generate carbonyl sulfide (COS), which is rapidly hydrolyzed to H₂S in the presence of carbonic anhydrase, a widely prevalent enzyme, is reported.

Full text of DOI:10.1021/acs.orglett.7b02259

Letter
pubs.acs.org/OrgLett
Visible-Light-Triggered Uncaging of Carbonyl Sulfide for Hydrogen
Sulfide (H2S) Release
Ajay Kumar Sharma,† Mrutyunjay Nair,† Preeti Chauhan,† Kavya Gupta,‡ Deepak K Saini,‡
and Harinath Chakrapani*,†
†Department of Chemistry, Indian Institute of Science Education and Research Pune, Dr. Homi Bhabha Road, Pashan, Pune 411 008,
Maharashtra, India
‡Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, Karnataka, India
S
* Supporting Information
ABSTRACT: Generation of hydrogen sulfide (H2S) is challenging and
few methods are capable of localized delivery of this gas. Here, a boron
dipyrromethene-based carbamothioate (BDP-H2S) that is uncaged by
visible light of 470 nm to generate carbonyl sulfide (COS), which is rapidly
hydrolyzed to H2S in the presence of carbonic anhydrase, a widely
prevalent enzyme, is reported.
aseous species derived from carbon, nitrogen, and sulfur
G
are emerging as major mediators of cellular processes.
Some have been termed as “gasotransmitters”, which are small
gaseous molecules that can act as signal transmitting agents
within cells as well as across cells. For example, hydrogen
sulfide (H2S) is known to mediate a number of physiological
processes.1 Due to its highly diffusible nature and multiple
targets within cells, cell signaling by H2S is often complex and
can depend on local concentrations of this species.2,3 This
underscores the need for spatiotemporally controlled gener-
ation of this gas under ambient conditions. A number of donors
of H2S with varying degrees of control over release of H2S are
known.4,5 Among these, light-triggered donors have distinct
advantages for localized delivery.6−8 On account of the ability
to direct the light source, generation of H2S in a spatially
controlled manner is possible. Although small molecule-based
light-triggered H2S generation methodologies are known, they
all are based on ultraviolet light. For example, Xian and co-
workers have reported a 2-nitrobenzyl-based donor that when
exposed to 365 nm light produces a geminal dithiol, which
undergoes hydrolysis to generate H2S (Figure 1a). Nakagawa
and co-workers have reported two scaffolds based on thioethers
that are triggered by UV light to produce H2S (Figure 1a).7,8
More recently, Connal and co-workers have reported photo-
generated thiobenzaldehydes as H2S donors.9 While these
methods offer excellent spatiotemporal control, the photo-
toxicity associated with UV light is a major limitation.10 Thus, a
method for photouncaging of a H2S source under ambient
visible light conditions is yet unavailable.
Figure 1. (a) Selected UV-light-activated methodologies for H2S
generation R1 = various alkyl groups; Ar = PhCOPh; R2 = aryl group.
(b) Design of a visible light-triggered COS/H2S donor. Visible-light-
mediated cleavage of the B−O bond produces an intermediate I that
undergoes self-immolation to generate COS that undergoes hydrolysis
in the presence of CA to generate H2S.
rapid hydrolysis in the presence of the widely occurring enzyme
carbonic anhydrase (CA),19 to produce H2S. Pluth and co-
workers have recently reported a UV light activated COS/H2S
Recently, 4-arylalkoxy-boron dipyrromethene (4-OAr-BOD-
IPY)11 has been used to deliver histamine, an important
mediator of inflammation and allergy.12 Our group13 and
others14−17 have shown that carbonyl sulfide (COS)18 is an
excellent donor of H2S. The gas COS is known to undergo
Received: July 23, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b02259
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Scheme 1. Synthesis of H2S Donor 1
donor (Figure 1a).20 Again, the potential for phototoxicity due
to prolonged UV exposure may be a limitation of this method.
Therefore, masking COS in the form of a carbamothioate with
attachment of this functional group to a BODIPY-based
photolabile group was considered (Figure 1b). Upon exposure
to visible light, this compound is expected to undergo cleavage
to produce COS/H2S.
Compound 2 (Scheme 1) was first synthesized using a
reported methodology (see Supporting Information (SI)).21
Next, 4-S-(hydroxybenzyl) ethanethioate 3 was prepared, and
reaction of 3 with 2 in the presence of a Lewis acid AlCl3 gave a
thioacetate (crude) as an intermediate, which was subsequently
hydrolyzed to the corresponding thiol (not isolated). Reaction
of the crude thiol with the 4-nitrophenylcarbamate 4 gave 5 in
75% yield over two steps. Due to the increased hydrophobicity
associated with BODIPY derivatives, incorporation of a short
oligo-ethylene glycol functional group that should increase
aqueous solubility was considered. Using a copper-mediated
click reaction, the desired compound, BDP-H2S 1, was
prepared in 21% yield.
Using a reported method, BDP-H2S was incubated in MeOH
and exposed to 470 nm light for 20 min. HPLC analysis
(detection wavelength, 500 nm) revealed nearly complete
decomposition of 1 (Figure 2a; also see Figure S1, SI). Urano
and co-workers have reported that uncaging of the 4-aryloxy-
BODIPY derivative12 is accomplished by a photoinduced
electron transfer (PeT)22,23 process that results in the
formation of a charge-separated intermediate, involving a
cation radical of the aryloxy group and the anion radical of
BODIPY. This results in the cleavage of the B−O bond by
solvolysis, and the expected product in methanol is the B−
OMe derivative. The time course of decomposition of 1 was
studied, and a first-order rate constant of 0.16 min−1 was
obtained (Figure 2b). During photolysis of 1, HPLC analysis
revealed the formation of a new product (Figure 2a), which was
characterized by mass spectrometry as the methoxy-derivative 7
(Figure 2d, Figure S5, SI). The time course for formation of 7
gave a first order rate constant of 0.20 min−1 (Figure 2b).
Together, these data suggest cleavage of 1 is accompanied by
rapid solvolysis to produce 7.
Figure 2. (a) HPLC traces for 1 before and after irradiation with 470
nm light (30 mW/cm2) for 20 min in MeOH showed nearly complete
disappearance of 1 with concomitant formation of an intermediate at
retention time of 13.2 min, which was later identified by mass
spectrometry analysis as BDP-OMe (7). (b) Time course of
disappearance of 1 and formation of intermediate 7 during the same
time period was determined by HPLC analysis. Curve fitting yielded
first-order rate constants for disappearance of 1 as 0.16 min−1 and
formation of 7 as 0.20 min−1. (c) Time course of disappearance of
intermediate I was followed by HPLC analysis. The rate constant for
decomposition was found as 0.02 min−1, (d) Proposed structure of the
intermediate 7 produced during photolysis of 1 in methanol and the
negative control 8.
when compared with the OMe group in 7. In a separate
experiment, the fluorescence enhancement was monitored by a
microwell plate reader and the fluorescence enhancement after
irradiation was >20-fold (Figure 3a). In order to test whether
this fluorescence enhancement also occurred within cells,
human cervical cancer HeLa cells were incubated with 1 for 2
min followed by imaging using a fluorescence microscope. A
small fluorescence signal at 540 nm (excitation 470 nm) was
seen under these conditions (Figure 3b, dark). However, when
HeLa cells pretreated with 1 were irradiated with a 470 nm
light source for 2 min, and subsequent fluorescence enhance-
ment was studied by microscopy, a significant enhancement in
fluorescence signal was recorded (Figure 3b and 3c, light versus
dark).
When photolysis of 1 was monitored by HPLC attached with
a fluorescence detector, a distinct peak attributable to 7 was
formed (see SI, Figure S2). Thus, photocleavage of 1, a weakly
fluorescent compound, in methanol produces 7, which is highly
fluorescent (excitation 470 nm, emission 540 nm).
A cell viability assay was next conducted to assess the
cytotoxicity during photocleavage of 1. HeLa cells were
independently incubated with 1 at various concentrations,
This enhancement in fluorescence is attributable to the
differences in the quenching ability of the O-aryl group in 1
B
DOI: 10.1021/acs.orglett.7b02259
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Organic Letters
Letter
Scheme 2. Proposed Mechanism for H2S Generation during
Photocleavage of BDP-H2S 1 in Methanol Followed by
a
Incubation in pH 7.4 Buffer
Figure 3. (a) Enhancement in fluorescence (excitation 470 nm,
emission 540 nm) when 1 (50 μM) was irradiated with 470 nm light;
(b) HeLa cells pretreated with 1 (10 μM) were irradiated for 2 min by
470 nm light and imaging showed increased fluorescence signal (YFP
channel); Scale bar = 50 μm (c) Fluorescence enhancement data for
the cellular experiment. **p-value = 0.002.
a
For further details, see Scheme S2, SI.
produce H2S was observed (Figure 5a). In the absence of CA or
when incubated in the dark, 1 was incapable of generating H2S
and these cells were exposed to 470 nm light for 5 min. We
found no evidence for inhibition of proliferation of HeLa cells
by 1 during photocleavage suggesting that this compound was
not cytotoxic (Figure 4). The compound itself was not toxic in
the dark (Figure S9a), and the absorbance at 570 nm from this
compound was not significant (Figure S9b).
Figure 5. (a) A methylene blue formation assay was used to determine
H2S release from 1 when irradiated with 470 nm light for 25 min
followed by exposure to carbonic anhydrase in pH 7.4 buffer. Inset,
time course of increase in absorbance at 676 nm during 2 h. Curve
fitting to a pseudo-first-order release gave a rate constant of 0.03 min−1
(see SI). (b) H2S generation during incubation of 1 after irradiation
with visible light followed by exposure to CA in pH 7.4 buffer was
assessed by a H2S-sensitive electrode. Dark: A similar experiment was
conducted in the absence of light.
Figure 4. Effect of 1 on cell viability was determined by two
independent assays: (a) HeLa cells with increasing doses of 1 followed
by irradiation with 470 nm light for 5 min (24 h incubation, MTT
assay). (b) A549 cells with increasing doses of 1 followed by
irradiation with 470 nm light for 5 min (24 h incubation, crystal violet
assay). The differences in viability are not significant (ns) as
determined by Student’s t-test.
in 2 h (Figure S6, SI). The time course of H2S release showed a
gradual increase over 2 h (Figure 5a, inset). The rate constant
for H2S release was found to be 0.03 min−1, which is
comparable with the disappearance of intermediate I (Figure
2c). The yield of H2S was found to be in the range 30%−40%;
this efficiency is comparable with the previously reported
photouncaging of histamine that produced a similar yield of the
amine.12
Next, a H2S-sensitive electrode was used to independently
verify the generation of H2S from 1. Here, the irradiated sample
of 1 in methanol was incubated in pH 7.4 buffer in the presence
of CA and H2S generation was monitored by the electrode. As
expected, a distinct signal attributable to H2S was recorded
(Figure 5b). No detectable signal was observed when the donor
was incubated under similar conditions in the absence of light.
BDP-H2S 1 was found to be stable in pH 7.4 buffer during 2.5 h
incubation in the dark as determined by HPLC analysis (see SI,
Figure S4). Together, these data confirm the ability of 1 to
generate H2S only when exposed to visible light of 470 nm.
Compound 8 was next synthesized (see Figure 2d, Scheme
S1, SI) and was similarly irradiated with 470 nm light in
methanol. The resultant reaction mixture was incubated in pH
7.4 buffer and was analyzed for H2S production by a methylene
A crystal violet assay was independently conducted using the
A549 human lung cancer cell line to validate our results: the
compound was not significantly cytotoxic (Figure 4b).
Together, these results indicate that 1 is nontoxic, is cell
permeable, and can be cleaved by a visible light source within
cells to generate a fluorescence signal.
Cleavage of the B−O bond results in the formation of a
phenolate-intermediate I, which has been previously reported
to generate COS (Scheme 2).13 The time course of
decomposition of I was monitored in pH 7.4 buffer (see
Figure S3, SI), and this intermediate disappeared within 2 h
(Figure 2c). The rate constant for decomposition was found to
be 0.02 min−1. In the presence of carbonic anhydrase (CA),
COS is known to generate H2S (Scheme 2). In order to test
this hypothesis, 1 was irradiated using visible light at 470 nm in
methanol and the resulting mixture was incubated in pH 7.4
buffer in the presence of CA. The formation of H2S was
monitored by a methylene blue assay. If H2S were produced, a
characteristic UV−visible absorption spectrum with absorption
maxima at 676 nm would be expected.
When this experiment was conducted, as predicted, an
increase in absorption at 676 nm confirming the ability of 1 to
C
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.7b02259.
■
S
Compound characterization data, spectra, and assay
protocols (PDF)
(27) Dharmaraja, A. T.; Ravikumar, G.; Chakrapani, H. Org. Lett.
2014, 16, 2610.
(28) Sharma, K.; Iyer, A.; Sengupta, K.; Chakrapani, H. Org. Lett.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: harinath@iiserpune.ac.in.
ORCID
2013, 15, 2636.
(29) Keefer, L. K. ACS Chem. Biol. 2011, 6, 1147.
(30) Khodade, V. S.; Kulkarni, A.; Gupta, A. S.; Sengupta, K.;
Chakrapani, H. Org. Lett. 2016, 18, 1274.
(31) Wu, L.; Wang, R. Pharmacol. Rev. 2005, 57, 585.
(32) Malwal, S. R.; Chakrapani, H. Org. Biomol. Chem. 2015, 13,
2399.
Harinath Chakrapani: 0000-0002-7267-0906
Notes
The authors declare no competing financial interest.
(33) Malwal, S. R.; Gudem, M.; Hazra, A.; Chakrapani, H. Org. Lett.
2013, 15, 1116.
ACKNOWLEDGMENTS
■
(34) Malwal, S. R.; Sriram, D.; Yogeeswari, P.; Konkimalla, V. B.;
Chakrapani, H. J. Med. Chem. 2012, 55, 553.
(35) Malwal, S. R.; Sriram, D.; Yogeeswari, P.; Chakrapani, H. Bioorg.
Med. Chem. Lett. 2012, 22, 3603.
(36) Day, J. J.; Yang, Z.; Chen, W.; Pacheco, A.; Xian, M. ACS Chem.
Biol. 2016, 11, 1647.
(37) Wang, W.; Ji, X.; Du, Z.; Wang, B. Chem. Commun. 2017, 53,
1370.
The authors thank the Department of Science and Technology
(DST, Grant No. EMR/2015/000668) and Council for
Scientific and Industrial Research (CSIR) for financial support.
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DOI: 10.1021/acs.orglett.7b02259
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