A Highly Selective Fluorescent Probe for Detection of Hydrogen Sulfide in Living Systems: In Vitro and in Vivo Applications

Article abstract of DOI:10.1002/chem.201701124

A fluorescein-based fluorescent probe has been designed and synthesised that selectively detects H₂S in aqueous medium, among various analytes tested. This fluorescein-based fluorescent probe has also been successfully utilised for real-time imaging of exo- and endogenously produced H₂S in cancer cells and normal cells. Moreover, the probe can also detect H₂S in the rat brain hippocampus at variable depths and in living nematodes.

Full text of DOI:10.1002/chem.201701124

A Journal of
Accepted Article
Title: A Highly Selective Fluorescent Probe for Detection of Hydrogen
Sulfide in Living Systems: in vitro and in vivo Applications
Authors: Shahi Imam Reja, Neetu Sharma, Muskan Gupta, Payal
Bajaj, Vandana Bhalla, Ripu D Parihar, Puja Ohri, Gurcharan
Kaur, and Manoj Sharma Kumar
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To be cited as: Chem. Eur. J. 10.1002/chem.201701124
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A Highly Selective Fluorescent Probe for Detection of Hydrogen
Sulfide in Living Systems: in vitro and in vivo Applications
Shahi Imam Reja[a]†, Neetu Sharma[a]†, Muskan Gupta[b]†, Payal Bajaj[b], Vandana Bhalla[a], Ripu D.
Parihar[c], Puja Ohri[c], Gurcharan Kaur[b]* and Manoj Kumar[a]*
Dedication ((optional))
Abstract: A fluorescein based fluorescent probe 3 has been
designed and synthesized which selectively detects H2S in aqueous
medium among the various analytes tested. The probe 3 has also
been successfully utilized for real time imaging of exogenous and
endogenously produced H2S in cancer cells and normal cells.
Moreover, the probe 3 can also detect H2S in rat brain hippocampus
at variable depth and in living nematodes.
Recently, we reported a bodipy based probe for detection of H2S,
but unfortunately the probe shows turn-off fluorescence
behaviour and did not work in aqueous medium.3b Keeping in
view the limitations of reported probes, we were then interested
to design a probe which could selectively and rapidly detect H2S
in aqueous medium with turn-on fluorescence response. For
achieving this target, we envisaged that if we could design a
molecule that has dual nucleophilic center then, it will be
possible to achieve selectivity and faster response and thus
designed and synthesized fluorescein based probes 2 and 3
with 2-bromoethyl moiety linked via carbonate ester and ester
linkages. We have introduced bromo groups in both the probes
as these groups can be easily replaced by H2S which will then
facilitate the ring opening of fluorescein moiety. However, out of
the two synthesized probes, only probe 3 shows selectivity
towards H2S and works well in aqueous medium. Moreover, the
designed probe 3 has several advantages: (i) It shows fast turn-
on response towards H2S. (ii) The probe can detect upto 0.13
µM of H2S which is sufficiently low for monitoring endogenously
produced H2S in living cells. (iii) It can also detect endogenously
generated H2S in living C6 and BV-2 cell lines. (iv) Moreover,
probe 3 is used for real time monitoring of H2S production in
normal phenotype vs cancer cells. (v) Probe 3 was also utilized
for the detection of H2S levels in the hippocampus region of rat
brain after stimulating the tissue with cysteine and for the
detection of H2S in nematodes. We believe that probe 3 will help
in real time visualization of H2S receptors in normal and cancer
cells.
Introduction
Hydrogen sulfide (H2S) recognized as a toxic and inflammable
gas regulates many biological processes in human body.1˗3 H2S
when present in low concentration can cause personal distress
but its high concentration above 250 ppm can lead to cell death.4
In mammals, H2S is primarily produced from three endogenous
enzymes: cystathionine γ-lyase (CSE), cystathionine β-synthase
(CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST) which
convert cysteine and their derivatives into H2S in different
tissues and organs. The endogenously produced H2S mediates
several physiological processes such as regulation of cell growth,
cardiovascular protection, vasodilation, anti-inflammation and
antioxidation effects.5
Recently, a number of fluorescent probes have been reported
for the detection of H2S6 but most of these probes suffer from
the limitations of poor detection limit, slow response time,
interference from biothiols and their limited biological
applications. Further, there is no report of utilization of these
probes for real time monitoring of endogenously produced H2S
in both cancer and normal cells. The monitoring of H2S
production in both types of cells is important as it helps in
visualizing the distribution of H2S receptors in these cells.
[a]
[b]
[c]
Dr. S. I. Reja, Dr. N. Sharma, Dr. V. Bhalla and Prof. M. Kumar
Department of Chemistry, UGC Centre for Advanced Studies-II
Guru Nanak Dev University
Amritsar, India
E-mail: mksharmaa@yahoo.co.in
Miss M. Gupta, Miss P. Bajaj and Prof. G. Kaur
Department of Biotechnology
Guru Nanak Dev University
Amritsar, India
E-mail: kgurcharan.neuro@yahoo.com
Mr. Ripu D. Parihar and Dr. Puja Ohri
Department of Zoology
Scheme 1. Synthesis of compound 2 and 3
Results and Discussion
The coupling of 4-bromobutyric acid with methyl protected
fluorescein 1 in the presence of DCC and DMAP furnished the
compound 2 in 37% yield (Scheme 1). The reaction of 2-
bromoethanol with methyl protected fluorescein
1 in the
Guru Nanak Dev University
presence of DIPEA and triphosgene furnished the compounds 3
in 32% yield (Scheme 1). The structure of compounds 2 and 3
were confirmed from their spectroscopic data.
Amritsar, India
†These authors contribute equally
Supporting information for this article is given via a link at the end of the
document.((Please delete this text if not appropriate))
The molecular recognition behaviour of probes 2 and 3 was
˗
˗
studied towards different analytes (H2S, GSH, Cys, HSO3 , SO3 ,
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S2O32˗, H2O2, BuO. and OH) in H2O: DMSO (99.5:0.5, v/v)
buffered with HEPES, pH = 7.4 by UV-vis and fluorescence
spectroscopy. The absorption spectrum of probe 3 (5.0 μM)
exhibits two very weak absorption bands at 497 nm and 460 nm
(Fig. S5). Upon addition of H2S (NaHS was used as H2S source)
to the solution of probe 3, an increase in the absorption band at
495 nm as a function of H2S concentration was observed.
Similar absorption behaviour was observed for probe 2 (Fig. S6).
The fluorescence spectrum of probe 3 exhibits no emission band
when excited at 470 nm (Fig. 1a). However, upon addition of
H2S (0–80 μM) to the solution of probe 3, a new emission band
presence of N2H4 (Fig. S8-S11). However, compound 3 does not
show any significant fluorescence enhancement upon
incremental addition of N2H4 which is probably due to the fact
that the carbonate ester is less reactive towards basic analytes
than the corresponding ester derivatives.10 To test the practical
applicability of probe 3 we also carried out the competitive
experiments in the presence of H2S; mixed with other analytes
but no noticeable change in the fluorescence emission was
observed in comparison with or without any other analytes (Fig.
S12-13). Thus, from these
t
.
900
(a)
(b)
800
700
600
500
400
300
200
100
0
80 µM
H2S
0 µM Buffer
only
Probe
only
1
2
3
4
5
6
7
8
9 10 11 12
Analytes
Figure 1. (a) Fluorescence emission spectra of probe 3 (1.0 μM) upon the
addition of increasing concentration of H2S (0–80 μM) in H2O (0.5% DMSO
used as co˗solvent) buffer with HEPES (50 mM), pH=7.4. (b) Fluorescence
change of probe 3 (1.0 μM) towards addition of different analytes (80 μM) in
Scheme 2. Possible mechanism of H2S induced ring opening of fluorescein
moiety
˗
˗
H2O; 12 = H2S, 11 = GSH (0.5 mM), 10 = Cys (0.5 mM), 9 = HSO3 , 8 = SO3 ,
˗
7 = S2O32˗, 6 = H2O2, 5 = tBuO•, 4= HCO3 , 3= HO•,2= ClOˉ, 1= 2-
aminothiophenol.
appears at 517 nm, the intensity of which increases with the
increase in the concentration of H2S. The fluorescence intensity
of probe 3 increased dramatically by 32-folds on addition of H2S
(Fig. 1a). Similar fluorescence behaviour was observed for
probe 2 (Fig. S7).
This increase in fluorescence emission at 517 nm with the
addition of H2S is attributed to double nucleophilic addition of
H2S on probe 3. Initially, nucleophilic substitution (SN2)
mechanism operates where H2S acts as a nucleophile and
bromo group acts as leaving group.7 After replacement of bromo
group by SH (intermediate 4), the 2nd nucleophilic substitution
reaction operates and now thiol group of intermediate 4 acts as
nucleophile which eliminates the thio-lactone and generates ring
opened fluorescein moiety which is responsible for emission
enhancement at 517 nm (Scheme 2).8
Scheme 3. Possible mechanism of cysteine induced thioether formation
studies it is clear that only probe 3 is selective for H2S. Thus, we
carried out further studies using probe 3 only. We also studied
the effect of pH on the recognition behaviour of probe 3 towards
H2S, which exists in equilibrium with HS-/S2- at pH 7. In basic pH
(pH 7-10), probe 3 did not show fluorescence emission however
fast fluorescence response was observed upon addition of H2S
as compared to fluorescence response in acidic pH (pH<6). This
fast enhancement in fluorescence emission at pH 7-10 is due to
the existence of H2S as HS- under basic conditions, which has
more nucleophilicity in comparison to H2S which exists in acidic
pH (Fig. S14). From these studies, we may conclude that probe
Under the same conditions as used for H2S, we also carried out
absorption and fluorescence studies of probes 2 and 3 with
cysteine, no significant change in fluorescence emission was
observed in the presence of cysteine. The selective response of
probes 2 and 3 towards H2S over cysteine is due to the rapid
intramolecular attack of sulphur atom of thiol moiety to carbonyl
carbon atom to form a five membered ring and ring opened
fluorescein moiety (SI page S10). On the other hand on reaction
with cysteine an intermediate 5 is formed in which intramolecular
attack of nitrogen atom of amino group to carbonyl carbon does
not takes place. This may be due to fact that formation of eight
membered ring (6) is not kinetically favorable (Scheme 3 and SI
page S10).9 We have also carried out studies with reactive sulfur
3
does not show any interference of pH. Further, to
experimentally verify the reaction mechanism, we carried out the
mass spectral studies of probe 3 in the presence of H2S. The
appearance of peaks at m/z 347.08 and 105.09 (Fig. S15) in the
mass spectrum confirms the formation of ring opened
fluorescein moiety and 1,3-oxathiolan-2-one. Further, the 1H
NMR spectrum of probe 3 in the presence of H2S also shows
splitting and up field shift of aromatic protons which supports the
formation of ring opened fluorescein moiety (Fig. S16). Next, we
studied the time-dependent fluorescence response of the probe
3 in the presence of H2S and cysteine. It was observed that
within 10 min maximum fluorescence enhancement takes place
in case of H2S whereas no significant fluorescence
˗
species like SO3 , S2O32˗ and SO42˗; reactive oxygen species like
H2O2, tBuO•, HO•, reducing agent like N2H4, NH3 etc.,
biologically relevant metal ions (Na+, K+, Mg2+ and Ca2+) (Fig.
1b) but no significant change was observed in the UV-visible
and fluorescence spectra of these probes in the presence of
these analytes except the response of probe 2 with N2H4 which
shows increase in fluorescence emission at 517 nm in the
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enhancement takes place in case of cysteine. This indicates the
Having done all this, we were then interested to explore the
fast response of probe 3 towards H2S (Fig. S17). The detection
applications of probe 3 in monitoring H2S levels under different
limit11 of probe 3 for H2S was found to be as low as 0.13 µM (Fig. physiological conditions in living cells. For this, we used both in
S18), which is sufficiently low for monitoring H2S level in
biological systems. Further, to demonstrate the efficiency of
probe 3 for quantitative detection of H2S, we treated probe 3 with
different concentrations of H2S to obtain a standard curve of
emission intensity vs H2S concentration and regression analysis
was done (Fig. S19). The concentration of probe 3 was
maintained at 1.0 µM, while the concentration of H2S varied from
0 to 80 µM. The regression curve indicates that the signal in the
fluorescence spectrum was indeed linearly related to the
concentration of H2S. Thus, from these results, we may
conclude that probe 3 can detect H2S both qualitatively and
quantitatively.
vitro (C6 Glioblastoma and BV-2 microglial cells) and in vivo rat
model systems. We performed the MTT assay12 to check
whether probe 3 is non-toxic in nature. Cytotoxicity assay of the
C6 glioma and BV-2 microglial cells treated with probe 3 showed
that IC50 of the probe was around 2-4 μM and 1-2 μM for C6
glioma and BV-2 microglial cells respectively (Fig. S20). Based
on this preliminary data, 1 and 0.75 μM concentrations of probe
3 were used for C6 glioma and BV-2 microglial cells respectively
for further experiments. Firstly, we carried out exogenous
detection of H2S using probe 3. For this, C6 glioma and BV-2
microglial cells were first exposed to 1.0 μM and 0.75 μM of
probe 3 respectively for 1 hr, then exposed to 20.0 μM
BV-2
C6
Probe + LPS
Probe +H2S
Control
Probe (1.0 μM)
Probe +H S
Control Probe (0.75 μM)
Probe + LPS
2
100
0
(A)
(B)
(C)
(D)
(A)
(B)
(C)
(D)
Figure 2. Confocal images of (I) C6 glioma and (II) BV-2 microglial cells treated with probe 3: (Column A) Control (Column B) Probe alone: C6 glioma and
BV-2 microglial cells were exposed to 1.0 μM and 0.75 μM of probe 3 respectively for 60 min at 37 °C. Column C: in it the probe 3 pre-treated cells were
incubated with 20 μM of NaHS (exogenous source of H2S) for another 60 min and column D: (I) C6 glioma and (II) BV-2 microglial cells were treated with
LPS (2.0 μg/ml) and (100 ng/ml) for 24 hours respectively then incubated with probe 3 (1.0 μM for C6 glioma cells and 0.75 μM for BV-2 microglial cells) for
another 60 min. Images were taken at λex = 488 nm and λem range = 500-550 nm. Images were captured using A1R Nikon Confocal Microscope at 488 nm
channel using 60X magnification.
exogenous H2S source (NaHS) for another 1 hr. Cells treated
with exogenous source of H2S showed high fluorescence
intensity than the probe alone as revealed by confocal
imaging and intensity analysis (Fig. 2 & S21). Next, we
studied the endogenous production of the H2S in both C6
glioma and BV-2 microglial cells using activated bacterial cell
wall component lipopolysaccharide (LPS). LPS is a known
activator13 that induces the production of H2S by increasing
cystathionine γ-lyase (CSE) expression.14 For this, cells were
pre-treated with LPS for 24 hrs and then exposed to probe 3
for 1 hr. Treatment with LPS increased the production of H2S
endogenously as indicated by increased fluorescent intensity
in LPS treated cells in comparison to probe alone (Fig. 2 &
S21).
Time dependent changes in the fluorescence of probe 3 were
further monitored in the presence of H2S in C6 glioma and
BV-2 microglial cell lines, cells were pre-treated with probe 3
for 1 hr and then exposed to exogenous H2S for different time
intervals ranging from 0-60 min. Confocal imaging and
intensity analysis showed the increase in fluorescence
intensity of the probe 3 with increase in the exposure time of
the exogenous H2S source (Fig. 3). These studies clearly
indicate that the probe 3 has the potential to monitor dynamic
changes in H2S levels in both cancer and normal cells.
Further the specificity and selectivity of the probe 3 for H2S
was further confirmed by analyzing the fluorescence intensity
of probe in the presence of the H2S scavenger NEM (N-
Ethylmaleimide). For this, H2S scavenger treated C6 glioma
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cells were exposed to probe 3 and change in fluorescent
intensity was recorded at intervals of 10 min, for 60 min. The
fluorescence intensity of the probe decreased by 56% in the
presence of H2S scavenger and was constant upto 50 min
and then slightly increased at 60 min (Fig. S22). This slight
increase in fluorescence emission at 60 min in comparison
fluorescence emission at 50 min may be due to the more
production of H2S with time which reduces the quenching
effect of the quencher and leads to higher availability of H2S
towards probe 3. H2S plays an important role in regulating
CNS functions and has been reported to exert
neuroprotective effects via various mechanisms like
antioxidative, anti-inflammatory and anti-apoptotic.15 In
addition to its physiological role as a neuromodulator and
neuroprotectant, higher concentration of H2S is involved in
the pathophysiology of the CNS16. For in vivo studies probe
3 was tested for monitoring the endogenous H2S levels in
dentate gyrus (DG) region of the hippocampus, a brain region
which plays an important role in stress, depression, memory
formation, and spatial behaviour.
Figure 3. Confocal images of the (Row A) C6 glioma cells and (Row B) BV-2 microglial cells representing time dependent expression of the probe 3. Cells
were first treated with probe 3, and then incubated with H2S (20 μM) for different time intervals of 10, 20, 30, 50, 60 min. Images were captured using the
A1R Nikon Confocal Microscope at λex = 488 nm (for probe) with λem range 500–550 nm.
To monitor H2S levels in brain tissue, the brain was dissected
and snap-frozen by using isopentane. Three groups were
chosen; I, Probe alone: Brain sections (thickness: 60
µm)were incubated with probe 3 (5.0 µM) for 1 hr at 37 °C, II,
Probe + H2S: the probe 3 incubated sections were further
treated with 20 µM of exogenous H2S source (NaHS) for
another 1 hr. III, Sections were first treated with 10 mM of
endogenous source of H2S, L-Cysteine (As Cysteine
promoted the production of hydrogen sulfide in the brain
tissue by stimulating CBS)17,18 for 1 hr then exposed to probe
for another 1 hr. Then the images were captured using
confocal microscope at 488 nm wavelength. Tissue-imaging
results showed increased fluorescence intensity in the
sections treated with L-Cysteine and exogenous H2S source
as compared to probe 3 alone (Fig. 4 & S23).
From these tissue-imaging studies, it may be concluded that
the probe 3 also has the potential to monitor hydrogen sulfide
levels in tissues. Since the brain tissue is heterogeneous
having different types of cells, optical sectioning was used to
capture images at intervals of 5.0 µm along the z-axis to
visualize the endogenous distribution of the H2S in the brain
tissue and the degree of penetration of probe 3 into the DG
region of the brain slices at different depths.19,20 The tissue
images indicate that probe 3 has the ability to penetrate
deeper inside the tissue. During sectioning, the cells on both
the top and bottom surfaces of the brain slices may have
been damaged due to slicing. An increase in fluorescence
intensity in sections that were exposed to cysteine and the
exogenous source of H2S was observed in almost all the
middle sections, thereby indicating that probe 3 effectively
Figure 4. Fluorescent images of the 60 µm rat hippocampal slice labelled
with 5.0 μM probe 3 (10X magnification). (a) Brain sections were incubated
with 5.0 μM probe 3 for 1hr. (b) Probe pre-treated sections were exposed
to 20 μM exogenous H2S for 1hr. (c) Sections treated with 10 mM L-
Cysteine for 1 hr then exposed to 5.0 μM probe 3 for 1hr. 3b represents the
3D images of the different treated groups.
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All reagents were purchased from Aldrich and were used without
further purification. HPLC grade DMSO was used in UV-vis and
fluorescence studies. UV-vis spectra were recorded on a SHIMADZU
UV-2450 spectrophotometer, with a quartz cuvette (path length 1 cm).
The cell holder was thermostatted at 25 °C. The fluorescence spectra
were recorded with a SHIMADZU 5301 PC spectrofluorimeter. 1H and
13C spectra were recorded on a Bruker Avance III HD 500 MHz
spectrophotometer using CDCl3 and DMSO˗d6 as solvents and
tetramethylsilane as the internal standard. Mass spectra were
recorded on a Bruker MicroTOF QII mass spectrometer. Data are
reported as follows: chemical shift in ppm (d), multiplicity (s = singlet,
d = doublet, t = triplet, m = multiplet, br = broad singlet), coupling
constants J (Hz).
monitored the production of hydrogen sulfide in the intact
cells (Fig. S24).
Since the probe 3 could detect H2S in living and intact cells at
a depth of 60 µm, we envisaged that probe 3 may have the
potential to detect H2S under in vivo conditions as well and
thus carried out in vivo studies on nematodes (Distolabrellus
species) as in vivo model system.21 To better understand the
capability of probe 3 in tracing the accumulation of H2S, in
vivo imaging was done in nematodes. Confocal fluorescence
imaging was examined by staining nematodes with probe 3
with different conditions. For detection of H2S in nematodes,
we choose three groups (i) probe alone: nematodes were
exposed to 10.0 µM of probe 3 alone (ii) probe pre-treated
nematodes were incubated with 0.3 mM exogenous H2S
source for 6 hrs; (iii) Firstly, the nematodes were incubated
with 0.3 mM exogenous H2S then 1 mM of NEM (N-
Ethylmaleimide) was added and incubated for 6 hrs and
thereafter exposed to the probe for another 3 hrs. The
confocal images indicate that probe alone treated nematodes
Synthesis of Probe 2
Compound 1 (0.400 g, 1.156 mmol) dissolved in THF (10 ml) was
added dropwise to a solution of 4-bromobutyric acid (0.212 g, 1.271
mmol), DCC (0.357 g, 1.734 mmol) and DMAP (0.014 g, 0.12 mmol)
in THF (20 ml). After the addition the reaction mixture was further
stirred at room temperature for overnight. The solvent was then
evaporated under reduced pressure and the crude product was
purified by column chromatography to give 2 as yellow solid in 37%
yield (212 mg). 1H NMR (DMSO-d6, 500 MHz): 2.28 (m, 2H, CH2),
2.81 (t, J = 7.5 Hz, 2 H, CH2), 3.63 (t, J = 5 Hz, 2H, CH2), 3.85 (s, 3H,
CH3), 6.70 (s, 1H, Ar-H), 6.76 (m, 1H, Ar-H), 6.85 (d, J = 10 Hz, 1H,
Ar-H), 6.94 (m, 2H, Ar-H), 7.28 (s, 1H, Ar-H), 7.33 (d, J = 5 Hz, 1H,
Ar-H), 7.76 (t, J = 7.5 Hz, 1H, Ar-H), 7.82 (t, J = 5 Hz, 1H, Ar-H), 8.04
(d, J = 5 Hz, 1H, Ar-H). 13C NMR (DMSO-d6, 125 MHz): 27.89, 32.61,
33.79, 56.19, 82.76, 101.18, 110.83, 110.99, 112.82, 116.88, 118.74,
124.49, 125.37, 126.10, 129.54, 130.89, 136.37, 151.48, 151.99,
152.26, 152.76, 161.68, 169.00, 171.13. MS (ESI) Calcd. for
C25H19O6Br: 494.03; Found: 495.0285 (81Br isotope) and 497.0279
(83Br isotope) [M+1].
Figure 5. Confocal images of nematodes treated with probe 3 alone and
probe 3 + H2S. λex= 488 nm channel using 10Xmagnification.
Synthesis of Probe 3:
To a stirred solution of 2˗bromoethanol (72 mg, 0.58 mmol) and
DIPEA (290 mg) in dry DCM (10.0 mL) was added triphosgene (172
mg, 0.58 mmol). The reaction mixture was stirred at 0 °C under N2
atmosphere. After 2 hours, the excess phosgene was removed from
the reaction mixture by N2 purge. Then the compound 1 (200 mg,
0.58 mmol) dissolved in dry dichloromethane with few drops of dry
DMF was added in the reaction mixture and kept stirring for 2 days.
After completion of the reaction (by TLC), the reaction mixture was
diluted with EtOAc, washed twice with brine, dried over sodium
sulphate, then filtered and the solvent was removed under reduced
pressure. The crude product was purified by column chromatography
to give probe 3 as orange yellowish solid in 32% yield (92 mg). 1H
NMR (500 MHz, DMSO˗d6): δ = 2.77 (t, J = 5 Hz, 2H, CH2Br), 3.83 (s,
3H, CH3), 4.26 (t, J = 5 Hz, 2H, CH2CO), 6.70˗6.75 (m, 2H, Ar-H),
6.86 (d, 1H, Ar-H), 6.94˗6.97 (m, 2H, Ar-H), 7.28 (d, J = 5 Hz, 1H, Ar-
H), 7.34 (d, J = 5 Hz, 1H, Ar-H), 7.75 (t, J = 5 Hz, 1H, Ar-H), 7.80 (d, J
= 7.5 Hz, 1H, Ar-H), 8.04 (d, J = 5 Hz, 1H, Ar-H). 13C NMR (DMSO-d6,
125 MHz): δ = 37.92, 56.20, 79.63, 101.30, 110.81, 111.05, 112.82,
116.99, 124.50, 125.36, 126.14, 129.48, 129.54, 130.88, 136.34,
151.53, 152.04, 152.28, 161.71, 168.96, 171.10. ESI-MS Calcd. for
C24H17O7Br: 496.0158 (81Br) and 498.0137(83Br); Found: 499.03
[M+1] for (83Br) and 497.03 (81Br).
show very weak fluorescence emission (Fig. 5c). Exposure to
exogenous H2S source caused the increase in the
fluorescence intensity of probe, which indicates that our
probe 3 can be used for real time monitoring of H2S in living
systems (Fig. 5f). Moreover, the treatment with NEM caused
the decrease in the fluorescence of probe 3 (Fig. S25). These
studies clearly indicate that the higher fluorescence is due to
the reaction with H2S. From the above studies it is clear that
probe is an efficient tool for monitoring H2S in in vivo system.
Conclusions
In conclusion, we designed and synthesized probe 3 which is
highly selective for H2S and is used for monitoring exogenous
and endogenous H2S in both cancer and normal cells. Further,
probe 3 was applied for imaging of H2S in living tissues at
variable depths and in nematodes. These studies designate
probe 3 as an efficient platform for monitoring H2S in living
systems and it may be a useful tool for monitoring H2S-
related pathological responses in tissues as well as in in vivo
system.
Optical Studies:
UV-vis and fluorescence titrations were performed on 5.0 and 1.0 μM
solution of ligand in H2O (buffered with HEPES, pH = 7.4; at 25 °C)
mixture. Typically, aliquots of freshly prepared M(ClO4)n (M = Cu2+
,
Zn2+, Fe3+, Fe2+, Mg2+, Ca2+, Na+ and K+; n = 1 or 2 or 3), anions
2
2
(SO3ˉ as NaSO3; S2O3 ˉ as Na2S2O3; SO4 ˉ as Na2SO4; HCO3ˉ as
Experimental Section
NaHCO3), sulphur species such as H2S as NaHS, cysteine,
glutathione and ROS such as H2O2, OClˉ standard solutions (10-1
M
to 10-3 M) were added to record the UV-vis and fluorescence spectra.
Materials and Instrumentation:
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In titration experiments, each time a 3 ml solution of ligand was taken
in a quartz cuvette (path length, 1 cm) and spectra were recorded
after the addition of appropriate analytes.
Coord. Chem. Rev. 2013, 257, 2335; c) F. Yu, X. Han, L. Chen,
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Imaging of H2S in living cells:
C6 glioma cells and BV-2 microglial cells was obtained from National
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Imaging of H2S in Tissue:
Wistar strain rat was decapitated and its brain was carefully dissected.
The freshly dissected brain was submerged in cryomatrix inside the
mold and then snap-frozen in chilled isopentane for 5 min. Then, the
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for 1 h; 4) the sections were first treated with 10 mM L-Cysteine for 1
hour and then exposed to probe 3 for another hour. After treatment,
images were recorded by using an A1R Nikon Laser Scanning
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Optical sectioning was performed to observe the changes in
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We are thankful to University Grant Commision (UGC) and
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A fluorescein based fluorescent probe
3 has been designed and synthesized
which selectively detects H2S in
aqueous medium among the various
analytes tested. The probe 3 was also
successfully utilized for real time
Author(s), Corresponding Author(s)*
Shahi Imam Reja[a]†, Neetu Sharma[a]†
,
Muskan Gupta[b]†, Payal Bajaj[b], Vandana
Page No. – Page No.
Bhalla[a], Ripu D. Parihar[c], Puja Ohri[c],
Gurcharan Kaur[b]* and Manoj Kumar[a]*
Title
A Highly Selective Fluorescent Probe for
Detection of Hydrogen Sulfide in Living
Systems: in vitro and in vivo Applications
((Insert TOC Graphic here: max.
width: 55 cm; max. height: 5.0 cm))
imaging of exogenous and
endogenously produced H2S in cancer
cells, normal cells. Moreover, the
probe 3 can also detects H2S in rat
brain hippocampus in variable depth
and living nematodes.
This article is protected by copyright. All rights reserved.