A highly selective ratiometric fluorescent probe for H2S based on new heterocyclic ring formation and detection in live cells

Article abstract of DOI:10.1080/10610278.2019.1590573

A 3-indolylacrylate derivative, 3-IA, prepared by connecting an ethyl acrylate in 3-position of indole has been synthesised and characterised. Ethyl acrylate moiety acts as the Michael acceptor towards H₂S, and the resultant addition product then participates in intramolecular cyclisation with the ester group at 2-position to form another new heterocyclic ring. Blue fluorescence of 3-IA turned into green in presence of H₂S, leading to ratiometric behaviour of the fluorescent sensor with large stokes shift of 55 nm. Probe 3-IA has excellent selectivity towards H₂S over other biothiols and other competing anions. Density function theory/time-dependent density function theory calculations were carried out to validate the reaction mechanism and the electronic properties of 3-IA. Importantly, the ratiometric probe 3-IA shows great promise in H₂S detection by simple visual fluorescent inspection in filter paper-based protocol. The probe shows its excellent ability to detect H₂S in different natural water samples. Furthermore, we have employed our probe to detect H₂S for ratiometric imaging in live Vero cell.

Full text of DOI:10.1080/10610278.2019.1590573

Supramolecular Chemistry
ISSN: 1061-0278 (Print) 1029-0478 (Online) Journal homepage: https://www.tandfonline.com/loi/gsch20
A highly selective ratiometric fluorescent probe for
H2S based on new heterocyclic ring formation and
detection in live cells
Sandip Kumar Samanta, Syed Samim Ali, Ankita Gangopadhyay, Kalipada
Maiti, Ajoy Kumar Pramanik, Uday Narayan Guria, Aritri Ghosh, Pallab Datta
& Ajit Kumar Mahapatra
To cite this article: Sandip Kumar Samanta, Syed Samim Ali, Ankita Gangopadhyay,
Kalipada Maiti, Ajoy Kumar Pramanik, Uday Narayan Guria, Aritri Ghosh, Pallab Datta & Ajit
Kumar Mahapatra (2019): A highly selective ratiometric fluorescent probe for H2S based on
new heterocyclic ring formation and detection in live cells, Supramolecular Chemistry, DOI:
10.1080/10610278.2019.1590573
To link to this article: https://doi.org/10.1080/10610278.2019.1590573
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SUPRAMOLECULAR CHEMISTRY
https://doi.org/10.1080/10610278.2019.1590573
ARTICLE
A highly selective ratiometric fluorescent probe for H2S based on new
heterocyclic ring formation and detection in live cells
Sandip Kumar Samantaa, Syed Samim Alia, Ankita Gangopadhyaya, Kalipada Maitia, Ajoy Kumar Pramanika,
Uday Narayan Guriaa, Aritri Ghoshb, Pallab Dattab and Ajit Kumar Mahapatraa
b
aDepartment of Chemistry, Indian Institute of Engineering Science and Technology, Shibpur,Howrah, India; Centre for Healthcare Science
and Technology, Indian Institute of Engineering Science and Technology, Shibpur, India
ABSTRACT
ARTICLE HISTORY
Received 28 August 2018
Accepted 25 February 2019
A 3-indolylacrylate derivative, 3-IA, prepared by connecting an ethyl acrylate in 3-position of
indole has been synthesised and characterised. Ethyl acrylate moiety acts as the Michael acceptor
towards H2S, and the resultant addition product then participates in intramolecular cyclisation
with the ester group at 2-position to form another new heterocyclic ring. Blue fluorescence of
3-IA turned into green in presence of H2S, leading to ratiometric behaviour of the fluorescent
sensor with large stokes shift of 55 nm. Probe 3-IA has excellent selectivity towards H2S over
other biothiols and other competing anions. Density function theory/time-dependent density
function theory calculations were carried out to validate the reaction mechanism and the
electronic properties of 3-IA. Importantly, the ratiometric probe 3-IA shows great promise in
H2S detection by simple visual fluorescent inspection in filter paper-based protocol. The probe
shows its excellent ability to detect H2S in different natural water samples. Furthermore, we have
employed our probe to detect H2S for ratiometric imaging in live Vero cell.
KEYWORDS
Ratiometric sensor;
hydrogen sulfide; DFT/
TDDFT calculations;
detection in various water
sample; cell imaging
COOEt
COOEt
H2S
S
C
O
N
COOEt
N
H
H
1. Introduction
derivatives. Thus, in this work, we have prepared a new
heterocyclic compound via hydrogen sulfide detection
technique.
A reaction-based chemosensor, named as chemodosi-
meter, has a proficient application due to irreversible
chemical reaction with the external stimuli. Upon inter-
action with those external stimuli, receptors unit change
their electronic properties which are the basis of the
molecular recognition. Although lots of derivatives of
indole and thiophene have been prepared, an indole-
conjugated thiophene compound is still in investigation.
For the best of our knowledge, till, not much devotion
has been employed around this field of chemistry. Thus,
our aim of this work mainly focused on designing
an organic protocol which upon treatment with an ana-
lyte will produce those indole–thiophene-coupled
Hydrogen sulfide (H2S), the simplest biothiol, has
been considered as the gaseous molecular messenger
in a variety of human biological systems (1–4). Like
other reactive small molecules (5–7) eg. carbon mon-
oxide (CO) and nitric oxide (NO), endogenous H2S is
used by various organisms extending from bacteria to
mammals for production of energy, signal transduc-
tion, and response in immune system (8–10).
3-Mercaptopyruvate sulfur transferase, cystathionine-
γ-lyase (CSE), and cystathionine β-synthase (CBS) are
such enzymes, responsible for the formation of endo-
genous H2S in a certain catalytic reaction (11, 12).
CONTACT Ajit Kumar Mahapatra
mahapatra574@gmail.com
Department of Chemistry, Indian Institute of Engineering Science and Technology,
Shibpur, Howrah 711103, India
Supplemental data for this article can be accessed here
© 2019 Informa UK Limited, trading as Taylor & Francis Group

2
S. K. SAMANTA ET AL.
Toxicity and hence lethal effect of H2S are attributed
as it is a colourless, highly water-soluble, and flam-
mable gas with an unpleasant rotten egg smell (13).
So, the irregular levels of H2S are strictly related to
many diseases, for example, slow growth, Alzheimer’s
disease, cardiovascular disease, Down’s syndrome, dia-
betes, osteoporosis, leucocyte loss, cancer, and liver
cirrhosis (14–20).
Therefore, to better comprehend the role of H2S in
pathological and physiological processes, the recognition
of H2S has become a vital topic of scientific research,
especially in biological systems (21). A number of analy-
tical techniques have been reported for the detection of
H2S, like gas chromatography and electrochemical (22)
and colorimetric (23) methods. But, due to the short life-
time of H2S in live cells, these approaches experience
complications in preserving precision and real-time
imaging.
Recently, fluorescent chemodosimeters have fascinated
a considerable devotion due to the benefit of high sensi-
tivity, selectivity, and real-time detection (24–26). To date,
lots of fluorescent sensors for H2S have been reported so
far. Mostly, reduction of azide and nitro functionality,
nucleophilic addition, and coppersulfide precipitation-
based fluorescent probes have been reported for H2
S detection (27–41). These sensors are highly efficient at
the task they are designed to perform. However, there is
still room for new ideas in this field. Cleavage of dinitro-
phenyl ether group has become an interesting approach
for H2S detection. Some design strategies relied on intra-
molecular cyclisation followed by regeneration fluorescent
moiety. In a few cases, S2- were recognised in terms of
secondary analyte detection from a metal-bound ensem-
ble. Recently, dinitrophenyl ether group incorporated into
a lysosome-targeted 1,8-naphthalimide fluorophore for
live cell imaging of H2S has been reported by Tang et al.
(42). A mitochondria-targeted fluorescent sensor for detec-
tion of H2S has been explored by Yoon et al., where the
response time is 40 min (43). Merocyanine-based fluores-
cent chemodosimeters for the detection of H2S over other
interfering biothiols have been synthesised by three inde-
pendent groups of authors, namely, Guo, Zhao and
Goswami et al. (44–46). In their strategy, an electrophilic
indolium part of merocyanine unit is responsible for the
facile nucleophilic reaction of S2-/SH− which is accompa-
nied by a change in optical properties. Recently our group
reported a merocyanine-based dual-reactive centre for H2
S, wherein the low the H2S concentration, the more reac-
tive indolium moiety was reacted and then the dinitrophe-
nyl-ether part was cleaved upon increasing the analyte
concentration (47). As manifested by Holmes et al. that at
a physiological pH, simple thiols can react readily with
some Michael acceptors. But, due to fast equilibrium, the
products could not be obtained or recognised from the
reaction mixture (48). Anchored in Holmes’ fallout, we
proposed that, due to the high reactivity of H2S over the
biothiols, certain Michael acceptors might be helpful to
distinguish H2S from biological thiols by stimulating the
intramolecular cyclisation reaction at physiological pH. For
instance, biothiols have a nucleophilic sulfhydryl group (–
SH) and the pKa of H2S (6.9) is lower than that of Hcy (8.9),
Cys (8.3), or GSH (9.2), demonstrating that H2S has a higher
nucleophilicity than other biothiols under physiological
conditions. Therefore, it is effortless to discriminate Hcy/
Cys/GSH from H2S simply via nucleophilic reaction-based
strategies in physiological media. In this respect, Xian
group has successfully developed this type of fluorescent
chemosensors for the selective detection and bioimaging
of H2S (49). Nevertheless, in Table S7, (50-57) we have
compared some recent H2S probes with our probe in
different aspects.
Detection of H2S through the formation of S-
heterocycle, coupled with indole in a ratiometric manner.
Herein, we have designed a 3-indolylacrylate deriva-
tive, 3-IA, by connecting an ethyl acrylate in 3-position of
indole as Michael acceptor and an ester group ortho to
ethyl acrylate was employed as a specific function for
cyclisation via intramolecular nucleophilic attack. In parti-
cular, ratiometric fluorescent probes are always favoured
to ‘on-off’ or ‘off-on’ response as they are able to evade
fake detection and improved signal-to-noise ratio as the
detection occurs by using the intensity ratio of two differ-
ent wavelengths. The philosophy of the design lies in that
the methylene malonate will trap H2S through Michael
reaction to yield an intermediate 3-IASH moiety, which
can undergo tandem intramolecular nucleophilic attack
on the reactive ester carbonyl group, present in suitable
position, through a five-membered cyclic transition state,
illuminating blue to green emission. Thus, the design
strategy revealed a route for the synthesis of the indole–
thiophene-coupled new heterocyclic compound. In
adddition to this no ratiometric fluorescent response
was observed when probe 3-IA reacts with other biothiols.
Based on the above reaction strategy, highly discrimina-
tive detection of H2S over other biothiols can be succes-
sively employed. However, the detection limit is not as
impressive as that of some other reported H2S probe (58).
The sensing mechanism involving nucleophilic attack at
electrophilic centre leading to cyclisation that ultimately
leads to ratiometric fluorescent spectral changes is an
important aspect of this work. Most importantly, our
design strategy is completely different from those

SUPRAMOLECULAR CHEMISTRY
3
Scheme 1. Conventional way for detection of H2S
previously reported techniques, especially in cases where
nucleophilic addition of H2S followed by regeneration of
fluorescent moiety was occurred. Li et al. (59) reported
a BODYPI-based sensor containing a conjugated ester unit
placed in a close vicinity of an aldehyde group which acts
as nucleophilic acceptor and in presence of H2S undergo
intermolecular cyclisation to form a new heterocyclic
compound which was responsible for turn-on fluores-
cence response. But the response time and limit of detec-
tion towards H2S was very high. Here, in our case, we have
changed the design scheme by keeping an ester group to
the close vicinity of the conjugated ester unit. Thus, we
observed that the reaction rate is increased and the limit
of detection of the probe to H2S is reduced. Not only this,
we have achieved a ratiometric fluorescence response
upon treatment with H2S which is, in turn, favourable
than a turn-on sensor. Also, by using this design plan,
we have able to synthesise a new heterocyclic compound,
i.e. an indole-conjugated thiophene which is still an undis-
covered compound to the chemist.
2. Results and discussion
2.1. Synthesis and characterisation
Probe 3-IA can be readily synthesised in three steps
starting from indole-2-carboxylic acid followed by the
preparation of its ester and the subsequent formylation
reaction according to the standard procedure outlined
in Scheme 2. The probe molecule 3-IA has been synthe-
sised by the condensation of 2 with diethyl malonate in
presence of piperidine. The structure of 3-IA was recog-
nised by 1H-NMR spectroscopy, 13C-NMR spectroscopy,
LC-MS, and single crystal x-ray analysis (Supporting
Information). Details of experimental procedure and
data of characterisation of different structures are
given in the Supporting Information.
2.2 UV-Vis study
Probe 3-IA [10.0 µM in DMSO–H2O (1:9), HEPES
buffer; pH 7.4, 10 mM] itself showed two main

4
S. K. SAMANTA ET AL.
Scheme 2. Reagent and condition: (a) EtOH, H+, reflux, 12 h, 70%; (b) DMF, POCl3, 60°C, 35%; and (c) DEM, EtOH, piperidine, reflux,
24 h, 48%.
absorption bands at 278 and 354 nm. Then, to
a fixed concentration (10.0 µM) of 3-IA, varying
concentrations of H2S (using Na2S as the equivalent
source of H2S) were added to evaluate the absorp-
tion titration. Addition of H2S (0–12 equiv.)
persuades a red shift in both the absorption peaks
(Figure 1), resulting in two new absorption bands
centred at around 309 and 414 nm, respectively,
accompanied with the colour of the solution of
3-IA changing from colourless to yellow (Figure
1(a)).
So, compound 3-IA can serve as a ‘naked-eye’
sensor for H2S. Three distinct isosbestic points at
290, 320, and 378 nm indicate the formation of
a new compound upon reaction of probe 3-IA with
H2S.
2.3 Fluorescence study
Upon excitation at 308 nm, sensor 3-IA [10.0 µM in
DMSO–H2O (1:9), HEPES buffer; pH 7.4, 10 mM] dis-
played an intense emission band at 408 nm.
However, as revealed in Figure 2(a), the addition of
H2S evoked a drastic decrease in the emission intensity
at 408 nm, and the concurrent appearance of a new
emission band at 463 nm accredited to the thiol-
mediated cyclisation. The free probe exhibited a blue
emission colour, and the addition of H2S elicited
a significant variation in emission colour from blue to
green. Thus, the probe provided a substantial ratio-
metric fluorescent response (I463/I408) from 0.3259 to
5.0746 (a 15.57-fold enhancement) upon addition of
12 equiv. of H2S (Figure 2). Figure 2(b) shows a graph
of the ratio of fluorescence intensity (F463/F408) of the
Figure 1. (colour online) (a) Absorption spectra of 3-IA (10.0 μM) upon addition of H2S (0–12 equiv.) (HEPES buffer, pH = 7.4). (b)
Absorbance intensity ratio changes (A414/A354) of 3-IA (10.0 μM) upon addition of various concentrations of H2S.

SUPRAMOLECULAR CHEMISTRY
5
Figure 2. (colour online) (a) Emission spectra of 3-IA (10.0 μM, λex = 308 nm) upon addition of H2S (0–12 equiv.). (b) Fluorescence
intensity ratio changes (F463/F408) of 3-IA (10.0 μM) upon concomitant addition of H2S (0–12 equiv.).
sensor 3-IA against the concentration of H2S starting
from 0 to 50.0 μM. From the above-mentioned graph,
the corresponding linear fit (R2 = 0.998) has been drawn
to the experimental data. From the linear fit of the
experimental data, the limit of detection 3-IA for H2
S was calculated to be 1.7 μM. (Figure S9 of the
Supplementary Information, available online).
3-IA could still hold on to the sensing response to
H2S in the incidence of different potential competi-
tive analytes, the probe 3-IA (10.0 μM) was treated
with H2S in the presence of F−, Cl−, Br−, I−, SO4
,
2−
−
2-
HSO3 , and S2O3 (Displayed in Figure S10 of the
Supplementary Information, available online). All the
pertinent analytes examined have almost no impact
on the fluorescent detection of H2S. Furthermore,
when biothiols (1 mM) and H2S (10 μM) have coex-
isted, we are able to notice ratiometric fluorescence
change using this probe. These results established
that the ratiometric response of probe 3-IA was
highly selective for H2S and could be served for the
detection and discrimination of H2S in presence of
a high concentration of interfering biothiols. Notably,
no visible variations were observed upon addition of
other biological thiols, but the huge ratiometric fluor-
escent response delivered the probe suitable for
detection of H2S by easy visual assessment and
2.4 Interference studies
Further, to test the efficient applications of sensor 3-IA as
a ratiometric fluorescent probe for H2S, the fluorescence
response of probe 3-IA to H2S in the presence of typical
competing species such as various anions F−, Cl −, Br−, I−,
−
2−
SO42−, HSO3 , and S2O3 and related thiol-containing
compounds (Cys, Hcy, and GHS) was studied (Figure 3).
As shown in Figure 3(b), during titration of allied
ions with the probe, no significant fluorescence
increase was observed. To examine whether probe
(A)
(B)
Figure 3. (colour online) (a) Absorption spectra of 3-IA (10.0 µM) in presence of various analytes (50.0 μM) in aqueous DMSO
(DMSO:H2O = 1:9 v/v, 10.0 mM HEPES buffer, pH = 7.4). (b) Fluorescence spectra of probe 3-IA (10.0 μM) in presence of various
analytes (50.0 μM) in aqueous DMSO (DMSO:H2O = 1:9 v/v, 10 mM HEPES buffer, pH = 7.4).

6
S. K. SAMANTA ET AL.
established the potential ability of the sensor 3-IA for
detection of H2S in biological systems.
2.5 Kinetic study
Next, we wish to look at the kinetic profiles of the
reaction in the fluorescence method (at 463 nm) in
different concentrations of Na2S at room temperature.
Initially, the pseudo-first-order rate constant (Kobs
)
was calculated by using the following equation: ln
(Fmax − Ft)/Fmax = – Kobs×t. For this purpose, the time-
dependent fluorescence experiments were also car-
ried out on probe 3-IA (10.0 μM) with various con-
centrations of Na2S (5, 10, 15, and 20 equiv.). As
observed from Figure 4, the fluorescence intensity at
408 nm decreases, while fluorescence intensity at 463
nm increases with reaction time.
Figure 5. (colour online) pH-dependent fluorescent spectra of 3-IA
in the absence (black line) and in the presence (red line) of H2S.
The higher concentration of Na2S resulted in a faster
reaction and pronounced fluorescence enhancement. The
ratio of fluorescence intensities (F463/F408) increases with
reaction time and gets saturated within 15 min. So, the H2
S-mediated cyclisation reaction was found to follow
a pseudo-first-order rate law of rate constant k’ = 0.292
min−1 (Figure S12 of the Supplementary Information, avail-
able online) under excess H2S concentration. In addition,
the second-order rate constant was also evaluated by
calculating the linear fit between kobs and the H2
S concentration, and it was to be 192.67 M−1Min−1 (Figure
S13 of the Supplementary Information, available online).
of H2S, is stable over a broad range of pH values from 4
to 11 and works well between pH 5.0 to 8.0 in response
to H2S (Figure 5).
However, with the addition of H2S, the fluorescence
intensity at 463 nm augmented appreciably in the pH
range of 6.5–7.8, which implies that the probe may work
well under these physiological conditions. When the pH
beat the value 8.0, the emission intensity drops down
because of poor reactivity of H2S at high pH, and at
lower pH, emission intensity sharply decreased because
of protonation of H2S. The results indicate that 3-IA
works well in physiological pH circumstances.
2.6. pH study
2.7 Study of the reaction mechanism
To be useful in the practical applicability, we also stu-
died the emission behaviour of 3-IA treated with H2S in
different pH environments. The probe, in the absence
Based on the observations on UV and fluorescent beha-
viour of the probe in presence of H2S, we suggest the
Figure 4. (colour online) F463/F408vs time plot 3-IA (10.0 μM) in DMSO–H2O (1:9, v/v) solution upon addition of different
concentrations of H2S λex = 308 nm; λem = 463 nm.

SUPRAMOLECULAR CHEMISTRY
7
COOEt
EtOOC
COOEt
H2S
H
S
S
N
COOEt
N
H
COOEt
N
H
H
O
3-IA
3-IAS
3-IASH
Figure 6. (colour online) Proposed sensing mechanism.
probable mechanism as shown in Figure 6. The product
of this process was extracted from the reaction mixture
and characterised in order to achieve evidence to sup-
port this mechanistic proposal.
product 3-IAS, 64.67 kcal/mol (Table S2 in
Supplementary Information), responsible for the batho-
chromic shift in the absorption spectra observed upon
the treatment of probe with H2S. Furthermore, TDDFT
calculations clarify the electronic properties of the probe
3-IA and its corresponding cyclic product 3-IAS. The
vertical main transitions, i.e. the calculated λmax and
oscillator strength (f), are scheduled in Table S1 in
Supplementary Information, available online.
The vertical transitions calculated by TDDFT of 3-IA
and its cyclic product show good concurrence with the
experimental data. The experiment recommends that
the vertical transitions observed at ∼359 and ∼319 nm
are analogous to those of the practically observed spec-
tra at ∼354 and ∼309 nm for 3-IA and its cyclic product,
3-IAS, respectively. The main fundamental transitions for
3-IA and its cyclic product, 3-IAS, are S0→S1 (~69.10%)
and S0→S2 (~68.50%), respectively, which corresponds
to energy states arises from HOMO→LUMO and HOMO-
1→LUMO, respectively. Thus, the calculated red shift is
accord with experimental results due to n → π*
transition.
1H-NMR analysis of the product revealed that it con-
tains the structure constituted by the ring formation
through Michael addition of H2S, and subsequently an
intramolecular cyclisation yielded compound 3-IAS. The
resonance signal analogous to the alkene protons at 8.62
and 6.11 ppm moved out along with the aspect of two
new peaks at 4.32 and 3.29 ppm designated to the
methane and methylene protons of the compound
3-IAS. LCMS spectrum analysis also confirmed the cyclisa-
tion reaction between H2S and 3-IA. For probe 3-IA,
a characteristic peak at m/z = 287.1 was obtained which
is responsible for [3-IA]+. Upon addition of H2S, a new
peak at m/z = 275.1 (assigned to the cyclise product)
appeared, whereas peak at m/z = 287.1 was disappeared
(Figures S4 andS8 of the Supplementary Information,
available online).
2.8 Theoretical study
The electronic behaviour of the compound 3-IA and its
corresponding cyclic product 3-IAS upon reaction with
H2S was explored using density function theory (DFT)
and time-dependent density function theory (TDDFT)
calculation at the B3LYP/6-31G(d) level of the Gaussian
09 program. The most effective geometries and calcu-
lated electron distributions in the frontier molecular orbi-
tal’s HOMO and LUMO of the probe 3-IA and its cyclic
product 3-IAS are shown in Figure 7(a). Main chromo-
phoric unit indole moiety exists in a nearly planar con-
formation with ethyl acrylate part. These planar
structures permit a proficient π-conjugation and esteem
the Internal Charged Transfer (ICT) transition between
the donor indole -NH and the acceptor acrylate group.
In particular, the HOMOs and LUMOs of the probe 3-IA
and its cyclic product are widely spread out over the
indolyl part. Additionally, the energy difference between
the HOMO and LUMO of the probe 3-IA was larger,
100.25 kcal/mol, than that of its corresponding cyclic
2.9 Practical application
To build the detection experiments operationally sim-
ple, user-friendly, and less expensive detection device
for the on-site analysis of H2S, cellulose paper Whatman
40 filter paper was used. It was immersed into the
aqueous CH3CN solution of 3-IA (0.1 mg/mL) and then
dried in air. The colourless white filter paper changed
into yellow after spraying of an aqueous solution of Na2
S (Figure 8).
As it has shown, an obvious immediate colour
change occurs under a UV hand lamp (Figure 8 bot-
tom row), and an enhanced, rapid, and ratiometric
colour change can be obtained from blue to green
fluorescence in a longer time (3 min) that it could be
clearly observed by the naked eye. These results
exemplify the efficient application of 3-IA for the
advance of simple fluorimetric strips for H2
S detection.

8
S. K. SAMANTA ET AL.
A)
B)
L U M O
E = 4 .3 4 7 1 e V
Δ
E = 2 .8 0 4 6 e V
Δ
H O M O
3 -IA
3 -IA S
Figure 7. (colour online) (a) Energy-optimised structures of 3-IA and 3-IAS. (b) HOMO–LUMO distribution and energy difference of
3-IA and 3-IAS.
Now, we extend the application of our probe 3-IA
in detecting hydrogensulfide in presence of various
industrial waste materials and different water sources.
Though several interfering agents are present in nat-
ural surface water sources, yet we have successfully
detected H2S by using our probe 3-IA. We collected
various surface water samples from diverse parts of
West Bengal, India (Supplementary Information, avail-
able online) and observed the potentiality of our
probe 3-IA in detecting H2S in various surface water
samples.
distinguished (Figure 9). No doubt these results
showed that whatever is the source of water, enhance-
ment in fluorescence of 3-IA is revealed in the pre-
sence of H2S only.
2.10 Cell imaging
Ideally, for cellular imaging experiments, higher exci-
tation wavelengths are desirable. However, we were
lucky enough to find that cells were able to survive our
experiments with probe 3-IA which required excitation
at 308 nm. Fluorescence imaging investigation was
accomplished in Vero cells to reveal the practical
applicability of the probe 3-IA in biological systems.
Prior to cell imaging, the MTT [3-(4,5-dimethyl-2-thia-
zolyl)-2,5-diphenyl-2- H-tetrazolium bromide] assay
To carry out this experiment, we prepared 10 μM
solutions of probe 3-IA which were added to different
water samples and emission spectral change observed
upon addition of H2S (12 equiv.). In case of all samples,
significant fluorescence (at 463 nm) improvement was

SUPRAMOLECULAR CHEMISTRY
9
emission inside cells was noticed (Figure 10(e,f)). This
surveillance reveals that 3-IA can detect the presence of
H2S in cells. Thus, the results indicate that 3-IA is cap-
able of detecting H2S in living cells with satisfactory cell
membrane permeability.
1)
2)
3. Synthesis and structure characterisation
3.1 Synthesis of 2
To a solution of indole-2-carboxylic acid (1) (1 g, 6.2
mmol) in 20 mL of ethanol, kept in an ice bath, four
drops of sulfuric acid was added. Then, the reaction
mixture was refluxed for 12 h with continuous TLC
monitoring. After that, the mixture was cooled down
to room temperature. After evaporation of the solvent
in vacuum, the solid residue was purified by column
chromatography on silica (eluted with 4% ethyl acetate/
hexane) to afford the indole-2- ethyl carboxylate (2)
(820 mg, 70%), a white solid.1H-NMR (CDCl3, 300
MHz): δ (ppm) 8.99 (s, 1H), 7.71 (d, 1H, J = 7.8 Hz),
7.44 (t, 1H, J = 6.9 Hz), 7.34 (m, 1H),7.26 (t, 1H,), 7.17
(m, 1H), 4.44 (q, 2H, J = 7.2 Hz), 1.44 (t, 3H, J = 7.2 Hz).
a)
b)
c)
d)
Figure 8. (colour online) (1) Naked eye colour changes visua-
lised on TLC plate strips of (a) 3-IA (c = 1.0 × 10−2M) alone and
after addition of H2S at (b)1.0 × 10−4M, (c) 1.0 × 10−3M, and (d)
1.0 × 10−2M in DMSO/H2O = 1:9 (v/v). (2) Fluorescence colour
changes visualised on TLC plate strips of (a) 3-IA (c = 1.0 ×
10−2M) alone and after addition of H2S at (b) 1.0 × 10−4M, (c)
1.0 × 10−3M, and (d) 1.0 × 10−2M in DMSO/H2O = 1:9 (v/v).
3.2 Synthesis of 3
In nitrogen atmosphere, 2 ml dry DMF was taken and
then freshly distilled 2 ml phosphorus oxychloride
(POCl3) was added dropwise under ice-cold condition.
The mixture was stirred until a red colouration arise.
Then, indole-2-ethyl carboxylate (2) (500 mg, 2.65
mmol) dissolved in least amount of dry DMF was
added to the previous mixture. Then, the whole mix-
ture was heated to 50–60°C for 8 h. After completion
of the reaction (evaluated by TLC monitoring), the
mixture was cooled and poured into ice with stirring
and then work up with ethyl acetate. The organic layer
was dehydrated over sodium sulphate, and the sol-
vents were evaporated under reduced pressure. The
crude product was purified by column chromatogra-
phy using (8% v/v) ethyl acetate-hexane to give pure
Figure 9. (colour online) Sensing of H2S from surface water
sample by using 3-IA.
using the Vero cell line was carried out to evaluate the
cytotoxicity of 3-IA. As displayed in Figure S1 of the
Supplementary Information, available online, it is
observed that more than 80% of cells were viable
even after 24 h incubation. The result stipulates little
cytotoxicity of 3-IA, while the concentration of 3-IA
was augmented up to 100 mg/mL.
1
aldehyde. White solid (200 mg, 35%). H-NMR (CDCl3,
300 MHz): δ (ppm) 10.67 (s, 1H), 8.34 (d, 1H, J = 8.1 Hz),
7.54 (d, 1H, J = 8.1 Hz), 7.32 (m, 2H),4.48 (q, 2H, J = 7.2
Hz), 3.06 (s, 1H), 1.46 (t, 3H, J = 7.2 Hz).
To image the cells, a fluorescence microscope was
used. Bright field images indicate that the cells were
not injured in presence of the probe (Figure 10(a)) and
H2S (Figure 10(d)). When Vero cells were incubated with
3-IA (1.0 µM) for 6 h, blue fluorescence was observed
inside cells (Figure 10(b,c)). Upon addition of Na2S (30.0
µM) to the above cells for another 8 h, a strong green
3.3 Synthesis of 3-IA
Compound 3 (100 mg, 0.46 mmol) was taken in dry
ethanol. Then, diethyl malonate (110 mg, 0.69 mmol)

10
S. K. SAMANTA ET AL.
C)
F)
B)
E)
A)
D)
Figure 10. (colour online) Fluorescence images of probe in Vero cells (20× objective lens): (a) Bright field image of only cells
treated with 3-IA (1.0 μM). (b) Blue channel fluorescence images of cells treated with 3-IA (1.0 μM) (blue channel, λex = 308 nm,
λem = 408 nm). (c) Merged image of 3-IA. (d) Bright field image of 3-IA (1.0 μM) treated with H2S (30.0 μM). (e) Fluorescence image
of 3-IA (green channel, λex = 308nm, λem = 463 nm) treated with 30.0 μMH2S. (f) Merged image of 3-IA treated with H2S (30.0 μM)
(scale bar: 100 µm).
and 0.5 ml piperidine were added and reflux for 24 h. In
the end of the reaction (TLC monitoring), the solvent was
removed under vacuum. Then, the solid product was
purified by column chromatography over silica (eluted
with 7% ethyl acetate/hexane) to afford the Light yellow
solid (63 mg, 48%), mp:190–195°C, MS (LCMS): (m/z, %):
J = 7.2 Hz), 4.06 (q, 3H, J = 7.2 Hz), 3.29 (d, 2H, J = 7.2
Hz), 1.02 (t, 3H, J = 7.2 Hz).
4. Conclusion
287.1 [100%]; calculated for C16H17NO4 : 287.06. 1H-NMR
+
In conclusion, we have planned and synthesised
3-indolylacrylate derivative, 3-IA, by connecting an
ethyl acrylate in 3-position. The probe itself showed
blue emission, but upon reaction with H2S, it ratio-
metrically turned into a compound which showed
green emission. The probe 3-IA is highly selective as
well as sensitive towards H2S and the sensing process
is governed by intramolecular cyclisation reaction.
X-ray data of the ratiometric probe showed that it
has lateral intramolecular hydrogen bonding along
with a pendant π. . .π stacking interactions. The for-
mation of 3-IAS has been recognised both experi-
mentally and theoretically (DFT/TDDFT). Importantly,
the ratiometric probe 3-IA to be best employed for
H2S detection by simple visual fluorescent inspection
in filter paper-based protocol. Additionally, further-
more, we have employed our probe to detect H2
S for ratiometric imaging in live Vero cell. Thus, this
molecule not only demonstrates H2S sensing effi-
ciency but also brings to light a simple method of
synthesising sulfur heterocycles fused with an indole
moiety. The possibility of such a specific targeted
chemical reaction can be explored in the design and
(DMSO-d6, 500 MHz): δ (ppm) 12.35 (s, 1H), 8.62 (d, 1H, J =
16.5 Hz), 8.0 (d, 1H, J = 8 Hz), 7.55 (d, 1H J = 3.5),7.37 (d, 1H,
J = 7.7 Hz), 7.23 (t, 1H, J = 7.7),6.11 (d, 1H, J = 16.5 Hz), 4.41
(q, 2H, J = 7 Hz), 4.2 (q, 2H,J = 7 Hz), 1.35 (t, 3H, J = 7), 1.23
(t, 3H, J = 7 Hz). 13C-NMR (DMSO-d6, 100 MHz): 166.63,
160.71, 136.87, 127.18, 125.05, 121.76, 117, 115.39, 113.21,
61, 59.66, 39.59, 14.07.
3.4. Synthesis of the receptor 3-IA + H2S adduct
(3-IAS)
3-IA (50 mg, 0.17 mol) was taken in acetonitrile. Na2
S (70 mg, 0.85 mol) and few drops of water were
added to it. The mixture was stirred about 30 min,
and the resultant product was purified by column
chromatography using 10% ethyl acetate in hexane
(v/v) to give a white compound 3-IAS (31 mg, 65%).
Mp. 160–165°C. LCMS: (m/z, %): [M]+calcd for C14H13
NO3S is 275.06; Found: 275.1. 1H-NMR (CDCl3, 300
MHz): δ (ppm): 11.89 (s, 1H), 7.35(m, 4H), 4.31 (t, 1H,

SUPRAMOLECULAR CHEMISTRY
11
synthesis of the new fluorescent probe in the future
for H2S
(15) Heafield, M.T.; Fearn, S.; Steventon, G.B.; Waring, R.H.;
Williams, A.C.; Sturman, S.G. Neurosci. Lett. 1990, 110,
16. DOI: 10.1016/0304-3940(90)90814-P.
(16) Sachdev, P. J. Neurol. Sci. 2004, 226, 25. DOI: 10.1016/j.
jns.2004.09.006.
Acknowledgements
(17) Giuliani, D.; Ottani, A.; Zaffe, D.; Galantucci, M.;
Strinati, F.; Lodi, R.; Guarini, S. Neurobiol. Learn. Mem.
2013, 104, 82. DOI: 10.1016/j.nlm.2013.05.006.
(18) Cao, Q.; Zhang, L.; Yang, G.; Xu, C.; Wang, R. Antioxid.
Redox Signaling. 2010, 12, 1101–1109. DOI: 10.1089/
ars.2009.2915.
We are grateful to DST-SERB [File No. EMR/2016/006640] for
financial support. SSA and Dr AKP wish to acknowledge UGC-
MANF (MANF-WES-67786) and SERB-NPDF, New Delhi, for the
financial and research support.
(19) Szabo, C. Antioxid. Redox Signaling. 2012, 17, 68–80.
DOI: 10.1089/ars.2011.4451.
Disclosure statement
(20) Szabo, C.; Coletta, C.; Chao,; Modis, C.; Szczesny, K.B.;
Pa-Papetropoulos, A.; Hellmich, M.R. Natl. Acad. Sci.
U. S. A. 2013, 110, 12474–12479. DOI: 10.1073/
pnas.1306241110.
No potential conflict of interest was reported by the authors.
Funding
(21) Liu, S.; Zhang, L.; Yang, T.; Yang, H.; Zhang, K.Y.;
Zhao, X.; Lv, W.; Yu, Q.; Zhang, X.; Zhao, Q.; Liu, X.;
Huang, W. ACS Appl. Mater. Interfaces. 2014, 6,
11013–11017. DOI: 10.1021/am5035158.
(22) Manibalan, K.; Mani, V.; Chang, P.C.; Huang, C.H.;
Huang, S.T.; Marchlewicz, K.; Neethirajan, S. Biosens.
Bioelectron. 2017, 96, 233–238. DOI: 10.1016/j.
bios.2017.05.006.
We are grateful to DST-SERB [File No. EMR/2016/006640] for
financial support. SSA and Dr AKP wish to acknowledge UGC-
MANF (MANF-WES-67786) and SERB-NPDF, New Delhi, for the
financial and research support.
References
(23) Ren, K.; Shang, X.; Chen, Y.; Zhang, X.; Fu, J.; Zhao, P.;
Zhang, J. Lumin. 2017, 32, 765–771. DOI: 10.1002/
bio.3248.
(24) Carter, K.P.; Young, A.M.; Palmer, A.E. Chem. Rev. 2014,
114, 4564. DOI: 10.1021/cr400546e.
(25) Zhu, H.; Fan, J.; Du, J.; Peng, X.A. Chem. Res. 2016, 49,
2115. DOI: 10.1021/acs.accounts.6b00292.
(26) Chen, X.; Pradhan, T.; Wang, F.; Kim, J.S.; Yoon, J. Chem.
Rev. 2012, 1910. DOI: 10.1021/cr200201z.
(27) Lin, V.S.; Chen, W.; Xian, M.; Chang, C.J. Chem. Soc. Rev.
2015, 44, 4596. DOI: 10.1039/C4CS00298A.
(28) Henthorn, H.A.; Pluth, M.D. J. Am. Chem. Soc. 2015, 137,
15330–15336. DOI: 10.1021/jacs.5b10675.
(29) Dai, C.G.; Liu, X.L.; Du, X.J.; Zhang, Y.; Song, Q.H. ACS
Sens. 2016, 1, 888–895. DOI: 10.1021/acssensors.
6b00291.
(30) Lippert, A.R.; New, E.J.; Chang, C.J. J. Am. Chem. Soc.
2011, 133, 10078–10080. DOI: 10.1021/ja203661j.
(31) Peng, H.; Cheng, Y.; Dai, C.; King, A.L.; Predmore, B.
L.; Lefer, D.J.; Wang, B. Angew. Chem. Int. Ed. 2011,
50, 9672–9675. DOI: 10.1002/anie.201104236.
(32) Chen, B.; Li, W.; Lv, C.; Zhao, M.; Jin, H.; Jin, H.; Du, J.;
Zhang, L.; Tang, X. Analyst. 2013, 138, 946–951. DOI:
10.1039/C2AN36113B.
(33) Yu, F.; Li, P.; Song, P.; Wang, B.; Zhao, J.; Han, K. Chem.
Commun. 2012, 48, 2852–2854. DOI: 10.1039/
c2cc17658k.
(34) Li, W.; Sun, W.; Yu, X. J. Fluoresc. 2013, 23, 181–186. DOI:
10.1007/s10895-012-1131-4.
(35) Zhang, L.; Zheng, X.E.; Zou, F.; Shang, Y.; Meng, W.;
Lai, E.; Xu, Z.; Liu, Y.; Zhao, J. Sci. Rep. 2016, 6, 18868.
DOI: 10.1038/srep18868.
(36) Montoya, L.A.; Pluth, M.D. Chem. Commun. 2012, 48,
4767–4769. DOI: 10.1039/c2cc30730h.
(37) Velusamy, N.; Binoy, A.; Bobba, K.N.; Nedungadi, D.;
Mishra, N.; Bhuniya, S. Chem. Commun. 2017, 53,
8802–8805. DOI: 10.1039/C7CC05339H.
(1) Kimura, H.; Nagai, Y.; Umemura, K.; Kimura, Y. Antioxid.
2005, 7, 795. doi: 10.1089/ars.2005.7.795
(2) He, J.; Huang, J.; He, Y.; Cao, P.; Zeller, M.; Hunter, A.D.;
Xu, Z. Chem. – Eur. J. 2016, 22, 1597. DOI: 10.1002/
chem.201504941.
(3) Bailey, T.S.; Pluth, M.D. J. Am. Chem. Soc. 2013, 135,
16697–16704. DOI: 10.1021/ja408909h.
(4) Yu, Q.; Gao, P.L.; Zhang, K.Y.; Tong, X.; Yang, H.R.; Liu, S.
J.; Du, J.; Zhao, Q.; Huang, W.light: Sci. Appl. 2017, 6,
e17107.
(5) Li, L.; Rose, P.; Moore, P.K. Rev. Pharmacol. Toxicol. 2011,
51, 169–187. DOI: 10.1146/annurev-pharmtox-010510-
100505.
(6) Wang, R. Physiol. Rev. 2012, 92, 791–896. DOI: 10.1152/
physrev.00017.2011.
(7) Fukuto, J.M.; Carrington, S.J.; Tantillo, D.J.; Harrison, J.
G.; Ignarro, L.J.; Freeman, B.A.; Chen, A.; Wink, D.A.
Chem. Res. Toxicol. 2012, 25, 769–793. DOI: 10.1021/
tx2005234.
(8) Wu, P.; Zhang, J.; Wang, S.; Zhu, A.; Hou, X. Chem. Eur. J.
2014, 20, 952–956. DOI: 10.1002/chem.201303753.
(9) Liu, J.; Sun, Y.Q.; Zhang, J.; Yang, T.; Cao, J.; Zhang, L.;
Guo, W. Chem. Eur. J. 2013, 19, 4717–4722. DOI:
10.1002/chem.201300455.
(10) Lin, V.S.; Lippert, A.R.; Chang, C.J. Proc. Natl. Acad. Sci.
U. S. A. 2013, 110, 7131–7135. DOI: 10.1073/
pnas.1302193110.
(11) Kimura., H. Amino Acids. 2011, 41, 113–121. DOI:
10.1007/s00726-010-0570-y.
(12) Singh, S.; Padovani, D.; Leslie, R.A.; Chiku, T.; Banerjee, R.
J. Biol. Chem. 2009, 284, 22457. DOI: 10.1074/jbc.
M109.010868.
(13) Reiffenstein, R.J.; Hulbert, W.C.; Roth, S.H. Annu. Rev.
Pharmacol. Toxicol. 1992, 32, 109. DOI: 10.1146/
annurev.pa.32.040192.000545.
(14) Shahrokhian, S. Anal. Chem. 2001, 73, 5972. DOI:
10.1021/ac010541m.

12
S. K. SAMANTA ET AL.
(38) Wu, M.Y.; Li, K.; Hou, J.T.; Huang, Z.; Yu, X.Q. Org. Biomol.
Chem. 2010, 10, 8342–8347. DOI: 10.1039/c2ob26235e.
(39) Reja, S.I.; Kumar, N.; Sachdeva, R.; Bhalla, V.V.; Kumar, M.
RSC Adv. 2013, 3, 17770–17774. DOI: 10.1039/c3ra42499e.
(40) Wang, R.; Yu, F.; Chen, L.; Chen, H.; Wanga, L.;
Zhanga, W. Chem. Commun. 2012, 48, 11757–11759.
DOI: 10.1039/c2cc36088h.
(50) Liu, C.; Pan, J.; Li, S.; Zhao, Y.; Wu, L.Y.; Berkman, C.E.;
Whorton, A.R.; Xian, M. Angew. Chem. Int. Ed. 2011,
50, 10327. DOI: 10.1002/anie.201104305.
(51) Peng, B.; Chen, W.; Liu, C.; Rosser, E.W.; Pacheco, A.;
Zhao, Y.; Aguilar, H.C.; Xian, M. Chem. Eur. J. 2014, 20,
1010. DOI: 10.1002/chem.201303757.
(52) Xu, Z.; Xu, L.; Zhou, J.; Xu, Y.; Zhu, W.; Qian, X. Chem.
Commun. 2012, 48, 10871. DOI: 10.1039/c2cc36141h.
(53) Choi, M.G.; Cha, S.; Lee, H.; Jeon, H.L.; Chang, S.-K. Chem.
Commun. 2009, 7390. DOI: 10.1039/b916476f.
(54) Sasakura, K.; Hanaoka, K.; Shibuya, N.; Mikami, Y.;
Kimura, Y.; Komatsu, T.; Ueno, T.; Terai, T.;
Kimura, H.; Nagano, T. J. Am. Chem. Soc. 2011, 133,
18003. DOI: 10.1021/ja207851s.
(41) Nickson, T.E. J. Org. Chem. 1986, 51, 3903–3904. DOI:
10.1021/jo00370a031.
(42) Wu, Z.; Liang, D.; Tang, X. Anal. Chem. 2016, 88,
9213–9218. DOI: 10.1021/acs.analchem.6b02459.
(43) Pak, Y.L.; Li, J.; Ko, K.C.; Kim, G.; Lee, J.Y.; Yoon, J. Anal.
Chem. 2016, 88, 5476–5481. DOI: 10.1021/acs.
analchem.6b00956.
(44) Chen, Y.; Zhu, C.; Yang, Z.; Chen, J.; He, Y.; Jiao, Y.;
He, W.; Qiu, L.; Cen, J.; Guo, Z. Angew. Chem. Int. Ed.
2013, 52, 1688. DOI: 10.1002/anie.201207701.
(45) Paul, S.; Goswami, S.; Mukhopadhyay, C.D. New J. Chem.
2015, 39, 8940. DOI: 10.1039/C5NJ01297J.
(46) Feng, X.; Zhang, T.; Liu, J.-T.; Miao, J.-Y.; Zhao, B.-X. Chem.
Commun. 2016, 52, 3131. DOI: 10.1039/C5CC09267A.
(47) Guria, U. N.; Maiti, K.; Ali, S. S.; Samanta, S. K.; Mandal, D.;
Sarkar, R.; Datta, P.; Ghosh, A. K.; Mahapatra, A. K. New
J. Chem. 2018, 42, 5367–5375. DOI: 10.1039/
C7NJ04632D.
(48) Pritchard, R.B.; Lough, C.E.; Currie, D.J.; Holmes,; Can., H.
L. J. Chem. 1968, 46, 775–781. doi: 10.1139/v68-128
(49) Liu, C.; Peng, B.; Li, S.; Park, C.M.; Whorton, A.R.;
Xian, M. Org. Lett. 2012, 14, 2184. DOI: 10.1021/
ol3008183.
(55) Hou, F.; Huang, L.; Xi, P.; Cheng, J.; Zhao, X.; Xie, G.;
Shi, Y.; Cheng, F.; Yao, X.; Bai, D.; Zeng, Z. Inorg. Chem.
2012, 51, 2454. DOI: 10.1021/ic2024082.
(56) Hou, F.; Cheng, J.; Xi, P.; Chen, F.; Huang, L.; Xie, G.;
Shi, Y.; Liu, H.; Bai, D.; Zeng, Z. Dalton Trans. 2012, 41,
5799. DOI: 10.1039/c1dt11660f.
(57) Santos-Figueroa, L.E.; de la Torre, C.S.; Sayed, E.;
Sanceno, F.; Martı´nez-Ma´n˜ez, R.; Costero, A.M.;
Gil, S.; Parra, M. Eur. J. Inorg. Chem. 2014, 41. doi:
10.1002/ejic.201301306
(58) Velusamya, N.; Thirumalaivasan, N.; Bobba, K.,.N.;
Wu, S.,.P.; Bhuniya, S. New J. Chem. 2018, 42,
1590–1594. DOI: 10.1039/C7NJ04119E.
(59) Li, X.; Zhang, S.; Cao, J.; Xie, N.; Liu, T.; Yang, B.; He, Q.;
Hu, Y. Chem. Comm. 2013, 49, 8656–8658. DOI: 10.1039/
c3cc44539a.