Alizarin red S-zinc(II) fluorescent ensemble for selective detection of hydrogen sulphide and assay with an H2S donor

Article abstract of DOI:10.1039/c5ra11901d

A new Alizarin Red S based fluorescent probe ARS-Zn(ii) has been reported for the detection of H₂S in aqueous buffer solution. In the presence of zinc ions, ARS displayed an increase in fluorescence intensity through the formation of an ARS-Zn(ii) ensemble. The addition of H₂S to the ensemble caused the disassembly of the metal and ARS, which restored the fluorescence intensity of ARS. This sensing ensemble exhibited H₂S-selectivity over other biothiols and biologically relevant analytes. The calculated detection limit of ARS-Zn(ii) with H₂S was 92 nM. ARS-Zn(ii) also detected H₂S in serum under physiological conditions. Moreover, as a practical application, it could be used for the real time monitoring of H₂S released from the H₂S-donor molecule benzoic (methyl carbonic) dithioperoxyanhydride and also applied to live cell imaging. The reported ensemble ARS-Zn(ii) has advantages like ready availability, high selectivity with good sensitivity, fast response time, higher excitation (520 nm) and emission (625 nm) wavelengths and real time fluorescence assay with H₂S donors.

Full text of DOI:10.1039/c5ra11901d

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ARTICLE
Alizarin red S –Zinc(II) fluorescent ensemble for selective
detection of Hydrogen Sulphide and assay with H2S donor
Received 00th January 20xx,
Accepted 00th January 20xx
Rahul Kaushika , Pawan Kumar a, Amrita Ghosh a Neha Guptab, Davinder Kaurc, Saroj Arorac and D.
Amilan Jose* a
DOI: 10.1039/x0xx00000x
Alizarin red S based new fluorescent probe ARS-Zn(II) has been reported for the detection of H2S in aqueous buffer
solution. In presence of zinc ion, ARS displayed an increase in fluorescent intensity through formation of ARS-Zn(II)
ensemble. Upon addition of H2S to the ensemble cause the disassembly of metal and ARS, this restored the fluorescence
intensity of ARS. This sensing ensemble exhibited H2S-selectivity over other biothiols and biological relevant analytes. The
calculated detecting limit of ARS-Zn(II) with H2S was 92 nM. ARS-Zn(II) was also detect H2S in serum under physiological
condition. Moreover, as a practical application it could be used for the real time monitoring of H2S released from H2S-
donor molecule benzoic (methyl carbonic) dithioperoxyanhydride and also applicable for live cell imaging. The reported
ensemble ARS-Zn(II) has advantages like readily available, high selectivity with good sensitivity, fast response time, higher
excitation (520 nm) and emission (625 nm) wavelength and real time fluorescence assay with H2S donor.
www.rsc.org/
Dye displacement approach is
a competitive binding
supramolecular sensor system,17-19 where an analyte displaces
a dye from a receptor, as a results colour or fluorescent
change was obtained, which can be related to the amount of
analyte present.20 In this regard, Alizarin red-S (ARS) has been
successfully employed as an indicator in competitive binding
assays for the detection of various analytes such as biological
Introduction
Hydrogen Sulfide (H2S) is an unpleasant gas with
a
characteristic smell of rotten eggs. Endogenously produced
H2S has been recognized as one of the three gasotransmitters
along with nitric oxide (NO) and carbon monoxide (CO).1 The
physiological levels of H2S appear to be involved in various
biological functions and essential for maintaining the human
health along with other roles.2 Unusual level of H2S in human
has been linked to diseases like Alzheimer’s disease,3 Down’s
syndrome4 and hypertension.5 The physiological and
therapeutic importance of H2S, leading to a quick progress in
research activity involving H2S.6
Number of fluorescent probes for H2S has been developed in
last decade.7-12 However, selectivity over competing biothiols
such as glutathione (GSH), cysteine (Cys) and homocysteine
(Hcy), sensitivity, response time, multistep synthesis and
biocompatibility are some of the serious limitations with the
existing fluorescent probes.13-16 These drawbacks call for a
need of H2S sensors with specific properties such as selective,
sensitive, fast response and readily available; so that they can
be widely used to tackle biological issues and pharmacological
application. To deal with those demanding issues, we wish to
report a displacement approach for the development of
practically useful fluorescent probe for H2S.
22
24
phosphates,21, saccharides,23, and anion.25 ARS also has
been known for the colorimetric detection of metal ions.21, 26,
27
Analyte mediated release of ARS from ARS-Copper complex
ensemble has been reported for the selective detection of
glutathione,28 tiopronin,29 proteins,30 and L-cysteine.31 Similar
to copper, ARS is also known to form complex with zinc ion
with high affinity.32 However ARS-Zinc ion based fluorescent
ensemble is not well documented for the detection of analytes
using dye displacement method.
Herein, we wish to report a new probe ARS-Zn(II) (Scheme 1)
for the selective detection of H2S under biological condition.
ARS shows ratiometric absorbance change and fluorescent
enhancement upon coordination with zinc to form ARS-Zn(II)
ensemble. In presence of H2S the emission intensity of the ARS
restored immediately as it separates from sensing ensemble
by formation of ZnS. We have also used this ensemble to
monitor the H2S released from H2S donor molecule in aqueous
solution.
Experimental Section
a.Department of Chemisry, NIT-Kurukshetra, Haryana-136119, India. Email:
amilanjosenit@nitkkr.ac.in Tel: +91-1744233559
b.Department of Chemistry, Guru Nanak Dev University, Amritsar.
c.Department of Botanical and Environmental Sciences, Guru Nanak Dev University,
Amritsar.
Materials and reagent
Methoxycarbonylsulfenyl chloride, Thiobenzoic acid, Sodium
sulfide were purchased from Sigma-Aldrich. Other chemicals
Electronic Supplementary Information (ESI) available: [Synthesis of H2S Donor,
Experimental methods and Some Uv-Vis and Fluorescence spectrum are available].
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were purchased from Loba Chemie or Merck limited unless (MeOH/PBS buffer, 3/1, v/v) at pH 7.4. The UV-Vis spectra
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DOI: 10.1039/C5RA11901D
otherwise mentioned. Solvents used were analytically pure resulting upon addition of different concentration of zinc are
and used without further purification.
shown in Figure. 1.
Instrumentation methods
1H NMR was measured on a Bruker Avance 400 MHz. Mass
spectrum was measured on Waters Q-Tof mass spectrometer.
Cary eclipse fluorescence spectrophotometer was used to
obtain the fluorescence spectra. UV absorption spectra were
recorded on a Evolution 201 UV-Vis spectrophotometer using
quartz cells of 1.0 cm path length. Live cell images were
recorded by using Nikon Air Laser Scanning Confocal
Microscope. Donor studies were performed on Synergy-HT
Multimode Plate reader (BIOTEK). Buffer solutions were
prepared by using deionized water and pH studies were done
on HACH sension 2.
Scheme 1: Proposed Sensing mechanism for the detection of H2S using ARS-
Zn(II) ensemble.
General procedures for spectroscopic measurements
As shown in figure 1, upon addition of Zn(II) to the ARS induces
a ratiometric change with increase in absorbance at 520 nm,
along with decrease in wavelength at 425 nm and appearance
of isosbestic point at 465 nm. Based on the UV–Vis spectral
change, the binding mode was determined by a Job’s plot
using mixtures of ARS and zinc in buffer solution.
The relative ARS–Zn(II) complex concentration reached
maximum when the molar fraction was 0.33, therefore the job
plot was confirmed that a 2:1 (ARS:Zn(II)) complex formation
(SI Fig 3), it was similar to the observation of ARS and Cu2+
coordination stoichiometry as reported earlier.31 At neutral pH
one of the phenolic OH groups of ARS has been deprotonated
and it forms ARS-Zn(II) complex as shown in Scheme 1.
Spectral study of ARS with Zinc: Absorbance and Fluorescence
study was performed in 10 mM MeOH-PBS buffer (3/1 (v/v),
pH = 7.4) at RT. For spectroscopic titrations 120
mM) stock solution was diluted to 2000 L by adding buffer.
For metal complex formation, the titration was performed
with different concentration of zinc perchlorate (0 – 30 M) in
l of ARS (0.5


buffer. Each spectrum was recorded in standard quartz cuvette
cell of length 1 cm. The change in absorption was monitored at
530 nm. Emission spectrum was recorded upon excitation at
530 nm with excitation and emission slit width 10/10
Spectral study of ARS-Zn(II) vs H2S: For the binding study with
H2S
,
120

l of ARS (0.5 mM) was mixed with 40


l of zinc
L to form
ARS-Zn(II) complex. The concentration of [ARS] = 30 M and
perchlorate (1.5 mM) in buffer and diluted to 2000
a
[Zn(II)] = 30 M was maintained for all titrations. UV-Vis and
Fluorescence titration was done by adding different
concentration of Na2S (0 – 44 M) to this solution. For all the

experiments Na2S has been used as a source for H2S gas.
Fluorescence assay with H2S Donor
For the fluorescence assay with H2S donor molecules, 100 l of
ARS-Zn(II) (60 
(3/1,(v/v), 10 mM , pH = 7.4, 0.04% THF used as a co-solvent)
and diluted by adding 134 l of buffer to this 6 l of glutathione
) was prepared in MeOH:PBS buffer


(0.1 M, 100 equivalent) was added and fluorescence emission
was monitored at 645 nm for 30 minutes at 35°C. The
excitation wavelength was 530 nm and emission wavelength
was 645/30 nm. The titration was done in 96 well- plate using
Synergy-HT multimode micro plate reader.
Fig 1: Absorption spectra of ARS (30 M) in MeOH/ PBS buffer (3:1, v/v, pH = 7.4) in the
presence of (0 – 30 M) equivalent of Zn(II).
The ability of ARS to form complex with zinc ion was also
established by using fluorescence measurement. ARS has been
a natural dye with weakly fluorescent property in aqueous
solution at neutral condition.36
As depicted in figure 2, with increasing the concentration of
Zn(II) ion the fluorescence intensity in the orange region at 625
nm was increased gradually upon excitation at 530 nm, which
was mainly due to a combination of chelation enhanced
Result and Discussion
Binding studies of ARS with Zn(II)
Alizarin and ARS are known for their complexation behaviour
with different metal ions.32-35 We have studied the complex
formation ability of ARS with Zn(II) by using Ultraviolet-visible
(UV-Vis) and fluorescent spectroscopy in aqueous buffer
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fluorescence (CHEF) and internal charge transfer (ICT) In addition to that, the original fluorescent intensity of ARS
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mechanism.
almost 100% recovered, when the concDeOntI:r1a0t.i1o0n39o/Cf5HRA21S19w01aDs
about 42 M. It confirmed that the H2S disassemble the Zn(II)

ion and ARS from the sensing ensemble. These results revealed
that the applicability of the ARS-Zn(II) system for the
detection of H2S in aqueous medium. The fluorescence
recovery could be used to plot calibration curve to afford
quantitative measurement of H2S in biological sample and H2S
releasing drugs.
Fig 2: Fluorescence spectrum of ARS (30 M) in MeOH/ PBS buffer (3:1, v/v, pH = 7.4)
upon addition of 0 - 30 M equivalent of Zn2+
.
Moreover, it was observed that Zn(II) ion induced almost 6 fold
increases in fluorescence intensity of ARS. The fluorescent
intensity of ARS reached maximum at 30 M of zinc, which
makes ARS as a more sensitive probe for zinc. But as described
earlier, when the Zn(II) was added more than 1 equivalent the
fluorescence intensity was dropped.32 The association constant
(Ka) was evaluated graphically by plotting 1/F against
1/[Zn(II)] as shown in SI Fig. 4. The data was linearly fitted
according to the Benesi–Hilderbrand equation and apparent
binding (Ka) value was obtained from the slope and intercept
of the line. The Ka value was found to be 13826 M-1. Zinc ion
induced increase in fluorescence intensity and ratiometric
change in the UV-Vis absorbance ensured the determination of
H2S with high sensitivity with ARS- Zn(II) sensing ensemble.
Effect of H2S on the optical signal of ARS- Zn(II) ensemble
H2S has been known for the formation of metal sulphides upon
reaction with metal ions. Zinc ions are known to react with
sulphide to form stable ZnS species,37 which has low solubility
products constant (Ksp) = 1.6 x 10-24. So it was expected that,
ARS-Zn(II) sensing ensemble would act as a potential sensor
for the detection of H2S through displacement of zinc thereby
recovery of original ARS optical (fluorescent and absorbance)
signal. Figure 3a illustrates the emission spectral changes of
probe with H2S at pH 7.4 in aqueous buffer solution. ARS-Zn(II)
emitted strong fluorescence at 625 nm, upon addition of
different concentration of H2S to this solution, the fluorescent
intensity was decreased. The decrease of fluorescence at 625
nm was achieved immediately (less than a minute) with a fast
response time. Real time detection are important for the
practical application, consequently the quick time response
time of the ARS-Zn(II) sensing ensemble has the advantage as
compared to DHAQ-Cu2+ ensemble reported earlier for H2S
detection, because it has response time about 4 minutes.38
Fig 3: (A) Fluorescence spectrum of ARS –Zn (II) ensemble in MeOH/PBS buffer (3:1,
v/v, pH = 7.4) upon addition of 0 - 42 M equivalent of H2S. (B) Detection limit plot
from fluorescent titration of ARS –Zn (II) with H2S.
Based on the change in emission intensity the detection limit
of H2S calculated for the sensing ensemble are 92 nM, which
was better than other probes reported for H2S, based on zinc
displacement approach.39-41 The detection limit was calculated
by the equation 3
plotting change in fluorescence intensity (
and = the standard deviation of the blank signal obtained
without H2S (Fig 3b).

/slope, where slope was obtained by

F) vs conc. of H2S

Further, we have also monitored the UV-Vis absorbance
change in presence of H2S. As shown in figure 4, upon addition
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of H2S in to the ARS-Zn(II) the absorption band at 525 nm or no interference with detection in the coexistence of various
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decreased gradually and restored to the original absorption species and H2S. Accordingly, probe DAORI:S1-0Z.n10(I3I9)/Cc5oRuA1ld190b1De
state of ARS after adding 44
ARS and Zn(II) has been dissociated from the sensing ensemble of other biological species like ATP, ADP, AMP, PPi, GSH and
in presence of H2S. cysteine. This result was also confirming the possible use of
M of H2S. It indicates that the dye applicable for selective determination of H2S even in presence
Hence the results obtained from fluorescent and absorbance the probe even when H2S co-exists with other biological
spectroscopy confirmed the convenience of using ARS-Zn(II) relevant biothiols like glutathione, cysteine, methionine and
sensing ensemble for the detection of H2S in aqueous buffer phosphates.
solution.
Fig 4: Absorption spectra of ARS-Zn(II) ensemble in MeOH/ PBS buffer (3:1, v/v, pH =
7.4) upon addition of 0–44 M of H2S.
Selectivity studies and effect of interference
The selectivity of the sensing ensemble ARS-Zn(II) for H2S was
examined by fluorescence spectroscopy under the same
condition as discussed above. As shown in figure 5a, the probe
checked with the range of physiological and biological
important analytes such as reactive sulphur species
(glutathione, cysteine, methionine, S2O32-) reactive nitrogen
species (NO., NO3 and N3 ), biological phosphates (ATP, ADP,
AMP, PPi, H2PO4¯ and HPO42-) and other anions (F¯, Cl¯, Br¯, I¯,
OH¯, HCO3¯and CH3COO¯). Among these biological phosphates
(PPi, AMP and H2PO4¯), GSH and cysteine displayed little and
other analytes exhibited insignificant change in the fluorescent
-
-
Fig 5: (a) Fluorescent spectra of ARS-Zn(II) ensemble in the presence of various
analytes in Me-OH/ PBS buffer (3:1, v/v, pH = 7.4) at RT upon excitation at 530 nm. (b)
Different analytes response of ARS-Zn(II) in buffer. [ARS] = 30 M, [Zn(II)] = 30 M,
[anion] = 300 M, [H2S] = 30M. Red colour bar indicates only probe ARS-Zn(II), dark
blue colour indicates ARS-Zn(II) + anions and the Sky blue bar indicates ARS-Zn(II) +
anions + H2S.
spectra of ARS-Zn(II) as shown in Fig. 5a
. At higher
concentration of cysteine (1mM) and glutathione (1mM)
notable change was obtained, but these change are less as
compared to H2S (30M) (SI Fig 11).
These results confirms that the higher selectivity of H2S among
other physiological and biological important analytes. The
decrease in fluorescence intensity with H2S was due to the
displacement of zinc and formation ZnS in aqueous solution.
But non-bonding interaction of glutathione (GSH), cysteine and
H2PO4¯ to the Zn(II) center could be responsible for the weak
binding and small change in fluorescent intensity with these
analytes.
Effect of pH
The maximum fluorescence of ARS has been greatly affected
by pH of the solution.42 The influence of pH on ARS-Zn(II) was
studied using fluorescence measurement. The nature of the
fluorescence profile shown in fig.6 could be a result of the
stability of the ARS–Zn(II) sensing ensemble. At pH values less
than 6, the competition between the OH-proton and the metal
Further, the selectivity of the probe with H2S was also tested in
presence of interfering species. As shown in Fig 5b. The
competitive experiments revealed that minimal interference
ion could occur. Therefore, the ARS–Zn(II) complex was less
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stable, this prevented the H2S detection, therefore, emission displacing the Zn(II) by H2S (42

M) and again, adding Zn(II)
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DOI: 10.1039/C5RA11901D
intensity does not change so much with H2S at lower pH lesser (30 M) to the ARS (30M). As shown in Fig. 7 even after 5

than 6. Due to the high concentration of hydroxide ions, at cycles, the change in fluorescent or decrease in fluorescent for
higher pH (pH > 9.0) it is expected that Zn(II) ion may ARS-Zn(II) solution with H2S were still relatively good. Thus,
precipitate and consequently higher pH sensing ensemble not switching between the fluorescent OFF-ON states could be
stable, therefore it may be not suitable for binding studies. repeated more than five times without decomposition of the
These results described that the detection of H2S using probe fluorescent indicator ARS dye. Therefore the results of high
ARS–Zn(II) was pH dependent and maximum change in reversibility will be in favour of the real-life application.
emission intensity with H2S was obtained in the pH range of
6.0-8.0.
Fluorescent study of ARS-Zn(II) with H2S in serum
After confirming the sensitivity and selectivity of the probe in
buffer solution, we have decided to explore the practical
application of probe for the quantization of H2S, in Human
serum(HS) and Bovine Serum albumin (BSA).
Fig 6: Effect of pH on the fluorescent intensity of ARS-Zn(II) ensemble and the
ensemble with H2S. [ARS] = 27 M, [Zn(II)] =27 M [H2S] = 27 M in Me-OH/ PBS
buffer (3:1, v/v, pH = 7.4).
Recycling Sensing Performance
The reusability of any sensor is very important for practical
application, which requires that the sensor device should be
reversible. Therefore, the recycling sensing performance of the
ARS-Zn(II) was studied through a disassembly and reassembly
of ARS by H2S and Zinc.
Fig 8: Fluorescent intensity of ARS-Zn(II) ensemble upon addition of H2S spiked (6 M,
12M, 18M, 24M, and 30M) buffer, Human serum and BSA.
As shown in Fig. 8 the background fluorescence of ARS-Zn(II)
with blank HS and BSA not much changed. However upon
addition of H2S spiked (6 - 30 M) HS or BSA, fluorescence
intensity of the probe ARS-Zn(II) changed significantly. A linear
calibration curve can be found between the change in
fluorescent intensity at 620 nm and the concentration of H2S.
The change in fluorescence intensity at 620 nm increased in a
linear fashion with a higher percentage H2S in the serum
solutions (SI fig. 12). This calibration curve could be useful for
estimating the concentration of H2S in biological fluid. These
results confirmed that probe ARS-Zn(II) can be used to detect
H2S rapidly and quantitatively in biological samples.
Real time monitoring of H2S from donor molecule
Recently development of novel H2S donors has become a
rapidly growing field, because synthetic H2S donors are useful
research tool as well as potential therapeutic agents.6, 43 These
donor molecules release H2S via different mechanism with
different rate. Therefore it is important to understand the rate
of H2S release from donor molecules. Various methods have
Fig 7: Fluorescent intensity of ARS-Zn(II) ensemble upon alternate addition of H2S and
Zn(II) [ARS]= 30 M, [Zn(II)]=30M, [H2S]=42M.
The reversibility study was carried out by monitoring the
change in fluorescence of ARS-Zn(II) ensemble in a buffered
solution. The fluorescent intensity was checked after
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been used to determine concentration of H2S released by GSH by using multimode micro plate reader. ThVeiewamArtoiculenOtnlionef
DOI: 10.1039/C5RA11901D
these donor molecules. Fluorescence assays were one of the H2S released was expressed as relative fluorescence unit (F/F0).
promising methods, in this regard researchers always looking The fluorescent assay showed that the spontaneous release of
for better probe that uses fluorescent technique to measure H2S by donor molecules easily monitored by new probe ARS-
and monitor the release of H2S for donor. The present probe Zn(II) ensemble based on fluorescent change.
ARS-Zn(II) has high selectivity, sensitivity and quick response We have also assessed that H2S release assay in
a
time for the detection of H2S in biological condition. Thus, we temperature-dependent experiment by varying assay
have decided to make use of this new probe to monitor the temperature from 30-50oC (SI Fig. 13), we found that the rate
H2S released from donor molecule.
of H2S release from donor was quite faster at higher
temperature; it confirmed the stability of probe at higher
temperature. Hence, the results demonstrated that the H2S
released from donor molecules can be monitored by probe
ARS-Zn(II) in a real time.
Scheme 2: Sensing mechanism for the detection of H2S released from H2S Donor
molecule.
In this regard, we have prepared, H2S donor benzoic (methyl
carbonic) dithioperoxyanhydride as describe in the literature.44
Fig 10: Fluorescence images of C6 cell lines. (a) DIC image of C6 cells treated with ARS
(30 μM) only. (b) Fluorescence image of cells in red channel treated with ARS (30 μM)
for 20 min at 37°C. (c) DIC image of cells treatment with ARS (30 μM) and then treated
with 30 μM of Zn(II) ions. (d) Fluorescence images of cells in red channel upon
treatment with 30 μM of ARS and then 30 μM Zn(II) ions for 20 min at 37°C. (e) DIC
image of cells treatment with 30 μM of ARS and then treated with 30 μM of Zn(II) ions
followed by addition of Na2S. (f) Fluorescence images of cells in red channel upon
treatment with 30 μM of ARS and then treated with 30 μM of Zn(II) ions followed by
addition of 44 μM of Na2S for 20 min at 37°C. Images were taken at λex = 543 nm with
40X objective.
Fig 9: H2S release monitored by the ARS-Zn(II) (2 M) ensemble with H2S donor (20
M) and GSH (200 M) at 35 oC.
The spectroscopic properties of ARS-Zn(II) and its selectivity
for H2S appeared to be suitable for live cell image studies. Thus
we have used cultured C6 cell lines to investigate the potential
application of ARS-Zn(II) for the detection of H2S in biological
cells. As shown in Figure 10, C6 cells images were recorded
after incubating with compound ARS for 30 min, and we did
not observe any fluorescent signal. But strong fluorescence in
the cells was induced after addition of Zinc and the
fluorescence was disappeared upon treatment with H2S. This
result proves that probe ARS-Zn(II) can be used for the
detection of H2S in cultured cells.
Our aim was to examine the feasibility of using ARS-Zn(II) to
monitor in situ production of H2S from donor molecules. In a
typical experimental setup, the sensing ensemble ARS-Zn(II) (1
mM, 100µL of the stock solution) in phosphate buffer (134 µL)
was mixed with GSH (0.1 M, 6 µL of the stock solution). Then
H2S donor (1 mM, 60 µL of the stock solution in THF) was
added just before starting the fluorescent measurement. Time
dependent fluorescence measurement was performed in
Synergy HT micro plate reader at 530/645nm as
excitation/emission wavelength for 30 minutes period of time.
As shown in figure 9, ARS-Zn(II) could determine the H2S
release associated with decomposition of donor in presence of
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Notes and references
View Article Online
DOI: 10.1039/C5RA11901D
Table 1: List of Zinc(II) based probes for the detection H2S available in literature
1. H. Kimura, Mol. Neurobiol., 2002, 26, 13-19.
2. R. Wang, Physiol.Rev., 2012, 92, 791-896.
Sensor Probe
LOD
Medium
Detectio
n
Reference
3. L. E. Rojo, J. A. Fernández, A. A. Maccioni, J. M. Jimenez and
R. B. Maccioni, Arch. Med. Res., 2008, 39, 1-16.
4. P. Kamoun, Med. Hypotheses, 2001, 57, 389-392.
5. G. Yang, L. Wu, S. Bryan, N. Khaper, S. Mani and R. Wang,
Cardiovasc Res., 2010, 86, 487-495.
time
8-aminoquinoline
based Zn-Complex
7-mercapto-4-
1.2
CH3CN-
Tris-HCl
Acetone-
HEPES
2 mins
40
39
M
1

M
ND
6. J. L. Wallace and R. Wang, Nat Rev Drug Discovery, 2015, 14
,
methylcoumarin
based Zn- Complex
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Acknowledgements
We acknowledge the Director and Head of the Chemistry
Department, NIT Kurukshetra, Haryana for the research
facilities. DAJ acknowledge the financial support of
Department of Science and Technology (DST) India for the
SERB-DST project grant SB/FT/CS-195/2013. AG thankful to the
financial support of DST, India for the SERB-DST young scientist
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