A reactive primary fluorescence switch-on sensor for Hg2+ and the generated fluorophore as secondary recognition receptor toward Cu2+ in aqueous acetonitrile solution

Article abstract of DOI:10.1016/j.jphotochem.2017.04.011

A new N-methylbenzoimidazole-based fluorescence “turn-on” chemodosimeter (VPBA) was synthesized and characterized for cation to cation relay recognition (CCRR) with high sequence specificity (Hg²⁺?→?Cu²⁺). The selectivity and sensiti

Full text of DOI:10.1016/j.jphotochem.2017.04.011

Journal of Photochemistry and Photobiology A: Chemistry 343 (2017) 7–16
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Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
A reactive primary fluorescence switch-on sensor for Hg2+ and the
generated fluorophore as secondary recognition receptor toward Cu2+
in aqueous acetonitrile solution
Srimanta Mannaa, Parthasarathi Karmakara, Kalipada Maitia, Syed Samim Alia,
Debasish Mandalb, Ajit Kumar Mahapatraa,
*
a
Department of Chemistry, Indian Institute of Engineering Science and Technology, Shibpur, Howrah, 711103, West Bengal, India
Institute of Chemistry, The Hebrew University of Jerusalem, 91904, Jerusalem, Israel
b
A R T I C L E I N F O
A B S T R A C T
Article history:
A new N-methylbenzoimidazole-based fluorescence “turn-on” chemodosimeter (VPBA) was synthesized
and characterized for cation to cation relay recognition (CCRR) with high sequence specificity (Hg2
Received 5 January 2017
Received in revised form 31 March 2017
Accepted 10 April 2017
+
! Cu2+). The selectivity and sensitivity of VPBA have been explored in aqueous acetonitrile solution
Available online 13 April 2017
through a specific cyclization reaction triggered by the mercury-promoted hydrolysis of the vinylether
group with a significant change of fluorescence color. The probe displayed a fast switch-on fluorescence
response with 164 fold toward Hg2+in aquoues acetonitrile solution. Further, the in situ system generated
from the sensing of Hg2+ showed good relay recognition ability for Cu2+ via fast fluorescence quenching
by the formation of a 1:2 complex in aquoues acetonitrile. The complexation of Cu2+ by VPBA has been
addressed by HR-MS, 1H NMR, and UVꢀvis spectra. The probe and their Cu-complex structures have been
established by DFT calculations. TDDFT calculations were also performed in order to demonstrate the
electronic properties of probe and their copper complex. By using the new strategy, the novel probe could
be used for the detection of Hg2+ in real-life water samples with good recovery. The probe could be
successfully used for the fluorescence image of Hg2+ in living cells. These results suggested that the probe
has promising applications for Hg2+ sensing in biological and environmental sciences.
Keywords:
Relay recognition
Metal ion!metal ion sequence
Combination of the chemodosimeter and
chemosensor
Mercury and copper
Living cells
© 2017 Elsevier B.V. All rights reserved.
1. Introduction
Mercury (Hg2+) is considered as one of the most hazardous
environmental pollutant exist as elemental, inorganic, and organic
There is great interest in the development of good bifunctional
chemosensors for the detection of heavy metal ions sequentially
based on a single host that can independently recognize two guest
species with distinct spectra responses [1]. Recently, this idea has
gradually become a new research focus in the field of colorimetric
and fluorescent chemosensors [2–9]. Compared to one-to-one
analysis methods, detection methods for numerous metal ions
with differential responses have advantages of being more efficient
and less expensive. Zhou et al. proposed, an excellent dual
chemosensor that can detect Cu2+ and Hg2+ by monitoring changes
in UV absorption and the fluorescence spectral pattern has been
developed [10]. Recently, we have also reported bifunctional probe
that include combination of Hg2+/Zn2+ [11].
mercury forms [12,13]. It is widely distributed in air, water, and soil
through different processes such as volcanic emissions, mining,
solid waste incineration, and the combustion of fossil fuels [14–18].
It is a considerably dangerous metal to human life and ecology
even at low concentrations. Among the serious health consequen-
ces due to the presence of Hg2+ in the human body are prenatal
brain damage, DNA damage, various cognitive and motion
disorders, Minamata disease, myocardial infarction, some kinds
of autism and damage of the brain, kidneys, central nervous
system, immune system and endocrine system [19–23]. On the
other hand, copper (Cu2+) is an essential trace element in humans,
and is found at low levels in a variety of cells and tissues with the
highest concentrations in the liver, and in the brain [24]. However,
the deficiency and the excess of Cu2+ lead to anemia, gastrointes-
tinal, Wilson disease and neurodegenerative syndromes [25–27].
Therefore, the development of
a new approach with high
sensitivity and specificity toward Hg2+ and Cu2+ is highly desirable
* Corresponding author.
E-mail address: mahapatra574@gmail.com (A.K. Mahapatra).
http://dx.doi.org/10.1016/j.jphotochem.2017.04.011
1010-6030/© 2017 Elsevier B.V. All rights reserved.

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S. Manna et al. / Journal of Photochemistry and Photobiology A: Chemistry 343 (2017) 7–16
as these two ions are spectroscopically and magnetically silent Hg2
+ and a paramagnetic Cu2+ deserves special attention by scientists
across different disciplines.
coordination, so it would be expected to serve as a relay probe for
the sensing of Cu2+ metal ion, by eliciting differences in their
absorption and emission spectroscopy.
In the past several years, considerable efforts have been made to
develop chemosensors for the detection of Hg2+ and Cu2+
,
2. Experimental
especially, for Hg2+ based on the coordination to heteroatom,
Hg2+ catalyzed desulfurization, and Hg2+ promoted hydrolysis of
2.1. Apparatus and reagents
the vinylether group and b-alkynyl ether group [28–36]. However,
most of them still have some limitations as Hg2+ ions are known to
produce fluorescence quenching when binding to fluoroionophore
molecules via the spin–orbit coupling effect (closed-shell d10
configuration) as well as interference from other coexisting metal
ions and poor water-solubility problem [37–40]. Therefore, for
practical applications, it is still desirable to develop simple Hg2+
sensors with turn-on response, good water solubility and high
selectivity and sensitivity. In this context, we propose sequential
cation to cation relay recognition (CCRR) of Hg2+ and Cu2+ based on
the specific chemical reaction (chemodosimeter) and followed by
chemosensor approaches [41,42].
Unless otherwise mentioned, materials were purchased from
Sigma Aldrich and were used without further purification. A
Brucker 400 MHz instrument was used to record 1H and 13C NMR
spectra. For NMR spectra, CDCl3 and DMSO-d6 were used as solvent
using TMS as an internal standard. Chemical shifts were reported
in
d
ppm units and 1H–1H and 1HꢀꢀC coupling constants in Hz. A
Waters QTOF Micro YA 263 mass spectrometer was used to record
Mass spectra. UV spectra were recorded on a JASCO V-530
spectrophotometer. Fluorescence spectra were obtained using
on a Perkin Elmer Model LS 55 spectrophotometer.
Our research group is actively engaged in the development of
novel selective and sensitive fluorescent chemodosimetric probes
for toxic ions [43–47]. Therefore, the present paper deals with the
reaction-based fluorescent sensing approach for inorganic mercu-
ry species (Hg2+) via Hg2+ ion-promoted hydrolysis of vinylether to
form the corresponding hydroxyl group. We proposed that the Hg2
2.2. Design and synthetic scheme of probe VPBA
Our underlying principle in the design of a colorimetric and
turn-on chemodosimeter probe VPBA was depicted in Scheme 1.
The oxymercuration–hydrolysis reaction of the vinyl ether group in
VPBA would generate the hydrolyzed product—alkylmercury
species HgCl(CH2CHO) and phenolate ion, which would undergo
+
ion promoted hydrolysis of the vinyl enol ether group in probe
VPBA would generate the hydroxy intermediate, which will readily
spontaneous cyclize to form iminocoumarinꢀN-methylbenzoimi-
dazole that demonstrates significant improvement of fluorescence
performance of the probe. As in situ generation of iminocoumarin
after Hg2+ reaction and it has several binding sites exist for
subsequent intramolecular nucleophilic attack on the a,b-
unsaturated nitrile moiety by 1,2-addition and in 6-exo-dig
fashion to produce imine anion intermediate. This transient
species would quickly abstract a proton from the solvent and
eventually lead to the cyclized product iminocoumarin-benzo-
Scheme 1. Synthesis of VPBA.

S. Manna et al. / Journal of Photochemistry and Photobiology A: Chemistry 343 (2017) 7–16
9
imidazole 5, which was responsible for the dramatic fluorescence
1HNMR (400 MHz, DMSO-d6):
d
(ppm) = 7.62 (d, J = 7.72 Hz, 1H),
enhancement. Because vinyl ether VPBA was almost non-fluores-
cent but the product iminocoumarin-benzo-imidazole 5 was
highly fluorescent, we could realize a turn-on-type sensing. All
the precursors and probe molecules were characterized by various
analytical and spectral techniques, such as 1H and 13C NMR and
LCMS (Supporting information).
The vinyl ether probe molecule, VPBA, has been synthesized in
three steps starting from commercially available 2-hydroxy-4-
methoxybenzaldehyde 1 by reacting with 1,2-dibromoethane to
form 2-(2-bromoethoxy)-4-methoxybenzaldehyde 2 and subse-
quent elimination process results in the formation of vinyl ether 3
(Scheme 1). Reagents & Conditions: (a) 1,2-dibromoethane, K2CO3,
acetone, reflux, 12 h, 35% (b) KOBut, DMSO, RT, 1 h, 41.6% (c)
Piperidine, ethanol, RT, 1 h, 44.8%
7.54 (d, J = 8.04, 1H), 7.28-7.19 (m, 2H), 4.52 (s, 2H), 3.76 (s, 3H). MS
(ESI): m/z calc. for C10H9N3: 171.08; found: 172.1[M + H]+.
2.3.4. Preparation of 3-(4-methoxy-2-vinyloxy-phenyl)-2-(1-methyl-
1H-benzoimidazol-2-yl)-acrylonitrile (VPBA)
A mixture of 1-methyl-1H benzoimidazol-2-yl)-acetonitrile (4)
(23 mg, 0.13 mmol) and 4-methoxy-2-vinyloxy-benzaldehyde (3)
(24 mg, 0.13 mmol) in ethanol (4 mL) were treated with one drop of
piperidine at RT. The solution was stirred for 1 h. No starting was
left in TLC. The reaction mixture was evaporated. The obtained
crude mass was purified by column chromatography on silica,
product eluted with 10–15% ethyl acetate in hexane to afford 3-(4-
methoxy-2-vinyloxy-phenyl)-2-(1-methyl-1H-benzoimidazol-2-
yl)-acrylonitrile(VPBA) (20 mg, 44.8%) as yellow solid. 1HNMR
(400 MHz, CDCl3):
d (ppm) = 8.39-8.35 (m, 2H), 7.78 (d, J = 7.8 Hz,
2.3. Synthesis of probe VPBA
1H), 7.38-7.30 (m, 3H), 6.75 (d, J = 8.8 Hz, 1H), 6.60-6.56 (m, 2H),
4.87 (d, J = 13.64 Hz, 1H), 4.58 (d, J = 5.76 Hz, 1H), 3.99 (s, 3H), 3.87
(s, 3H). 13C NMR (100 MHz, CDCl3): 164.03, 157.61, 148.34, 146.96,
145.08, 142.46, 136.71, 129.98, 123.58, 122.97, 119.95, 117.32, 115.98,
109.68, 108.89, 102.37, 98.23, 97.37, 55.77, 31.63. MS (ESI): m/z calc.
for C20H17N3O2: 331.13; found: 332.14 [M + H]+.
2.3.1. Preparation of 2-(2-Bromo-ethoxy)-4-methoxy-benzaldehyde
(2)
A mixture of 2-Hydroxy-4-methoxy-benzaldehyde (1) (500 mg,
3.28 mmol) in acetone (22 mL) was added K2CO3 (453 mg,
3.28 mmol) at RT. To this reaction mixture, 1, 2-dibromoethane
(3.2 g, 17.05 mmol) was added. The resulting reaction mixture was
heated at 60 ꢁC for 12 h. TLC showed that majority of the starting
material was consumed. The reaction mixture was partitioned
between ethyl acetate and water. The combined organic solution
was washed with brine, dried over sodium sulfate and then
concentrated. The crude mixture was purified by column
chromatography on silica, product eluted with 5% ethyl acetate
in hexane to afford 2-(2-Bromo-ethoxy)-4-methoxy-benzaldehyde
2.3.5. Preparation of 7-Methoxy-3-(1-methyl-1H-benzoimidazol-2-
yl)-chromen-2-ylideneamine (5)
A mixture of 3-(4-methoxy-2-vinyloxy-phenyl)-2-(1-methyl-
1H-benzoimidazol-2-yl)-acrylonitrile (VPBA) (50 mg, 0.15 mmol)
and mercuric chloride (40.7 mg, 0.13 mmol) in acetonitrile (4 mL)/
water(1 mL) at RT. The reaction mixture was stirred for 1–2 h. The
resultant solid was filtered and dried to afford 7-Methoxy-3-(1-
methyl-1H-benzoimidazol-2-yl)-chromen-2-ylideneamine(5)
(2) (300 mg, 35%) as white solid. 1HNMR (400 MHz, CDCl3):
(ppm) = 10.35 (s, 1H), 7.83 (d, J = 8.72 Hz, 1H), 6.59 (d, J = 8.4 Hz, 1H),
6.40 (s,1H), 4.37 (t, J = 5.92 Hz, 2H), 3.86 (s, 3H), 3.69 (t, J = 5 Hz, 2H).
MS (ESI): m/z calc. for C10H11Br O3: 259.10; found: 261.09 [M + H]+.
d
(20 mg, 43%) as brown solid. 1HNMR (400 MHz, CDCl3):
7.79 (d, J = 7.2 Hz, 1H), 7.5 (s, 2H), 7.38 (d, J = 7.2, 1H), 7.34-7.28 (m,
3H), 6.73 (dd, J = 17.6, and 20.0 Hz, 2H), 3.87 (s, 3H), 3.83 (s, 3H). 13
d (ppm) =
C
NMR (100 MHz, CDCl3):163.05,155.52,142.8,136.29,129.27,123.11,
122.37, 119.98, 112.27, 111.17, 109.72, 100.64, 55.78, 31.69. MS (ESI):
m/z calc. for C18H15N3O2: 305.12; found: 306.0 [M + H]+.
2.3.2. Preparation of 4-methoxy-2-vinyloxy-benzaldehyde (3)
Under cooling condition, potassium tert butoxide (100 mg,
0.89 mmol) was taken in DMSO (0.3 mL). To this reaction mixture, a
solution of 2-(2-Bromo-ethoxy)-4-methoxy-benzaldehyde (2)
(210 mg, 0.81 mmol) in DMSO (1.4 mL) was added drop wise at
0 ꢁC. The reaction mixture was then stirred at RT for 1 h. TLC
showed SM was consumed and the RM was partitioned between
ethyl acetate and water. The combined organic solution was
washed with brine, dried over sodium sulfate and concentrated.
The crude mixture was purified by column chromatography on
silica, product eluted with 10% ethyl acetate in hexane to 4-
methoxy-2-vinyloxy-benzaldehyde (3) (60 mg, 41.6%) as sticky
3. Results and discussion
To understand the recognition behaviour of VPBA and the
generated fluorophore, we carried out absorption and emission
spectroscopic studies, 1H NMR spectroscopy and computations
based on the density functional theory (DFT). Spectroscopic
experiments were carried out in aqueous acetonitrile (CH3CN:
H2O = 2:3 v/v, 10 mM HEPES buffer, pH = 7.4).
3.1. UV–vis and fluorescence spectroscopic studies
colorless liquid. 1HNMR (400 MHz, CDCl3):
d (ppm) = 10.27 (s, 1H),
7.84 (d, J = 8.72 Hz, 1H), 6.69-6.63 (m, 2H), 6.52 (d, J = 2.2 Hz, 1H),
4.90-4.86 (m,1H), 4.62-4.60 (m,1H), 3.86 (s, 3H). MS (ESI): m/z calc.
for C10H10O3: 178.06; found: 179.0 [M + H]+.
In order to confirm the reactivity of different metal ions with
probe VPBA, and their preferential reactivity toward Hg2+ over the
other ions has been studied by absorption and fluorescence
titrations. The UV–vis absorption spectra of chemosensor VPBA in
aqueous acetonitrile (CH3CN:H2O = 2:3 v/v, 10 mM HEPES buffer,
pH = 7.4) were moderately displayed by two absorption bands at
284 and 345 nm, respectively. During the titration, the concentra-
2.3.3. Preparation of (1-Methyl-1H-benzoimidazol-2-yl)-acetonitrile
(4)
A
mixture of N-Methyl-benzene-1,2-diamine (250 mg,
2.04 mmol) and cyanoacetic acid (348 mg, 4.09 mmol) in ethylene
glycol (10 mL) was heated at 165 ꢁC for 2 h. TLC showed SM was
consumed and formed a desired polar spot with major undesired.
The reaction mixture was diluted with ethyl acetate. The organic
layer was washed with water followed by the brine washing. The
combined organic solution was dried over sodium sulfate and then
concentrated. The crude mass was purified by column chromatog-
raphy on silica, eluted with 1% methanol in DCM to afford (1-
Methyl-1H-benzoimidazol-2-yl)-acetonitrile (4) (50 mg, 14%) as
pink solid.
tion of probe VPBA was kept constant at 10 mM and the mole ratio
of Hg2+ was varied. Upon addition of Hg2+ (0–2 equiv.), the
absorption band at 345 nm decreased and a new band at 375 nm
appeared (Fig. 1). Instantly with an isosbestic point at 355 nm,
which is owing to the loss of vinyl enol ether group and the
formation of cyclic compound 5 (Scheme 2).
The fluorescence properties of VPBA were investigated in
aqueous acetonitrile (CH3CN:H2O = 2:3 v/v, 10 mM HEPES buffer,
pH = 7.4). As expected, the probe VPBA exhibited very weak
emission i.e. almost non-fluorescent (F= 0.004) when excited at

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S. Manna et al. / Journal of Photochemistry and Photobiology A: Chemistry 343 (2017) 7–16
when 2.5 equiv. of Hg2+ was added. Spectra were recorded after
10 min of each addition of Hg2+ (100
M) solution.
m
Thus, the new peak appeared at 455 nm can be recognized to
the fluorescence emission of the reaction product. Hence these
results indicated that the probe VPBA reacted with Hg2+ at room
temperature to generate iminocoumarin fluorophore 5. In addi-
tion, the fluorescent color of the VPBA solution upon interaction
with Hg2+ changed from colorless to turquoise blue under UV light
at 365 nm (see Fig. 2(a) inset), which would allow fluorescence
naked eye detection of Hg2+.
3.2. Selectivity in sensing
In order to check whether VPBA was selective to only Hg2+ or
even to the other ions, fluorescence titrations were carried out in
the same medium with 16 different metal ions (apart from Hg2+),
Fig. 1. Absorption spectra of VPBA (10 mM) upon addition of increasing
viz. Al3+, Na+, Co2+, Cr3+, Cu2+, Fe2+, Fe3+, Mg2+, Mn2+, Ni2+, Pb2+, Sn4+
,
concentration of Hg+2 (0–2 equiv.) in aqueous acetonitrile (CH3CN:H2O = 2:3 v/v,
10 mM HEPES buffer, pH = 7.4). Spectra were recorded after incubation with Hg+2 for
10 min at room temperature.
Au3+
, , , and no significant fluorescence
Ag+, Ca2+ and Zn2+
enhancement was found in the presence of these ions (Fig. 3a).
Scheme 2. Sensing of Hg2+ and Cu2+
.
Fig. 2. (a) Fluorescence spectra of VPBA (10
(0- 2.5 equiv.). Each spectrum was recorded after 10 min of addition of Hg2+ solution. (b) Change in fluorescence intensity of VPBA with respect to concentration of Hg2+.
m
M) in aqueous acetonitrile (CH3CN:H2O = 2:3 v/v,10 mM HEPES buffer, pH = 7.4) upon addition of different concentration of Hg2+
355 nm in neutral aqueous solution. Titration of probe VPBA
Much to our delight, the turn-on response of VPBA is highly specific
for Hg2+. Fluorescence response of VPBA towards Hg2+ in presence
of the said competing metal ions were also studied and found no
significant interference Fig. 3b).
(10 m
M) with Hg2+ resulted in the enhancement of fluorescence
intensity at 455 nm as a function of the added Hg2+ concentration
(Fig. 2) and the enhancement approaches 164-fold at saturation

S. Manna et al. / Journal of Photochemistry and Photobiology A: Chemistry 343 (2017) 7–16
11
3.3. Selectivity and sensitivity of the generated fluorophore in-situ and
isolated compound 5 towards Cu2+
To the solution (VPBA + Hg2+) aqueous solution of copper
perchlorate was added and a new absorption peak observed, at
about 421 nm, and its intensity gradually increased upon the
addition of Cu2+ (Supporting information, Fig. S15). The color of the
solution gradually changed from colorless to light yellowish-green
upon the addition Cu2+ to probe VPBA+ Hg2+. The absorption
changed at 375 nm was indicated that relay recognition of these
two ions were realized through UVꢀVis spectroscopy as well.
Finally, we studied the UV–vis titration of the pure isolated
compound 5 with Cu2+ ions which exhibited similar type of result
to what happened when VPBA + Hg2+ system was titrated with Cu2
+, but the obtained spectra was more prominent and sharp
(Supporting information, Fig. S15b). The initial peak at ꢂ375 nm
was due to the formation of compound 5, intensity of which
decreased upon gradual addition of Cu2+ ions. A bathochromic shift
at 439 nm was observed due to ligand-to-metal charge transfer
(LMCT). It is important to note that when UV–vis titration with Cu2
+
was carried out on generated compound 5 in-situ, the UV–vis
spectra did not exactly match (especially in higher wavelength
region) with that of isolated compound 5. It may be due to
presence of other species e.g. by-product due to loss of vinyl ether,
excess Hg2+ etc. in the reaction medium of the former.
Fig. 3. (a) Fluorescence responses of VPBA (10 mM) to various metal ions, including
2. Al3+ 3. Na+ 4. Cu2+ 5. Cr3+ 6. Co2+ 7. Fe2+ 8. Fe3+ 9. Mg2+ 10. Mn2+ 11. Ni2+ 12. Pb2+ 13.
Sn4+ 14. Au3+ 15. Ag+ 16. Ca2+ 17. Zn2+ 18. Hg2+. Excitation was at 355 nm and
emission was at 455 nm. (b) Fluorescence responses of the receptor VPBA (10 mM)
to various metal ions, including 1. Al3+ 2. Na+ 3. Cu2+ 4.Cr3+ 5. Co2+ 6. Fe2+ 7. Fe3+ 8.
Mg2+ 9. Mn2+ 10. Ni2+ 11.Pb2 12. Sn4+ 13.Au3+ 14.Ag+ 15.Ca2+ 16.Zn2+ (10 mM).
Excitation was at 355 nm and emission was at 455 nm. The blue bars represent the
emission intensity of receptor in the presence of other cations. The red bars
represent the emission intensity that occurs upon the subsequent addition of Hg2+
to the above solutions. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.).
The in situ generated iminocoumarin 5 has several binding sites
exist for coordination, so, the highly fluorescent compound 5 has
been used to study for their relay sensing property toward selective
cations. Upon the addition of Cu2+ (100 M) to the VPBA + Hg2+
m
solution, fluorescence quenching was indeed observed at 455 nm
when excited at 355 nm (Fig. 5).
The total decrease of fluorescence intensity is 64% in the VPBA+
Hg2+ system when more than 2.0 equivalent of Cu2+ was present.
The observed fluorescence quench is due to paramagnetic nature
of Cu2+ and LMCT between the reaction product and Cu2+ by
coordination, and this behaviour has been further revealed by DFT
calculations next section in this paper.
Minimum detectable concentration was calculated to be
0.39 mM and it has been detected by the fluorescence titrations
carried out by keeping the probe to Hg2+ mole ratio as 1/1
(Supporting information, Fig. S17). Therefore, VPBA can be used for
the selective recognition of Hg2+ among the 16 different metal ions
studied. It should be mentioned that VPBA still responds to Hg2+
sensitively even in the presence of other relevant competing ions
(Fig. 3b).
The fast and selective maximum quenching response of 5
toward Cu2+ as compared to many cations, including other allied
cations viz. Co2+, Cr3+, Cu2+, Fe2+, Fe3+, Mg2+, Mn2+, Ni2+, Pb2+, Sn4+
,
Ag+, Ca2+, and Zn2+ does not show any significant fluorescence
change toward any of these cation. Cyclic compound 5 was found to
be sensitive to Cu2+ even in the presence of other relevant
competing ions (Supporting information, Fig. S16). These results
clearly demonstrated the resulting VPBA + Hg2+ solution showed
high selectivity for Cu2+ in the same media as well. In this way the
designed relay recognition of these two ions was achieved with
sequence specificity (Hg2+ ! Cu2+) via a fluorescence “off-on-off”
switching mechanism.
The fluorescent quantum yield (F) of VPBA was found to be
0.004 and the isolated intermediate compound 5 show higher (
value of 0.65.
F)
The observed 164-fold increase in the quantum yield of the
compound 5, the presence of water and buffer in the medium of
measurements, and the observed low detection limit seem to
qualify VPBA for sensing Hg2+ by switch-on fluorescence.
Under UV light at 365 nm, the solution of VPBA is non-
fluorescent, whereas in the presence of Hg2+ a turquoise blue
fluorescent color was observed (Fig. 4) and no such fluorescent
color was observed in case of the other metal ions. Thus, Hg2+ can
easily be differentiated by its fluorescent color change from the
other metal ions.
The selectivity was shown on the basis of fluorescence and
absorption spectroscopy studies. It was important to note that the
above relay recognition of two species (Hg2+ ! Cu2+) could be
defined as “metal ion ! metal ion sequence” with a combination of
the chemodosimeter and chemosensor approach that provided a
complementary sensing advance to previous bifunctional probes
[48,49].
The 1:2 stoichiometry of the 5-Cu2+ was established from the
measurements of emission intensity as
a
function of Cu2+
concentration. Plot of relative fluorescence intensity (A-A0) versus
[Cu2+]/[Cu2+] + Ligand (5) mole ratio showed a clear bend of the
curve at 0.65 which suggested that the formation of a 1:2 complex
and hence was stoichiometric (Fig. 6).
This stoichiometry in the solution state was also further
supported by HR-MS studies. The molecular ion peak appear at m/z
763.1444, corresponds to the mass of [dimer of
(Supporting information, Fig. S13). The binding constant for the
Fig. 4. Change in fluorescence of VPBA (10
sequentially form left to right 1. VPBA 2. Al3+ 3. Na+ 4. Cu2+ 5. Cr3+ 6. Co2+ 7. Fe3+ 8.
Mn2+ 9. Hg2+ 10. Ni+2 11. Pb+2 12. Sn+4 13. Au+3 14. Ag+1 15. Ca+2 16.Zn+2 17. Mg+2
mM) on addition of various cations
5 ]
+Cu2+
.

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S. Manna et al. / Journal of Photochemistry and Photobiology A: Chemistry 343 (2017) 7–16
Fig. 5. (a) Fluorescence spectra of VPBA (10 m m mM) in aqueous acetonitrile (CH3CN:H2O = 2:3 v/v, 10 mM HEPES buffer,
M) + Hg2+ (100 M) upon addition of Cu2+ (100
pH = 7.4). (b) Change in fluorescence intensity of 5 with respect to concentration of Cu2+
.
column and then was subjected to 1H NMR analysis. The partial 1H
NMR spectra of VPBA and the isolated product 5 are shown in
Figure (Supporting information, Fig. S10) The characteristic
resonance signal corresponding to the vinyl group of alkenyl
protons Ha and Hb from 4.58 to 6.59 ppm completely disappeared
and at the same time, the appearance of new peak at 7.59 ppm
assigned to the iminocoumarin ꢀꢀNH proton, which indicated that
VPBA undergoes oxymercuration followed by hydrolysis to
generate the compound 5. To confirm the formation of new
molecules, TLC analysis was performed. Interestingly, a fluorescent
spot with higher polarity was observed. Moreover, the reaction
mechanism was also confirmed by LCMS spectrum analysis, and a
peak at m/z 306.00 corresponding to product 5 was obtained
(Supporting information, Fig. S11). For pure probe VPBA, a
characteristic peak at m/z = 332.14 was obtained which corre-
sponds to the species [VPBA + H]+. Therefore, 1H NMR experiments
and mass spectrometry results suggested the validity of the
detection mechanism.
Fig. 6. Job’s plot for 5 with Cu2+ in aqueous acetonitrile (CH3CN:H2O = 2:3 v/v,
10 mM HEPES buffer, pH = 7.4).
3.5. Kinetic study
formation of 5-Cu2+ complex was calculated on the basis of change
of emission at 455 nm by considering 1:2 binding stoichiometry.
The binding constant (K) determined by the BenesiꢀHildbrand plot
experiments was found to be 1.87 ꢃ105 M-1(Supporting informa-
tion, Fig. S20). The detection limit of 5 for Cu2+ ions was found to be
We studied time-dependent response of probe VPBA towards
Hg2+ (Fig. 7). In this set of experiments, several concentrations of
Hg2+ ions were tested with a fixed concentration of probe VPBA
(10 mM). These consequences showed that, upon addition of
0.74 mM (Supporting information, Fig. S21).
increasing concentrations of Hg2+, a dramatic increase of emission
intensity at 455 nm was observed within 15 min, and the emission
signal essentially reached maximum within 26 min. It was
observed that 80% of fluorescence enhancing takes place within
3.4. Reaction mechanism studied by 1H NMR and mass spectral
analysis
15 min of addition of 500 m mM) at a time. The time
L of Hg2+(100
dependent fluorescence enhancing indicated that probe VPBA was
an efficient probe for monitoring the changes in the Hg2+ level in
living systems.
Furthermore, to confirm the hydrolysis of vinyl enol ether and
subsequent cyclization product 5 of probe VPBA, we also carried
out the 1HNMR and mass studies of probe VPBA in the presence of
Hg2+. The probe VPBA was treated with Hg2+, and the reaction
product was isolated by column chromatography over silica gel
We then examined the kinetic profiles of the reaction under
pseudo-first-order conditions with
a
large excess of Hg2+
Fig. 7. Time dependent fluorescence spectra (a) and plot (b) of probe VPBA in aqueous acetonitrile (CH3CN:H2O = 2:3 v/v, 10 mM HEPES buffer, pH = 7.4) in presence of Hg2+
.

S. Manna et al. / Journal of Photochemistry and Photobiology A: Chemistry 343 (2017) 7–16
13
(500.0
m
M) over probe VPBA (10.0
m
M) in aqueous acetonitrile
fashion starting from semiempirical PM2 followed by ab initio HF
to DFT B3LYP by using various basis sets, viz., PM2 ! HF/STO-
3G ! HF/3-21G ! HF/6-31G ! B3LYP/6-31G(d,p). For Cu complex,
a starting model was generated by taking the DFT optimized 5 and
placing the Cu2+ ion well in the core of the iminocoumarin N and
(CH3CN:H2O = 2:3 v/v, 10 mM HEPES buffer, pH = 7.4) at room
temperature. The pseudo-first-order rate constant k/was calculat-
ed according to Eq. (1):
ln[(Fmax-Ft)/Fmax] = -K/t . . . . . . (1)
benzoimidazole
N as donors moieties at a non-interacting
distance. This model was then optimized initially using HF/3-
21G level of calculations, and the output structure from this was
taken as input for DFT calculations performed using B3LYP with
LANL2DZ relativistic pseudopotentials basis set for Cu2+ and 6–
31 + G(d,p) for all other atoms in the complex.
where Ft and Fmax are the fluorescence intensities at 455 nm at
time t and the maximum value obtained after the reaction is
complete, respectively, and k/is the observed pseudo-first-order
rate constant and here it is k/ = 0.02561 sꢀ1 (Supporting informa-
tion, Fig. S18).
Based on the results at different concentrations of Hg2+, the
second-order rate constant was calculated to be 3.684 Mꢀ1minꢀ1
(Supporting information, Fig. S19).
The geometry optimization of VPBA resulted in the nearly
planar conformation at the two sides of C
benzoimidazole parts and that can be estimated through the
dihedral angles (N13-C7-C19-C18 = 177.1; N12-C7-C19-
¼C i.e.; phenyl and
C20 = ꢀ170.4) of the two sides. By contrast, the cyclic product
iminocoumarin and benzoimidazole moieties in 5 are essentially
non planar, with a dihedral angle of ꢂ1450, ultimately planarity
deviate by 350 due to presence of NꢀꢀCH3 group in benzoimidazole
part. The spatial distributions and orbital energies of HOMO and
LUMO of VPBA and cyclization product 5 were also determined
(Fig. 8).
3.6. Effect of pH
Moreover, fluorescence titrations of probe VPBA with Hg2+ at a
wide range of pH values was investigated. In the pH range of 1.0–
11.0, VPBA alone was inert i.e. exhibited no variation in the
fluorescence intensity. (Supporting information, Fig. S14). Also, the
fluorescence responses of the probe VPBA toward mercury was
also found to be almost pH-independent over pH range of 1–11. So,
satisfactory Hg2+ ꢀsensing abilities were exhibited in the range of
pH from 1.0 to 11.0, indicating that VPBA could be used in neutral
natural systems, or a mildly acidic or basic environment. This
indicates that the probe may be suitable for bio-applications at the
physiological pH.
The
p electrons distribution on the HOMO and the LUMO of 5
are essentially distributed in the entire iminocoumar-
inꢀbenzoimidazole backbone. By contrast, in the case of VPBA,
the
p electrons on HOMO is mainly distributed at the benzene ring
and benzoimidazole moieties, whereas on the LUMO is mainly
located on the electron withdrawing cyano group. This indicates
that VPBA bears electron transfer from the benzene/benzoimida-
zole moiety to the cyano group, thus rendering the fluorescence
3.7. Theoretical study
relatively weak (f= 0.004).
By contrast, the nearly complete overlap of electrons on the
To get insight into the optical response of probe VPBA and their
corresponding cyclization product to Hg2+ and Cu2+, we carried out
density function theory (DFT) and time-dependent density
function theory (TD-DFT) calculations at the B3LYP/6-31G(d) level
of the Gaussian 09 program. All element except Cu were assigned
6–31 + G(d) basis set. The geometry optimizations for VPBA, cyclic
compound 5 and their dimeric Cu-complex were done in a cascade
transition orbitals may induce the strong fluorescence emission for
5 (
f
= 0.65). Moreover, the HOMOꢀLUMO energy gap of 5 become
smaller relative to that of probe VPBA, and hence the ICT character
of the 5 is only modest as a result an obvious changes in the
absorption spectra observed at 375 nm upon treatment of VPBA
with Hg2+ cations. The energy gaps between HOMO and LUMO in
the probe VPBA and 5 were 3.83 eV and 3.68 eV respectively (Fig. 8)
Fig. 8. HOMO-LUMO distribution of VPBA and Compound 5.

14
S. Manna et al. / Journal of Photochemistry and Photobiology A: Chemistry 343 (2017) 7–16
As the stiochiometry of the complex formed between Cu2+ and
Table 1
Determination of Hg2+ from natural water sample.
5 was found to be 1:2 based on emission, absorption, and HR-MS
studies, so its dimeric Cu-complex structure was optimized. The
optimized structure of Cu-complex shows that the copper is bound
by two molecules of cyclic compound 5 to satisfy the need for
saturated coordination through centro-symmetric four nitrogen
atoms (two iminocoumarin nitrogens and two imidazole nitro-
gens) in the binding core N4 leads to highly regular square planar
geometry, and the whole molecular system forms a nearly planar
structure. The optimized complex of Cu2+ with 5 associated with
four bonds (2 ꢃ NimineꢀCu and 2 ꢃ NimidazoleꢀCu) to the central
ion with their distances of 1.973 and 1.983 Å respectively
(Supporting information, Fig. S24c). Moreover, the HOMOꢀLUMO
energy gap of complex becomes much smaller relative to that of
both probe VPBA and cyclization product 5.
Sample
River Water. 5.0
10.0
5.0
Added Hg2+
(
m
M) Found Hg2+
(mM) Recover (%) RSD (%)
5.12
102.4
102.3
97.4
1.62
1.78
1.82
0.89
1.70
1.12
10.23
4.87
9.92
5.20
10.19
Pond Water
Tap Water
10.0
5.0
10.0
99.2
104.0
101.9
3.8. Practical application of VPBA
The practicability of the probe VPBA for detecting Hg2+ in real
samples was explored by analysis of natural water. To do this, we
collected different water samples (river water, pond water and tap
water) and detect Hg2+ using our probe VPBA. The water samples
were spiked with standard solutions containing different concen-
The energy gaps between HOMO and LUMO in the Cu-complex
and cyclic compound 5 were 2.42 eV and 3.68 eV respectively
(Supporting information, Table S1). The initial peak of compound 5
at 373 nm was due to HOMO to LUMO electronic transition as
evident from DFT calculation (Supporting information, Table S1).
trations of Hg2+
.
The results are displayed in Table 1. For the three samples,
similar results were obtained and good recovery rates range from
97.4 to 104.0% was found. The relative standard deviation (RSD) of
three measurements was less than 1.82%. These results suggested
that the probe VBPA was suitable for detection of Hg2+ in real
water sample.
The
p electrons on the HOMO of Cu-complex is mainly located on
the iminocoumarin and benzoimidazole framework, but the LUMO
is mostly positioned at the center of the guest Cu2+ ion, which
indicated a strong ligand-to-metal charge transfer (LMCT) process
(Fig. 9). Thus, 5 acts as a quenching fluorescent probe toward Cu2+
ion. The origin of the bathochromic shift (lmax 372 nm ! 439 nm)
in the absorption spectra of the Cu-complex compared with that
for 5 is attributed to LMCT between the 5 and Cu2+ by effective
coordination.
In addition, we also performed time-dependent density
function theory (TD-DFT) calculations to explain the electronic
properties of these complexes in their ground and excited states.
The vertical transitions i.e, the calculated lmax, main orbital
transition, and oscillator strength (f) are listed in Table S1 in the
Supporting information. The vertical transitions calculated by TD-
DFT were compared with the experimental UVꢀVis spectra of
VPBA, 5 and Cu-complex and were found to have good agreement
with the experimental data.
3.8.1. Cytotoxicity assay
To demonstrate the practical application of the probe (VPBA)
we have done the live cell imaging to detect even trace amount of
Hg2+. Vero cells were used to detect Hg2+ ions in live cells. In this
context, we have also detected trace amount of Cu2+ in live cells by
using compound 5. However, to materialize this objective we have
probed the cytotoxic effect of VPBA, Hg2+, Compound 5, Cu2+ and 5-
Cu2+ on live cells. We have performed MTT assay, which is based on
mitochondrial dehydrogenase activity of viable cells to study
cytotoxicity of above mentioned compounds at varying concen-
trations mentioned in method section (Supporting information,
Fig. S22 and S23). The experiment shows that probe VPBA and
Fig. 9. HOMO-LUMO distribution of Compound 5 and 5 + Cu2+.

S. Manna et al. / Journal of Photochemistry and Photobiology A: Chemistry 343 (2017) 7–16
15
compound 5 did not exert any significant effect on cell viability;
4. Conclusions
however the Hg2+ and Cu2+ ions have dose dependent adverse
effect when cells were treated with varying concentrations of Hg2
+and Cu2+. The metal-probe complex has the significant adverse
In conclusion we may state that our probe VPBA is an efficient,
selective and sensitive probe that successfully demonstrates both
chemodosimetry and traditional co-ordination based sensing for
two different analytes with sequence specificity (Hg2+ ! Cu2+) via a
fluorescence “off-on-off” mechanism. The well-known hydrolysis
reaction of vinyl enol ether by mercury is exploited in the
chemodosimetric approach and the sensing response was quanti-
fied through the intensity of turn-on fluorescence response. This
hydrolysis reaction led to formation of highly fluorescent
iminocoumarin species which participated in 1:2 co-ordination
with Cu2+. Especially, iminocoumarin 5 is also potentially useful for
quantitative determination of Cu2+ by absorption and fluorescence
titration in aqueous acetonitrile (CH3CN:H2O = 2:3 v/v, 10 mM
HEPES buffer, pH = 7.4). The spectral changes have been corrobo-
rated by DFT studies as well as mass and 1H NMR spectra. Further,
the presence of the coumarin moiety makes this probe an ideal
candidate for applications in intracellular imaging studies which
has also been successfully demonstrated in this work.
effect on cell viability beyond 75 mM. The effect is more
pronounced in higher concentration and has showed an adverse
cytotoxic effect in a dose-dependent manner. The viability of vero
cells is not influenced by the solvent (DMSO).
3.8.2. Application of VPBA in live cell imaging
The results obtained in the in vitro cytotoxic assay has revealed
that, in order to pursue fluorescence imaging studies of metal-
probe complex in live cells, it would be cautious to choose a
working concentration of 10–20
assess the effectiveness of compound, first cells are treated with
VPBA, followed by 20
M Hg2+ for 1 h. Fluorescent microscopic
mM for probe compound. So, to
m
studies has revealed a lack of fluorescence in vero cells when
treated with Hg2+ (Fig. 10a) and the probe alone (Fig. 10c). When
the cells are treated with the probe VPBA in presence of Hg2+, a
striking Green fluorescence is observed inside the cells, which
indicates the formation of compound 5, as observed earlier in
solution studies (Fig. 10e&f). Consequently, quenching of fluores-
cence intensity was observed after addition of Cu2+ ((Fig. 10g&h).
The fluorescent microscopic studies suggested that probe VPBA
could eagerly cross the membrane barrier of the vero cells, and
quickly sense intracellular Hg2+ and Cu2+ in very lowconcentration.
Most importantly, bright field images of treated cells did not
expose any gross morphological changes, which suggested that
vero cells are viable. These results open up the path for future in
vivo biomedical applications of this sensor.
Acknowledgment
We acknowledge DAE-BRNS, India [Project No. 36(1)/14/12/
2016-BRNS/36012] for financial support.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/j.
jphotochem.2017.04.011.
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