Hg2+-selective "turn-on" fluorescent chemodosimeter derived from glycine and living cell imaging

Article abstract of DOI:10.1016/j.jphotochem.2012.05.001

A new nonfluorescent benzthiazole derivative of dithio-N-phthaloylglycine was prepared, and its fluorogenic chemodosimetric behaviors toward transition metal ions were investigated. The dithio-N-phthaloylglycine derivative showed highly Hg²⁺-selective fluorescence enhancing ("turn-on") properties in 20% aqueous acetonitrile solution (H₂O/CH₃CN = 80:20, v/v). The chemodosimetric behavior is based on the Hg²⁺ triggered desulfurization of dithio-N-phthaloylglycine derivative into its oxygen analogue. To observe the cell permeability of 3 into Pollen grains, we also employed it for the fluorescence detection of the changes of intracellular Hg²⁺ in cultured cells.

Full text of DOI:10.1016/j.jphotochem.2012.05.001

Journal of Photochemistry and Photobiology A: Chemistry 240 (2012) 26–32
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Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Hg2+-selective “turn-on” fluorescent chemodosimeter derived from glycine and
living cell imaging
Ajit Kumar Mahapatraa,∗, Jagannath Roya, Saikat Kumar Mannaa, Supratim Kundua, Prithidipa Sahoob,
Subhra Kanti Mukhopadhyayc, Avishek Banikc
a Department of Chemistry, Bengal Engineering and Science University, Shibpur, Howrah 711 103, India
b Chemistry and Chemical Biology Department, Rutgers University, 610 Taylor Road, Piscataway, NJ 08854, USA
c Department of Microbiology, The University of Burdwan, Burdwan, West Bengal, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new nonfluorescent benzthiazole derivative of dithio-N-phthaloylglycine was prepared, and its
fluorogenic chemodosimetric behaviors toward transition metal ions were investigated. The dithio-N-
phthaloylglycine derivative showed highly Hg2+-selective fluorescence enhancing (“turn-on”) properties
in 20% aqueous acetonitrile solution (H2O/CH3CN = 80:20, v/v). The chemodosimetric behavior is based
on the Hg2+ triggered desulfurization of dithio-N-phthaloylglycine derivative into its oxygen analogue.
To observe the cell permeability of 3 into Pollen grains, we also employed it for the fluorescence detection
of the changes of intracellular Hg2+ in cultured cells.
Received 5 January 2012
Received in revised form 14 March 2012
Accepted 1 May 2012
Available online 9 May 2012
Keywords:
Fluorescent chemodosimeter
Glycine
© 2012 Elsevier B.V. All rights reserved.
Mercury
Turn-on fluorescent probes
Pollen cell
1. Introduction
that detect mercury ions [6–9]. Among them, a chemodosimet-
ric approach frequently yields successful results in the selective
Mercury is one of the most dangerous and widespread global
pollutant [1,2], as seen in such unfortunate incidents as Mina-
mata disease [3] and mercury poisoning in Iraq [4]. As these cases
showed, methylmercury is extremely toxic to aquatic organisms
and birds than inorganic mercury, which eventually reaches the top
of the food chain and accumulates in higher organisms, especially
in large edible fish [5]. When consumed by humans, methylmer-
cury triggers several serious disorders including sensory, motor,
and neurological damage. The development of methods for the
determination mercury is, therefore, of significant importance for
environment and human health. Traditional analytical techniques
for Hg2+ include atomic absorption spectroscopy, cold vapor atomic
fluorescence spectrometry and gas chromatography. These meth-
ods, however, require not only complicated instrumentation but
also a long measuring time. Therefore, it is urgent to develop new
methods for monitoring Hg2+ with a low limit of detection, as
well as rapid and facile detection. In particular, fluorescence has
been regarded as the most powerful optical technique for detect-
ing low concentrations of metal ions, and considerable efforts have
been devoted to the development of fluorescent chemical sensors
and sensitive signaling of Hg2+ ions [10–15]. Hg2+-triggered desul-
furization and/or transformation of thioamide [16], selenolactone
[17] and thione has been successfully utilized for the development
of Hg2+-selective chemodosimeters, beginning with the classi-
cal anthracene-based dosimeter [18] developed by Czarnik and
coworkers.
These methods may facilitate mercury analyses by the conver-
sion of thiocarbonyl compounds into their carbonyl analogues via
oxidative procedures that involve inorganic and organic reagents,
as well as hydrolytic reactions [19]. Because mercury is a soft Lewis
acid, a vast majority of the chemosensors and chemodosimeters
for mercury contain sulfur atom(s) that can tightly coordinate the
metal, thus weakening the C S bond and as part of off–on fluo-
rescence switches. So the probes for Hg2+ generally consist of a
chromophore and a reactive region that can be chelated [20–22]
or reacted [16,23–30] with Hg2+ ions. The absorption or emission
properties of the probes change with the interactions between
the reactive region and Hg2+ ions. The development of novel
chromophores with appropriate reactive regions for selective, sen-
sitive detection remains an important goal for ion sensors. During
our ongoing research on both cation [31,32] and anion [33,34]
recognition, herein, we report the synthesis and application of a
probe based on benzthiazole, 2-benzthiazol-2-ylmethylisoindole-
1,3-dithione (3), for the selective, sensitive detection of Hg2+ ions in
∗
Corresponding author. Tel.: +91 33 2668 4561; fax: +91 33 26684564.
E-mail address: mahapatra574@gmail.com (A.K. Mahapatra).
1010-6030/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotochem.2012.05.001

A.K. Mahapatra et al. / Journal of Photochemistry and Photobiology A: Chemistry 240 (2012) 26–32
27
O
O
OH
Et3N, toluene
reflux, 4 h
OH
O
O
+ H2N
2-Aminothiophenol
PCl3, toluene
N
O
O
O
1
S
O
S
N
Lawesson's reagent
S
N
N
O
toluene, reflux
N
S
S
O
3
2
N
N
S
4
Scheme 1. Synthesis of chemodosimeters 3 and 4.
aqueous acetonitrile H2O/CH3CN = 80:20, v/v. The desulfurization
reaction of carbonyl sulfide takes advantage of the high affinity of
Hg2+ for sulfur. A simple substitution of the oxygen atom with sul-
fur results in a significant change in the optical properties of the
compounds.
under a dry atmosphere. All experiments were carried out in HEPES
(20 mM, pH 7.05) buffer solution (containing 10% CH3CN as co-
solvent).
2.3. Synthesis and characterization
In the present case, our strategy for designing a chemical sensor
for mercury was glycine amino acid to N-phthaloylglycine scaffold
having an appended benzthiazole. As amino acids are water-soluble
and biologically and environmentally compatible, new chemical
sensors based on amino acids are a promising route of develop-
ment. However, as none of the natural amino acids except Trp
and Tyr have fluorescent properties [35,36], conjugation of a flu-
orophore into the amino acid is a critical step for the synthesis
of fluorescent chemical sensors based on amino acids. Therefore,
we speculated that the introduction of the benzthiazole ring to
the dithio-N-phthaloylglycine-based probe based on the following
considerations: (i) increase the affinity to Hg2+ ions in competitive
aqueous media; (ii) change the spatial effects within one molecule;
(iii) realize the real-time detection (quickly induce the fluorescent
response); (iv) improve the sensitivity of Hg2+ ions.
The receptor 3 was synthesized according to Scheme 1. N-
phthaloylglycine (1) was prepared in 82% yield from the reaction
of glycine with phthalic anhydride in toluene at 100 ◦C in the pres-
ence of triethylamine. Benzothiazole derivative 2 was prepared by
the reaction of N-phthaloylglycine with 2-aminothiophenol (PCl3,
toluene, 75%). Dithio-derivative of 3 was prepared from the 2 by
the reaction with Lawesson’s reagent in good yield (62%) [37].
Compound 2 was reacted with Lawesson’s reagent in refluxing
dry toluene, and the resulting material was purified by column
chromatography with silica gel using chloroform as the eluent.
Compound 3 was obtained as chocolate brown solid with a yield
of 62% (Scheme 1). The structures of 2 and 3 were fully confirmed
by NMR spectroscopy and mass spectrometry (MS) (Figs. S1–S7).
The structure of probe 2 was also confirmed by X-ray analysis
[38] (Fig. 1) of its single crystal that was grown in acetonitrile.
Thiocarbonyl derivative of 4 was similarly prepared from their N-
phthaloylglycine (1) precursor.
2. Experimental
2.1. Materials and apparatus
2.3.1. Synthesis and characterization of receptor 2:
All the solvents were of analytic grade. All cations, in the form
of perchlorate salts, were purchased from Sigma–Aldrich Chemical
Co., stored in a desiccator under vacuum containing self-indicating
silica, and used without any further purification. Solvents were
dried according to standard procedures. Unless stated otherwise,
commercial grade chemicals were used without further purifica-
tion. All reactions were magnetically stirred and monitored by
thin-layer chromatography (TLC) using Spectrochem GF254 silica
gel coated plates. The 1H NMR spectra were recorded on Bruker-
AM-400 spectrometer. The 1H NMR chemical shift values are
expressed in ppm (ı) relative to CHCl3 (ı = 7.26 ppm). UV–vis and
fluorescence spectra measurements were performed on a JASCO
V530 and a PerkinElmer LS-55 spectrofluorimeter respectively.
2-benzothiazole-2-yl methyl-isoindole-1,3-dione
2-Aminothiophenol (138 mg, 1.1 mmol) and compound
1(189 mg, 1 mmol) were dispersed in dry toluene. The solu-
tion was warmed to 40 ◦C and phosphorus trichloride (0.1 mL,
1.1 mmol) was added dropwise to the reaction mixture. The
mixture was heated 100 ◦C for 4 h and then cooled to room tem-
perature. The reaction mixture was extracted with 20% sodium
carbonate solution and washed with distilled water, and then
evaporated to produce solid residue. The crude product was
crystallized in dichloromethane/hexane to give compound 2 in
75% yield. Mp 189–191 ◦C. The structure of probe 2 was confirmed
by X-ray analysis of its single crystal (CCDC 832376) that was
grown in acetonitrile.
2.2. General method
2.3.2. Synthesis and characterization of receptor 3:
2-benzothiazol-2-yl methyl-isoindole-1,3-dithione
A 2.0 × 10−5 M solution of the probe 3 in CH3CN/H2O (20:80,
v/v) was. Solutions of 5.0 × 10−4 M perchlorate salts of the respec-
tive cation were prepared in distilled CH3CN and were stored
Lawesson’s reagent (203 mg, 0.5 mmol) was added to the solu-
tion of compound 2 (196 mg, 0.5 mmol) in dry toluene. The mixture
was heated at 110 ◦C for 36 h and then allowed to cool slowly to

28
A.K. Mahapatra et al. / Journal of Photochemistry and Photobiology A: Chemistry 240 (2012) 26–32
Fig. 1. X-ray crystal structure of 2. Hydrogen atoms are not shown for clarity.
room temperature. The coloured solid product obtained was fil-
tered and purified by column chromatography using chloroform as
the eluent to yield chocolate brown 3 (62%). Mp 173 ◦C.
1H NMR (CDCl3, 400 MHz) ı 7.94 (d, J = 10.9 Hz, 1H), 7.84 (d,
J = 13.4 Hz, 1H), 7.65 (m, 4H), 7.40 (t, J = 8.16 Hz, 1H), 7.35 (t,
J = 9.76 Hz, 1H), 6.03 (s, 2H). 13C NMR (CDCl3, 100 MHz) ı 171.57,
167.61, 167.59,134.63, 134.61, 134.53, 132.17,132.16, 126.57,
125.73,124.04, 123.93, 123.51, 121.87, 39.75, 38.76. ESI-MS: m/z
327.10 [M+1]+.
chromogenic change from brown to colorless was observed. Upon
interaction with various metal ions, significant changes in absorp-
tion spectra were observed particularly with Hg2+. Other metal
ions caused relatively minor changes in the absorption spectra of
3 (Fig. S8). Judging from the titrations, the stoichiometries of the
complexes were ascertained from the break of the UV–vis titration
curves on the basis of the Job’s plot method [39] (as shown in the
inset of Fig. 2).
The stoichiometry of the Hg2+ complex formed with 3 was deter-
mined by Job’s plot (Fig. 2, inset) at 243 nm, and it was found to be
1:1. In the plot, a maximum at 0.5 mol fraction was observed that
the large absorption change occurs via the 1:1 reaction. In addition,
the absorption data shows clear isosbestic point.
3. Results and discussion
3.1. UV–vis studies
The UV–vis spectroscopic behavior of 3 toward representative
metal ions was investigated by treating it with physiologically
important transition metal perchlorates in 20% aqueous acetoni-
trile solution (H2O/CH3CN = 80:20, v/v, 50 mM HEPES at pH 7.4).
The absorption spectrum of 3 exhibited strong absorption bands at
243 and 360 nm. Fig. 2 shows the gradual increase in the UV–vis
absorption spectra of 3 in 20% aqueous acetonitrile solution con-
taining various concentrations of Hg2+ at room temperature. A clear
isosbestic point was observed at 270 nm. The clear isosbestic point
at 270 nm in the absorption spectra (Fig. 2) indicate that the reac-
tion of 3 with Hg2+produces a single component. A concomitant
3.2. Fluorescent studies
To examine the sensitivity of 3, fluorescence emission spectra
were recorded in the presence of 50 equiv. of representative metal
perchlorates in the same solvent system. It is expected that the
designed probe 3 will display weak fluorescent. However, when
it reacts with excess Hg2+ to yield 2, strong fluorescence should
be observed gradually. As shown in Fig. 3a, 3 displays relatively
weak fluorescence centered at about 360 nm in the absence of Hg2+
with ꢀex = 243 nm, and a pronounced ‘off–on’ type signaling toward
Hg2+ ions. Upon interaction with various metal ions, the fluores-
cence intensity at 360 nm was markedly enhanced with Hg2+ ions
(Fig. 3b), while other metal ions (Zn2+, Mn2+, Ni2+, Co2+, Cu2+, Cd2+
,
and Pb2+) did not cause any noticeable responses (Fig. S9). The
only exception was Cu2+, which gave rise to a small fluorescent
quenching response (Fig. S9b). The fluorescence titration profile
of 3 versus Hg2+revealed that the maximum enhancement of the
emission from 3 was obtained when 50 equiv. Hg2+ was added to
the solution (Fig. 3b).
The fluorescence ratio (I0 − I)/I0 is displayed in Fig. 4a. For Hg2+
,
the value was almost 3 times. In contrast, a range of other ions
showed ratios of less than 1. It can be concluded that 3 displays
much higher selectivity for Hg2+ ions than the other ions screened.
Furthermore, the emission color of probe 3 is displayed blue flu-
orescence and also metal-ion-dependent (Fig. 4b), demonstrating
that the probe could be employed for a convenient visual sensing
of Hg2+
.
The signaling mechanism is based on Hg2+-induced desulfur-
ization of the thio derivative (Scheme 2). The thioketone group of
3 is readily transformed to its ketone function by reaction with
Hg2+ ions [40] and the liberated sulfur atom forms a stable species
of HgS with Hg2+ ions [41]. As a result of this transformation, the
Fig. 2. UV–vis absorption spectra of 3 (10 ␮M) in H2O/CH3CN (H2O/CH3CN = 80:20,
v/v, 50 mM HEPES at pH 7.4) containing different concentrations of Hg2+ (0, 0.2, 0.4,
0.8, 1.0, 2.0, 4.0, 8.0, 10, 20, 40, 80, 100, 120, 150, 200 ␮M). Inset: UV–vis Job plot of
3 with Hg2+ by using continuous variation method.

A.K. Mahapatra et al. / Journal of Photochemistry and Photobiology A: Chemistry 240 (2012) 26–32
29
Fig. 3. (a) Fluorescence titration of 3 with Hg2+ ions. [c] = 1.0 × 10−5 M, H2O/CH3CN (80:20) at pH 7.4 (50 mM HEPES). ꢀex = 243 nm. Inset: the photograph shows the fluores-
cence color of probe 3 before and after addition of 2 equiv. Hg2+ ions. (b) Emission spectra of 3 (10 ␮M) in the presence of 50 equiv. of Hg2+ ions and 100 equiv. of each of Zn2+
Mn2+, Ni2+, Co2+, Cu2+, Cd2+, and Pb2+ ions. ꢀex = 243 nm.
,
Fig. 4. (a) The fluorescence ratio of [It/I0 = (I0 − I)/I0] of 3 at 360 nm upon addition of different metal ions (50 equiv. of each) in H2O/CH3CN = 80:20, v/v. (b) Visual fluorescence
emission of probe 3 to metal ions (50 equiv. except 20 equiv. Hg2+) in H2O/CH3CN = 80:20, v/v (50 mM HEPES at pH 7.4).
observations demonstrated that the 3·Hg2+ signaling system oper-
weakly fluorescent dithio-N-phthoylglycine derivative 3 (˚ = 0.002
in 20% aqueous acetonitrile) is transformed into a strongly emitting
ates based on an irreversible chemodosimetric process.
phthoyl derivatve of 2 (˚ = 0.113).
3.4. Mass spectral analysis
3.3. 1H NMR studies
The another evidence for the 1:1 stoichiometry was suggested
by ESI-MS analysis. In the absence of Hg2+, a peak was observed at
m/z 327.1 corresponding to [3+H]+ (Fig. S4). After the addition of
2 equiv. of Hg2+ to the solution of 3, the peak at m/z 327.1 disap-
peared. At the same time, a new peak at m/z 527.6 appeared, which
is consistent with the compound [3+ Hg2++H]+ (Fig. S5). In addition,
changes in the fluorescence spectra of 3 in response to Hg2+ ions
also confirmed the proposed Hg2+-induced desulfurization process.
To obtain further information about the proposed transforma-
tion of sensor 3, 1H NMR titration experiments were performed
in CD3CN. Changes in the 1H NMR spectra of sensor 3 before and
after addition of Hg2+ are shown in Fig. 5. In general, Hg2+ induced
resonance signals of the sensor molecule to become broad and
slightly shifted to downfield region. For instance, the resonance
signal corresponding to the aromatic protons were shifted down-
field from 7.93 to 8.26 and 7.88 to 7.88 ppm upon addition of Hg2+
.
These changes indicated that indeed the sensor complexed with the
metal ions. The chemodosimetric nature of the signaling was fur-
ther verified by testing reversibility of the 3·Hg2+system with EDTA
treatment. The enhanced fluorescence of the 3·Hg2+system was
not significantly affected by treatment with chelating chemicals
of EDTA (Fig. S10), but in the presence of EDTA, the fluores-
cence of 3 was not enhanced upon addition of Hg2+ ions. These
3.5. DFT calculation
To understand the interactions between probe 3 and the Hg2+
metal center, the quantum chemical calculations using at the DFT
level were performed (Fig. S11). The molecular modeling showed
(Fig. 6) that the cation probe 3 has concave structure. The ring
S of benzthiazole and one of the S of dithio-N-phthaloylglycine
O
S
S
Hg2+ (excess)
HEPES buffer (pH 7.4)
-HgS
S
N
N
S
N
N
O
2
3
Strongly fluorescent
Weakly fluorescent
Scheme 2. Fluorescence enhancement of 3 in response to the Hg2+ ion induced desulfurization.

30
A.K. Mahapatra et al. / Journal of Photochemistry and Photobiology A: Chemistry 240 (2012) 26–32
Fig. 7. Time trace of the Hg2+-signaling of 3. [c] = 5.0 × 10−6 M, [Hg2+] = 5.0 × 10−4
M
in H2O/CH3CN (H2O/CH3CN = 80:20, v/v, 50 mM HEPES at pH 7.4). ꢀex = 243 nm.
derivative of probe 3 are oriented into the cavity for the com-
plexation of Hg2+, owing to thiophilic character of Hg2+. Due
to the open-shell nature of the Hg2+-containing complexes, the
structure for the [3·Hg2+] complex was calculated in a first step,
following the experimentally observed 1:1 ligand/cation ratio. The
2:2 stoichiometry [32·Hg2]4+ was also checked but displayed a
very endergonic complexation energy.
Fig. 5. Partial 1H NMR spectra of 3 in the absence (a) and in presence of 1 equiv.
Hg2+ ions. (b) 2 equiv. Hg2+ ions (c) in CD3CN.
4. Analytical application
To examine the sensitivity of 3 for Hg2+ ion sensing, its kinetic
study was monitored for the reaction of Hg2+-promoted desulfu-
rization of compound 3. Fluorescence titration was carried out to
gain quantitative insight into the signaling behavior of 3 toward
Hg2+ ions. From the Hg2+ concentration dependency of the signal-
ing behavior, the detection limit of 3 for the analysis of Hg2+ ions
was estimated to be 1.0 × 10−5 M. The Hg2+ signaling of 3 was rel-
atively slow compared with that of other chemo dosimeters based
on sulfur derivatives. A time trace revealed that signaling was com-
plete within about 30 min at temperature of 25 ◦C (Fig. 7), these
observation are some what undesirable for a practical applicability
of sensor 3 as a Hg2+-selective chemodosimeter.
Fig. 6. Calculated optimized structure (B3LYP/6-31G*) of 3-Hg2+ (E = −798.201 a.u.).
.
Fig. 8. (a) Fluorescence microscope images of Pollen cells only, (b) images of cells + Hg2+(25 mM), (c) images of cells +Hg2+ + 3 (5 ␮M), and fluorescence images (d–f) of Pollen
cells incubated with 10 ␮M, 20 ␮M, 40 ␮M of 3 for 20 min, respectively.

A.K. Mahapatra et al. / Journal of Photochemistry and Photobiology A: Chemistry 240 (2012) 26–32
31
On the other hand, imidazole 4, which has a closely related
structure to 3, are very slowly photodegraded under the same
experimental conditions about 0.5% after 30 min (Fig. S12). These
observations suggest that the selective determination of Hg2+ ions
by fluorescence enhancement could be practically feasible with
sensor 3.
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