Carbazole phenylthiosemicarbazone-based ensemble of Hg2+ as selective fluorescence turn-on sensor toward cysteine in water

Article abstract of DOI:10.1016/j.tetlet.2013.03.111

A carbazole-phenylthiosemicarbazone-based open chemosensor has been designed and synthesized, which exhibits high selectivity toward Hg²⁺ by forming a 1:1 complex. In its Hg²⁺ensemble, it is reported as a highly sensitive and selective probe for the detection of mercapto biomolecules in aqueous solution. The addition of Cys (Cysteine) to a 99% aqueous solution of carbazole-phenylthiosemicarbazone-Hg²⁺ ensemble resulted in a rapid and remarkable fluorescence OFF-ON (emission at 436 nm). The IMP logic gate has also been generated by choosing Hg²⁺ and Cys as input and by monitoring the output signal at 436 nm that originates from the emission spectra of chemosensor in the presence of Hg²⁺.

Full text of DOI:10.1016/j.tetlet.2013.03.111

Tetrahedron Letters 54 (2013) 2946–2951
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Carbazole phenylthiosemicarbazone-based ensemble of Hg2+ as selective
fluorescence turn-on sensor toward cysteine in water
Ajit Kumar Mahapatra a, , Jagannath Roy a, Prithidipa Sahoo a, Subhra Kanti Mukhopadhyay b,
⇑
Avishek Banik b, Debasish Mandal c
a Department of Chemistry, Bengal Engineering and Science University, Shibpur, Howrah 711103, India
b Department of Microbiology, The University of Burdwan, Burdwan, West Bengal, India
c Department of Spectroscopy, India Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A carbazole-phenylthiosemicarbazone-based open chemosensor has been designed and synthesized,
which exhibits high selectivity toward Hg2+ by forming a 1:1 complex. In its Hg2+ensemble, it is reported
as a highly sensitive and selective probe for the detection of mercapto biomolecules in aqueous solution.
The addition of Cys (Cysteine) to a 99% aqueous solution of carbazole–phenylthiosemicarbazone–Hg2+
ensemble resulted in a rapid and remarkable fluorescence OFF–ON (emission at 436 nm). The IMP logic
gate has also been generated by choosing Hg2+ and Cys as input and by monitoring the output signal at
Received 11 December 2012
Revised 21 March 2013
Accepted 23 March 2013
Available online 6 April 2013
Keywords:
Biothiols
Carbazole
Cysteine
436 nm that originates from the emission spectra of chemosensor in the presence of Hg2+
.
Ó 2013 Elsevier Ltd. All rights reserved.
Ensemble
Logic gate
Candida cell
Mercapto amino acids play several important roles in biological
systems.1,2 The important mercapto amino acids are cysteine (Cys),
homocysteine (Hcy), and glutathione (GSH). These are essential
biological materials required for the growth of cells and tissues
in living systems. Specifically, Cys deficiency is involved in many
syndromes for instances, slow growth in children, hair depigmen-
tation, edema, lethargy, neurotoxicity, liver damage, loss of muscle
and fat, skin lesions, and weakness.3 Because of its important role
in biological systems, the optical method for the detection and
sensing of Cys, Hcy, and GSH with high sensitivity is of current
interest in the chemosensor research field. Up to now, various
biothiol chemosensors based on Michael addition,4 cyclization
reaction with aldehyde,5 cleavage reaction by thiols,6 or many ana-
lytical techniques, including UV–vis detection assays, mass spec-
trometry (MS),7 gas chromatography,8 high-performance liquid
chromatography,9 and electrochemical methods,10 are the avail-
able techniques. Among these techniques, the fluorescent probes
have been widely used to sense mercapto biomolecules11 because
of their simplicity, high selectivity, and sensitivity. However, there
are relatively few examples of fluorescent chemosensors for Cys/
Hcy/GSH, which are capable with dual monitoring of metal ions
and amino acids in water. Among the thiophilic metal ions, Hg2+
has attracted much more attention because it could cause a variety
of symptoms in vivo, including digestive, cardiac, kidney, and
neurological diseases.12,13 Therefore, detecting and sensing mer-
cury and cysteine based on one fluorescent sensor with dual func-
tionality is feasible. It is more economical, convenient and also of
great significance to cell imaging as it is a reversible probe. Such
detection may be achievable in such a way of Hg2+ coordinated thi-
osemicarbazone moiety followed by demetalization to generate
fluorescence ON. Here, we designed a carbazole–thiosemicarba-
zone–Hg2+ ensemble based first displacement approach fluores-
cence sensor for detecting Cys in aqueous media.
In this work, we developed a new carbazole-based imine, C1,
bearing two thiourea moieties with an extensively blue emission
(Scheme 1). The thiourea unit was incorporated into C1 to increase
its water compatibility and emitting ability in the hydrophilic envi-
ronment. As expected, C1 gives rise to an extensive emission at
436 nm in aqueous solution with a quantum yield of 0.63. The
emission can be completely quenched (over 97%) by 1 equiv of
Hg2+ and recovered again (over 96%) with the addition of Cys. Both
C1 and its ensemble with Hg2+ exhibit good stabilities under a wide
pH span from 6 to 10, covering physiological conditions. These
properties of C1 including high water solubility, longer emission
wavelength (436 nm), and fluorescence OFF–ON to the guests are
comparable with those of coumarin/anthracene based fluorescent
probes.14
With admirable properties of C1 in hand, herein, we further
developed the ensemble complex carbazole–diphenyldithiosemic-
⇑
Corresponding author. Tel.: +91 33 2668 4561; fax: +91 33 26684564.
E-mail address: mahapatra574@gmail.com (A.K. Mahapatra).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.03.111

A. K. Mahapatra et al. / Tetrahedron Letters 54 (2013) 2946–2951
2947
R
N
S
R
N
R
NH2
N
PhHN
N
H
POCl3, DMF
63%
CH3OH, 70 0C, 12h
N
N
OHC
CHO
85%
HN
NH
S
S
2
R = C4H9
3
PhHN
NHPh
C1
Scheme 1. Synthetic route to compound C1.
arbazone–Hg2+ as an exchangeable fluorescent probe, which exhib-
ited high sensitivity and selectivity to mercapto biomolecules via a
reversible decomplexation. Moreover, the complex of C1 and Hg2+
was utilized as a chemosensing ensemble for Cys, Hcy and GSH
detection, which showed highly sensitive and selective colorimetric
rect visualization of color change makes the detection straight
forward. No obvious responses could be observed upon the
addition of Na+, K+, Cu2+, Co2+, Pb2+, Al3+, Mg2+, Ni2+, Cd2+, Fe3+
,
and Zn2+, respectively, (Fig. S1). These results clearly suggested
that the metal complexation of C1 shows a great preference for
mercury ion over other cations. The binding constant was deter-
mined to be 1.98 Â 104 MÀ1(Fig. S4).
response to thiols among the tested a-amino acids. Confocal fluo-
rescence imaging reveals that C1/C1-Hg2+ can be applied to monitor
the intracellular self-detoxification of foreign Hg2+ ions in candida
cells.
Corresponding to its absorption spectra, the fluorescence spec-
trum of C1 (c = 1.0 Â 10À5(M), kex = 324 nm) in 99% aqueous solu-
tion exhibited an intensive emission band at 436 nm and its
fluorescence was completely quenched (over 98%) immediately
upon the addition of 1 equiv of Hg2+ (Fig. 2).
The target compound C1 was readily synthesized in two steps
as shown in Scheme 1. Compound C1 was synthesized by conden-
sation of N-butylcarbazoledicarbaldehyde 2 and phenylthiosemic-
arbazide in high yield, and its structure has been proved by various
spectroscopic characterizations. The detailed experimental proce-
dures and 1H and 13C NMR spectra are summarized in Supporting
Information.
The photophysical properties of C1 were evaluated with physi-
ologically important transition metal perchlorates/chlorides in 99%
aqueous solution (containing 1% DMSO for complete dissolution of
sample, 30 mM HEPES at pH 7.4). Generally, imine derived mole-
cules are pH sensitive15 upon irradiation. In this work, we found
that carbazole-derived imine and its complex, C1/C1–Hg2+, were
stable enough even within a wide pH range (6–10). So, all the spec-
troscopic studies were performed in 99% aqueous solution (50 mM
HEPES at pH 7.4) in which compound C1 formed a colorless solu-
tion that was stable for more than three months. The ligand C1
exhibited three absorption bands centered at 324, 365, and
382 nm; all of these bands showed a decrease in the absorbance
upon addition of Hg2+. Two clear isosbestic points were observed
at 289 and 398 nm (Fig. 1), which are consistent with the presence
of only two species, free ligand, and Hg2+–ligand complex. The col-
or of the solution changes from nearly colorless to yellow. This di-
As a result, excess Hg2+ cannot achieve further quenching of
fluorescence. Based on the mole ratio method, mole ratio between
C1 and Hg2+ is 1:1, meanwhile, Job’s plots highly indicated a 1:1
mol ratio. The detection limit of Hg2+ is about 2.54 Â 10À10 M.
Based on the 1:1 binding mode, the binding constant16 derived
from the fluorescence titration data was found to be
3.04 Â 104 MÀ1 (Fig. S5). The 1:1 binding model of Hg2+and C1
can be further confirmed by mass spectra. The ESI mass spectrum
of complex C1–Hg2+ has a major peak with m/z of 836.9674
[C1ÀHg2++NaCl], which corresponds to 1:1 complex (Fig. S6).
Upon interaction with various metal ions, the fluorescence
intensity at 436 nm was markedly quenched with Hg2+ ions, while
other metal ions (Na+, K+, Cu2+, Co2+, Pb2+, Al3+, Mg2+, Ni2+, Cd2+
,
Fe3+, and Zn2+) did not cause any noticeable responses(Fig. S7a).
To find out whether C1 can detect Hg2+ selectively even in the pres-
ence of other metal ions, competitive metal ion titrations were car-
ried out. Of practical significance is that even 20 equiv each of
these metal ions did not interfere in the sensing of Hg2+, and the
results of the competition experiments are shown in (Fig. S8). It
is generally believed that, in most fluorescent molecules, the intro-
Fig. 1. UV–vis spectra of compound C1(10
of Hg2+ ions (0–2 equiv) in pH 7.4 HEPES buffer (25 mM, pH 7.4, containing 1.0%
DMSO). Inset shows the change in color of compound C1 (10 M) upon addition of
Hg2+ ions (10
M).
l
M) with the increasing concentrations
Fig. 2. Fluorescence spectra of compound C1 (5 lM) with the increasing concen-
trations of Hg2+ ions (0–1.0 equiv) in pH 7.4 HEPES buffer (25 mM, pH 7.4,
containing 1.0% DMSO). The inset shows Fluorescence Job plot of C1 with Hg2+ at
436 nm.
l
l

2948
A. K. Mahapatra et al. / Tetrahedron Letters 54 (2013) 2946–2951
duction of an extended conjugation structure to the rigid aryl ring
will result in a red shift of the emission wavelength, as well as a
drastic decrease of the fluorescent quantum yield because of its
weaker coplanar effect.17 Here, the origin of the fluorescence
quenching may result from the electron or energy transfer from
the excited carbazole fluorescence to the Hg2+ ion or the fluores-
cence quenching of C1 at 436 nm (Fig. S7b) bound with Hg2+
may arise due to the MLCT-based18 heavy metal ion effect. Alkali
and alkaline earth metal ions showed no interaction with C1,
which may be due to their hard acid properties. Thus, Hg2+ ion
can be detected quantitatively in the presence of a number of bio-
logically relevant metal ions.
C1–Hg2+ were also determined (Fig. S20). The
HOMO of C1–Hg2+ complex are mainly located on the whole
-conjugated carbazole framework (excluding the n-butyl group),
but the LUMO is mostly positioned at the center of the guest
Hg2+ ion. Moreover, the HOMOÀLUMO energy gap of complex be-
comes smaller relative to that of probe C1.
p electrons on the
p
The energy gaps between HOMO and LUMO in the probe C1 and
C1–Hg2+ complex were 84.45 kcal molÀ1 and 53.02 kcal molÀ1
,
respectively (see the supplementary data). The result clearly sug-
gests that the two thiosemicarbazone moieties undergo conforma-
tional changes to bind to the Hg2+ in a symmetrical fashion.
Based on the results of emission, absorption titration experi-
ments and ESI MS suggest that the probe C1 clearly recognizes
Hg2+, the utility of [C1+Hg2+] complex toward selective recognition
of amino acid has been studied so that this complex can act as a
secondary sensor for specific amino acid. To test this idea, the
probe C1–Hg2+ ensemble based sensor was prepared by mixing
equal equivalents of probe C1 and mercuric chloride in the solution
of HEPES buffer (50 mM, pH 7.4, containing 1% DMSO). We found
that C1–Hg2+ ensemble is initially nonfluorescent (Ufl = 0.03), the
addition of mercapto biomolecules such as Cys, Hcy, or GSH to
the aqueous solution of C1–Hg2+ ensemble resulted in obvious
spectral changes.
As the stoichiometry of the complex formed between Hg2+ and
C1 was found to be 1:1 based on emission, absorption, and ESI MS
studies, the nature of Hg2+ coordination with C1 was studied by
computational studies using the Gaussian 2003(B3LYP/6-
31G(d,p)) software package.19 The geometry optimizations for C1
and C1–Hg2+ complex were done in a cascade 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 C1–Hg2+ complex, a starting model
was generated by taking the DFT optimized C1 and placing the
Hg2+ ion in between the diphenyldithiosemicarbazone moieties
at a non-interacting distance. This model was then optimized ini-
tially using HF/3-21G level of calculations, and the output struc-
ture from this was taken as input for DFT calculations performed
using B3LYP with SDDAll basis set for Hg2+ and 6-31+G(d,p) for
all other atoms in the complex. The optimized complex of Hg2+
with C1 (Fig. S19) showed that a distinct complexation occurs be-
tween Hg2+ and thiosemicarbazone sulfurs where two thiosemi-
carbazone sulfurs are bonded to the Hg2+ ion with a symmetrical
Hg–S distance of 2.36 Å. These Hg–S distances are comparable with
the experimental ones reported in the literature.20The spatial dis-
tributions and orbital energies of HOMO and LUMO of C1 and
The fluorescence increase (about 40-fold) was observed, and the
fluorescence quantum yield could be revived to 0.61, indicating
that C1–Hg2+ ensemble could be applied as a fluorescent OFF–ON
probe for Cys in aqueous solution. This is exactly reverse to what
happens when probe C1 is titrated with Hg2+, as reported in this
Letter, the Hg2+ is being removed from the complex by cysteine
to release free C1 and thus the recognition of cysteine by
[C1+Hg2+] ensemble as a secondary sensor.
Fluorescence titration of [C1+Hg2+] ensemble was conducted in
the solution of HEPES buffer (50 mM, pH 7.4, containing 1% DMSO)
by the addition of cysteine from 0 to 1.0 Â 10À5 M. About 2.1 equiv
Fig. 3. (a) Fluorescence spectra of the C1-Hg2+ ensemble (5
lM) in pH 7.0 HEPES buffer (25 mM, pH 7.4, containing 1.0% DMSO) in the presence of Cysteine (0–2 equiv). (b)
Fluorescence intensity changes at 436 nm of the ensemble (5
lM) in the presence of increasing concentrations of cysteine .
R
N
R
N
HgCl2
N
N
N
N
HN
NH
NHPh
Hg2+
HN
NH
Cys
S
S
S
S
PhHN
PhHN
NHPh
C1-Hg2+ Complex
Cys-Hg-Cys
C1
Scheme 2. Proposed displacement mechanism for sensing of cysteine.

A. K. Mahapatra et al. / Tetrahedron Letters 54 (2013) 2946–2951
2949
of cysteine makes the quenched fluorescence restore to the largest
at 436 nm. Excess cysteine cannot achieve further enhancing of
fluorescence. It is found that cysteine increases the FL intensity
of [C1+Hg2+] ensemble in a concentration dependence (Fig. 3b).
The release of probe C1 in the titration of cysteine is followed by
the formation of [Cys–Hg–Cys] complex in Scheme 2 (Fig. S11),
as it can form a stable complex with the thiol functionality.21
Both C1 and [C1ÀHg2+] ensemble exhibit water solubility and
biocompatibility, as well as good stability under a wide pH span
from 6 to 10 covering physiological conditions (Fig. S12). These
features of C1 and [C1ÀHg2+] ensemble facilitate their practical
applications for the determination of Cys. [C1ÀHg2+] ensemble dis-
plays a high sensitivity to Cys (the reaction can be completed with-
in several seconds). Cys was added at different concentrations from
of HgCl2 in DMSO-d6 and returned to the original values with the
subsequent addition of 1.0 equiv of Cys. The titration of
[C1+Hg2+] ensemble with Cys could not be continued beyond
1 equiv owing to precipitation of [Cys+Hg2+] complex. The family
of 1H-NMR spectra of probe 1 obtained by the titration of Hg2+ is
shown in Fig. 4. The continuous addition of Hg2+ (from 0.5 to
1.5 equiv.) to the solution of probe C1 caused minimal to marginal
changes of d values, a downfield shift by 0.46 ppm in the signal cor-
responding to –NHa (–CH@N–NHa–), whereas imine-H (–CH@N–)
is shifted downfield by 0.19 ppm. These 1H NMR changes, espe-
cially the move of thiosemicarbazone proton Ha, suggest that the
Hg2+ ion of the [C1+Hg2+] ensemble may be coordinated to the lone
pair electrons on the sulfur atom and imine nitrogen. It is well-
known that Hg2+ ions (a soft acid) can preferentially interact with
sulfur (a soft base) according to Pearson’s hard and soft acids and
bases theory.22 These results are also fully in line with the fluores-
cence spectrum changes (Fig. 2), which were observed for the
[C1+Hg2+] ensemble.
0 to 30
(5.0
l
M, and the fluorescence intensity of [C1ÀHg2+] ensemble
l
M) at 436 nm was recorded to generate a calibration curve. A
good linearity (R = 0.995) was found between the fluorescence
intensity of the solution and the Cys concentration (Fig. 3b). This
linear fitting analysis reveals that [C1ÀHg2+] is suitable for deter-
With the exception of Cys, other mercapto biomolecules such as
Hcy and GSH also induced similar variations in the absorption and
fluorescence spectra of [C1+Hg2+]. Among these, Cys led to the larg-
est fluorescence increase, whereas Hcy and GSH gave relatively
smaller increases in fluorescence (Fig. S9).
mining Cys from 0.5 to 30 lM. The detection limit of cysteine is
about 9.6 Â 10À11 M.
NMR experiments were performed to explore the coordination
sites and sensing mechanism between probe C1 and Hg2+. The pro-
ton chemical shifts of the dithiosemicarbazone moieties in probe
C1 were distinctly shifted downfield upon the addition of 1.5 equiv
For practical applications, an important consideration is its
selective detection in the presence of other amino acids without
R
N
-CHimine
N
N
a
HN
NH
NHPh
b
S
S
PhHN
C1
Fig. 4. 1H NMR (400 MHz) spectra of Compound C1 (a), C1+0.5 equiv Hg2+ (b), C1+1.0 equiv Hg2+ (c), and C1+1.5 equiv Hg2+ (d), C1+Hg2+ ensamble+2.0 equiv cysteine and (e)
in d6 DMSO-D20, it is appeared same as (a).
Entry
InA
InB
Output
Hg+2 Cys Emission at
436nm
1.
2.
3.
4.
0
1
0
1
0
0
1
1
1
0
1
1
In
In
A
Hg2+
Cys
I436
B
Fig. 5. Truth table and molecular circuit generated upon the addition of Hg2+ and cysteine to C1.

2950
A. K. Mahapatra et al. / Tetrahedron Letters 54 (2013) 2946–2951
with various amounts of Cys (upto 200 lM) for 12 min, blue fluo-
(a)
(b)
(e)
(c)
rescence imaging was recovered (Fig. 6e and f). The recurrent
imaging indicated that the uptake of Cys resulted in the decom-
plexation of intracellular [C1+Hg2+] ensemble to fluorescent C1.
Through reversible fluorescence imaging, intracellular interconver-
sion of [C1+Hg2+] ensemble was explicitly illustrated. Therefore,
the ON–OFF–ON fluorescence imaging of probe C1 was accom-
plished in Candida cell lines by the intracellular complexation/
decomplexation interaction modulated by Hg2+/Cys. These results
also indicate that probe C1 is cell membrane permeable and able
to response to Cys in the living cells.
(d)
(f)
In summary, we have synthesized a new carbazole-derived
imine probe C1, which exhibits high selectivity toward Hg2+. The
selectivity has been demonstrated by fluorescence, absorption,
and ESI MS spectroscopy. Most significantly, the in situ prepared
mercury ensemble of C1, viz., [C1+Hg2+], was able to detect selec-
tively Cys among the naturally occurring amino acids to a lowest
concentration of 9.6 Â 10À11 M and it is exactly reverse to what
happens when Hg2+ is added to probe C1 in fluorescence spectros-
copy. Hence probe C1 acts as secondary sensor toward Cys.
Fig. 6. (a) Fluorescence microscope images of Candida albicans cells only, (b) images
of cells+C1, (c) images of cells+C1+Hg2+(5 M), (d) images of cells+C1+Hg2+ (25
M)
(e) images of cells+C1-Hg2+ ensemble+Cys and (f) same as (e) after 12 min.
l
l
thiol groups, such as glycine, alanine, valine, leucine, isoleucine,
methionine, proline, tyrosine, lysine, histidine, serine, and
tryptophan. As shown in Figure S10, the addition of other compet-
itive amino acids to the solution of [C1+Hg2+] did not result in any
notable fluorescent increases. However, when Cys was added to
the aqueous solution of [C1+Hg2+] containing other competitive
amino acids, an obvious increase in fluorescence was observed.
This result indicates that [C1+Hg2+] can detect Cys with a high
selectivity over other coexisting amino acids.
Acknowledgment
We thank CSIR [Project No. 01(2460)/11/EMR-II] for the finan-
cial support. JR thanks the CSIR, New Delhi for fellowship. We also
acknowledge DST, AICTE, UGC-SAP, and MHRD for funding instru-
mental facilities in our department.
Since fluorescence changes occur only in the presence of Cys
and not with the other amino acids, the role of the –SH function
in Hg2+ binding was further confirmed by studying the fluores-
cence response of [C1+Hg2+] with different sulfur-containing sys-
tems, viz., Cys, GSH, and methionine (Met) (Fig. S9). Cys and GSH
exhibited changes in the fluorescence owing to the presence of
the –SH functionality, but Met does not show changes in the fluo-
rescence owing to the presence of R–S–Me and not the –SH func-
tion. This experiment clearly suggests that Hg2+ mainly interacts
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2013.03.
111.
through the –SH function of cysteine, and hence the [C1+Hg2+
]
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dida cells were incubated with probe C1 (10
buffer containing 1/500 DMSO for 30 min at 30 °C and then it
was treated with HgCl2 (100 M) for12 min. Their fluorescence
lM; Fig. 6b) in PBS
l
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