Histidine based fluorescence sensor detects Hg2+ in solution, paper strips, and in cells

Article abstract of DOI:10.1021/ic300530f

A chemosensor having a bipodal thiocarbamate scaffold attached to histidine moieties senses Hg²⁺ with a remarkable selectivity. The binding results in a 50 nm blue shift in the fluorescence spectra and a 19-fold enhancement of the fluorescence quantum yield of the ligand. In addition to the detection of Hg²⁺ visually under UV light in solution, the chemosensor was used for fabrication of paper strips that detected Hg ²⁺ in aqueous samples. The sensor was also used for imaging Hg ²⁺ in adult zebrafish and in human epithelial carcinoma HeLa S3 cells.

Full text of DOI:10.1021/ic300530f

Article
pubs.acs.org/IC
Histidine Based Fluorescence Sensor Detects Hg2+ in Solution, Paper
Strips, and in Cells
Joydev Hatai, Suman Pal, Gregor P. Jose, and Subhajit Bandyopadhyay*
Department of Chemical Sciences, Indian Institute of Science Education and Research Kolkata, BCKV Campus PO, Mohanpur,
Nadia, WB 741252, India
S
* Supporting Information
ABSTRACT: A chemosensor having a bipodal thiocarbamate scaffold
attached to histidine moieties senses Hg2+ with a remarkable selectivity. The
binding results in a 50 nm blue shift in the fluorescence spectra and a 19-fold
enhancement of the fluorescence quantum yield of the ligand. In addition to
the detection of Hg2+ visually under UV light in solution, the chemosensor was
used for fabrication of paper strips that detected Hg2+ in aqueous samples. The
sensor was also used for imaging Hg2+ in adult zebrafish and in human
epithelial carcinoma HeLa S3 cells.
in terms of selectivity for different charged states of an element.
AAS suffers from the non linearity of the calibration curves,
particularly at higher absorbance range. Highly sensitive
voltammetric methods such as the one with hanging drop
mercury electrode have several disadvantages. It has a low
surface area to volume ratio that causes a reduction of the
plating efficiency, and the large volume yields a low
concentration of metal in mercury.9
INTRODUCTION
■
Mercury is an important analyte because of its toxic effect in the
ecosystem.1,2 Apart from anthropogenic activities, environ-
mental contamination by mercury occurs from volcanic
eruptions, geothermal vents, and leaching of mercury from
ores, such as cinnabar, into natural sources of water.3
Depending on the environment, Hg2+ can be transformed to
Hg0 and also to highly toxic organomercury compounds by
several strains of aerobic bacteria.4 The metal and its
compounds are bioconcentrated and biomagnified by sea
creatures, and subsequently consumed by humans, giving rise
to mercury poisoning. Dental amalgam is also a source of
mercury toxicity.5 Organomercury compounds can easily cross
the cell membranes and the blood brain barrier impairing
nefrological and neurological functions. Because of this high
toxicity, the U.S.-EPA directive for the highest concentration of
mercury in drinking water is 2 ppb (10 nM).6
Detection and quantitative determination of mercury in its
most stable Hg2+ form has received much attention, perhaps,
more than any other toxic heavy metal ions. Quantitative
detection of samples containing both organomercury species
and inorganic mercury is possible with several analytical
methods.7 Atomic absorption spectrometry (AAS), atomic
emission, and fluorescence spectrometry, inductively coupled
plasma-mass spectrometry (ICPMS), inductively coupled
plasma-atomic emission spectrophotometry (ICP-AES), and
electrochemical techniques (such as ion-selective potentiometry
and anodic stripping voltammetry) have high sensitivity that
can even reach the submicrogram to nanogram per liter range.8
Although these methods are unparallel in terms of sensitivity,
each of these techniques bear certain disadvantages.7 The
instrumentation cost of the ICPMS is high. Moreover, it suffers
Although the sensitivity of fluorogenic detection is lower by
several orders in magnitude, it has certain advantages because
of its low cost, facile sample preparation, high selectivity, and
easy detection by both visual and instrumental methods. The
importance of detection of mercury ion has prompted
researchers to detect it fluorogenically through a diverse
range of approaches. The most popular ones are based on
optical responses with small molecules having luminescent
probes.10 Often the luminescent probes are based on
nanoparticles,11 semiconductor nanocrystals,12 polymer−oligo-
nucleotide composites,13 metal regulatory proteins,14 and
nucleotide conjugates such as DNAzymes,15 DNA hydrogels,16
and DNA-functionalized silica nanoparticles.17 There are
reports of chemodosimeters where the effect of the ion cannot
be reversed.18 The detection methodologies of these sensors
rely on the changes in the fluorescence intensity and color and
hence the sensing can be carried out visually with the naked
eye, or with a spectrometer. Lippard19 and later, Kim20 have
extensively reviewed optical sensors for mercury ions.
Luminescence responses involve several energy or electron
transfer mechanisms that have been discussed systematically in
Received: March 12, 2012
Published: September 14, 2012
© 2012 American Chemical Society
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these articles. The sensors, based on photoinduced electron
transfer (PET) mechanism, exhibit enhancement of fluores-
cence intensity with small shifts in the emission wavelengths,
whereas, pronounced signal enhancement accompanying a
large blue-shift in the emission wavelength is typically observed
where the addition of the ion inhibits internal charge transfer
(ICT) process.21 Many of the ICT-based mercury sensors only
work with organic solvents and lack selectivity from competing
metal ions.19,22
To date there are reports of only a few amino acid based
Hg2+ sensors, including lysine,23a aspartic acid-BSA conjuga-
te,23b tryptophan23c and methionine based.23d In all cases the
detection of mercury occurred by appending a fluorophore
directly to the amino acid. Further modification of the amino
acid structure was not required. In this article, we report a
histidine based turn-on fluorescence sensor for Hg2+, which is
unique because histidine normally has a strong affinity for
Cu2+.24 We have appended histidine residues to a bipodal
thiocarbamate scaffold to provide sulfur atoms as donors and
alter its selectivity. Dithiocarbamates with simple amines have
been used by Kamata et al. as ionophores for Hg2+ and Ag+ ions
and in ion selective electrodes for heavy metals.25 We have
recently demonstrated the use of the thiocarbamate scaffold
attached to a salicyl aldehyde based aryl system and coumarin
fluorophores for differential detection of multiple metal ions by
tuning of solvents.26 In this case, the histidine based sensor
displays a turn-on fluorescence behavior with high selectivity
for mercury with a large emission shift (Δλ = 50 nm, from 590
to 540 nm) and a 19-fold enhancement of the fluorescence
quantum yield. The sensor works in mixed aqueous−organic
media (MeOH/H2O, 80:20, v/v with 1% acetonitrile as a
cosolvent, buffered at pH 7.0 with HEPES) at 25 °C and is
highly selective for Hg2+ ions, which is remarkable given that
mercury sensors are commonly known to respond to other ions
along with Hg2+, namely, Ag+, Cd2+, Fe2+, and Pb2+.27
Interestingly, Cu2+ ion responded to the sensor with a
quenching of the fluorescence. Additionally, fabrication of a
simple dip-strip that works in real time scale with aqueous
samples is always desirable for convenient in-field detection of
Hg2+ contaminated samples. Paper based strips were fabricated
with the chemosensor that were able to detect Hg2+ in aqueous
solutions. The probe was also successfully used for detection of
mercury in adult zebrafish samples and in carcinoma cells.
The bipodal chemosensor 1 (Figure 1A) consists of two
dithiocarbamate moieties appended to histidine units bearing
dansyl fluorophores. The central aryl unit, which under our
excitation conditions is nonfluorescent, serves as the linker. The
synthetic route for the sensor 1 is shown in Scheme 1. The key
step of the synthesis was performed under aqueous conditions
where the methyl ester of histidine 328 upon treatment with
carbon disulfide and the bromomethyl compound 229 in
dioxane−water (1:4, v/v) mixture afforded the thiocarbamate
compound 4 in a single step. Dansylation of compound 4 in
presence of 2 equiv of triethylamine in dry dichloromethane
afforded 1 in 45% yields. The structure of 1 was confirmed by
1H and 13C NMR, IR, and MS data.
Figure 1. (A) Structure of 1. (B) Fluorescence spectra (λex = 340 nm,
excitation and emission slit widths = 4.5 nm) of 1 (10 μM) and
addition of different metal salts (60 μM) in mixed solvent media
(MeOH/H2O, 80:20, v/v with 1% acetonitrile as a cosolvent, buffered
with HEPES) at pH 7.0 at 25 °C.
Scheme 1. Synthesis of the Bipodal Chemosensor 1
Figure 2. Change in the absorption spectra of chemosensor 1 (10 μM)
with increasing concentration of Hg(NO3)2 (0 to 30 μM) in mixed
solvent media (MeOH/H2O, 80:20, v/v with 1% acetonitrile as a
cosolvent, buffered with 1 mM HEPES) at pH 7.0 at 25 °C (Inset:
Hg2+ titration profile at 295 nm).
The metal affinity of chemosensor 1 toward a variety of
cations: Cr3+, Mn2+, Fe2+, Fe3+, Co2+, Hg2+, Ni2+, Cu2+, Zn2+,
Pb2+, Ag+, Al3+, Ba2+, Ca2+, Cd2+, K+, Na+, Li+ was investigated
by absorption and fluorescence spectroscopy in mixed
aqueous−organic solvent conditions mentioned earlier.
On addition of Hg2+, the absorption band at 373 nm
diminished, and a new band at 295 nm was observed. The
change in the absorption spectra was significant up to addition
of 1 equiv of Hg2+ (inset, Figure 2). This indicates that the
binding of Hg2+ with 1 takes place in a 1:1 ratio.
Compound 1 in the absence of any metal ion showed an
absorption band centered at 373 nm and at 260 nm (Figure 2).
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The fluorescence spectrum (λex at 340 nm, excitation and
emission slit widths = 4.5 nm) of 1 (10 μM) displays with a
weak fluorescence band at 590 nm (quantum yield, Φ = 0.022)
in mixed solvent media (MeOH/H2O, 80:20, v/v with 1%
acetonitrile as a cosolvent, buffered at pH 7.0 with 1 mM
HEPES) at 25 °C. Under the same conditions, addition of Hg2+
(0−40 μM) to ligand (10 μM), afforded a 17-fold enhancement
of the fluorescence intensity and 19-fold enhancement of
quantum yield (Φ = 0.402). A large blue shift of 50 nm of the
fluorescence band was also observed (Figure 1B).
The visual appearance of the samples under 366 nm light
changed from dark orange to greenish-yellow (Figure 4A). The
fluorescence response of chemosensor 1 (10 μM) was also
tested with different metal ions such as Cr3+, Mn2+, Fe2+, Fe3+,
Co2+, Ba2+, Ni2+, Cu2+, Zn2+, Pb2+, Ag+, Ca2+, Cd2+, K+, Na+, Li+,
and Al3+ (Figure 1B). While no significant change of
fluorescence emission was observed in the presence of these
metal ions (60 μM), Cu2+ displayed a quenching effect with no
shift of the fluorescence band. The fluorescence turn-on with
probe 1 is therefore highly specific toward Hg2+.
A peak at m/z at 1346 in electrospray ionization mass
spectrometry corresponding to one Hg2+ ions associating with
one molecule of 1 under these conditions further supported the
binding stoichiometry (Supporting Information, Figure S6B).
To test the practical applications of chemosensor 1 as a Hg2+
selective fluorescence sensor, competition experiments were
carried out to investigate the effect of other coexisting cations
with Hg2+ in the presence of chemosensor 1. The emission
spectra containing 1 (10 μM) and Cr3+, Mn2+, Fe2+, Fe3+, Ba2+,
Co2+, Ni2+, Zn2+, Pb2+, Ag+, Ca2+, Cd2+, K+, Na+, Li+, and Al3+
(60 μM) followed by addition of Hg2+ (40 μM) were recorded.
As shown in the Figure 4C, no significant variation of
fluorescence intensity was observed with other metal ions
except for copper. The enhancement of fluorescence was
affected significantly on addition of Hg2+ in presence of Cu2+,
and under the given conditions the enhancement was only
about 40% compared to the ones without it (last bar, Figure
4C).
To elucidate the binding mode of chemosensor 1 with Hg2+,
1
an H NMR spectroscopic titration experiment was performed
by addition of Hg2+ to a CD3CN solution of 1 (1 × 10−3 M)
(Figure 3C). The protons attached to the chiral carbon shifted
upfield (δ 5.23 to 4.64), indicating its proximity to the
coordination site involving the thiocarbamate unit. The
characteristic signal corresponding to the imidazole 2-H
experienced a significant downfield shift (δ 8.13 to 8.64),
indicating coordination to Hg2+. The imidazole 5-H shifted
upfield (δ 7.48 to 6.99). As a result of the histidine
coordination, the charge transfer from the sulphonamide
nitrogen to the naphthyl ring of the dansyl is reduced. This
is supported in the downfield shift of the naphthalene protons.
The −OMe protons had a very little upfield shift (<0.05 ppm)
probably because of its out of the place orientation toward the
shielding zone of the central aryl ring to avoid steric interaction
with the metal ion. Taking these results into account, we
propose a putative binding mode of Hg2+ with the ligand 1 as
shown in Figure 3D.
Additionally, the observed fluorescence response (Support-
ing Information, Figure S1) of chemosensor 1 to various metal
ions indicates that 1 can be employed for detection of Hg2+ ion
by simple visual inspection at this concentration range. For
quantitative investigation of 1·Hg2+ binding, fluorescence
titration was performed by varying the concentration of Hg2+
to a fixed amount of 1 (10 μM) (Figure 3A). The plot of
Thus, it may be possible that in the absence of any mercury
ion the weak emission intensity of the free chemosensor 1 is
due to the flexibility and rotation of the imidazole ring attached
to the dansyl fluorophore. This, in turn, causes an efficient ICT
process from the imidazole nitrogen to the dansyl unit.30
Coordination of Hg2+ restricts the ICT process and results in
fluorescence enhancement. An alternate possibility is that the
ligand is not fully solvated because of its hydrophobic nature.
This might give rise to stacking of the chromophores causing
formation of excimers, and thus quenching the probe’s
luminescence. Coordination of Hg2+ results in a positively
charged complex that has a better solubility. Consequently, this
circumvents the stacking and excimer formation resulting in
fluorescence enhancement. This mechanism was recently
observed by Fahrni with a fluorescent probe for Cu(I).31
To check the reversibility of the chemosensor 1, three metal
ion chelators: EDTA, N,N,N′,N′-tetrakis(2-pyridylmethyl)-
ethylenediamine (TPEN),32 and 8-hydroxyquinoline (100 μM
each) were added separately to three different solutions
containing 1 (10 μM) and Hg(NO3)2 (40 μM, corresponding
to the saturation of fluorescence intensity) in MeOH/H2O
(80:20, v/v with 1% acetonitrile as a cosolvent, buffered with
HEPES at pH 7.0) at 25 °C. The recorded fluorescence spectra
revealed that TPEN successfully decomplexed the metal ion
from 1 and activated the enhanced fluorescence caused by the
addition of Hg2+. 8-Hydroxyquinoline was less effective at low
Figure 3. (A) Enhancement of fluorescence intensity (λex at 340 nm,
excitation and emission slit widths of 4.5 nm) of 1 (10 μM) with
addition of Hg2+ (0−60 μM) in mixed solvent media (MeOH/H2O,
80:20, v/v with 1% acetonitrile as a cosolvent, buffered with HEPES at
pH 7.0) at 25 °C. (B) Enhancement of fluorescence intensity with
1
addition of 0−6 equiv of Hg2+. (C) H NMR spectra of 1 (1 × 10−3
M) with Hg2+ in CD3CN at 25 °C. (D) Putative binding mode of 1
with Hg2+ (see text).
fluorescence intensity of 1 at 540 nm against the added [Hg2+]
revealed that the fluorescence intensity increased up to addition
of 1 equiv of Hg2+ (Figure 3B) and reached a saturation at
about 4 equiv of the metal ion. A 1:1 association was
established from the Job’s plot experiment (Supporting
Information, Figure S2). From the titration data, the binding
constant was found to be 1.25 ( 0.4) × 106 M−1 (Figure 5B).
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concentration in reverting the fluorescence turn-on; however, at
a much higher concentrations (100 mM), it was able to
decomplex the Hg2+. EDTA minimally affected the fluorescence
spectra (Supporting Information, Figure S4).
The maximum fluorescence response of 1 was observed
between pH 5.5 to 8.0 (Figure 5A), indicating that our sensor
can be used at a biologically relevant pH.
Although there are a large number of reports of Hg2+ sensors,
simple and effective detection of the ion still remains a
challenge. Encouraged by the positive results of our sensor to
detect Hg2+, we fabricated a simple paper strip that could detect
Hg2+.33 The operation principle of our paper based strip is
given in Supporting Information, Figure S3. Whatmann 1 filter
papers of the dimension 5 cm ×0.75 cm was coated with a
solution of 1 in acetonitrile at a height of 1 cm from the bottom
of the strip (sensor zone, see Supporting Information, Figure
S3) and was air-dried for one day. The strips were dipped
directly into aqueous solutions containing Hg2+. The Hg2+ was
carried upward by a capillary action and the complex that
formed in the sensor zone was carried to the detection zone by
the solvent. After the solvent front reached a certain height, the
strip was taken out, dried with a blow-drier, and the paper was
held under 366 nm fluorescent light. In presence of Hg2+, we
envision that sensor 1 formed complex with Hg2+ that has a
higher solubility in water compared to the hydrophobic ligand.
Consequently, it moved further up in the detection zone
displaying a bluish green fluorescence (Figure 4B). In the
Figure 5. (A) Change in the fluorescence intensity of the emission
band at 550 nm of the chemosensor 1 at different pH. (B) Benesi−
Hildebrand plot for the determination of binding constant of Hg2+
with chemosensor 1 in mixed solvent media (MeOH/H2O, 80:20, v/v
with 1% acetonitrile as a cosolvent, buffered with 1 mM HEPES at pH
7.0) at 25 °C.
In solution phase (MeOH/H2O, 80:20, v/v with 1%
acetonitrile as a cosolvent, buffered with 1 mM HEPES at
pH 7.0), the analytical detection limit34 of Hg2+ with 1 was
found to be 1.03 × 10−7 M (0.02 ppm) (Supporting
Information, Figure S5). This value is 10 times higher than
the acceptable value mandated by the U.S.-EPA for the
concentration of mercury in drinking water.6 However, the
sensor can be useful in detecting inorganic mercury in samples
of biological products, drugs, fish, and also in samples where
regulations for mercury are less stringent.35 The U.S. FDA has
enlisted a large number of drugs and biological products such as
nasal spray and ophthalmic solutions, where the concentration
of inorganic mercury is allowed at the ppm level or more.36
Probe 1 can also be used to detect mercury in certain fish when
the levels are above the detection limit.37
Figure 4. (A) Fluorescence images under 366 nm UV-light. (B)
Photographs of the paper strip based sensing with HgNO3 solutions
(left to right): no Hg2+, 10 μM, 100 μM, and 1 mM Hg2+ in aqueous
medium buffered at pH 7.0 with HEPES (1 mM) at 25 °C. (C). Green
bars: Changes in fluorescence intensity (λex at 340 nm, excitation and
emission slit widths of 4.5 nm) of the 1 (10 μM) upon addition of
various metal salts (60 μM). Violet bars: Restoration of fluorescence
(at same λex and slit-widths) of 1 (10 μM) toward Hg2+ (40 μM) in
the presence of competitive cations (60 μM) in mixed solvent media
(MeOH/H2O, 80:20, v/v with 1% acetonitrile as a cosolvent, buffered
at pH 7.0 with 1 mM HEPES) at 25 °C.
Chemosensor 1 was used for the imaging of Hg2+ ion in cells
with a fluorescence imaging experiment using an epifluor-
escence microscope. Human epithelial carcinoma cells, HeLa
S3, were cultured in Dulbecco’s Modified Eagle’s Medium
(DMEM), supplemented with 10% fetal bovine serum (FBS),
100 units/mL penicillin, and 100 μg/mL streptomycin, at 37
°C and 5% CO2. Cells (0.4 × 106 per mL) were plated on 12
mm coverslips and were allowed to adhere for 24 h. The culture
cells were exposed to Hg(NO3)2 (10 μM) in DMEM for 1 h at
37 °C. Immediately, before the experiments, the cells were
washed twice with PBS to remove the remaining Hg2+ ions, and
incubated with 1 (10 μM in DMSO) in PBS for 10 min at 25
°C; followed by washing the cells using PBS to remove residual
dye from the cells. The series of fluororescence images revealed
absence of mercury, such coloration was not detected in the
detection zone. With this method, up to 10 μM concentration
of Hg2+ was visually detected with the naked eye. With lower
concentrations, the changes were visually less discernible. We
are working on the improvement of the probe to achieve better
detection limits.
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marked differences in brightness of the Hg2+ strained cells
compared to the controls (Figure 6). The fluorescence image of
Figure 7. Images of full grown zebrafish under 366 nm light. (A) Only
fish. (B) Fish incubated with Hg(NO3)2 (10 μM). (C) Fish stained
with 1 (10 μM in DMSO) for 20 min. (D) Fish incubated with Hg2+
(10 μM) (10 min) and subsequently stained with 1 (20 min).
In conclusion, we have reported a chemosensor that senses
Hg2+ with a large blue shift and a 19-fold enhancement of the
fluorescence quantum yield in mixed aqueous−organic solvent.
The sensor displayed a high selectivity for mercury ions. This
selectivity of 1 inspired us to fabricate paper strips for detection
of Hg2+ which were successful in detecting the ion in the
aqueous media. The sensor also detected Hg2+ in fish and
human epithelial carcinoma cells. The analytical detection limit
of the sensor is 20 ppb, which is higher than the US-EPA
mandated limit for mercury in drinking water. Development of
the scaffold with improved sensitivity and better water solubility
is under progress.
Figure 6. Fluorescence microscope images of HeLa S3. (A) Only cells.
(B) Cells loaded with 1 (10 μM). (C) Cell incubated with Hg2+ (10
μM, 1 h, 37 °C) exposed to 1 (10 μM) for 10 min. (D) Cells stained
with 1 and Hg2+ and subsequently treated with TPEN (100 μM) for
20 min.
HeLa S3 loaded with 1 (10 μM) under the same conditions
showed weak intracellular fluorescence (Figure 6B). In contrast,
cells treated with Hg2+ and 1 displayed vivid intracellular
fluorescence (Figure 6C). To ensure that the fluorescence turn-
on was due to the coordination of mercury and not some
artifact or photoactivation of the probe, the stained cells were
exposed to an excess (10 equiv) of metal-ion chelator
N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN)
in DMEM medium for 30 min,32 followed by washing with
PBS. On addition of the membrane permeable Hg-chelator, a
gradual decrease in the fluorescence intensity was observed
(Figure 6D). These results demonstrate that 1 can be used to
detect intracellular Hg2+.
To find out whether sensor 1 can be used for visual detection
of mercury in fish, a series of experiments was carried out with
adult zebrafish. The adult fishes with a mean length of 3 cm
were treated with aqueous Hg(NO3)2 (10 μM) for 30 min,
washed with PBS to remove any Hg2+ adhering to the surface,
and were subsequently treated with a solution of sensor 1 (10
μM in DMSO) for 20 min, washed with PBS and observed
under 366 nm light. The visual images taken with a digital
camera (Figure 7) shows striking differences of the samples
compared to the controls. The control zebrafish (control A)
and the fish in the presence of Hg2+ (control B) were faintly
illuminated (Figure 7A and 7B). In contrast, the fish exposed
only to dye 1 displayed an orange color (Figure 7C) akin to the
color of the dye under 366 nm light (Figure 4A). In certain
parts of the fish: the abdomen, fins, and head, the accumulation
of the color was higher compared to that in the body. In the
fish exposed to both the dye and Hg2+, a vividly illuminated
greenish color was observed (Figure 7D). The reversibility of
the green luminescence was slow even with an excess of TPEN
(100 μM).
EXPERIMENTAL SECTION
■
General Information. Structures of the compounds were
determined by nuclear magnetic resonance (NMR).1H and 13C
NMR spectra were recorded on a 400 MHz Jeol and 500 MHz Bruker
respectively. Chemical shifts are reported in δ values relative to an
internal reference of tetramethylsilane (TMS) or the solvent peak for
1H NMR and the solvent peak for 13C NMR. IR data were obtained
with a Bruker-Optics-Alpha-T spectrophotometer. Mass spectrometry
data were obtained from an Acquity Ultra Performance LC.
Fluorescence imaging experiments were carried out using an Olympus
IX 51 inverted microscope with UV excitation. pH data were recorded
with a Sartorius Basic Meter PB-11 calibrated at pH 4, 7, and 10.
Solvents used were purified and dried by standard methods. Reactions
were monitored by thin layer chromatography using Merck plates
(TLC Silica Gel 60 F254). Developed TLC plates were visualized with
UV light. Silica gel (100−200 mesh, Merck) was used for column
chromatography. Yields indicate the chromatographically and
spectroscopically pure compounds, except as otherwise indicated.
The solvents for the spectroscopy experiments were of the
spectroscopic grades and were free from any fluorescent impurities.
Double distilled water was used for the experiments. The solutions of
metal ions were prepared with Al(NO3)3·9H2O, LiClO4·3H2O,
NaClO4, KClO4, Ba(NO3)2·4H2O, Mn(ClO4)2, Fe(ClO4)2·xH2O,
Fe(ClO4)3·xH2O, Co(ClO4)2·6H2O, Cr(ClO4)3·6H2O, Cd(NO3)2,
AgNO3, Hg(NO3)2, Pb(ClO4)2, Ca(ClO4)2·4H2O, Cu(ClO4)2.6H2O,
Ni(ClO4), and Zn(ClO4)2·6H2O for the respective metal ions.
Synthesis. The dibromo compound 2 and the histidine methyl
ester 3 were prepared following the reported methods.26,27 The
spectral and physical data were consistent with those reported.
2-{5-tert-Butyl-3-[2-(1H-imidazol-4-yl)-1-methoxycarbonyl-ethyl-
thiocarbamoylsulfanylmethyl]-2-methoxy-benzylsulfanylthiocarbo-
nylamino}-3-(1H-imidazol-4-yl)-propionic Acid Methyl Ester (4).
The dihydrochloride salt of 3 (0.86 g, 3.6 mmol) and K2CO3 (1.38 g,
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10 mmol) in dioxane/water (1:4) (20 mL) was stirred for 5 min at 0
°C. To it carbon disulfide (2.72 g, 9.05 mL, 36 mmol) was added, and
it was stirred for another 10 min. Compound 2 (0.50 g, 1.4 mmol)
dissolved in dioxane (15 mL) was added dropwise to the reaction
mixture over a period of 10 min. The solution was warmed to room
temperature and stirred for 2 h until the spot for the starting materials
completely disappeared on the TLC plate. The solution was
concentrated under reduced pressure. The solid residue was extracted
in methylene chloride (20 mL) and washed with water. The organic
layer was dried over anhydrous Na2SO4 and volatiles were removed
under reduced pressure. The residue on chromatography with
methylene chloride/acetonitrile, (9:1, v/v) yielded 0.80 g (85%) of
compound 4 as a white foamy substance. 1H NMR (500 MHz,
CDCl3) δ 9.22 (b, 2H, ArNH), 7.49 (s, 2H, imid-2-H), 7.35 (s, 2H,
ArH), 6.75 (s, 2H, imid-5-H), 5.43 (t, J = 5 Hz, 2H, −CHCO2Me),
4.48 (s, 4H, −CH2S), 3.83 (s, 3H, -OCH3), 3.69 (s, 6H,-CO2CH3),
3.24 (t, J = 5.0 Hz, 4H, −CH2CH), 1.24 (s, 9H, t-Bu); 13C NMR
(500 MHz, CDCl3) δ 198.01, 170.01, 154.02, 147.76, 135.29, 133.93,
128.60, 127.98, 115.48, 62.40, 58.99, 52.57, 34.69, 34.34, 31.20, 28.47;
FT−IR (KBr, cm-1) 3221, 2954, 1738, 1483, 1362. ESI-MS cal. for
C29H38N6O5S4H+, 679.18, found 679.09
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: sb1@iiserkol.ac.in.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge two anonymous reviewers
for their constructive feedback and helpful suggestions, Prof.
Tapas K. Sengupta for his help with the cell imaging
experiments, and Dr. Anuradha Bhat for providing the zebrafish
samples. This work is funded by DST (SR/S1/OC-26/2010).
J.H. thanks CSIR for a Senior Research Fellowship.
ABBREVIATIONS
■
Chemosensor 1. Dansyl chloride (0.076 g, 0.29 mmol) in
methylene chloride (5 mL) was added dropwise to a solution of 4
(0.10 g, 0.146 mmol) in methylene chloride (5 mL) and triethylamine
(0.04 mL, 0.29 mmol) at 0 °C. After stirring for 4 h at room
temperature the reaction mixture was extracted with methylene
chloride (15 mL) and washed with distilled water (15 mL × 2). The
organic phase was dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The residue on chromatography with
methylene chloride/ethyl acetate (9:1, v/v) yielded 0.07 g (45%) of
PBS, Phosphate buffered saline; TLC, thin layer chromatog-
raphy; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid
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1
compound 1 as a fluorescent yellowish foam. H NMR (500 MHz,
CDCl3) δ 8.64 (d, J = 10 Hz, 2H, ArH), 8.57 (d, J = 8 Hz, 2H, ArH),
8.29 (d, J = 7.5 Hz, 2H, ArH), 8.19 (d, J = 8.5 Hz, 2H, ArH), 8.00 (s,
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CH2CH), 2.85 (s, 12H, -NCH3), 1.23 (s, 9H, t-Bu). 13C NMR (500
MHz, CDCl3) δ 198.20, 170.24, 154.35, 152.35, 147.55, 138.93,
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ESI-MS Calc.for C53 H60N8O9S6H+, 1145.28, found 1145.27
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concentrations of Hg(NO3)2 (0 to 30 μM) in mixed solvent media
(MeOH/H2O, 80:20, v/v with 1% acetonitrile as a cosolvent, buffered
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fluorescence measurements. Fluorescence data was recorded in mixed
solvent media (MeOH/H2O, 80:20, v/v with 1% acetonitrile as a
cosolvent, buffered at pH 7.0 with 1 mM HEPES) at 25 °C. Excitation
was performed at 340 nm with all excitation and emission slit widths at
4.5 nm unless otherwise indicated. Quantum yield data reported here
were measured relative to fluorescein in 0.1 N NaOH (Φ = 0.95).10d
The integration of the emission spectra were obtained from the
Fluoromax-3 instrument directly.
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* Supporting Information
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