Highly sensitive and selective rhodamine-based "off-on" reversible chemosensor for tin (Sn4+) and imaging in living cells

Article abstract of DOI:10.1021/ic4007026

A structurally characterized new oxo-chromene functionalized rhodamine derivative L1 exhibits high selectivity toward Sn⁴⁺ by forming a 1:1 complex, among other biologically important metal ions, as studied by fluorescence, absorption, and HRMS spectroscopy. Complexing with Sn⁴⁺ triggers the formation of a highly fluorescent ring-open form which is pink in color. The sensor shows extremely high fluorescence enhancement upon complexation with Sn⁴⁺, and it can be used as a "naked-eye" sensor. DFT computational studies carried out in mimicking the formation of a 1:1 complex between L1 and Sn⁴⁺ resulted in a nearly planar pentacoordinate Sn(IV) complex. Studies reveal that the in situ prepared L1-Sn complex is selectively and fully reversible in presence of sulfide anions. Further, confocal microscopic studies confirmed that the receptor shows in vitro detection of Sn⁴⁺ ions in RAW cells.

Full text of DOI:10.1021/ic4007026

Article
pubs.acs.org/IC
Highly Sensitive and Selective Rhodamine-Based “Off−On”
Reversible Chemosensor for Tin (Sn4+) and Imaging in Living Cells
Ajit Kumar Mahapatra,*,† Saikat Kumar Manna,† Debasish Mandal,§
and Chitrangada Das Mukhopadhyay‡
†Department of Chemistry and ‡Centre for Healthcare Science & Technology, Bengal Engineering and Science University, Shibpur,
Howrah711103, India
§Department of Spectroscopy, Indian Association for The Cultivation of Science, Jadavpur, Kolkata700032, India
S
* Supporting Information
ABSTRACT: A structurally characterized new oxo-chromene function-
alized rhodamine derivative L1 exhibits high selectivity toward Sn4+ by
forming a 1:1 complex, among other biologically important metal ions, as
studied by fluorescence, absorption, and HRMS spectroscopy. Complexing
with Sn4+ triggers the formation of a highly fluorescent ring-open form
which is pink in color. The sensor shows extremely high fluorescence
enhancement upon complexation with Sn4+, and it can be used as a “naked-
eye” sensor. DFT computational studies carried out in mimicking the
formation of a 1:1 complex between L1 and Sn4+ resulted in a nearly planar
pentacoordinate Sn(IV) complex. Studies reveal that the in situ prepared
L1−Sn complex is selectively and fully reversible in presence of sulfide
anions. Further, confocal microscopic studies confirmed that the receptor
shows in vitro detection of Sn4+ ions in RAW cells.
It is familiar that rhodamine B is a classic fluorescent dye and
its derivatives (RhB) are extensively used as a fluorescent
chemosensor because of their excellent photophysical proper-
ties, such as high extinction coefficients (>75 000 cm−1 M−1),
excellent quantum yields,5 great photostability,6 and relatively
long emission wavelengths in the visible region.7 In addition,
the equilibrium between the nonfluorescent spirocyclic form
and the highly fluorescent ring-open form provides a better
model for the development of turn-on sensors. So, rhodamine
B is widely used as a fluorescent probe for the detection of
cysteine8 and metal ions,9 including Cu2+,10 Cr3+,11 Fe3+,12
Hg2+,13 Pb2+,14 Zn2+,15 Au3+,16 Ba2+,17 and Al3+,18 due to the
ring-opening reaction of rhodamine.
INTRODUCTION
■
The design and development of optical sensors for selective
recognition and sensing of environmentally and biologically
important metal ions is an important and contemporary
research area. In this regard, metal ions that are known to
have harmful effects on living organisms or the environment are
generally more common as target metal ions for such studies.1
Among the different metal ions, tetravalent tin, Sn4+, is an
important trace mineral for human and animal biology as it is
involved in several biochemical processes at the cellular level.2a
Tin deficiency can increase the risk factors associated with poor
growth, hearing loss, and cancer prevention. Some severe
immunotoxic and neurotoxic effects of tin have been reported,
with symptoms mostly associated to gastrointestinal complaints
such as nausea, abdominal pain, and vomiting.2b In addition, tin
compound is a known environmental pollutant due to its
widespread use as chemical reagent, agricultural, chemical
weapon, and industrial activities.3 Therefore, great importance
is attached to developing selective chemosensors for tin. In this
aspect, fluorogenic methods with suitable probes are preferable
approaches for the measurement of these analytes because
fluorimetry is rapidly performed, is nondestructive, is highly
sensitive, is suitable for high-throughput screening applications,
and can afford real information on the localization and quantity
of the targets of interest. In recent years, significant emphasis
has been placed on the development of new, highly selective
fluorescent chemosensors of different architectures for metal
cations because of their potential applications in biochemistry
and environmental research.4
With the above-mentioned criteria in mind, herein we
introduced oxo-benzopyran or oxo-chromene moiety to
rhodamine, and we synthesized a chemosensor, L1 (Scheme
1),19 which can give highly selective and rapid responses to
Sn4+ in living cells. As far as we are aware, L1 is the first oxo-
chromene based Sn4+ sensor on rhodamine derivative. The
1
structure of chemosensor L1 was verified by H NMR, 13C
NMR, mass spectra, and elemental analyses (Figures S1−S3,
Supporting Information). Our sensor shows extremely high
sensitivity compared to the recently developed9d Sn4+ sensors
attributed to the very high association constants for the binding
of Sn4+.
Received: March 21, 2013
© XXXX American Chemical Society
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CH3OH/H2O solution (CH3OH/H2O = 4:1, v/v, 10 μM HEPES
Scheme 1. Synthetic Scheme for Compound L1
buffer, pH = 7.4). In titration experiments, each time a 4 × 10−5
M
solution of L1 was filled in a quartz optical cell of 1 cm optical path
length, and the ion stock solutions were added into the quartz optical
cell gradually by using a micropipet. Spectral data were recorded at 1
min after the addition of the ions. In selectivity experiments, the test
samples were prepared by placing appropriate amounts of the anions/
cations stock into 2 mL of solution of L1 (4 × 10−5 M). For
fluorescence measurements, excitation was provided at 555 nm, and
emission was collected from 560 to 700 nm.
Evaluation of the Association Constant for the Formation of
L1·Sn4+. Receptor L1 with an effective concentration of 4 × 10−5 M in
CH3OH/H2O solution (CH3OH/H2O = 4:1, v/v, 10 μM HEPES
buffer, pH = 7.4) was used for the emission titration studies with a
Sn4+ solution. A stock solution of SnCl4·5H2O, having a concentration
of 2 × 10−4 M in an aqueous HEPES buffer (3:2, v/v; pH 7.4)
solution, was used.
Calculations for the Binding Constants Using Spectropho-
tometric Titration Data. The association constant and stoichiometry
for the formation of the respective complexes were evaluated using the
Benesi−Hildebrand (B−H) plot (eq 1).20
EXPERIMENTAL SECTION
1/(I − I0) = 1/K(Imax − I0)[Mn+] + 1/(Imax − I0)
■
(1)
General Information and Materials. All the solvents were of
analytic grade. All cationic compounds such as perchlorate of Hg2+,
Cu2+, Cd2+, Fe2+, Co2+, Ni2+, Mn2+, Zn2+, Mg2+, nitrate of Ag+,
chlorides of Cr3+, Ca2+, Al3+, Fe3+, Pb2+, and SnCl4·5H2O were
purchased from Sigma−Aldrich Chemical Co., stored in a desiccators
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 purification. Elix Millipore water was used
throughout all experiments. All reactions were magnetically stirred and
monitored by thin-layer chromatography (TLC) using Spectrochem
GF254 silica gel coated plates. Column chromatography was
performed with activated neutral aluminum oxide. FTIR spectra
were recorded as KBr pellets using a JASCO FTIR spectrometer
(model FTIR-460 plus). The 1H NMR spectra were recorded on
Bruker 400 MHz spectrometer. Mass spectra were carried out using a
Waters QTOF Micro YA 263 mass spectrometer. The 1H NMR
chemical shift values are expressed in ppm (δ) relative to CHCl3 (δ =
7.26 ppm). UV−vis and fluorescence spectra measurements were
performed on a JASCO V530 and a Photon Technology International
(PTI-LPS-220B) spectrofluorimeter, respectively. The following
abbreviations are used to describe spin multiplicities in 1H NMR
spectra: s = singlet; d = doublet; t = triplet; m = multiplet. Elemental
analyses were performed with a Perkin-Elmer 2400 elemental analyzer.
Synthesis of L1. Rhodamine B hydrazide (0.40 g, 0.88 mmol) and
2-amino-6,7-dimethyl-4-oxo-4H-1-benzopyran-3-carbaldehyde (0.19 g,
0.88 mmol) were dissolved in 20 mL of dry methanol, and the
resulting solution was refluxed for 12 h. The solvent was then
evaporated in vacuo, and the residue was extracted with water/
dichloromethane. The organic layer was dried over sodium sulfate.
After filtration and evaporation of the solvent in vacuo, the residue was
chromatographed on silica gel with hexane/ethyl acetate (7:3, v/v) as
eluent to give L1 as a white solid in 85% yield. Mp > 250 °C. 1H NMR
(d6-DMSO, 400 MHz) δ (ppm): 9.26 (1H, s, CHN), 7.88 (1H, d, J
= 8.00 Hz), 7.56−7.59 (2H, m), 7.21 (1H, s), 7.13 (1H, s), 7.09(1H,
d, J = 6.8 Hz), 6.32−6.39 (6H, m), 4.03 (2H, s), 3.30 (q, 8H, J = 6.9
Hz, NCH2CH3), 2.29 (3H, s), 2.24 (3H, s), 1.07 (12H, t, J = 6.9 Hz,
NCH2CH3). 13C NMR (CDCl3, 400 MHz) δ (ppm): 14.11, 19.15,
20.19, 22.67, 29.34, 29.68, 31.91, 46.11, 60.39, 96.68, 114.19, 116.70,
117.08, 119.77, 123.62, 123.74, 125.80, 126.06, 12 8.82, 133.22,
133.70, 142.79, 151.33, 162.41, 174.79, 189.85. TOF MS ES+, m/z =
656.06 [M + 1]+, calcd for C40H41N5O4 = 655.78. Anal. Calcd for
C40H41N5O4: C, 73.26; H, 6.30; N, 10.68. Found: C, 73.17; H, 6.27;
N, 10.71.
Here I0, Imax, and I represent the emission intensity of free L1, the
maximum emission intensity observed in the presence of added metal
ion at 580 nm for Sn4+ (λext = 555 nm), and the emission intensity at a
certain concentration of the metal ion added, respectively. Binding
stoichiometries for the respective complex formations were also
confirmed from Job’s plot.
Finding the Detection Limit. The detection limit was calculated
on the basis of the fluorescence titration (Supporting Information).
The fluorescence emission spectrum of L1 was measured 14 times, and
the standard deviation of blank measurement was achieved. To gain
the slope, the ratio of the fluorescence intensity at 580 nm was plotted
as a concentration of Sn4+. So the detection limit was calculated with
the following equation10b,21
(2)
detection limit = 3Sb1/S
where Sbl is the standard deviation of blank measurement and S is the
slope of the calibration curve.
Cell Culture. Frozen human colorectal carcinoma cell line HCT
116 (ATCC: CCL-247) was obtained from the American Type
Culture Collection (Rockville, MD) and maintained in Dulbecco’s
modified Eagle’s medium (DMEM, Sigma Chemical Co., St. Louis,
MO) supplemented with 10% fetal bovine serum (Invitrogen),
penicillin (100 μg/mL), and streptomycin (100 μg/mL). The RAW
264.7 macrophages were obtained from NCCS, Pune, India, and
maintained in DMEM containing 10% (v/v) fetal calf serum and
antibiotics in a CO2 incubator. Cells were initially propagated in 25
cm2 tissue culture flask in an atmosphere of 5% CO2 and 95% air at 37
°C humidified air till 70−80% confluency.
Cell Imaging Study. For confocal imaging studies, RAW cells, 1 ×
10−5 cells in 150 μL of medium, were seeded on sterile 12 mm
diameter Poly L lysine coated coverslip, kept in a sterile 35 mm
covered Petri dish, and incubated at 37 °C in a CO2 incubator for 24−
30 h. The next day, cells were washed three times with phosphate
buffered saline (pH 7.4) and fixed using 4% paraformaldehyde in PBS
(pH 7.4) for 10 min at room temperature washed with PBS followed
by permeabilization using 0.1% saponin for 10 min. Then the cells
were incubated with 2.0 × 10−4 M SnCl4 dissolved in 100 μL DMEM
at 37 °C for 1 h in a CO2 incubator and observed under Andor
spinning disk confocal microscope with excitation at 561 nm
monochromatic laser beam, and the collected range of emission
wavelength was between 630 and 650 nm. The cells were again washed
thrice with PBS (pH 7.4) to remove any free metal and incubated in
DMEM containing probe to a final concentration of 1 × 10−6
M
UV−Vis and Fluorescence Spectral Studies. A stock solution of
the probe L1 (4.0 × 10−5 M) was prepared in CH3OH/H2O (4:1, v/
v). Solutions of 2.0 × 10−4 M salts of the respective cation were
prepared in Millipore water. All experiments were carried out in
followed by washing with PBS (pH 7.4) three times to remove excess
probe outside the cells. Again, images were captured using EMCCD
camera. In a separate coverslip undergoing the same treatment the
cells were then treated with 30 μM of Na2S solution for 1 h; the cells
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Figure 1. (A) UV−vis absorption titration spectra of spectra of L1 (c = 4× 10−5 M) in aq CH3OH (CH3OH/H2O = 4:1, v/v, 10 μM HEPES buffer,
pH = 7.4) upon addition of Sn4+ (c = 2× 10−4 M). Inset: Job’s plot that indicates 1:1 stoichiometry and photographs of L1 and L1 + Sn4+. (B)
Competitive absorption spectra of L1 in the presence of different metal ions (perchlorate, chloride, or nitrate salts of Cr3+, Mg2+, Ca2+, Al3+, Mn2+,
Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Ag+, and Pb2+) in aq CH3OH (CH3OH/H2O = 4:1, v/v, 10 μM HEPES buffer, pH = 7.4).
Figure 2. (A) Fluorescence emission spectra of L1 (c = 4× 10−5 M) in aq CH3OH (CH3OH/H2O = 4:1, v/v, 10 μM HEPES buffer, pH = 7.4) upon
addition of Sn4+ (c = 2 × 10−4 M). Inset: Change of emission intensity at 580 nm with incremental addition of Sn4+ (λext = 555 nm) and fluorescence
photographs of L1 and L1 + Sn4+. (B) Competitive fluorescence spectra of L1 in the presence of different metal ions (perchlorate, chloride, or
nitrate salts of Cr3+, Mg2+, Ca2+, Al3+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Ag+, and Pb2+) in aq CH3OH (CH3OH/H2O = 4/1, v/v,
10 μM HEPES buffer, pH = 7.4).
were washed with PBS three times to remove free compound and ions
before analysis. Before microscopic imaging, all the solutions were
aspirated and mounted on slides in a mounting medium containing
DAPI (1 μg/mL) and stored in dark before microscopic images are
acquired.
solubilized in DMSO, and its absorbance was measured in a microtiter
plate reader (Perkin-Elmer) at 570 nm. The assay was performed in
triplicate for each concentration of L1, SnCl4, and L1−SnCl4 complex.
The OD value of wells containing only DMEM medium was
subtracted from all readings to get rid of the background influence.
Data analysis and calculation of standard deviation were performed
with Microsoft Excel 2007 (Microsoft Corporation).
Cytotoxic Effect on Cells. The cytotoxic effects of probe L1,
SnCl4, and L1−SnCl4 complex were determined by an MTT assay
following the manufacturer’s instruction (MTT 2003, Sigma-Aldrich,
MO). HCT cells were cultured into 96-well plates (approximately 104
cells per well) for 24 h. The next day, the medium was removed, and
various concentrations of L1, SnCl4, and L1−SnCl4 complex (0, 15,
25, 50, 75, and 100 μM) made in DMEM were added to the cells and
incubated for 24 h. Solvent control samples (cells treated with DMSO
in DMEM), no cells, and cells in DMEM without any treatment were
also included in the study. Following incubation, the growth medium
was removed, and fresh DMEM containing MTT solution was added.
The plate was incubated for 3−4 h at 37 °C. Subsequently, the
supernatant was removed, the insoluble colored formazan product was
Computational Studies. All geometries for L1 and L1·Sn4+ were
optimized by density functional theory (DFT) calculations using
Gaussian 2003(B3LYP/6-31G(d,p)) software package.22
RESULTS AND DISCUSSION
■
UV−Vis Spectroscopic Studies of L1 in Presence of
Sn4+. The absorption spectra of L1 with Sn4+ was investigated
by spectrophotometric titration in CH3OH/aqueous HEPES
buffer (1 mM, pH 7.4; 4:1, v/v). When no Sn4+ ions were
added to the solution of L1, free L1, as expected, exhibited
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Figure 3. (A) UV−vis titration spectra of L1 (c = 4× 10−5 M) with 3 equiv of Sn4+ upon addition of sodium sulfide (c = 2× 10−4 M) in aq CH3OH
(CH3OH/H2O = 4:1, v/v, 10 μM HEPES buffer, pH = 7.4). Inset: Photographs of L1, L1 + Sn4+, L1 + Sn4+, + S2‑ in color changes. (B) Changes in
the absorption spectra of L1−Sn complex in presence of different anions. (C) Fluorescence spectra of L1 (c = 4× 10−5 M) with 3 equiv of Sn4+ upon
addition of sodium sulfide (c = 2 × 10−4 M) in aq CH3OH (CH3OH/H2O = 4:1, v/v, 10 μM HEPES buffer, pH = 7.4) Inset: Change in the
fluorescence intensity at 580 nm with incremental addition of Sn4+ (λext = 555 nm). (D) Changes in the fluorescence spectra of L1−Sn complex in
presence of different anions.
almost no absorption in the visible region, indicating that L1
exists as a spirocyclic form. It can be seen from Figure 1A that
the solution of L1 exhibits almost no absorption peak in the
visible region. However, upon addition of Sn4+ ion, a new
absorption band at 555 nm appeared with a shoulder at 518
nm, and absorbance increased with increasing Sn4+ concen-
tration (0−5 equiv of Sn4+), which can be ascribed to the
formation of the ring-opened amide form of L1 upon Sn4+ ion
binding.
Meanwhile, the titration solution turned pink instantaneously
as a result of the ring-open structure formation caused by Sn4+
binding (Figure 1A, inset), suggesting that chemosensor L1 can
serve as a “naked-eye” indicator for Sn4+ ions. The binding
ability of L1 was checked with perchlorate, chloride, and nitrate
salts of Cr3+, Mg2+, Ca2+, Al3+, Mn2+, Fe2+, Fe3+,Co2+, Ni2+,
Cu2+, Zn2+, Cd2+, Hg2+, Ag+, and Pb2+ ions in CH3OH/aqueous
HEPES buffer (1 mM, pH 7.4; 4:1, v/v). A significant change in
electronic spectral pattern was observed only in the presence of
Sn4+, among all the metal ions used (Figure 1B). The
absorbance peak was no longer changed when the concen-
tration of Sn4+ increased from 5 to 6 equiv, implicating a 1:1
complexation of the ligand with the Sn4+ ion. From the Job’s
plot, we can observe that the absorbance went through a
maximum at a molar fraction of about 0.5, indicating that a 1:1
stoichiometry was most possible for the binding mode of Sn4+
and L1 (Figure 1A, inset).
Fluorescence Spectroscopic Studies of L1 in Presence
of Sn4+. Fluorescence titrations of chemosensor L1 with Sn4+
in CH3OH/aqueous HEPES buffer (1 mM, pH 7.4; 4:1, v/v)
were performed. The fluorescence spectra were obtained by
excitation at 555 nm. As shown in Figure 2A, free probe L1
showed almost no fluorescence due to the spirocyclic structure.
However, the treatment with Sn4+ induced a dramatic change at
580 nm in the fluorescence spectra (14-fold) which was higher
than the result obtained with the rhodamine-based Sn4+ sensors
reported earlier.23 Upon addition of Sn4+, the fluorescence
intensity increased in a Sn4+ concentration-dependent way as
shown in Figure 2A (inset), accompanied with an obvious
orange fluorescence enhancement . The results indicated that
the spirolactam ring was opened and conjugated structure was
formed. The recognition interaction was completed immedi-
ately after the addition of Sn4+ within 1 min, and hence, L1
could be used in real-time determination of Sn4+ in environ-
mental and biological conditions. There was a significant
emission intensity enhancement (14-fold) with 3.0 equiv of
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Scheme 2. Proposed Mechanism for the Fluorescence Changes of L1 upon Addition of Sn4+
Sn4+ which indicates that compound L1 is an excellent turn-on
sensor for Sn4+.
adding S2− to the solution containing L1−Sn. Commom
− −
anions, such as F−, Br−, I−, NO3 , SO42−, CH3COO−, HSO4 ,
−
From the emission titration experiment, the association
constant24 (Ka) of L1 with Sn4+ was estimated to be 4.31 × 104
M−1 (Figure S9, Supporting Information). The detection limit
of Sn4+ is about 2.58 μM (Supporting Information). The
selectivity of L1 to the various metal ions were investigated. On
addition of different metal ions, viz., Cr3+, Mg2+, Ca2+, Al3+,
Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Ag+, and
Pb2+, to solution of L1, there is no significant change in its
fluorescence spectrum except in the case of addition of Sn4+
ions. As shown in Figure 2A, only the addition of Sn4+ resulted
in a prominent enhancement of fluorescence at 580 nm
(absorbance at 555 nm), which obviously implied the high
selectivity of L1 to Sn4+. The competition experiments (Figure
2B), which were carried out by adding Sn4+ to L1 solution in
the presence of other metal ions, revealed that there was no
significant interference of Sn4+-induced fluorescent responses
by the commonly coexistent metal ions (Figure S6, Supporting
Information).
Interestingly, a solution of L1 in optimized CH3OH/H2O
solution (4:1, v/v, HEPES buffer, pH = 7.2) is colorless and
emits no orange fluorescent light, but during the fluorometric
titration of L1 with Sn4+ ions the colorless solution of the
receptor became deep orange (Figure 2A, inset). This orange
fluorescent color is attributed to the opening of the spirolactam
ring and generation of the delocalized xanthene moiety,25
whereas compound L1 shows obvious pink color in buffered
CH3OH/HEPES solution upon addition of Sn4+ under visible
light. This was not observed with other metal ions (Figure S8,
Supporting Information). The results support our expectation
that L1 could serve as a sensitive naked-eye probe for Sn4+.
Selective Optical Response of L1−Sn Complex to
Various Anions. Hence, from the UV−vis and fluorescence
studies we can conclude that L1 selectively binds with Sn4+ to
form L1−Sn complex with considerable change in its spectral
properties. An important property of the chemosensor is high
selectivity toward the analyte as well as the reversibility in the
complexation of any probe to be employed as a chemical sensor
for detection of specific metal ions. Therefore, we have further
studied the influence of different anions on the rupture of this
metal−ligand complex and their effect on the reversibility of
L1−Sn complex to regenerate L1. It is very exciting and
noteworthy that compound L1 could be regenerated only by
and H2PO4 , and other forms of sulfate, did not generate the
same results. Addition of Na2S to the solution of complex of
L1−Sn brought the reverse change in the absorption spectra
(Figure 3A).
A similar finding was observed in emission (Figure 3C). The
L1−Sn system revealed remarkably selective fluorescence “off”
behavior exclusively with S2−. As shown in Figure 3C, upon the
addition of various amounts of sulfide ions (0−3 equiv) in the
presence of 3 equiv of Sn4+, both the intensity and shape of the
system’s emission spectrum return to those of the native L1
state. Figure 3C shows that the intensity of the fluorescence
emission decreases with increasing concentration of sulfide
anion, and on addition of nearly about 3 equiv of S2− anion
both the intensity and overall pattern of emission spectrum
closely match those of compound L1 (Figure 2A), so the
fluorescence intensity along with the maximum emission peak
are totally regenerated. However, there were nearly no
differences in the presence and absence of other anions (Figure
3B,D). This showed that Sn4+ was released from complex L1−
Sn, and SnS2 formed. The calibration curve of the fluorescence
intensity at 580 nm of L1 (Figure 3C, inset) elucidated that
sulfide anion interacted with Sn4+. For further understanding,
we carried out UV−vis titration in this complex. On addition of
the aqueous solution of Na2S to the deep pink solution of L1−
Sn, spectral bands (absorption band with λmax at 555 nm), as
well as the color of the solution, disappeared (Figure 3A, inset).
Again, on addition of Sn4+ to this solution mixture having Na2S,
the absorbance band at 555 nm and emission band at 580 nm
reappeared (Figure S11, Supporting Information). Simulta-
neously, the pink color of the solution was also restored.
Preferential binding of the S2− ion to the Sn4+ ion led to the
formation of SnS2 and the regeneration of the cyclic lactam
form of the reagent, a process which is well documented for
demonstrating the reversible binding of rhodamine derivatives
to Sn4+. This signifies that signal transduction is reversible;
therefore, the chemosensor would be recyclable (Figure S11,
Supporting Information), which is a highly efficient one.
Reversible binding of Sn4+ to L1 was also established through
spectral studies in presence of 3 equiv of Na2EDTA (Figure
S10, Supporting Information).
Sensing Mechanism. In order to gain insight into the
sensing mechanism, we decided to study the sensing process by
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Figure 4. Calculated energy-minimized structure of (A) L1 and (B) L1−Sn complex.
Figure 5. HOMO and LUMO distributions of L1 and L1−Sn4+ complex.
FTIR, HRMS, and the density functional theory (DFT)
calculation. The complex formed between L1 and Sn4+ is
found to be 1:1 in stoichiometry, which is confirmed from the
Job’s plot for binding of Sn4+ to L1 by UV−vis spectrometry.
The result was also confirmed through HRMS analysis, and it is
well matched with theoretical mass spectral simulation. The
peak at m/z 844.0773 in the mass spectrum is assignable to the
mass of [(L1 + Sn4+ + 2Cl− − 1)+] (Figure S4, Supporting
Information). The role of the CO amide bond on binding
Sn4+ cations was examined using FTIR techniques. FTIR
spectra of L1 revealed that the peak at 1682 cm−1, the
characteristic stretching frequency for the CO amide bond of
the rhodamine unit, shifted to 1634 cm−1 in presence of 1.5
equiv of the Sn4+ ion (Figure S12, Supporting Information).
Such a shift in the stretching frequency of CO amide bond of
the rhodamine unit on binding to a metal ion has been reported
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Figure 6. MTT assay to determine the cytotoxic effect of L1 and L1−Sn complex on HCT cells.
earlier.26 Thus, FTIR studies suggest that the shift in the
stretching frequency of CO bond was due to its participation
in the binding event of the metal cation. Furthermore, in order
to investigate the “on−off” property, the HRMS spectrum of
the [L1 + Sn4+ + S2−] system was obtained also. The parent
peak [M + Na+]+ at m/z 678.2179 (Figure S5, Supporting
Information) is equivalent to that of compound L1 on addition
of S2−. Therefore, it elucidated the mechanism of the sensing of
sulfide anions. From our Sn4+ binding studies it was also
evident that the binding induced breakage of the spirolactum
ring of L1 initiates the turn on fluorescence. As illustrated in
Scheme 2, the addition of Sn4+ ion can cause fluorescence on
(Φ = 0.132), simultaneously inducing a color change from
colorless to deep pink. Also, the added sulfide can capture Sn4+
ion, resulting in fluorescence quenching (Φ = 0.061) and
inducing a deep pink to colorless colorimetric change (Figure
3A, inset). This fluorescence enhancement can be attributed to
complex L1−Sn formation upon the addition of Sn4+, as shown
in Scheme 2, and then the tin ions were captured by sulfide
ions, resulting in the revival of L1, which is accompanied by
fluorescence off and disappear of color. Therefore, the above-
mentioned fact provides strong evidence of the dissociation of
L1−Sn complex in the presence of S2− anions to restore the
native structure of L1.
Density Functional Theory (DFT) Calculations. As the
stoichiometry of the complex formed between L1 and Sn4+ was
found to be 1:1 on the basis of emission, absorption, and
HRMS studies, for better understanding, the nature of Sn4+
coordination with L1 was studied by computational studies
using the Gaussian 2003(B3LYP/6-31G(d,p)) software pack-
age.22 The geometry optimizations for L1 and L1−Sn 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 L1−Sn complex, a starting
model was generated by taking the DFT optimized L1 and
placing the Sn4+ ion well in the core of the imidecarbonyl O,
enamine N, benzopyrancarbonyl O, and/benzopyranamine N
as donor moieties at a noninteracting 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 SDDAll basis set for
Sn4+ and 6-31+G(d,p) for all other atoms in the complex.
The spatial distributions and orbital energies of HOMO and
LUMO of L1 and L1−Sn were also determined (Figure 5).
The π electrons on the HOMO of L1−Sn complex are mainly
located on the imidecarbonyl of rhodamine part and
unsaturated carbonyl of benzopyran framework, but the
LUMO is mostly positioned at the center of the guest Sn4+
ion. Moreover, the HOMO−LUMO energy gap of the complex
becomes much smaller relative to that of probe L1.
The energy gaps between HOMO and LUMO in the probe
L1 and L1−Sn complex were 86.76 and 44.50 kcal mol−1,
respectively (Supporting Information). The results clearly
suggest that the Sn4+ ion binds to L1 very well through five
coordination sites, and the whole molecular system forms a
nearly planar structure. The optimized complex of Sn4+ with L1
(Figure 5) showed that a distinct low energy complexation
occurs between Sn4+ and benzopyrancarbonyl O sites of oxo-
chromene due to resonance effect of NH2 and the ring oxygen
atom (Table S1, Supporting Information). Hence, the
interaction of the benzopyrancarbonyl O atom with Sn4+ can
change the orbital energy level, realizing the optical detection.
The optimized complex of Sn4+ with L1 at this stage showed a
nearly planar pentacoordinated geometry around Sn4+ where all
five bonds (2 × Sn−O, Sn−N, and 2 × Sn−Cl) are bonded to
the central ion with their distances of 2.02, 2.05, 2.14, and 2.30
Å, respectively (Figure 4B).
These Sn−O, Sn−Cl, and Sn−N distances are comparable
with the experimental ones reported in the literature for
pentacoordinate Sn(IV) complexes.27
To study the practical applicability, the effects of pH on the
fluorescence response of L1 in the absence and presence of
Sn4+ ions were evaluated. In the absence of Sn4+, almost no
change in fluorescence intensity was observed in the free sensor
over a wide pH range of 5.5−11.0, indicating that the free
sensor L1 was stable in the wide pH range. Experimental results
show that, for free L1, at acidic conditions (pH < 5), an obvious
G
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Figure 7. Confocal microscopic images of probe in RAW 264.7 cells pretreated with SnCl4: (A) bright field image of the cells, (B) only SnCl4 at 2.0
× 10−4 M concentration, nuclei counterstained with DAPI (1 μg/mL), (C) stained with probe L1 at concentration 1.1 × 10−6 M, (D) overlay image
in dark field, (E) and cells treated with SnCl4 at 2.0 × 10−4 M, probe at concentration 1.1 × 10−6 M and Na2S (30 μM), no red signal is detected . All
images were acquired with a 40× objective lens.
off−on fluorescence appeared due to the formation of the
open-ring state because of the strong protonation. In the pH
range from 4.0 to 6.0, little fluorescence signal (excited at 555
nm) could be observed for free L1, suggesting that the
molecules prefer the spirocyclic form. Upon the addition of
Sn4+ ions, there was an obvious fluorescence off−on change of
L1 under different pH values. Thus, the sensor L1 has the
maximal sensing response at physiological pH, indicating that
the sensor L1 is promising for biological applications (Figure
S7, Supporting Information).
The results obtained in the in vitro cytotoxic assay suggested
that, in order to pursue confocal imaging studies of L1−Sn
complex in live cells, it would be prudent to choose a working
concentration of 20 μM for probe compound. Hence, to assess
the effectiveness of compound L1 as a probe for intracellular
detection of Sn4+ by confocal microscopy, RAW cells were
treated with 20 μM SnCl4 for 1 h followed by 10 μM probe
solution to promote formation of L1−Sn complex. Confocal
microscopic studies revealed a lack of fluorescence for RAW
cells when treated with either probe compound or SnCl4 alone
(Figure 7B). Upon incubation with SnCl4 followed by probe
L1, a striking switch-on fluorescence was observed inside RAW
cells, which indicated the formation of L1−Sn complex, as
observed earlier in solution studies. Further, an intense red
fluorescence was conspicuous in the perinuclear region of RAW
cells (Figure 7C).
Interestingly, when the cells were treated with Na2S solution
(Figure 7E) this bright red fluorescence was significantly gone.
Sulfide sensing inside RAW cells by L1−SnCl4 complex could
also be pursued as evident from this remarkable switch-off of
the red fluorescence emission inside cells following incubation
with Na2S solution.
The confocal microscopic analysis strongly suggested that
probe L1 could readily cross the membrane barrier of the RAW
cells, and rapidly sense intracellular Sn4+ and S2−. It is
significant to mention here that bright field images of treated
cells did not reveal any gross morphological changes, which
suggested that RAW cells were viable. These findings open up
the avenue for future in vivo biomedical applications of the
sensor.
Biological Studies of L1 in Presence of Sn4+ and S2‑.
Due to its favorable binding properties with Sn4+ and its
complex L1−Sn showing intense emission in visible region,
practical bioimaging application of the probe L1 could be
exploited for sensitive detection of intracellular tin. However, to
materialize this objective it is a prerequisite to assess the
cytotoxic effect of probe L1, Sn4+, and the complex on live cells.
The well-established MTT assay, which is based on
mitochondrial dehydrogenase activity of viable cells, was
adopted to study cytotoxicity of the above-mentioned
compounds at varying concentrations mentioned in the
Experimental Section. Figure 6 shows that probe L1 did not
exert any adverse effect on cell viability, and the same is the case
when cells were treated with varying concentrations of Sn4+.
However, exposure of HCT cells to L1−Sn complex resulted in
a decline in cell viability above 20 μM concentration. The effect
was more pronounced in higher concentration and showed an
adverse cytotoxic effect in a dose-dependent manner which is in
agreement with previous literature reports suggesting cytotoxic
and antiproliferative effects of L1−Sn complex on cancer
cells.28
CONCLUSION
The viability of HCT cells was not influenced by the solvent
(DMSO) as evidenced in Figure 6, leading to the conclusion
that the observed cytotoxic effect could be attributed to L1−Sn
complex. Also, Na2S does not significantly affect the cell
viability either, even in 100 μM (Figure 6), and thus, the probe
can also detect sulfide in biological systems.
■
A structurally characterized oxo-chromene rhodamine B
derivative of L1 exhibits high selectivity toward Sn4+. The
selectivity has been demonstrated by fluorescence, absorption,
and HRMS spectroscopy. Interaction of Sn4+ with L1 enhances
the fluorescence emission at 580 nm and induces a turn-on
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response in electronic and fluorescence spectra in the visible
region. L1 is sensitive and selective toward Sn4+ over other
biologically important ions studied, viz., Cr3+, Mg2+, Ca2+, Al3+,
Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Ag+, and
Pb2+, as demonstrated by individual as well as competitive
metal ion titrations. Thus, these receptors could be used as a
dual probe for visual detection through change in color and
fluorescence. Whereas the fluorescence and absorption spec-
troscopy provided information for the formation of 1:1
complex between Sn4+and L1, viz., L1−Sn, the HRMS
confirmed the same unambiguously by exhibiting correct peak
pattern for the presence of tin in the complex. The tin-binding
capabilities of the nitrogen−oxygen binding core of oxo-
chromene rhodamine moieties available in L1 has been
examined by computational calculations at the DFT level.
The theoretical studies supported that Sn4+ coordinating with
the O atom of the oxo-chromene moiety, accompanied by the
transferring of electrons of the oxo-chromene, resulted in the
opening of the spiro-ring. The in situ prepared tin complex of
L1, viz., L1−Sn, was able to detect S2− in exactly the reverse
manner compared to what happens when Sn4+ is added to L1
in fluorescence spectroscopy. Thus, upon addition of S2− to
L1−Sn, the 580 nm band intensity decreases, suggesting the
release of L1 from the tin complex. The receptor L1 shows
intense change in its fluorescence emission when bound to Sn4+
in physiological conditions. Hence, the effectiveness of
compound L1 as a probe for intracellular detection of Sn4+
by confocal microscopy was also studied. Moreover, the
confocal microscopic analysis strongly suggested that com-
pound L1 could readily cross the membrane barrier of the
RAW cells, and rapidly sense intracellular Sn4+ and S2−.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR, 13C NMR, mass, and IR spectra as characterization
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presence and absence of Sn4+ ions. Additional tables. This
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AUTHOR INFORMATION
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Corresponding Author
*E-mail: mahapatra574@gmail.com. Fax: +91 33 26684564.
Phone: +91 33 2668 4561.
Author Contributions
The manuscript was written through contributions of all
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the DST-New Delhi (Project SR/S1/OC-44/2012)
for financial support. S.K.M. thanks UGC, New Delhi, India, for
a fellowship and also Bhaskar Pramanik for his support. We are
also thankful to Annesur Rahaman Centre for High Perform-
ance Computing, IACS, Kolkata, for providing us with
computational time.
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