Intracellular detection of hazardous Cd2+ through a fluorescence imaging technique by using a nontoxic coumarin based sensor

Article abstract of DOI:10.1039/c6dt04833a

A new coumarin based turn on fluorescent sensor (R1) was reported for the detection of highly hazardous Cd²⁺ with excellent selectivity and sensitivity without any interference of other metal ions. The single crystal X-ray structure analysis of

Full text of DOI:10.1039/c6dt04833a

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Intracellular detection of hazardous Cd2+ through
a fluorescence imaging technique by using a non-
toxic coumarin based sensor†
Cite this: DOI: 10.1039/c6dt04833a
Chanda Kumari,a Dibyendu Sain,a,b Ashish Kumar,a Sushanta Debnath,c Partha Sahac
and Swapan Dey*a
A new coumarin based turn on fluorescent sensor (R1) was reported for the detection of highly hazardous
Cd2+ with excellent selectivity and sensitivity without any interference of other metal ions. The single
crystal X-ray structure analysis of the sensor showed the actual geometry of the molecule. For the first
time, a Cd2+ induced FRET mechanism was observed and explained accordingly. Instant naked eye detec-
tion of Cd2+ through a sharp colour change signified the practical applicability of R1. The sensor of high
quantum yield was applied in the intracellular detection of poisonous Cd2+ in living HeLa S3 cells.
Received 22nd December 2016,
Accepted 26th January 2017
DOI: 10.1039/c6dt04833a
rsc.li/dalton
of rapid and reliable methods for monitoring traces of
cadmium in the environment or in vivo biological media is a
Introduction
Cadmium has adverse power of causing severe environmental great challenging field with an aspiration of considerable
and human health problems due to its toxic effect including significance.7,8
carcinogenicity.1 So, it is an enlisted element of seventh rank
In recent years, fluorescent sensors are considered as an
in the Agency for Toxic Substances and Disease Registry attractive alternative to monitor extremely poisonous metal
(ATSDR).2 The level of cadmium accumulation in the environ- ions from biological media (in vivo or in vitro) because of their
ment is increasing dramatically due to various human activi- great potentiality to afford a highly sensitive, selective, and
ties such as mining, metallurgy, smelting, military affairs, economical online detection method.9 Among them, turn on
fossil fuel combustion, use of phosphate fertilizers in agricul- fluorescent sensors have great advantages in molecular reco-
ture, production of nickel cadmium rechargeable batteries and gnition and materials chemistry.10 Although a lot of fluo-
other industrial use.3 All these cadmium sources result in rescent sensors have already been reported with some success-
dietary exposure and increasing percentage of cadmium ful applications for sensing Cd2+ in living cells, most of them
content in our daily food.4 Biological effects of such cadmium are deficient in selectivity and sensitivity.11 The heaviest chal-
accumulation in our body include calcium metabolism dis- lenge for sensing of Cd2+ arises from the interference of other
orders, renal dysfunction, pneumonitis, pulmonary edema, metal ions like Zn2+ and Hg2+ which exhibit similar spectral
emphysema and even some types of cancers of the kidney, and chemical properties.12 So, the development of a highly
prostate, lung and pancreas.5 For these toxic effects, the World specific and selective sensor for Cd2+ without interference of
Health Organization has fixed the limit of cadmium level at other metal ions is still a challenging real time thorny problem
3.0 μg per liter in drinking water.6 Therefore, the development for modern researchers.13
In recent days, rhodamine and coumarin moieties are fre-
quently used as fluorescent probes for the detection of
aDepartment of Applied Chemistry, Indian Institute of Technology (ISM), Dhanbad-
different metal ions.14 Generally, coumarin and rhodamine
826004, India. E-mail: dey.s.ac@ismdhanbad.ac.in; Fax: +91 326 2296563;
moieties are used as the donor and acceptor respectively for
Tel: +91 326 2235607
showing the different energy transfer process because a large
overlap can be observed between the absorption spectrum of
rhodamine and the emission spectrum of coumarin.15 For a
successful Fourier resonance energy transfer (FRET) process,
the spectral overlap between the emission of the donor part
and the absorption of the acceptor part of the sensor molecule
is necessary and the distance between these two parts is also a
deciding factor for this process.16 In continuation of our
bDepartment of Chemistry, Indian Institute of Engineering Science and Technology,
Shibpur, Howrah, West Bengal-711103, India
cCrystallography and Molecular Biology Division, Saha Institute of Nuclear Physics,
1/AF Bidhannagar, Kolkata 700064, India
†Electronic supplementary information (ESI) available: 1H, 13C NMR, MS, FTIR
spectra; all photo-physical analysis (UV-Vis and fluorescence): quantum yield,
LOD, bar diagram, binding constant calculation, and single crystal X-ray analysis
of R1. CCDC 1524036 for R1. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/c6dt04833a
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work,17 we have described here a coumarin linked rhodamine Complexation studies of R1 with different metal ions through
based molecule as a FRET based turn-on fluorescence sensor visual detection and UV-Vis spectroscopy
system for Cd2+ with high selectivity and sensitivity in a semi-
The photo-physical complexation study of R1 was explored
aqueous environment. According to our knowledge, it is one of
with a series of perchlorate salts of different metal ions such
the very few previous reports of Cd2+ recognition using the
as Li+, Fe3+, Ni2+, Co2+, Cu2+, Hg2+, Cd2+, Zn2+, Pb2+, and Mg2+
FRET based mechanism.18
in MeOH/H2O (2/1, v/v, 1 mM HEPES buffer, pH = 7.2) solvent.
The colourless solution of R1 (8.0 × 10−6 M) turned pink
immediately upon addition of 1.0 equivalent of Cd2+ (8.0 ×
10−5 M) (Fig. 2a, inset). In comparison, other cations did not
Results and discussion
exhibit any changes of R1.
The UV-Vis absorbance spectrum of R1 (8.0 × 10−6 M) in
At first, 7-amino-4-methylcoumarin (compound 3) was syn-
thesized by using 3-aminophenol and ethylacetoacetate. After
MeOH/H2O (2/1, v/v, 1 mM HEPES buffer, pH = 7.2) showed no
characteristic peak in the visible region, but after gradual
that, it was coupled with a rhodamine moiety to obtain the
addition of Cd2+, a peak at 561 nm was visualized with increas-
designed sensor R1 (Scheme 1). The structure of R1 was
characterized by NMR, mass and IR spectroscopic techniques
ing peak intensity. The spirolactam ring opening by the influ-
ence of Cd2+ was the main reason behind the visual colour
(see the ESI†). The colourless crystal was developed in ethyl-
acetate solvent using a slow evaporation technique. The solid
change. Other metal ions were almost inactive towards R1 and
hence they did not show any type of spectral change (Fig. 2b).
Moreover, no interference effect of any other metal ions in UV-
state structure of R1 was determined by X-ray crystallographic
analysis. R1 was a triclinic system with the P1 space group.
The solid state ORTEP diagram confirmed that the spirolactam
ring was almost perpendicular to the xanthene moiety and the
coumarin moiety made an angle of 116° with that ring (Fig. 1).
The crystal packing structure showed the molecular
arrangement.
vis absorption spectra was observed when the solution of R1
was treated with Cd2+ in the presence of other metal ions
(Fig. 2b).
The complexation study of R1 with all the above mentioned
metal ions was also carried out using a fluorescence spectro-
scopic technique. The solution of R1 (8.0 × 10−6 M) in MeOH/
H2O (2/1, v/v, 1 mM HEPES buffer, pH = 7.2) exhibited the
fluorescence emission peak at 430 nm and after addition of
Cd2+, a new peak at 589 nm was observed when it was excited
at 340 nm. The intensity of that new peak enhanced with the
increase in the concentration of Cd2+ up to 1.0 equivalent
(Fig. 3a). This fluorescence spectral change was also observed
visually under long wavelength UV light, where the non-fluo-
rescent solution of R1 turned to bright red fluorescent
immediately in the presence of Cd2+ (Fig. 3a, inset). No such
spectral or visual fluorescence change and any interference
effect were observed for other metal ions (Fig. 3b). So, the
above results concluded the strong complexation between R1
and Cd2+.
The receptor showed a strong binding efficiency towards
Cd2+. By using Benesi–Hildebrand linear regression analysis,19
Scheme 1 Reagents and conditions: (a) ethylacetate, rt., 4 h; (b) ethyl-
acetoacetate, 70% H2SO4, rt, 4 h; (c) H2SO4, acetic acid, 110 °C, 4 h; (d)
a high binding constant value (Ka = 7.8 × 105 M−3) of R1 with
POCl3, 1,2-dichloroethane, 6 h, reflux; (e) dry Et3N, dry CH3CN, 10 h,
Cd2+ was calculated (see the ESI†). The fluorescence quantum
reflux.
Fig. 2 UV-Vis absorption spectra of R1 in the presence of (a) 1.0 equi-
Fig. 1 Single crystal X-ray (a) ORTEP structure of R1 and its (b) packing
structure along the b axis.
valent Cd2+ (inset: colour change of R1 with Cd2+), (b) other metal ions,
in MeOH/H2O (2/1, v/v), 1 mM HEPES buffer, pH = 7.2.
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Fig. 3 Fluorescence emission spectra of R1 (λexc = 340 nm) in the pres-
ence of (a) 1.0 equivalent of Cd2+ (inset: visual fluorescence change of
R1 with Cd2+), (b) other metal ions, in MeOH/H2O (2/1, v/v), 1 mM HEPES
buffer, pH = 7.2.
Fig. 5 Overlap plot of absorption spectra of rhodamine b and fluor-
escence emission spectra of compound 3.
yields20 (ϕ) for R1 and R1 : Cd2+ were found to be 0.011 and
0.19 respectively with respect to standard rhodamine B (litera-
ture ϕ = 0.49 ). The limit of detection (LOD) calculation21 was
carried out using fluorescence titration data. A very low LOD
value (1.01 × 10−8 M) obtained by this method implied the
high sensitivity of R1 towards Cd2+ (see the ESI†).
To investigate the practical applicability, the effect of pH on
visual colour change and fluorescence emission of R1 itself
and R1 with Hg2+ were examined with a wide range of pH (pH
1.0 to 14.0). R1 showed pink colour and red fluorescence
below pH 6.5, where the spirolactam ring opening form of R1
persisted. The stability of its ring closure form was found
above pH 6.5, where the colourless and non-fluorescence pro-
perties were observed. The maximum fluorescence emission
intensity of the R1 : Cd2+ complex was observed at 589 nm in
acidic medium (pH 2). The complex exhibited noticeable fluo-
rescence intensity from pH 1.0 to 11.0 suggesting a wide range
of practical usability of the sensor (Fig. 4). So the application
of R1 in complexation covered a wide range of pH.
observed (Fig. 5). The spectral overlap confirmed the FRET
mechanism for this molecule. In the absence of Cd2+, energy
transfer between the donor coumarin and the acceptor rhoda-
mine was impossible. But, when Cd2+ was introduced, the
spirolactam ring of the rhodamine moiety opened up through
the interaction of Cd2+ with two ‘O’ atoms of the coumarin
moiety and one ‘O’ atom of rhodamine. In this way, the whole
system of R1 turned a conjugated system by the influence of
Cd2+ and for this reason the energy of the coumarin moiety
transferred easily to the rhodamine moiety via ‘CvN’. Thus
the complexation between R1 and Cd2+ was established
through the FRET mechanism.
By the test strip experiment,22 R1 also displayed its sensing
applicability very easily without using any costly instrument to
detect Cd2+. For this experiment, R1 coated strips were pre-
pared by dipping the TLC plate in R1 (3 × 10−5 M) solution.
Firstly, R1 coated strips were colourless but after sinking in
Cd2+ solution, they turned into coloured strips. With the
increase in concentration of Cd2+, the test strips became more
intense pink colour (Fig. 6). So in future, the detection of Cd2+
could be possible without using any expensive instrument by
this method.
To establish the FRET mechanism, the emission spectra of
the donor coumarin moiety (λmax = 430 nm) and the absorp-
tion spectra of the acceptor rhodamine moiety (λmax = 555 nm)
were merged in a same graph, where a spectral overlap was
Probable binding mode of R1 with Cd2+
A peak of R1 was found at 600.2794 in mass spectroscopy
which was considered as the M + H+ peak and for the R1 : Cd2+
complex, it was obtained at 812.6. This value could be
assigned for the complex as [2R1 + 2Cd2+ + 2ClO4−]2+. The
Fig. 4 Influence of pH values on the fluorescence intensities of R1 and
the R1 : Cd2+ complex at 589 nm.
Fig. 6 Images of test strips from left to right: R1, R1 + Cd2+ (1.5 × 10−3 M),
R1 + Cd2+ (3 × 10−3 M), R1 + Cd2+ (6 × 10−3 M).
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Fig. 7 Probable binding mode of R1 with Cd2+
.
Fig. 9 Checking of viability of cervical cancer cells (HeLa S3 cells) in
the presence of R1. (Percentage of viability was calculated considering
the growth of the HeLa cells without R1 to be 100%).
probable binding mode of complexation is shown in Fig. 7,
where two Cd2+ ions were connected by two R1 molecules in
such a way that each Cd2+ was coordinated with two O centers
of the coumarin moiety of one R1 and one O center of the
spirolactam moiety of another R1. Thus a beautiful cavity was
formed by two R1 molecules for encapsulating two Cd2+ ions
and this binding mode was also supported by the theoretical
DFT calculation.
future this technique can also be applied for in vivo detection
of Cd2+ infected cells in the human body.
Cytotoxicity test
A cytotoxicity test of R1 was performed to measure its toxicity
level and practical applicability in the human body. The test
was carried out using the MTT assay of R1 (Fig. 9). This experi-
ment confirmed that about 90% of these cells remained viable
even upon exposure to a 10.0 μM concentration of R1 for 24 h.
Fluorescence bio-imaging study
The long wavelength sharp fluorescence emission of the
complex was successfully implemented in the intracellular
detection of poisonous Cd2+ in living HeLa S3 cells, which was
the real time most promising practical application of the
sensor. To identify Cd2+ in living HeLa S3 cells, R1 was incu-
bated into the cells for half an hour at 37 °C with 5.0 μM 5%
EtOH in H2O in Dulbecco’s modified Eagle’s medium
(DMEM). After that a 5.0 μM solution of Cd2+ was inserted into
the cell and incubated for another half an hour. Then the cells
were processed in a proper way and the fluorescence images
were captured. The cells containing only R1 did not show any
fluorescence image in the red channel of a fluorescence micro-
scope (Fig. 8c), but a bright red fluorescence image of the cells
incubated with both R1 and Cd2+ was observed (Fig. 8g). This
observation concluded that the sensor R1 could be used as a
real time practical kit for detecting Cd2+ in living cells and in
Quantum chemical DFT calculation
For clarification of the binding mode of R1 with Cd2+,
quantum chemical DFT calculation was performed using the
Gaussian 09 program with the help of the Gauss-View 5.0 visua-
lization program.23 The structures were optimized theoretically
using the B3LYP/6-311G+(d, p) basis set for R1 and the B3LYP/
LanL2MB basis set for the complex. The energy optimized
structure of R1 suggested that the coumarin moiety was
almost perpendicular to the xanthene part of the rhodamine
moiety to sustain stability of the system (Fig. 10a) and this
structure was also found to be similar to the crystal structure.
From the molecular electrostatic potential (MEP) diagram, it
was seen that the molecule is mainly divided into two colours,
where blue colour was the indication of positive regions and
red colour indicated the electronegative regions (Fig. 10b).
Electron rich red regions were mainly distributed around the
most electronegative centre of the molecule such as N and O
atoms. These regions were responsible for easy coordination
with the electron deficient Cd2+ ion. In the complex structure
(Fig. 10c), two R1 molecules were arranged in such a way that
it created a cavity for encapsulating two Cd2+ ions by the
strong electrostatic attraction with three O atoms for each
Cd2+. The optimization energy value of the R1 : Cd2+ complex
(−106 639.57 eV) suggested its very high stability compared to
R1 (−52 653.79 eV) (Table 1).
Fig. 8 Fluorescence bio-images of HeLa cells incubated with 5.0 μM R1
(left side) and the cells incubated with R1 with 5.0 μM Cd2+ (right side):
the corresponding bright-field transmission images (a & e); red channel
fluorescence images (c & g); blue channel images of DAPI stained cells
(b & f); overlay images of red and blue channels (d & h).
Theoretically calculated energy level diagrams displayed the
highest occupied molecular orbital (HOMO) and lowest un-
occupied molecular orbital (LUMO) of R1 and its complex
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Fig. 11 Energy level HOMO and LUMO diagrams of R1 and its complex
with Cd2+
.
recorded on a Bruker AV400 instrument using CDCl3 solvent
with TMS as an internal standard. ESI-MS measurements were
carried out using a microTOF-Q II 10337 mass spectrometer
instrument. IR spectra were recorded using a Spectrum 2000
Perkin-Elmer spectrometer. UV–Vis spectra and fluorescence
spectra were recorded using a UV-1800 Shimadzu spectro-
photometer (1.0 cm quartz cell) and a Perkin-Elmer LS 55
Fluorescence spectrometer, respectively. A Thermo Scientific
(FLASH 2000) CHN elemental analyser has been used for
elemental analysis. Melting points were determined using a
Remco hot-coil stage melting point apparatus and are un-
corrected. Fluorescence images of HeLa S3 cells were captured
using a Zeiss Axio Observer Z.1 fluorescence microscope.
Fig. 10 Optimized structure of (a) R1 (DFT/B3LYP/6-311G+(d,p)
method), (b) MEP map diagram of R1 and (c) R1 : Cd2+ complex by the
DFT/B3LYP/LanL2MB method.
along with their spatial distributions. The diagram of R1
showed that the xanthene moiety contained HOMO orbitals
around it whereas coumarin and spirolactam rings contained
LUMO orbitals (Fig. 11). On the other hand, HOMO orbitals
were mainly situated around the metal ion and LUMO orbitals
were placed around the coumarin moiety in the R1 : Cd2+
complex. The corresponding energy values suggested that both
the HOMO and LUMO of the R1 : Cd2+ complex were more
stable than that of R1. It was also observed that the energy gap
of HOMO and LUMO orbitals decreased after complexation
with Cd2+, which was the preferable condition for complexa-
tion of R1 with Cd2+ to show a bathochromic shift in the
UV-Vis absorption spectra.24
Cell culture and fluorescence imaging procedure
HeLa cells were grown on cover slips placed in 8 cell cultured
disks for 24 hours. Then the cells were treated with R1
(5.0 μM) for half an hour. The medium containing DMEM,
FBS, and PSM (penicillin–streptomycin) was then exchanged
with fresh medium. R1 stained cancer related HeLa cells were
then incubated with Cd2+ (5.0 μM) for 20 min. After that the
cells were washed with saline (1× PBS) and 4% PFA (para-
Experimental section
All reagents (AR grade) for synthesis were purchased from formaldehyde). After 20 minutes, the cells were washed again
Sigma-Aldrich chemicals commercially and used without twice with 1× PBS. The cells were then dried and DAPI was
further purification. All metal perchlorate salts were prepared used for staining the cells on the slides. Nail paint was used to
from the corresponding metal carbonates. Solvents were dried seal the cover slips mounted on the glass slides. Images were
following standard procedures. UV-grade CH3OH was used for obtained on
UV-vis and fluorescence titration. 1H NMR spectra were microscope.
a
Zeiss Axio Observer Z.1 fluorescence
Table 1 Optimization energy of R1 and its complexes with Cd2+ calculated using the DFT method
Comp.
Basis set
Optimization energy (eV)
EHOMO (eV)
ELUMO (eV)
ΔEHOMO–LUMO (eV)
R1
6-311G+(d,p)
LanL2MB
−52 653.79
−106 639.57
−5.2708
−2.4980
−1.6354
−0.2395
6.6354
2.2585
R1 : Hg2+
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Cell cytotoxicity assay
Synthesis of 6-nitro-7-amino-4-methyl-2H-chromen-2-one
(compound 3)
The cytotoxicity of R1 was measured in HeLa cells by the con-
ventional MTT assay.25 HeLa S3 cells in their exponential 6-Nitro-7-carbethoxyamino-4-methylcoumarin (1 g, 3.42 mmol)
growth phase were seeded in 96-well flat-bottom culture plates was refluxed in a mixture of conc. H2SO4 (4.0 ml) and acetic
at a density of 2.6 × 104 cells per well in 100 μl DMEM com- acid (3.0 ml) for 4 h. After cooling, the mixture was poured
plete medium (Himedia, India). The cells were allowed to stay into ice water (50 mL). By using NaOH followed by NH4OH
and develop for 24 h at 37 °C in a CO2 incubator (New solution, the resulting suspension was made a little basic.
Brunswick Scientific, USA). The medium was then exchanged Finally, the yellow precipitate was isolated and washed with
with 100 μl fresh medium containing various concentrations water to obtain a pure compound.
of R1 (0 to 100 μM). The assay was performed in triplicate for
each concentration. The cells were then incubated for 24 h,
Characteristic experimental data of compound 3
after which the culture medium was removed, and 100 μl of Melting point: above 250 °C; 1H NMR (400 MHz, CDCl3): δ
1 mg ml−1 MTT reagent in serum free media was added to 7.34 (d, J = 7.6 Hz, 1H), 6.56 (s, 2H), 6.00 (s, 1H), 4.30 (bs, 2H),
each well. After 4 h of incubation, MTT solubilization solution 2.33 (s, 3H).
(10% HCl–10% Triton X 100 in anhydrous isopropanol) was
added to each well. The resultant solution was calculated
Synthesis of R1
spectrophotometrically using a microplate reader at 570 nm Rhodamine b (125 mg, 0.261 mmol) was taken in dry 1,2-
and 630 nm. The cytotoxic effect of each treatment was dichloroethane (10.0 ml) and POCl3 was added dropwise into
expressed as a percentage of cell viability relative to the this solution for 15 minutes. Then the reaction mixture was
untreated control cells. [MTT = (3-(4,5-dimethylthiazol-2-yl)- refluxed for 6 h. After removal of the solvent, the reaction
2,5-diphenyltetrazolium bromide), a yellow tetrazole].
mixture was used directly for the next step. Compound 3
(68.96 mg, 0.392 mmol) and Et3N (1.0 ml) were taken in aceto-
nitrile (6.0 ml) for 10 minutes. Now, this solution was added
dropwise to the previous reaction mixture and then refluxed
for 8 h. By checking TLC, the completion of the reaction was
monitored. After evaporating the solvent under vacuum, the
crude residue was purified by column chromatography using
40% EtOAc in pet ether to obtain pure R1 (solid, white colour,
27% yield).
Synthesis of 3-carbethoxyaminophenol (compound 1)
Ethylchloroformate (0.87 mL, 9.16 mmol) was added dropwise
in a stirred solution of m-aminophenol (1.0 g, 9.16 mmol) in
ethylacetate (15 mL) solvent. Instantly, a white precipitate
appeared and consequently, the reaction mixture was stirred at
room temperature for 2 h. The precipitate of amine hydro-
chloride was filtered and it was evaporated under reduced
pressure to get the colorless crystal of the desired Characteristic experimental data of R1
compound.26
1
Melting point: 275–280 °C; H NMR (400 MHz, CDCl3): δ 7.99
(d, J = 8.0 Hz, 1H), 7.48 (m, 2H), 7.40 (s, 1H), 7.38 (s, 1H), 7.25
(d, J = 2.4 Hz, 1H), 7.11 (d, J = 7.2 Hz, 1H), 7.02 (s, 1H), 6.61 (d,
J = 8.8 Hz, 2H), 6.32 (s, 2H), 6.27 (dd, J = 6.4 Hz, 2H), 3.49 (s,
3H), 3.19 (q, J = 7.2 Hz, 8H), 1.15 (t, J = 7.2 Hz, 12H); 13C NMR
(100 MHz, CDCl3): δ 168.4, 161.2, 153.9, 153.7, 152.7, 148.9,
140.8., 133.6, 129.3, 128.2, 124.4, 123.8, 121.5, 117.5, 114.2,
113.2, 108.2, 105.9, 97.8, 67.6, 44.3, 18.5, 12.6; ESI-MS
(+ve mode, m/z): found 600.2794 for M + H+ (Calc. for
C38H37N3O4 is 600.2857); FT-IR (KBr, cm−1): 2972 (Ar C–H
Characteristic experimental data of compound 1
1
Melting point: 94–96 °C; H NMR (400 MHz, CDCl3): δ 7.32 (s,
1H), 7.12 (t, J = 8.0 Hz, 1H), 6.64 (d, J = 7.6 Hz, 2H), 6.55 (d, J =
7.6 Hz, 1H), 4.21 (q, J = 7.2 Hz, 2H), 1.30 (t, J = 7.2 Hz, 3H).
Synthesis of 7-carbethoxyamino-4-methylcoumarin
(compound 2)
3-Carbethoxyaminophenol (610 mg, 3.15 mmol) was taken in a str.), 1729 (CvO str.), 1698 (amide CvO str.), 1614 (Ar CvC
round bottom flask and ethylacetoacetate (0.48 mL, str.), 1265 (aliphatic C–H str.), 1221 (C–N str.), 1119 (C–O–C
13.25 mmol) was added into it. After that, 70% sulfuric acid str.); Anal. Calcd for R1 (C38H37N3O4): C, 76.10; H, 6.22; N,
(9 mL) was added and the reaction mixture was stirred for 4 h 7.01; O, 10.67. Found C, 76.02; H, 6.25; N, 7.00; O, 10.73.
at room temperature. After 4 h, the reaction mixture was
Characteristic experimental data of complexes
poured into an ice–water mixture (50 mL). The precipitate was
then formed, which was filtered to get 7-carbethoxyamino-4- ESI-MS (+ve mode, m/z) of the R1 : Cd2+ complex; found 812.13
methylcoumarin.26
for [2R1 + 2Cd2+ + 2ClO4−]2+.
Characteristic experimental data of compound 2
Conclusions
1
Melting point: 184–187 °C; H NMR (400 MHz, CDCl3): δ 7.50
(d, J = 8.4 Hz, 1H), 7.42 (d, J = 4.8 Hz, 1H), 7.37 (s, 1H), 6.87 (s, In summary, a new coumarin linked rhodamine based sensor
1H), 6.18 (s, 1H), 4.25 (q, J = 7.2 Hz, 2H), 2.39 (s, 3H), 1.32 (t, J was developed for the detection of highly toxic Cd2+ with
= 7.2 Hz, 3H).
superb selectivity and sensitivity. Single crystal X-ray analysis
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exhibited the actual structure of the molecule. Instant naked
eye detection of Cd2+ along with a new absorption peak in the
visible region was observed. Probably, this is one of the latest
reports of a FRET based turn-on fluorescence sensor system
for Cd2+. The nontoxic nature of the molecule in human cells
increased its importance to practical life and it was success-
fully applied in intracellular in vivo detection of poisonous
Cd2+ in living HeLa S3 cells. The probable binding mode and
spatial arrangement of R1 and R1 : Cd2+ complex were calcu-
lated by quantum chemical DFT calculation. The detection
level of Cd2+ was found to be 10.1 nM, hence this molecule
could also be used for the detection of excess Cd2+ from the
environment.
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