Unusual absence of FRET in triazole bridged coumarin-hydroxyquinoline, an active sensor forHg2+detection

Article abstract of DOI:10.1039/d0pp00140f

A triazole-bridged coumarin conjugated quinoline sensor has been ‘click’-synthesized by Cu(i) catalyzed Huisgen cycloaddition, and it exhibited high selectivity for toxicHg²⁺. Surprisingly, no evidence of energy transfer from the quinoline moiety to coumarin has been found, substantiated by time-resolved fluorescence study. The possible binding mode of this sensor toHg²⁺has been establishedviaNMR study, steady-state and time-resolved fluorescence spectroscopy, which is further supported by TDDFT calculations. The sensor has been found to be cell membrane permeable and non-toxic, and hence is suitable for intracellularHg²⁺detection.

Full text of DOI:10.1039/d0pp00140f

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Unusual absence of FRET in triazole bridged
coumarin–hydroxyquinoline, an active sensor
Cite this: DOI: 10.1039/d0pp00140f
for Hg2+ detection†
b
Surajit Mondal,a Niladri Patra,*a Hari Pada Nayek, a Sumit K. Hira,
a
Soumit Chatterjee *a and Swapan Dey
*
A triazole-bridged coumarin conjugated quinoline sensor has been ‘click’-synthesized by Cu(I) catalyzed
Huisgen cycloaddition, and it exhibited high selectivity for toxic Hg2+. Surprisingly, no evidence of energy
transfer from the quinoline moiety to coumarin has been found, substantiated by time-resolved fluor-
escence study. The possible binding mode of this sensor to Hg2+ has been established via NMR study,
steady-state and time-resolved fluorescence spectroscopy, which is further supported by TDDFT calcu-
lations. The sensor has been found to be cell membrane permeable and non-toxic, and hence is suitable
for intracellular Hg2+ detection.
Received 10th April 2020,
Accepted 8th July 2020
DOI: 10.1039/d0pp00140f
rsc.li/pps
On the other hand, the use of suitable fluorophores for the
Introduction
detection of Hg2+ is beneficial due to their sensitivity, simpli-
Mercury contamination through the cell membrane creates city and low cost.14–16
severe harmful effects on human health.1,2 Organic methyl
The basis of fluorometric detection of metal ions lies on
mercury (CH3Hg+), generated from both elemental and ionic the nature of metal ion binding with the fluorophore, resulting
mercury by bacterial action, causes several adverse effects to in the enhancement or quenching of the quantum yield of the
the central nervous system, endocrine system, kidneys, skin latter by virtue of various associated photophysical processes.
and DNA.3–7 Mercury ion is also highly responsible for environ- In general, the possible phenomena related to these kinds of
mental pollution. Therefore, quantitative recognition of adducts can be fluorescence resonance energy transfer (FRET),
elemental or ionic mercury is imperative in the current scen- photoinduced electron transfer (PET), through bond energy
ario. A variety of techniques like atomic absorption spec- transfer (TBET) etc. Coumarin derivatives have been widely
trometry (AAS) and inductively coupled plasma-mass spec- used for designing sensory systems due to their sensible
trometry (ICPMS)8,9 have been used for the qualitative detec- photophysical properties with a large Stokes shift and visible
tion of Hg2+ in the recent past. In addition to this, a supramo- emission wavelength.17–19 They are frequently used in the
lecular gel-based sensor,10,11 especially Hg2+ promoted sol–gel development of chemosensors and chemodosimeters due to
transformation, has been considerably used. Yet this strategy their high solubility in polar protic solvents.20–25 In addition,
has suffered from selectivity issues.12,13 However, these pro- 8-hydroxyquinoline (8-HQ) has also been extensively used in
cesses are expensive, time-consuming and require specific designing chemosensors for the detection of metal ions such
technical skills for machine operation and are only limited to as Cu2+,26 Cd2+,27 Mg2+,28 Zn2+,29,30 Al3+,31 and Hg2+,32 due to
scientific research.
their photostability and strong complexation ability. For
example, Ho and co-workers have reported three coumarin
based triazole fluorophores for the detection of Hg2+.33 A con-
formationally constrained quinoline coupled coumarin fluoro-
phore moiety has also been reported for the selective detection
of Al3+.34,35 However, in all these cases, the triazole was not
directly attached to coumarin. Therefore in continuation of
our recent development on designing chemosensors,36–40 we
present, herein, the synthesis and characterization of a three
triazole cross-linked simple chemosensor (R1) which exhibits a
large Stokes shift and quick response to Hg2+ with high sensi-
tivity and excellent selectivity.
aDepartment of Chemistry, Indian Institute of Technology (Indian School of Mines),
Dhanbad, Jharkhand, India. E-mail: swapan@iitism.ac.in
bDepartment of Zoology, The University of Burdwan, Burdwan, West Bengal 713104,
India
†Electronic supplementary information (ESI) available: Characterisation data of
all compounds, ESI mass and FT-IR of complex, binding constant calculation
table, LOD calculation, reversibility test and bar diagram, synthetic procedure of
coumarin amine. CCDC 1944133, 1904920 and 1904921. For ESI and crystallo-
graphic data in CIF or other electronic format see DOI: 10.1039/d0pp00140f
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coumarin moiety with torsional angles of 11.15 (1)°, 6.72 (1)°
and 11.23 (2)°, respectively and for all the compounds the tri-
azole rings were found to be coplanar to coumarin.
Results and discussion
Synthesis of R1, R2 and R3
All three sensors (R1, R2 and R3), in which a 1,2,3-triazolyl
ring is flanked by coumarin and 8-hydroxyquinoline (8-HQ)
(for R1), phenyl (R2) and naphthalene (R3) moieties respect-
ively, had been synthesized based on the synthetic variations
of the “click” reaction. The excellent complexation properties
of the resulting electron-rich 1,2,3-triazolyl moiety,41–45 facili-
tated by Cu(I)-catalyzed 1,3-dipolar cycloaddition of terminal
alkynes and azides. Coumarin azide has been synthesized fol-
lowing a known procedure and used as a precursor for the
syntheses of three different compounds R1, R2 and R3.46 The
synthetic routes of the sensors are shown in Scheme 1 and the
detailed procedures are given in the Experimental section. The
single crystals of the sensors were generated from a different
proportion of the ethyl acetate–pet ether mixture. Single-crystal
X-Ray studies revealed that both R1 and R3 were triclinic
Steady-state spectroscopy
The solutions of all three sensors (R1, R2 and R3) were pre-
pared in ethanol and were found to exhibit green fluorescence
upon long-wavelength UV exposure. Upon the addition of
Hg2+, the fluorescence of R1 was dramatically quenched. Other
metal ions such as Li+, Mg2+, Al3+, Fe3+, Co2+, Ni2+, Zn2+, Ag+,
Cd2+, Ba2+, and Pb2+ did not show any remarkable response
(Fig. 3). However, with Cu2+, the emission intensity was
quenched, but, to a much lesser extent than Hg2+. The com-
plete absorption and emission studies are discussed in the fol-
lowing paragraphs. R2 and R3, on the other hand, did not
show any detectable changes upon the addition of Hg2+
(Fig. S1, ESI†). R1 (3.4 × 10−5 M in EtOH) displayed an absorp-
tion maximum at 412 nm and another strong benzenoid band
at around 250 nm. After the gradual addition of Hg2+ up to
10.0 equivalents, the peak position of the 412 nm band
remained unchanged. Still, the optical density reduced to half
of that of pure R1 (Fig. 2a), suggesting an alteration in the
molar absorptivity coefficient of the R1–Hg2+ complex. On the
other hand, R2 and R3 solutions were almost non-responsive
upon treatment with Hg2+ (Fig. S1, ESI†).
ˉ
systems with the P1 space group, while R2 was monoclinic
with the P21/c space group. The ORTEP diagram (Fig. 1) con-
firmed that the 8-HQ (R1), phenyl (R2) and naphthalene moi-
eties (R3) were oriented almost perpendicular to that of the
The fluorescence study of R1 showed an emission
maximum at 485 nm, when excited at 410 nm. No excitation
dependent emission wavelength shift was observed for R1. The
fluorescence quantum yield (Φf) of R1 was found to be 0.15
using Lucifer yellow CH (Φf = 0.27 in water) as the reference.
When the Hg2+ solution was added gradually up to 10.0
equivalents, fluorescence quenching was caused, thereby
resulting in a 16 fold decrease in the quantum yield (Φf =
0.009). The complexation of R1 with Hg2+, creating a drastic
change in the fluorescence intensity, can be considered to be
an on–off sensing response. This analysis suggested the better
binding ability of R1 to Hg2+. The limit of detection (LOD)
calculation47,48 using fluorescence titration data showed that
Scheme 1 Reagents and conditions:(a) CuSO4, sodium ascorbate,
DCM : EtOH (1 : 1), rt, 12 h.
Fig. 1 Single crystal structure of R1 (a) ORTEP along b and (b) packing
diagram along a, R2 (c) ORTEP and (d) packing diagram along a and of
R3 (e) ORTEP and (f) packing diagram along a (30% probability).
Fig. 2 (a) Absorption and (b) fluorescence titration spectra of R1 with
Hg2+.
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Gaussian in nature, with the maximum intensity being
between pH 6 and 7. In contrast, for the R1–Hg2+ complex, the
sharp peak in between pH 9 and 12, centered around pH 10,
indicated that the stability of the R1–Hg2+ complex remained
unaffected in the entire acidic region and well above neutral
pH. Actually, for the whole pH range of 5–9, R1–Hg2+ remained
quite stable and thus could be found to be compatible with
the human physiological system.
Time-resolved fluorescence measurement
Upon gradual addition of Hg2+ to R1, the emission intensities
at 420 nm remained unchanged, while a steady quenching was
observed at 485 nm. Fluorescence decays were measured using
a time-correlated single photon counting system from IBH,
using 340 nm (λmax of the quinoline absorption band) as the
excitation wavelength. The FWHM of the excitation pulse was
1.23 ns, and a temporal resolution of 7.14 ps was used.
Fluorescence decay traces were recorded at 485 nm which
showed that upon addition of Hg2+ (R1 : Hg2+ = 1 : 9), the
single exponential decay of R1 (Fig. 5) transformed into a bi-
modal form with the faster component found to be of the
order of 600 ps and the slower one 2.5 ns (similar to the R1
lifetime at 485 nm). Upon gradual addition of Hg2+, the faster
component (600 ps) remained unchanged, but its contribution
increased steadily (Table T2, ESI†), while the slower com-
ponent (the emissive state) became even slightly slower with
Fig. 3 Comparative fluorescence titration spectra of R1 with Hg2+ and
other metal ions in EtOH.
R1 could be used to sense very low-level concentration
(1.72 × 10−7 M) of Hg2+ (Fig. S7, ESI†). A high binding
efficiency of R1 to Hg2+ (Ka = 5.6 × 104 M−1) found from the
Benesi–Hildebrand linear regression analysis49,50 also substan-
tiated the significant affinity of R1 towards Hg2+ (Fig. S6, ESI†).
The addition of other metal ions (except for Cu2+ and Zn2+)
up to 10.0 equivalents did not show any detectable change of
quenching in the emission spectra of R1. However, the extent
of quenching in the presence of both the ions was found to be
much less than Hg2+ (Fig. 3). On the other side, emissions of
R2 and R3 remained unresponsive upon the addition of either
Hg2+ or other metal ions (Fig. S1, ESI†).
its contribution being decreased by ∼10% (up to R1 : Hg2+
=
The reversibility of complexation between the sensor and
the metal ion was carried out using EDTA as a chelating agent.
Upon the addition of 1.0 equivalent of EDTA, the emission
intensity gradually increased, leading to the formation of the
EDTA–Hg2+ complex (Fig. S8, ESI†). However, the intensity
decreased again upon the addition of excess Hg2+, confirming
the reversible complexation nature of the sensor with Hg2+. To
reproduce the reversible complexation of the receptor, the
same experiment was performed using I−, which showed a
similar result (Fig. S9, ESI†). The pH-responsive emissions of
R1 and R1–Hg2+ complex were performed in order to have a
comparison with the human physiological system. The respect-
ive intensities of the emission maxima (485 nm) were plotted
against pH (Fig. 4). For R1, the correlation was found to be
1 : 1). It was suggested that upon gradual addition of Hg2+,
more R1–Hg2+ complex, involved in the non-radiative process,
was formed and the increase in population was reflected by
the rise in the contribution of the faster component, while the
complex formation somewhat stabilized the emissive state (the
lifetime of the emissive state was 3.3 ns). Similarly, steady-state
and time-resolved studies were performed using Cu2+, Cd2+
and Zn2+. Upon the addition of Cu2+, substantial quenching
was observed in the steady-state emission of R1. However, the
decay traces of pure R1 were unaltered (Fig. S4, ESI†), indicat-
Fig. 4 Influence of pH of R1 (λem = 485 nm) and R1–Hg2+ complex Fig. 5 Fluorescence decay traces of R1 with and without Hg2+ (λex
=
(λex = 410 nm).
315 nm and λem = 485 nm).
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ing that an ultrafast pathway may be associated with the
quenching process. However, there was no change in the emis-
sion spectra of R1 upon addition of Cd2+, but decay traces
were found to be faster in R1 itself. This hints about the poss-
ible missing out of the predominant component, unresolvable
by our time-resolved experimental setup. On the other hand,
steady-state and time-resolved data of R1, in the presence of
Zn2+corroborated each other and demonstrated similar obser-
vations found in the case of Hg2+
.
It was also imperative to perform time-resolved fluorescence
study of the sensor to establish if PET (photoinduced electron
transfer) or FRET (fluorescence resonance energy transfer) was
responsible for the excited state dynamics in order to under-
stand the origin of the quenching process in the presence of
Hg2+. The sensor R1 is composed of quinoline and coumarin
moiety. As the emission of quinoline overlaps with the absorp-
tion band of coumarin (Fig. 6a), it is expected that energy
transfer will take place via the dipole–dipole interaction from
quinoline to coumarin. This has been proposed for a number
of incidents, bearing similar moieties.41 However, it was essen-
tial to check if there is indeed an energy transfer from quino-
line to coumarin. For an effective FRET process, upon addition
of coumarin to the quinoline solution, the fluorescence inten-
sity of the former should increase with a concomitant decrease
in the fluorescence intensity of the latter. In a steady-state
experiment upon constant addition of coumarin to quinoline,
the fluorescence intensity of quinoline indeed decreased with
an increase in coumarin emission (Fig. 6b). In time-resolved
fluorescence, on the other hand, FRET would have been estab-
lished if the fluorescence decays of quinoline become faster,
while those of coumarin became slower when the latter was
added to the former. However, no such characteristic peak was
observed in this particular case. Quinoline decay at its emis-
sion maximum (420 nm) was bimodal, whereas coumarin (λem
Fig. 7 Fluorescence decay traces of quinoline with varying concen-
trations of coumarin (λex = 340 nm).
= 485 nm) decay was found to be single exponential in nature
(Table T1, ESI†). On the addition of coumarin and monitoring
at the quinoline emission maximum (420 nm), faster decays
were not observed and while monitored at the coumarin emis-
sion maximum (485 nm), the decays remained unchanged
(Fig. 7). To elaborate, upon gradual addition of coumarin,
while keeping the quinoline concentration constant, and
monitoring at quinoline emission, the longer component of
the decay traces became slower and the shortest component
was unaffected (Fig. 7). However, there was no change in the
decay traces monitored at coumarin emission with varying
concentrations of coumarin. This nullifies the possibility of
FRET and suggests that if there is indeed any energy transfer
through the dipole–dipole interaction, it is within an ultrafast
time scale and thus cannot be resolved by our system.
However, the computational calculation (explained later under
‘quantum chemical TDDFT calculation’ heading) reveals PET
to be the origin of the non-radiative channel for this com-
pound. The decay traces of R1, monitored at 420 nm and
485 nm respectively (i.e. at the quinoline and coumarin emis-
sion maximum respectively) were found to be following the
characteristics of the quinoline–coumarin (2 : 1) mixture (at
respective emission wavelengths), suggesting almost unaltered
nature of quinoline and coumarin in R1 (Fig. S5, ESI†).
However, it was also found that in R1, the quinoline moiety
got stabilized with respect to free quinoline, substantiated by a
longer lifetime (Table T1, ESI†), indicating that triazole
bridged quinoline experiences more rigidity.
Stern–Volmer plot
In order to find out the efficiency and the nature of the
quenching process in the presence of Hg2+, Stern–Volmer plots
were obtained from the steady-state and time-resolved fluo-
rescence studies. K0SV, the steady-state rate constant, and the
corresponding k0q were calculated from the lower part of the
S–V plot (Table 1, Fig. S3, ESI†) using eqn (1). However, it was
Fig. 6 (a) Normalized absorption and emission spectra of quinoline and
coumarin amine in ethanol (black and blue straight lines for absorptions
and dotted lines for emissions of quinoline and coumarin respectively).
(b) Steady state emission spectra of quinoline with increasing concen-
tration of coumarin.
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Table 1 The values of S–V constant and bimolecular quenching rate
constant
S–V constant K0sv
Bimolecular quenching rate
Sr. no.
(×105 M−1
)
constant kq (×1014 mol−1 L s−1
)
1
2
2.3
1.5
0.958
0.625
Fig. 9 1H-NMR spectra of R1 with varying amounts of Hg2+ in d6-
DMSO.
Fig. 8 Modified S–V plot.
Fig. 10 Probable binding mode of complexation of R1 with Hg2+.
found that at higher concentration of Hg2+, S–V plot deviated
from linearity (the inset plot of Fig. S3, ESI†), indicating the
onset of static quenching. Therefore, the dynamic quenching
constant (KSV) and the bimolecular quenching rate constant
(kq, as KSV = kqτ0) parameters were obtained using the linear
part of the modified S–V equation (eqn (2)) following the
‘sphere of action static quenching model’51 (Fig. 8). The
dynamic quenching constant and bimolecular quenching rate
constants are given in Table 1.
due to the paramagnetic effect which overwhelmed the spec-
trum with increasing the concentration of Hg2+ 52
.
Possible binding mode
The possible binding mode of R1 is depicted in Fig. 10, where
the suitable cavity for Hg2+ encapsulation was formed by the
electronegative ‘O’ atom, ring ‘N’ atom of quinoline and one
‘N-3’ atom of triazole. On the other hand, no cavity for Hg2+
was formed by R2 and R3 due to the absence of quinoline ‘N’.
DFT calculations and the NMR study also supported the prob-
able binding mode of R1. The FT-IR spectra of R1 and R1–
Hg2+ were obtained (ESI†). Upon addition of Hg2+ to R1, the
C–H band of triazole was shifted from 3133 cm−1 to 3120 cm−1
and the C–O band of quinoline was shifted sharply from
1129 cm−1 to 1060 cm−1 indicating the participation of triazole
and the ether subunit in R1–Hg2+ complexation. In addition to
that, a new band was found at 610 cm−1, which again
suggested the participation of quinoline ‘N’ in complexation.
The MS value of the sensor R1 was detected at m/z 441.1801,
whereas the value of m/z 678.1401 was regarded as [R1 + Hg +
2H2O–H].
I0
I
¼ 1 þ KSV½Qꢀ
ð1Þ
ð2Þ
I
I0
ꢀ ꢁ
1 ꢁ
I
ð1 ꢁ wÞ
¼ KSV
þ
½Qꢀ
I0
½Qꢀ
NMR study
The 1H NMR study of R1 with Hg2+ at room temperature
(298 K) has been performed to have an insight into complexa-
tion. Fig. 9 represents the distinguished chemical shift of the
proton adjacent to quinoline N (Ha), methylene proton (Hb),
and triazole ring hydrogen (Hc) at δ 8.85, 5.46, and 8.72 ppm
respectively (as mentioned in Fig. 10). Upon addition of 0.5
Quantum chemical (TDDFT) calculations
equivalent of Hg2+, the aforementioned peaks shifted to δ 8.97, Quantum chemical calculations were performed to understand
5.70, and 8.86 ppm, respectively. Upon addition of 1.0 equi- the nature of metal binding with R1. The geometrical optimi-
valent of Hg2+, the peaks again showed a downfield shift from zations of R1 and its complex were carried out using the
the previous observation, and the respective values were δ 9.04, B3LYP functional, and the 6-311g** basis set was used for C,
5.73, and 8.87 ppm. Therefore, it can be revealed that both the H, N, and O atoms, whereas LANL2DZ effective core potential
quinoline and triazole moieties actively take part in the com- (ECP) was used for Hg2+, as implemented using the TeraChem
plexation. The signal broadening occurred after a certain point Software.53–55 All calculations were performed in the gas
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phase. The initial coordinates for the geometry optimization
were obtained from ab initio molecular dynamics (AIMD) simu-
lations. AIMD simulations were performed using the 3-21g
basis set at the HF level and the temperature was set to 300 K.
The binding energy (EB) between the Hg2+ and R1 was calcu-
lated using the following equation:
2þ
EB ¼ Ecomp ꢁ ER1 ꢁ EHg
2þ
where, Ecomp, ER1, and EHg are the ground state optimized
energies of the R1–Hg2+ complex, R1, and Hg2+, respectively.
Two binding modes have been observed during the geometry
optimizations, as shown in Fig. 11. The binding mode A was
found to be more stable than the binding mode B by
20.867 kcal mol−1 (see Table T3, ESI†).
In order to reveal the intricate details of the electronic
structure of both the binding modes, the excited state highest
occupied molecular orbital (HOMO) and the lowest unoccu-
pied molecular orbital (LUMO) for the R1 and R1–Hg2+ com-
plexes (mode A and mode B) under vacuum were calculated
from their optimized structures by the TDDFT method using
the 6-311g** basis set, as shown in Fig. 12. In the case of R1,
the HOMO was spread mainly over coumarin and triazole,
whereas the LUMO is partially on the coumarin and triazole
ring. After the complexation with Hg2+ (binding mode A), the
HOMO now resided on the Hg2+ ion and partially on quinoline
and triazole nitrogen atoms, whereas the LUMO was over the
coumarin ring. The energy difference between the HOMO and
the LUMO of R1 and R1–Hg2+ (binding mode A) were
83.207 kcal mol−1 and 14.495 kcal mol−1, respectively. These
results showed that the binding of Hg2+ with R1 (mode A)
lowered the energy gap between the HOMOand the LUMO of
the complex and stabilized the system. The energy gap of the
R1–Hg2+ complex on the A-mode (14.495 kcal mol−1) is higher
than that on the B-mode (7.216 kcal mol−1), revealing that the
excitation of Hg2+ ion attached to R1 on the B-mode occurred
more easily than that on the A-mode. This indicates that the
R1–Hg2+ complex on the A-mode has higher stability than on
Fig. 12 Excited-state HOMO–LUMO diagrams; (a) free sensor, R1; (b)
R1–Hg2+ complex on A binding mode; (c) R1–Hg2+ complex on B
binding mode.
the B-mode at the ground state which is also realized by the
binding energy difference between the two modes (Table T3,
ESI†). The HOMO to LUMO excitation of R1 was found to be
charge transfer with the largest CI coefficient of −0.686911.
To confirm the exact mechanism of fluorescence quenching
upon addition of Hg2+, the TDDFT study was carried out.
Generally, the PET-based fluorescent probes contained three
units as fluorophore and chelator, which was separated by the
third unit i.e., a short aliphatic spacer. As shown in Fig. S12
(ESI†), the LUMO of the electron-poor chelator (quinoline
moiety) lies just below the LUMO of the excited fluorophore
(coumarin moiety) so that the singly excited LUMO of the cou-
marin moiety donated an electron to the LUMO of quinoline,
an example of typical d-PET mechanism.56 Upon complexation
of R1 with Hg2+, the chelator became more electron-poor and
the corresponding energy of the LUMO of the chelator was
further reduced, and thus the PET was accelerated, leading to
fluorescence quenching.
Biological application
Cell culture. U-2 OS (ATCC® HTB-96™) cells were grown in
RPMI-1640 supplemented with 10% FBS at 37 °C under 5%
CO2. Cells were placed in a 48 or 96 well culture plate and
allowed to adhere for 48 hours before experimentation.
Cell viability assay. First, stock solutions of R1 and R2 were
prepared in H2O/ethanol solution (1 : 1, v/v) in such a way that
during experimentation, it was further diluted in water to
maintain the ratio of 1 : 5 (stock : water). This minimized the
cellular toxicity caused by ethanol.
The methyl thiazolyl tetrazolium (MTT) assay has been
used to measure the cytotoxic potential of R1 and R2 in U-2 OS
cells. These cells were seeded in a 96-well cell-culture plate.
Various concentrations (5, 10, 25 and 50 µM) of R1 and R2
Fig. 11 Optimized structures of (a) sensor, R1; (b) R1–Hg2+ complex
on A mode: (c) R1–Hg2+ complex on B mode.
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were added to the cells. Then, these were incubated at 37 °C detectable fluorescence (Fig. 14d–f). However, upon treatment
under 5% CO2 for 24 hours. The MTT solution (10 µL, 5 mg with R1 or R2, a bright green fluorescence was observed in the
mL−1) was added to each well and incubated at 37 °C under U-2OS cells (Fig. 14a–c & 15a–e). Overlaying of fluorescence
5% CO2 for 4 hours. The MTT solution was removed and the
yellow precipitates (formazan) observed in plates were dis-
solved in 200 µL of acidic isopropanol. A Multiskan™ GO
Microplate Spectrophotometer was used to measure the absor-
bance at 570 nm for each well. The viability of cells was calcu-
lated according to the following equation:
%Cell viability ¼ ½experimental OD570=control OD570ꢀ ꢂ 100
Fluorescence imaging in cells
U-2 OS cells were placed in a 48 well cell-culture plate and
allowed to adhere for 48 hours. To assess the Hg2+ sensing
capacity of R1 and R2, initially uptakes of R1 and R2 were pre-
pared in phosphate-buffered saline (PBS). Then the cells were
treated with 35 µM of R1 and R2 respectively and incubated
for 2 h. After the culture medium was removed, the treated
cells were washed with PBS before observation. Fluorescence
images of the cells were obtained using the EVOS® FL Cell
Imaging System. Then Hg(ClO4)2, dissolved in sterile PBS (pH
= 7.4), was added at 1.5 μM concentration to the desired experi-
mental wells and incubated at 37 °C for another 30 minutes.
The treated cells were again washed with PBS to wash out the
remaining metal ions. Again, the fluorescence images of the
cells were obtained in an interval of 30 min and 60 min using
Fig. 14 Fluorescence images of U-2-OS cells treated with R1 (a–c) and
the EVOS® FL Cell Imaging System (Life Technologies, USA).
The potential of 8-hydroxy quinoline appended coumarin–tri-
azole chemosensor (R1) for sensing Hg2+ in living cells was
Hg2+ after 0 min (d–f), 30 min (g–e) & 60 min ( j–l). (Left) Bright field
images; (middle) fluorescence images in green channel and (right) over-
laid images. Magnification 400×.
also examined. The MTT assay for U-2 OS (osteosarcoma cell
line) cells was used to determine the cyto-compatibility of R1.
The cell viability was found to be more than 80% (Fig. 13) fol-
lowing treatment with the compound for 24 hours. This result
indicated that R1 was safe and induced low cytotoxicity even at
higher concentration (50 µM).
Imaging analyses were performed for demonstrating the
bio-distribution of the compound R1 and sensing Hg2+ in
living cells using the EVOS® FL Cell Imaging System. U-2 OS
cells that were incubated with Hg2+ only did not produce any
Fig. 15 Fluorescence images of U-2-OS cells treated with R2 (1–e) and
R2+Hg2+ (f–j). (a–f) bright field images, images taken in blue (b–g) and
Fig. 13 MTT assay of U-2-OS cells treated in the presence of R1 and R2
(0–50 µM) incubated at 37 °C for 24 hours.
green (c–h) channel, images of U2OS cells after merging all the chan-
nels (d–i) and after overlay with a bright field (e–j). Magnification 400×.
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and bright-field images confirmed that the fluorescence G8910B. Melting points were determined using a Remco hot-
signals were localized in the intracellular areas, which indi- coil stage melting point apparatus.
cated efficient cell-membrane permeability for R1 and R2. The
Preparation of 3-azido-7-(diethylamino)-2H-chromen-2-one
(compound 1)
specificity of R1 towards Hg2+ was demonstrated by perform-
ing similar experiments using Hg2+. Upon the addition of
Initially, 7-diethylamino-3-amino coumarin was prepared accord-
ing to the reported procedure46 (see details in the Experimental
section, ESI†), and then it (100 mg, 0.43 mmol) was dissolved
slowly in aq. HCl (17.2%, 4.0 mL) at room temperature. After
being cooled at 0 °C–5 °C and consequent addition of NaNO2
solution (30 mg, 0.43 mmol), the reaction mixture was continu-
ously stirred for 1.0 hour at 0–5 °C. This was followed by the
addition of potassium acetate (2.0 g) in water (5.0 mL) to adjust
the pH of the resulting solution to 4.0. Sodium azide (57 mg,
0.88 mmol) was added in portions at 0–5 °C, and the mixture
was stirred for another five hours maintaining the same temp-
erature. Then, the precipitated product was rapidly filtered,
washed with ice-cold water (10.0 mL) and dried under vacuum
to yield the final product as a yellow solid (yield: 84%).
Hg2+ treated with R1 and R2, the fluorescence images were
obtained at intervals of 30 min and 60 min. Due to the pres-
ence of Hg2+, the bright green fluorescence intensity of R1
was reduced in a time-dependent manner (Fig. 14g–i). After
60 minutes of addition, there was almost no or very low level
of fluorescence (Fig. 14j–i). But in the case of R2 the attenu-
ation of fluorescence intensity in U-2-OS cells was found to be
negligibly lower than that in the case of R1 (Fig. 15f–j). In con-
trast to R1, R2 displayed blue fluorescence rather than green
colour at a concentration of 35 µM being tested (Fig. 14b and
15g). These observations demonstrated that R1 was much
more selective than R2 to detect Hg2+ in living cells.
Conclusions
General procedure for the preparation of compound 2, 3, and 4
For the preparation of compound 2, 3, and 4, to the solutions
of 8-hydroxy quinoline, benzyl alcohol, and 1-naphthol
respectively (1.0 equiv.) in acetone (20 mL), anhydrous potass-
ium carbonate (1.5 equiv.) was added and the solution was
stirred at room temperature under an argon atmosphere.
Propargyl bromide (1.5 equiv.) was added to the solution and
the resulting mixture was refluxed for 5 h. After completion of
the reaction, the solvent was dried, and the crude solid was
partitioned between ethyl acetate and water. The ethyl acetate
layer was collected and the process was repeated three times.
The combined organic extract was dried over anhydrous
Na2SO4, concentrated under vacuum and purified by column
chromatography.
A hydroxyquinoline appended coumarin–triazole conjugated
chemosensor (R1) has been developed for selective sensing of
Hg2+, which exhibits a turn-off response. The study of the
excited state processes of this compound revealed an unusual
PET (photoinduced energy transfer), and not FRET (Förster reso-
nance energy transfer), unlike similar types of compounds, to be
the key associated photophysical process. This chemosensor can
potentially be applied for the fluorescence imaging of living
cells. Moreover, it exhibits low cytotoxicity and, therefore, can be
used for in vivo detection of Hg2+. The ‘N’ atom of quinoline
(R1) has been found to play a crucial role in forming a suitable
cavity for Hg2+ detection, as evidenced by the absence of the ‘N’
atom of quinoline (quinoline was replaced by benzene and
naphthalene respectively) in R2 and R3, for which any detectable
change in the presence of Hg2+ was not observed.
8-(Prop-2-ynyloxy) quinoline (compound 2). 1H NMR
(400 MHz, CDCl3): δ 8.60 (s, 1H), 7.76 (d, J = 4.0 Hz, 1H), 7.10
(m, J = 8.0 Hz, 3H), 6.92 (d, J = 8.0 Hz, 1H), 4.70 (s, 2H), 2.24
(s, 1H); 13C NMR (CDCl3, 100 MHz) δ 149.36, 135.86, 126.37,
121.65, 120.59, 109.86, 78.26, 76.12, 56.41; FTIR (KBr, cm−1):
3303, 1502, 1490, (yield 85%).
Experimental
General
1-(Prop2-ynyloxy) benzene (compound 3). 1H NMR
(400 MHz, CDCl3): δ 7.161 (t, J = 8.4 Hz, 3H), 6.85 (q, J = 8.0
Hz, 3H), 4.51 (s, 2H), 2.37 (s, 1H); 13C NMR (CDCl3, 100 MHz)
δ 157.63, 129.59, 121.66, 114.98, 78.78, 75.66, 55.75; FTIR
(KBr, cm−1): 3296, 1599, 1494, (yield 80%).
All reagents (AR grade) for synthesis were purchased from
Sigma-Aldrich chemicals and used without further purifi-
cation. All metal perchlorate salts were prepared from the
corresponding metal carbonates. Solvents were dried following
standard procedures. UV-grade ethanol was used for UV-Vis
and fluorescence titration. 1H NMR spectra were recorded on a
Bruker AV400 instrument using CDCl3 and d6-DMSO solvents
with TMS as the internal standard. ESI-MS measurements were
carried out using a microTOF-Q II 10337 mass spectrometer
instrument. FTIR spectra were measured using a Spectrum
2000 Perkin-Elmer Spectrometer. UV–Vis spectra and fluo-
rescence spectra were recorded using a UV-Lambda 365
PerkinElmer Spectrophotometer (1.0 cm quartz cell) and a
1-(Prop2-ynyloxy) naphthalene (compound 4). 1H NMR
(400 MHz, CDCl3): δ 8.37 (s, 1H), 7.89 (s, 1H), 7.56 (m, 3H),
7.45 (t, J = 7.6 Hz, 1H), 7.00 (s, 1H), 4.94 (s, 1H), 2.62 (t, J = 2.4
Hz, 1H); 13C NMR (CDCl3, 100 MHz): δ 155.2, 134.4, 127.4,
126.4, 125.4, 125.3, 121.9, 121.13, 105.4, 78.5, 75.5, 56.0, (yield
90%).
General procedure for the preparation of compound R1, R2
and R3
Perkin-Elmer LS 55 Fluorescence spectrometer, respectively. For the preparation of R1, R2 and R3, compound 2, 3 and 4
Single-crystal XRD data were collected using a SuperNova (1 equiv.) were dissolved separately in 80 ml of the 1 : 1 DCM/
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EtOH mixture. Then compound 1 (1 equiv.), sodium ascorbate
(1.5 equiv.) and copper sulfate (1.5 equiv.) were added to this
solution. The resulting mixture was stirred for 24 h at room
temperature. Upon completion of the reaction, the mixture
was filtered, extracted with ethyl acetate, concentrated under
vacuum and then subjected to column chromatography to
obtain the desired product.
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Acknowledgements
SD acknowledges SERB-DST (project no. EMR/2016/002183),
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