A Quninolylthiazole Derivatives as an ICT-Based Fluorescent Probe of Hg(II) and its Application in Ratiometric Imaging in Live HeLa Cells

Article abstract of DOI:10.1007/s10895-018-2241-4

As a structural analogue of pyridylthiazole, 2-(2-benzothiazoyl)-phenylethynylquinoline (QBT) was designed as a fluorescent probe for Hg(II) based on an intramolecular charge transfer (ICT) mechanism. The compound was synthesized in three steps starting from 6-bromo-2-methylquinoline, with moderate yield. Corresponding studies on the optical properties of QBT indicate that changes in the fluorescence ratio of QBT in response to Hg(II) could be quantified based on dual-emission changes. More specifically, the emission spectrum of QBT before and after interactions with Hg(II) exhibited a remarkable red shift of about 120?nm, which is rarely reported in ICT-based fluorescent sensors. Finally, QBT was applied in the two-channel imaging of Hg(II) in live HeLa cells.

Full text of DOI:10.1007/s10895-018-2241-4

Journal of Fluorescence
https://doi.org/10.1007/s10895-018-2241-4
ORIGINAL ARTICLE
A Quninolylthiazole Derivatives as an ICT-Based Fluorescent Probe
of Hg(II) and its Application in Ratiometric Imaging in Live HeLa Cells
Jian-Ying Bai1,2 & Yu-Zhong Xie1,3 & Chang-Jiang Wang4 & Shu-Qing Fang4 & Lin-Nan Cao1 & Ling-Li Wang1 & Jing-Yi Jin1,2
Received: 9 March 2018 /Accepted: 11 May 2018
#
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
As a structural analogue of pyridylthiazole, 2-(2-benzothiazoyl)-phenylethynylquinoline (QBT) was designed as a fluorescent
probe for Hg(II) based on an intramolecular charge transfer (ICT) mechanism. The compound was synthesized in three steps
starting from 6-bromo-2-methylquinoline, with moderate yield. Corresponding studies on the optical properties of QBT indicate
that changes in the fluorescence ratio of QBT in response to Hg(II) could be quantified based on dual-emission changes. More
specifically, the emission spectrum of QBT before and after interactions with Hg(II) exhibited a remarkable red shift of about
120 nm, which is rarely reported in ICT-based fluorescent sensors. Finally, QBT was applied in the two-channel imaging of
Hg(II) in live HeLa cells.
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Keywords Fluorescence Pyridylthiazole Ratiometric sensor Mercury ion ICT Imaging
Introduction
sensitivity, fluorometric methods are advantageous for the
analysis of biological and environmental samples [6]. Many
fluorescent sensors for Hg(II) have been developed based on
various organic fluorophores [7].
Mercury is one of the most toxic metal species, due to its
bioaccumulation and persistence in the environment [1–3]. It
is known to adversely affect the neurological and endocrine
systems, and thus lead to various malignant diseases, such as
Minamata disease [4, 5]. Therefore, the detection of Hg(II) is
essential for public safety and environmental protection, and it
has been the subject of research studies for many decades. Due
to the ease of operation, non-invasiveness, and high
Compared with the Boff-on^ and Bon-off^ single-emission
types of fluorescent sensors, ratiometric fluorescent probes
with dual emission can eliminate most of ambiguities associ-
ated with fluorescent probes, including probe concentrations,
photon bleaching, and instrument efficiency [8, 9]. Generally,
the emission peak detected in the presence of target analytes
should be clearly discriminated from the original emission
peak of the probe, which means that a large red shift in the
emission peak is preferable for ratiometric probes. Förster
resonance energy transfer (FRET) is the mechanism that de-
scribes energy transfer from a donor to an acceptor
fluorophore, and it was proposed as a feasible strategy to
achieve two resolved emission peaks [10]. Most ratiometric
fluorescent probes for Hg(II) have been developed utilizing
the FRET mechanism [11–16].
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10895-018-2241-4) contains supplementary
material, which is available to authorized users.
* Jing-Yi Jin
jyjin-chem@ybu.edu.cn
1
Research Centre of Chemical Biology, Yanbian University,
Intramolecular charge transfer (ICT), an alternative mech-
anism to ratiometric probes, has been rarely applied in the
detection of Hg(II) because ICT-based fluorophores often ex-
hibit very broad emission peaks, which makes peak resolution
challenging [10]. ICT-based probes generally possess simpler
structures than FRET-based sensors, it implied a more straight
synthesis with convenience [17]. Thus, it is a challenge to
develop an ICT-based probe with a large red shift (>
Yanji 133002, China
2
National Demonstration Centre for Experimental Chemistry
Education (Yanbian University), Department of Chemistry, Yanbian
University, Yanji 133002, China
3
College of Chemistry, Northeast Normal University,
Changchun 130024, China
4
Department of Nephrology, The Central Hospital of Jiamusi,
Jiamusi 154002, China

J Fluoresc
100 nm) in the two emission peaks, before and after interac-
tion with Hg(II).
Experimental
Our previous works verified pyridylthiazole (PT) as an
ICT-type fluorophore, and we developed a series of PT deriv-
atives as ratiometric fluorescent probes for various analytes
[18–24]. Considering that 2-(2-quinolinyl)benzothiazole
(QT) is a structural analogue of PT, we explored the possibil-
ities of QT derivatives as probes in ICT-based fluorescence
sensors in the present paper. To improve the ICTeffects of QT,
a phenyl group was introduced at the 6-position of the quino-
line ring through a triple bond, according to the literature
[25–27], which was further examined by the corresponding
theoretical calculations as described (Chart 1). The designed
QT derivative, 2-(2-benzothiazoyl)-6-phenylethynylquinoline
(QBT), was employed as an ICT-based ratiometric probe for
Hg(II) in this study, with a large red shift before and after
interaction with Hg(II). The two-channel based imaging was
then performed in live HeLa cells.
Materials and Apparatus
All chemicals were purchased from Acros and other
commercial suppliers and used as received without fur-
ther purification. All reactions were performed under
argon atmosphere. Silica gel (100–200 mesh), used
for flash column chromatography, and thin-layer chro-
matography (TLC) plates were purchased from
Qingdao, China. 6-Bromo-2-methylquinoline (com-
pound 1) was synthesized according to a previously
published method [25].
Melting points were taken on a Thomas-Hoover capil-
lary melting point apparatus and were uncorrected. H
1
nuclear magnetic resonance (NMR) and 13C NMR spectra
were recorded with a Bruker AV 300 (300 MHz) instru-
ment using tetramethylsilane as the internal standard. IR
spectra were recorded on a Perkin-Elmer 1200 FT-IR
spectrometer. Both high-resolution mass spectra and ele-
mental analyses were performed at Changchun Institute of
Applied Chemistry, Chinese Academy of Sciences,
Changchun, China.
For all spectrometric measurements, HPLC grade sol-
vents were used. Solutions of metal ions were prepared
from their perchlorate salts (except for NaCl, KCl, and
CaCl2) and dissolved in Tris buffer (50 mM, pH = 7.4).
A stock solution of QBT (0.1 mM) was prepared in
MeCN. UV-Vis absorption spectra were measured with a
Hitachi UV-3010 spectrophotometer, and the fluorescence
spectra were recorded on a Hitachi F-4500 fluorescence
spectrophotometer. For fluorescence measurements, the
slit widths of the excitation and emission were both set
as 5 nm.
Cell Culture and Imaging
HeLa cells were purchased from Zeye Biotechnology Co.
Ltd. of China and cultured in DMEM containing 10%
fetal bovine serum and 1 × 104 μg·mL−1 streptomycin at
37 °C in 5% CO2/95% air incubator. The cells were
digested with trypsin when the cell density reached ap-
proximately 80%. For live cell imaging, the cells were
washed twice with PBS to remove the growth medium
and dead cells. Live HeLa cells were treated with
2.0 μM QBT and incubated in PBS (containing 1%
DMSO) at 37 °C for 10 min. The cells were washed three
times with PBS and incubated with 2.0 eq. Hg(II) at 37 °C
for another 10 min. Finally, the cells were washed three
times with PBS, and cell images were obtained on a
Nikon C2 confocal laser scanning microscope with an
objective lens (×20).
Chart 1 Design of a QT-based fluorophore, 2-(2-benzothiazoyl)-6-
phenylethynylquinoline (QBT), as a structural analogue of PT and the
corresponding HOMO and LUMO of QBT at the B3LYP 6-31G(d,p)
level of theory predicated by Gaussian 09. Gray: carbon atom; blue:
nitrogen atom; yellow: sulphur atom. To clarify the representation,
hydrogen atoms are omitted

J Fluoresc
Synthesis
and 50 mL of CH2Cl2 were added. The mixture was washed
with water and brine, and the organic solvent was dried over
anhydrous Na2SO4. Evaporation under reduced pressure pro-
vided the crude product, which was further purified via flash
column chromatography using a mixture of n-hexane and
EtOAc as eluents.
Synthesis of 6-Bromoquinoline-2-Carbaldehyde
(Compound 2)
To a solution of compound 1 (1 g, 4.52 mmol) in 12 mL
1,4-dioxane, selenium dioxide (550 mg, 1.1 eq.) was added
incrementally, and the solution was maintained at 60 °C.
The reaction temperature was increased to 80 °C, and the
reaction proceeded with stirring for 1.5 h. After cooling to
room temperature, the precipitate was filtered and washed
with CH2Cl2. Combined the organic phase, and the sol-
vents were removed under reduced pressure to yield the
crude product. The product was further purified via flash
column chromatography using a mixture of n-hexane and
CH2Cl2 as eluents.
1
Yield: 65.1%. Mp: 261.0–261.5 °C. H NMR (CDCl3,
300 MHz, ppm) δ = 7.31–7.36 (m, 3H), 7.40 (dd, 1H, J =
0.9, 7.8 Hz), 7.46 (d, 1H, J = 7.8 Hz), 7.52 (d, 1H, J =
5.6 Hz), 7.53 (d, 1H, J = 7.2 Hz), 7.79 (dd, 1H, J = 1.6,
8.8 Hz), 7.93 (d, 1H, J = 7.8 Hz), 7.98 (1H, d, J = 0.9 Hz),
8.08 (1H, d, J = 7.6 Hz), 8.09 (1H, d, J = 8.6 Hz), 8.19 (1H, d,
J = 8.6 Hz), 8.44 (1H, d, J = 8.6 Hz). 13C NMR (CDCl3,
75 MHz, ppm) δ = 88.10, 90.61, 118.13, 121.17, 121.73,
121.93, 122.95, 125.10, 125.47, 127.58, 127.82, 127.86,
128.91, 130.00, 130.86, 131.51, 131.98, 135.64, 135.74,
150.84, 153.49, 168.63. IR (film, cm−1): 688.6, 729.1,
759.5, 835.2, 916.2, 997.2, 1311.6, 1344.4, 1492.9, 1591.3.
ESI-MS: [M + 1]+: 362.13. Elemental analysis: C24H14N2S,
Calcd: C 79.53, H 3.89, N 7.73. Found: C 79.37, H 4.06, N
8.31.
1
Yield: 85.3%. Mp: 110–110.5 °C. H NMR (CDCl3,
300 MHz, ppm) δ = 7.84 (dd, 1H, J = 2.0, 9.0 Hz), 8.00 (d,
1H, J = 8.4 Hz), 8.03 (d, 1H, J = 2.0 Hz), 8.08 (d, 1H, J =
9.0 Hz), 8.19 (d, 1H, J = 8.4 Hz), 10.17 (s, 1H).
Synthesis of 2-(2-Benzothiazoyl)-6-Bromoquinoline
(Compound 3)
Results and Discussion
To a solution of compound 2 (200 mg, 0.84 mmol) in
20 mL EtOH was added 2-amino benzenethiol (211 mg,
2.0 eq.) at room temperature. After the addition of a few
drops of concentrated sulfuric acid, the reaction mixture
refluxed for 1 h. After cooling to room temperature, the
precipitate was filtered and washed with MeOH.
Recrystallization in CH2Cl2 provided the product as a
white solid.
To examine the electronic structure of QBT, density functional
theory (DFT) calculations was first performed at the B3LYP/
6-31G(d,p) level using Gaussian 09 [31–35]. The predicated
HOMO and LUMO electron densities of QBT are shown in
Chart 1. As depicted, the phenyl group is electron-rich at the
HOMO level and electron-deficient at the LUMO level. This
suggests that an ICT process should occur from the 6-phenyl
group as the electron donor to the 2-quinolyl group as the
acceptor upon photo-excitation of the QBT molecule.
Further, if the nitrogen atom of the quinoline moiety is in-
volved in the coordination to Hg(II), the electron-
withdrawing abilities of the acceptor unit of QBT should be
enhanced and a large red shift in the emission peak can be thus
expected.
The free ligand (QBT) can be synthesized as shown in
Scheme 1. Starting from 6-bromo-2-methylquinoline (com-
pound 1), oxidization using SeO2 gave the aldehyde (com-
pound 2), which was easily transformed to benzothiazole
(compound 3). Finally, Sonogashira coupling of compound
3 with phenylacetylene provided the target compound in mod-
erate yield.
Yield: 65.8%. Mp: 227.0–227.5 °C. 1H NMR (DMSO-d6,
300 MHz, ppm) δ = 7.55–7.62 (m, 2H), 7.99 (dd, 1H, J = 2.0,
9.0 Hz), 8.14 (d, 1H, J = 8.4 Hz), 8.19–8.22 (m, 2H), 8.44 (d,
1H, J = 2.0 Hz), 8.51 (d, 1H, J = 9.0 Hz), 8.59 (d, 1H, J =
8.4 Hz). 13C NMR (DMSO-d6, 300 MHz, ppm) δ = 121.31,
122.00, 122.10, 123.42, 126.75, 129.37, 129.57, 130.40,
130.98, 135.93, 137.9, 140.87, 144.01, 147.44, 151.81,
156.99. IR (film, cm−1): 763.8, 825.5, 871.8, 999.1, 1327.0,
1485.2, 1589.3.
Synthesis of 2-(2-Benzothiazoyl)-6-Phenylethynylquinoline
(QBT)
According to a standard method [28–30], compound 3
(100 mg, 0.293 mmol), Pd(PPh3)2Cl2 (16.5 mg, 0.08 eq.),
and CuI (4.5 mg, 0.08 eq.) were added to a 50 mL Schlenk
flask containing 5 mL N,N-dimethyl foramide. The flask was
subsequently pump-purged with argon three times.
Phenylacetylene (75 mg, 2.5 eq.) and triethyl amine (15 mL)
were added via syringe to the system. After continuous stirring
at 80 °C for 24 h, the mixture was cooled to room temperature
QBT exhibited an absorption maximum at 338 nm. The
addition of 10.0 equiv. of Hg(II) resulted in a significant
bathochromic shift to 396 nm (Figure S1). The UV-vis titra-
tion experiment disclosed that the absorbance at 338 nm de-
clined gradually with increasing absorption at 396 nm upon
the addition of 1.0 equiv. of Hg(II) (Figure S2). A clear and
fixed isosbestic point was found at 379 nm, which was

J Fluoresc
Scheme 1 Synthesis of the
designed QBT probe
ascribed to a simple coordination equilibrium between the
ligand and Hg(II).
The effects of Hg(II) on the fluorescence of QBT in an
aqueous system (MeCN:Tris buffer = 1:9, v/v) were also
investigated. The fluorescence titration experiment exhibited
a fluorescence ratio-dependent response to Hg(II), as expect-
ed. The decreasing emission at 417 nm was accompanied by
an emerging peak at 538 nm (Fig. 1a). Here, it should be noted
that the observed red shift of the emission peak was about
120 nm, which is significant for ICT-based fluorescent probes.
This indicates that QBT is a suitable probe for two-channel
ratiometric fluorescence imaging in biological samples. Other
metal ions, including Na(I), K(I), Ca(II), Mg(II), Cr(III),
Mn(II), Fe(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), Sr(II),
Cd(II), Pb(II), and Ag(I), exhibited negligible effects on the
fluorescence of the ligand (Fig. 1b, Figure S3-S4).
According to the Job plot (Figure S5), the stoichiometry
between QBT and Hg(II) is determined as 2:1. Based on the
fluorescence titration experiment, [(F-Fmin)/(Fmax-Fmin)]
was plotted versus log[Hg(II)], where Fmin and Fmax were
the fluorescence intensities at 538 nm in the absence of
Hg(II) and in the presence of a saturated concentration of
Hg(II), respectively (Fig. 2). The binding constant, Kas, was
Fig. 1 a Fluorescence spectral changes of QBT (1.0 μM) excited at
393 nm in an aqueous system (MeCN/Tris buffer = 1:9 (v/v), Tris
buffer: 50 mM, pH = 7.4) in the presence of 0–4.0 equiv. of Hg(II) at
20 °C; (b) Fluorescence intensity at 417 nm (meshed bar) and 538 nm
(solid bar) of QBT after the addition of 10.0 equiv. of different metal ions
under the same conditions
Fig. 2 Relative fluorescence ratio, (F-Fmin)/(Fmax-Fmin), as a function of
log[Hg(II)] (M) in an aqueous system (MeCN/Tris buffer = 1:9 (v/v), Tris
buffer: 50 mM, pH = 7.4) at 20 °C. The curve fitting for experimental data
was calculated using a least-squares method (■, experimentally observed
data points)

J Fluoresc
Fig. 3 a Bright-field image of HeLa cells; (b) image of HeLa cells
incubated with 2.0 μM QBT for 10 min, washed with PBS buffer, and
excited at 405 nm (blue channel); (c) image of HeLa cells with QBT
(green channel); (d) overlay of (a), (b) and (c); (e) bright-field image of
HeLa cells incubated with QBT and 2.0 equiv. of Hg(II), washed with
PBS buffer; (f) image of HeLa cells incubated with QBT followed by
Hg(II), excited at 405 nm (blue channel); (g) image of HeLa cells with
QBT and Hg(II) (green channel); (h) overlay of (e), (f) and (g)
then fitted as 5.25 × 105 M−2 with R2 = 0.991 [21, 24]. The
detection limit of the sensor for Hg(II) was 1.26 × 10−6 M,
which is comparable to the tolerated concentrations of
Hg(II) in human serum [36, 37].
Finally, we explored the application of QBT as a
ratiometric probe for Hg(II) in biological imaging. HeLa cells
were selected as the model cell line. The cells were incubated
with QBT (2.0 μM) at 37 °C for 30 min. It was found that the
QBT-cultured cells exhibited an emission peak at 417 nm in
the blue channel. After the addition of 2.0 eq. of Hg(II) in the
form of mercury chloride, fluorescence in the blue channel
decreased, while emission in the green channel increased
(Fig. 3).
Acknowledgements We thank the financial support from the National
Natural Science Foundation of China (NSFC 21062023 and 21768004).
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