Z. Lu, et al.
DyesandPigments171(2019)107715
Usually, organic solvents such as CH3CN and DMF are applied to in-
crease the solubility of iridium (III) complexes in aqueous solution as
co-solvents, while causes high toxicity to biological tissues [30]. To
solve these problems, diverse methods have been developed, including
incorporation of solubilising groups into ligands [28] and combination
with either surfactants or soluble polymers [31–34].
obtained by filtration and washed with water and ether, which was
used directly without further purification. Compound
3 (60 mg,
0.04 mmol) and auxiliary ligand compound 2 (35 mg, 0.08 mmol) were
added into flask contained methanol (10 mL) and dichloromethane
(20 mL) under reflux with stirring for 24 h. The reaction solution was
evaporated under vacuum and subsequently a small quantity of water
was added. Filtered to obtain final solution and evaporated the water
under vacuum, the product Ir-1 was acquired (31 mg, 80%, red solid).
1H NMR (500 MHz, D2O) δ 8.78 (d, J = 5.1 Hz, 2H), 8.25 (dd, J = 36.6,
7.3 Hz, 4H), 8.03 (d, J = 7.9 Hz, 2H), 7.88 (d, J = 4.4 Hz, 2H), 7.67 (d,
J = 3.8 Hz, 4H), 7.64–7.56 (m, 2H), 7.50 (d, J = 6.0 Hz, 2H), 7.00 (d,
J = 6.4 Hz, 2H), 6.83 (d, J = 8.5 Hz, 2H), 6.15 (s, 2H), 4.61 (s, 4H),
3.15 (s, 19H), 1.85 (s, 6H). HR-MS (ESI): m/z2+ [M]2+ calcd for
In previous work, the modification of the quaternary ammonium
groups on iridium (III) complexes not only endowed their water solu-
bility but also tailored their fluorescent properties [35]. Inspired by our
previous work, herein, we designed and synthesized a new cyclome-
talated iridium (III) complexes ClO− probe (Ir-1), which includes two
moieties: a water solubility auxiliary ligand (N∧N) modified with qua-
ternary ammonium groups and the response units attached into cyclo-
metalated ligands (C∧N). The C–O bonds in probe Ir-1 exhibited specific
cleavage by ClO− and quench the fluorescence to “turn-off”. The probe
showed ultrasensitivity and accurate response toward ClO− (detection
limit was 0.41 μM) within short responses time (< 30 s). Based on the
excellent sensitivity and selectivity for detecting ClO− in vitro, we
further investigated the ability to monitor ClO− in cellular environ-
ment. The results indicated that Ir-1 exhibits promising performance
for detecting ClO− in bio-system. In this contribution, we further expect
that these modification methods will open a protocol for regulating the
water solubility of transition metal complexes, which can be used in
profound study around the biomedicine science area such as cell ima-
ging, drug delivery and targeting therapy toward tumor.
C
48H52N6O4Ir, 484.6901; found, 484.6813.
2.3. Spectroscopic measurements
Spectrophotometric titrations of the probe Ir-1 were investigated in
aqueous solution containing PBS (10 mM, pH = 7.4). The testing solu-
2−
tions of various species (NaClO, H2O2, GSH, NO2−, NO3−, SO4
,
SO32−, Fe3+, Cu2+, F−, Br−, ONOO−, •OH, •O2−) were prepared in
deionized water. For fluorescence titration experiment, the excitation
wavelength was 370 nm with slit widths at 5 nm.
2. Experimental
2.4. Cytotoxicity assay
2.1. Materials and apparatus
The toxicity of probe Ir-1 toward HepG2 cells was evaluated
through MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetra-
zolium bromide) assays. 96-well plates were used to seed cells at a
concentration of 5 × 103 cells per well and the cells were incubated in
an incubator (37 °C, 5% CO2) for 24 h. The cells were further cultivated
for another 24 h after treated with a series of different concentrations of
probes (0,10,20,30 and 40 μM). MTT solution was then added into
plates and the cells were cultured for another 4 h. Before the addition of
150 μL DMSO, the residual MTT solution was discarded. The absor-
bance of separated well was measured using a microplate reader
(Varioskan Flash 1510, Thermo Fisher Scientific, Finland) at the wa-
velength of 490 nm. Cells viability rates were obtained according to the
following equation: Cell viability (%) = (mean Abs. of treatment
group/mean Abs. of control) × 100.
All chemical materials and solvents were purchased commercially
without further purification. 1H NMR spectra of compounds were per-
formed on a Bruker Avance III 500 MHz instrument. Mass spectra were
recorded on Agilent HPLC-MS. Fluorescent spectra and UV–vis ab-
sorption spectra were determined on
a Hitachi F-4600 spectro-
fluorometer and on Lambda 750 UV–vis spectrophotometer.
a
Fluorescent images were conducted with a Leica TCS SP8 Laser
Scanning Confocal Microscope.
2.2. Synthesis and characterization
2.2.1. Synthesis of compound 1 (4-(pyridin-2-yl)phenyl methacrylate)
Sodium hydride (14 mg, 0.6 mmol) was added slowly to a solution
of 2-(4-hydroxypenyl)pyridine (100 mg, 0.58 mmol) in THF (20 mL)
while stirred at 0 °C for 30 min. Then, methacryloyl chloride (90 mg,
0.86 mmol) in THF (1 mL) was added into the solution for additional 6 h
stirring at room temperature. The reaction mixture was quenched with
water (20 mL) and extracted with ethyl acetate (20 mL) three times. The
organic layer was dried over MgSO4 and the solvent was removed.
Finally, the crude product was purified by chromatography on silica gel
using dichloromethane as eluent to obtain the product 1 (108 mg, 78%,
yellow solid). 1H NMR (500 MHz, CDCl3) δ 8.66 (d, J = 4.6 Hz, 1H),
8.04 (s, 2H), 7.75 (d, J = 7.6 Hz, 1H), 7.28 (s, 4H), 6.97 (d, J = 8.7 Hz,
1H), 6.40 (s, 1H), 5.83–5.76 (m, 1H), 2.10 (s, 3H). HR-MS (ESI): m/z [M
+H]+ calcd for C15H14NO2, 240.1019; found, 240.1013.
2.5. Cell imaging of probe Ir-1
HepG2 cells were cultivated under a humid atmosphere containing
5% CO2 at 37 °C for 24 h in Dulbecco's modified Eagle's medium
(DMEM) with 10% FBS fetal bovine serum (FBS). The well-grown cells
were pretreated with probe Ir-1 (10 μM) for 30 min and the residual
probes were washed with PBS buffer for 3 times. Several plates were
incubated for extra 10 min after adding ClO− (50 μM), and the HepG2
cells were washed by PBS before imaging. Cell imaging were recorded
with a laser scanning confocal microscope. The excitation wavelength
was fixed at 405 nm.
2.2.2. Synthesis of compound Ir-1
The auxiliary ligand (N∧N) of iridium complex: compound 2 (4,4′-
bis [(trimethylamino)methyl]-2,2′-bipyridine) was synthesized fol-
lowing the method previously reported [36,37]. The synthetic routes of
IrCl3 (58 mg, 0.18 mmol) and compound 1 (78 mg, 0.32 mmol) were
poured into a flask including a solvent mixture of 2-ethoxyethanol
(30 mL) and H2O (10 mL). The mixture was heated at 125 °C with
stirring for 24 h. After cooling to room temperature, reddish-orange
compound 3 (cyclometalated iridium (III) chloro-bridge dimer) was
2.6. Determination of quantum yield
Fluorescent quantum yield (QY) was determined using an aqueous
solution of [Ru (bpy)3]Cl2 (QY = 0.028) as a reference by optically
dilute method. The quantum yield was calculated from the following
equation: Фf = (ArIf/AfIr) × Фr, where Фf and Фr are quantum yield of
sample and that of the reference respectively; If and Ir are the integrated
areas of emission bands, Af and Ar are the absorbance of the sample and
the reference.
2