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Cell imaging of dopamine receptor using agonist labeling iridium(iii) complex.
Chemical
Science
Volume 9 Number 5 7 February 2018 Pages 1075–1394
rsc.li/chemical-science
ISSN 2041-6539
EDGE ARTICLE
Wai-Jing Kwong, Weihong Tan, Chung-Hang Leung, Dik-Lung Ma et al.
Cell imaging of dopamine receptor using agonist labeling iridium(iii)
complex
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Cite this: Chem. Sci., 2018, 9, 1119
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Cell imaging of dopamine receptor using agonist
labeling iridium(III) complex†
Kasipandi Vellaisamy,‡a Guodong Li,‡b Chung-Nga Ko,‡a Hai-Jing Zhong,b
Sarwat Fatima,c Hiu-Yee Kwan,c Chun-Yuen Wong, d Wai-Jing Kwong,*a
Weihong Tan, *ef Chung-Hang Leung *b and Dik-Lung Ma *a
Dopamine receptor expression is correlated with certain types of cancers, including lung, breast and colon
cancers. In this study, we report luminescent iridium(III) complexes (11–14) as intracellular dopamine
receptor (D1R/D2R) cell imaging agents. Complexes 11 and 13, which are conjugated with a dopamine
receptor agonist, showed superior cell imaging characteristics, high stability and low cytotoxicity (>100
mM) in A549 lung cancer cells. siRNA knockdown and dopamine competitive assays indicated that
complexes 11 and 13 could selectively bind to dopamine receptors (D1R/D2R) in A549 cells.
Fluorescence lifetime microscopy demonstrated that complex 13 has a longer luminescence lifetime at
the wavelength of 560–650 nm than DAPI and other chromophores in biological fluids. The long
luminescence lifetime of complex 13 not only provides an opportunity for efficient dopamine receptor
tracking in biological media, but also enables the temporal separation of the probe signal from the
Received 6th November 2017
Accepted 9th December 2017
intense background signal by fluorescence lifetime microscopy for efficient analysis. Complex 13 also
shows high photostability, which could allow it to be employed for long-term cellular imaging.
Furthermore, complex 13 could selectively track the internalization process of dopamine receptors (D1R/
DOI: 10.1039/c7sc04798c
D2R) in living cells. To the best of our knowledge, complex 13 is the first metal-based compound that
rsc.li/chemical-science
has been used to monitor intracellular dopamine receptors in living cells.
Introduction
Lung cancer is a leading cause of death in both developed and
developing countries.1 Non-small cell lung cancer (NSCLC) is
the most common subtype of lung cancer, with a 5 year survival
rate less than 15%.2 Angiogenesis, mediated by vascular endothelial growth factor (VEGF) and the VEGF receptor (VEGFR), is
a
Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong,
China. E-mail: edmondma@hkbu.edu.hk; dkwong@hkbu.edu.hk
b
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese
Medical Sciences, University of Macau, Macau, China. E-mail: duncanleung@umac.
mo
c
School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong,
China
d
Department of Biology and Chemistry, City University of Hong Kong, Kowloon Tong,
Hong Kong, China
e
Department of Chemistry, Department of Physiology and Functional Genomics, Center
for Research at the Bio/Nano Interface, Shands Cancer Center, UF Genetics Institute,
McKnight Brain Institute, University of Florida, Gainesville, USA. E-mail: tan@
chem.u.edu
f
Molecular Sciences and Biomedicine Laboratory, State Key Laboratory for Chemo/
Biosensing and Chemometrics, College of Chemistry and Chemical Engineering,
College of Biology, Hunan University, Changsha, China
† Electronic supplementary
10.1039/c7sc04798c
information
(ESI)
‡ These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 2018
available.
See
DOI:
important for cancer cell proliferation.3 Recently, Basu and
coworkers have reported that a dopamine 2 receptor (D2R)
agonist is able to inhibit angiogenesis.4 Studies with a D2Rknockout mouse model have indicated the molecular mechanism through which D2R/VEGFR-2 crosstalk can mediate the
phosphorylation of VEGFR-2. Dopamine receptors also serve as
good targets for breast and colon cancers.5
Early-stage identication of cancer is a high priority in
research.5,6 Therefore, the development of methods for the
detection of cancer biomarkers in living systems has attracted
tremendous interest.7–9 Studies have shown that dopamine
receptor (D1R/D2R) expression is higher in NCI-H69 NSCLC
cells.10 Previously, the expression of the dopamine (D1/D2)
receptor in these cells has been monitored using an iodosulpride isotope probe11 which is subjected to biases caused by
the incubation of isotope. Fluorescent probes12–14 and biotin
derivatives15 of dopamine agonists have also been developed as
probes of D1R and D2R, whereas nitrobenzoxadiazole (NBD) and
uorescein-coupled dopamine agonists have been applied in the
uorescent labelling of D1R and D2R sites in monkey pituitary
gland. However, these probes are generally limited by their short
luminescence lifetimes (1–10 ns) and the phenomenon of selfquenching, photobleaching and uorescence resonance energy
transfer (FRET). Although radiolabelled probes for dopamine
receptors include S-labeled cRNA probes and [123I] N3-NAP have
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been extensively reported,16–21 great caution is required when
handling these radioactive materials. To the best of our knowledge, metal complex probes capable of identifying dopamine
receptors (D1R and D2R) in living cells have not yet been
developed. In particular, the long luminescence lifetime, large
Stokes shi, high luminescence quantum yield and high photostability properties of iridium(III) complexes render them an
excellent alternative agent for dopamine tracking.30,31
In this study, four cyclometalated iridium(III) complexes 11–
14 with general structure [Ir(N–C)2(N–N)](PF6) (where N–N ¼ 3(3,4-dihydroxyphenylagonistsagonists)-N-(1,10-phenanthrolin5-yl) propanamide (6) or 4-(2-(1,10-phenanthroline-5carboxamido)ethyl)-1,2-phenylene diacetate (9) and N–C ¼ 2phenylpyridine (ppy), 2-(2,4-diuorophenyl)pyridine (dfppy), or
2-phenylquinoline (pq)) were designed and synthesised
(Scheme 1). As ligands 6 and 9 are derived from dopamine
agonists, we hypothesized that the conjugated complexes would
be able to effectively recognize dopamine receptors (D2R/D1R).
Notably, complexes 11 and 13, showed superior cell imaging
characteristics, high stability and low cytotoxicity (>100 mM) in
A549 lung cancer cells. siRNA knockdown and dopamine
competitive assays indicated that complexes 11 and 13 could
selectively bind to dopamine receptors (D1R/D2R) in A549 cells.
Furthermore, complex 13 possesses useful photophysical
properties including long luminescence lifetimes, high photostability and high luminescence quantum yield. Since most of
the background uorescence in cell medium has a luminescence lifetime of less than 3 ns, the relatively longer luminescence lifetime for complex 13 should enable the temporal
separation of the probe signal from the intense background
signal by uorescence lifetime microscopy. To the best of our
knowledge, this is the rst application of iridium(III) complexes
for imaging D2R/D1R within living cells.
Results and discussions
Synthesis of N^N ligands 6 and 9
We envisaged that conjugating dopamine agonists, such as
dopamine or 3-(3,4-dihydroxyphenyl)propanoic acid (1),22 to an
iridium(III) scaffold could generate effective probes for
Scheme 1
Iridium(III) dopamine complexes as D1R and D2R probes.
1120 | Chem. Sci., 2018, 9, 1119–1125
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dopamine receptors (D1R and D2R). In our designed strategy,
the dopamine agonists were conjugated to N^N ligands 6 and 9.
To synthesize ligand 6, compound 1 was rst converted to the
corresponding methyl ester (2) in acidic conditions using MeOH
as solvent (Scheme S1†).23–25 Compound 2 was protected as the
tetrahydropyranyl ethers (THP) using pyridinium p-toluenesulfonate (PPTS) in dry DCM to generate compound 3 in 62%
yield.26 Subsequently, the THP-protected methyl ester (3) was
selectively hydrolyzed using LiOH to furnish the corresponding
THP-protected acid (4) aer acidic workup. Compound 4 was
then coupled with 1,10-phenanthrolin-5-amine using standard
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) coupling
to generate compound 5. Finally, THP deprotection with a catalytic amount of PPTS in EtOH produced ligand 6 with a yield of
94%. Ligand 6 was characterized by NMR spectroscopy and
HRMS (Fig. S1 and S2†).
Ligand 9 was generated in 62% yield from an acetylated
dopamine derivative (8) and 1,10-phenanthroline-5-carboxylic
acid (7) using EDCI coupling (Scheme S2†). Ligand 9 was
characterized by NMR spectroscopy and HRMS (Fig. S9 and
S10†).
Iridium(III) complexes synthesis
Complexes 11–14 were synthesised in good yields (85–91%) by
reacting two equivalents of N^N ligands (6 or 9) and the corresponding cyclometalated iridium(III) dimers in CH2Cl2/CH3OH
(1 : 1, v/v), followed by chloride anion exchange with NH4PF6.
Complexes 11–14 were puried by silica gel column chromatography and characterized by 1H, 13C NMR spectroscopy and
MALDI-HRMS. The photophysical properties of complexes 11–
14, including their luminescence quantum yields, emission
properties and UV-vis absorption properties were measured in
ACN and are reported in Table S1.† Excitation of 13 (10 mM) at
334 nm produced a maximum emission at 558 nm which is
assigned to the metal-to-ligand charge-transfer (MLCT) state
and is typical for iridium(III) complexes. Complex 13 also
displays large Stokes shi of 215 nm, which is considerably
larger than those generally displayed by organic molecules and
can efficiently prevent self-quenching. UV-vis absorption
spectra of complexes 11–14 are presented in Fig. S13.† Complex
13 exhibited an intense absorption at 255 and 280 nm and
a moderate peak at 331 and 446 nm in CH2Cl2. The bands are
assigned to the promotion of electrons based on the ligandcentered (p–p*) transition and the metal-to-ligand charge
transfer (MLCT) transition respectively. Furthermore,
complexes 13 and 14 were both stable in a DMSO-d6 and D2O
mixture (9 : 1) at 25 C for seven days, as revealed by NMR
spectroscopy, indicating that the acetyl groups of 14 are sufficiently stable to hydrolysis under ambient conditions (Fig. S14
and S15†). Among the four complexes, complex 13 showed the
highest luminescence quantum yield of 0.245 and a long
luminescence lifetime of 4.61 ms. Complexes 11 and 14 also
showed long luminescence lifetimes of 4.36 and 4.65 ms
respectively. In contrast, complex 12 was non-emissive. The
long luminescence lifetimes exhibited by iridium(III) complexes
11, 13 and 14 could enable their emission to be distinguished
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from background uorescence by the use of time-resolved
emission spectroscopy (TRES).
To validate this hypothesis, coumarin 460 (Cm-460) or thioavin S (THS) was employed as model matrix interferents. In
contrast to the iridium(III) complexes, organic uorophores
typically show nanosecond luminescence lifetimes. When the
luminescence spectra were recorded directly aer the excitation
pulse without any delay (lexc ¼ 355 nm), Cm-460 exhibited
a strong emission peak at 455 nm while THS exhibited
a moderate peak at 540 nm. Therefore, the peak of complex 13
was partially obscured by the trailing edge and the overlapping
emission peaks of Cm-460 and THS, respectively (Fig. 1a and c).
In contrast, when the luminescence spectra were recorded with
a delay of 333 ns aer the excitation pulse, the short-lived
uorescence of Cm-460 and THS were eliminated, and the
emission of complex 13 became more evident (Fig. 1b and d).
Cytotoxicity and cell staining in A549 cells
In consideration of the promising luminescent behaviour shown
by transition-metal complexes, the cytotoxicity of complexes 11–14
was measured in A549 cells, a human NSCLC cell line with dopamine receptor expression, using the MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) assay. The results revealed
that complexes 11–13 exhibited IC50 values above 100 mM, whereas
complex 14 showed an IC50 value of 70.79 mM (Fig. S16†). This
indicates that all of complexes are relatively nontoxic to cells,
making them suitable for cell staining experiments.
We next investigated the application of the iridium(III)
complexes for cell staining. A549 cells were incubated with
complexes 11–14 (30 mM) for 1 h and then washed with phosphate buffer. Luminescence imaging using a confocal laser
scanning microscope with excitation at 488 nm revealed that
complexes 11, 13 and 14 showed strong luminescence in A549
cells (Fig. 2), with luminescence intensity increasing with
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complex concentration (Fig. S18–S20†). In contrast, minimal
luminescence was observed with complex 12, even when the
concentration of the complex was increased to 60 mM
(Fig. S17†). We presume that complexes 11, 13 and 14 could
interact strongly with the dopamine receptor (D1R/D2R) via
their dopamine agonist moieties, thereby leading to an
enhanced luminescence of the cell.
Validation of dopamine receptor binding in A549 cells
In order to verify that the complexes were interacting with
dopamine receptors (D1R/D2R) in cells, siRNA knockdown
experiments were performed with D1R/D2R siRNA in A549.27–29
As shown by Western blotting experiments, the levels of both
D1R (Fig. 3a) and D2R (Fig. 3b) were reduced signicantly in the
presence of D1R/D2R siRNA, with a greater reduction for D2R.
Next, complexes 11 and 13 were introduced into D1R/D2R
knockdown cells. As depicted in Fig. 3c, the luminescence of
complexes 11 and 13 was reduced signicantly, indicating that
the emission enhancement of the complexes required the presence of dopamine receptors in living cells. On the other hand, the
luminescence intensity of complex 14 was relatively unaffected in
the presence of D1R/D2R siRNA (Fig. S21†). This suggests that
complex 14 may show nonspecic binding to molecules other
than dopamine receptors in the intracellular environment.
To provide further evidence that complexes 11 and 13
interacted with dopamine receptors (D1R/D2R) in living cells,
we preincubated A549 cells with an excess of dopamine (1 mM)
for 1 h, before staining with complexes 11 and 13 (30 mM) for
1 h. Interestingly, the luminescence intensity of complexes 11
and 13 was decreased in the presence of dopamine (Fig. 4),
suggesting that the presence of dopamine blocks the interaction between complexes and dopamine receptors (D1R/D2R) in
living cells, presumably via competitive binding to dopamine
receptors. Overall, complex 13 showed the highest luminescence enhancement in the cell imaging experiments, indicating
that it could serve as a useful scaffold for developing probes for
dopamine receptors in living cells.
Photostability of 13 in cellulo
Considering that high photostability is very important for the
practical application of a cellular probe for bioimaging,
Fig. 1 Time-resolved fluorescence emission spectra of complex 13 in
PBS buffer (pH ¼ 7.0, 25 C) in the presence of Cm-460 at (a) delay ¼
0 ns and (b) delay ¼ 333 ns or with THS at (c) delay ¼ 0 ns and (d) delay
¼ 333 ns. The peak of complex 13 was partially obscured by the
emission peaks of the fluorescent media when delay ¼ 0 ns, while the
peak of complex 13 became more evident when delay ¼ 333 ns.
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Fig. 2 Luminescence and bright-field images of A549 cells stained
with 30 mM of complexes 11–14 for 1 h. Scale bar ¼ 15 mm.
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(a) The relative amount of D1R in A549 cells before or after D1R/
D2R siRNA stimulation. (b) The relative amount of D2R in A549 cells
before or after D1R/D2R siRNA stimulation. (c) Luminescence and
bright-field images of A549 cells with D1R/D2R knockdown using
siRNA. A549 cells were stained with or without complexes 11 and 13
(30 mM) for 1 h. Scale bar ¼ 15 mm.
Fig. 3
Fig. 4 Luminescence and bright-field images of A549 cells. A549 cells
were pretreated with or without dopamine (1 mM) for 1 h, followed by
staining with complexes 11 and 13 (30 mM) for 1 h. Scale bar ¼ 15 mm.
a photobleaching assay was performed with iridium(III) complex
13 in paraformaldehyde-xed A549 cells. 40 ,6-Diamidino-2phenylindole (DAPI), an organic and commercial dye for
staining nuclei in cells, was used as a benchmark for photostability. Aer continuous excitation at 405 nm for 700 s, the
mean luminescence intensity of complex 13 (550–650 nm,
region 2) only decreased by 4.9%. Whereas the luminescence of
DAPI (430–480 nm, region 1) decreased by 39.6% (Fig. 5). This
result demonstrates that 13 exhibits higher photostability
compared to DAPI, indicating that 13 could be employed as
a potential bioimaging luminescent probe for continuous
tracking studies over a long period of time.
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Comparison of DAPI and 13 for resistance to photobleaching.
(a) Confocal luminescence images of fixed A549 cells stained with
DAPI and 13 under continuous irradiation at 405 nm with different
laser scan times (0, 350, 700 s). Scale bar ¼ 30 mm. (b) The relative
mean luminescence intensity of DAPI and 13 collected from region 1
(green, 430 to 480 nm) and 2 (red, 550 to 650 nm).
Fig. 5
within 60 min (Fig. 6). However, at 180 min, the luminescence
of cell boundaries was signicantly reduced while that of the
cytoplasm was enhanced in a punctuated pattern (Fig. 6),
indicating that complex 13 may be an agonist of dopamine
receptors and has the potential to monitor the internalization of
D1/D2-receptors in A549 cells. This result was also conrmed by
assessing the luminescence in D1R/D2R knockdown A549 cells
aer 180 min incubation, which showed a decrease in the
luminescence of the cytoplasm as expected (Fig. S22†). Collectively, these results suggest that complex 13 could not only
target dopamine receptors (D1R/D2R), but also monitor the
internalization of dopamine receptors (D1R/D2R) in living cells.
Lifetime imaging
Considering that the long luminescence lifetime of transition
metal complexes could overcome issues of an endogenous
uorescent background signal, 13-stained A549 cells were visualized with uorescence lifetime imaging microscopy (FLIM) to
demonstrate the merits of 13 in bioimaging. Most of the background uorescence has a luminescence lifetime of less than 3
ns, which is in contrast to the long luminescence lifetimes of
iridium(III) complexes. This large difference enables the
temporal separation of the probe uorescence signal from the
Complex 13 could selectively monitor the internalization of
dopamine receptors in cellulo
The internalization of dopamine receptor is important for
maintaining homeostatic control in the cell. We therefore
explored the application of complex 13 to track the internalization of dopamine receptors (D1R/D2R) in lung cancer cells.
The intracellular luminescence intensities of A549 cells treated
with complex 13 (30 mM) for different times (0, 10, 30, 60 and
180 min) were monitored by using uorescent microscopy. The
results showed that luminescence of the cells increased over
time and was predominantly localized in the cell boundaries
1122 | Chem. Sci., 2018, 9, 1119–1125
A549 cells were stained by complex 13 for 0, 10, 30, 60 and
180 min. The white arrow indicates the punctuated pattern. Scale bar
¼ 15 mm.
Fig. 6
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with 4.5 mL (5.1 mmol) of THP in 5 mL of DCM. The mixture
became homogeneous aer about 1 h, and the reaction was
essentially complete. When the TLC indicated the absence of
starting material, the reaction mixture was washed twice with
water, dried (Na2SO4), and evaporated to give 1.8 g of 3 (98%) as
a mixture of diastereoisomers, which were used in the next step
without further purication.
Fig. 7 Fluorescence lifetime confocal images of A549 cells treated
with (a) complex 13, (b) DAPI and (c) complex 13 + DAPI. The excitation
light of 800 nm from the MP laser (80 MHz pulse repetition rate) was
focused onto the sample with a 40 objective lens for two-photon
excitation. The luminescence signals were collected with the range of
550–650 nm.
intense background signal in cell media. As shown in Fig. 7a,
a long luminescence lifetime about 22 ns of complex 13 in cell
boundaries and the cytoplasm (blue) was detected by using
a Leica TCS SP5 confocal laser scanning microscope system in
A549 cells aer 1 h incubation. An MP laser with two-photon
excitation light of 800 nm was used for excitation. The FLIM
images consisted of 512 512 pixels were scanned with a scan
speed of 400 Hz. In contrast, DAPI only had a short luminescence lifetime in the cell nucleus (orange) in which its signal
decay in 2.1 ns aer the pulsed excitation (Fig. 7b). To validate
the potential of 13 in bioimaging, FLIM imaging of A549 cells coincubated with 13 and DAPI was also performed. A short excitation pulse with 80 MHz repetition rate excites all chromophores in the cell that absorb at the excitation wavelength,
including complex 13 and DAPI in the cell medium. As observed
from Fig. 7c, complex 13 showed a longer apparent lifetime than
DAPI in A549 cells (scale range from 0 ns (red) to 24 ns (blue)),
and displayed a longer uorescence intensity within cell
boundaries and the cytoplasm than that of DAPI in the cell
nucleus (orange). The FLIM results indicate that 13 has a longer
apparent lifetime within the cell than DAPI, suggesting that 13 is
suitable for the long-lived luminescence imaging even in the
presence of an endogenous uorescence background signal.
Experimental
Synthesis of 3,4-dihydroxyphenylacetic acid methyl ester (2)1
A solution of 1 g (0.0054 mol) of 3,4-dihydroxyphenylacetic acid
(1) in 50 mL of MeOH containing 1 mL of concentrated sulphuric acid (H2SO4) was reuxed overnight. The completion of
reaction was monitored by TLC. Aer completion, the MeOH
was evaporated, and the residue was dissolved in 50 mL of
EtOAc. The ethyl acetate layer was washed with dilute NaHCO3,
water, and brine, followed by drying with Na2SO4. The organic
layer was evaporated to give 1.2 g of the ester 2 (97%) that was
used in the next step without further purication.
Synthesis of 3,4-bis-(tetrahydropyranyloxy)phenylacetic acid
methyl ester (3)
A solution of 1 g (5.1 mmol) of 2 and 120 mg (0.102 mmol) of
pyridinium p-tosylate in 30 mL of DCM was treated dropwise
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Synthesis of 3,4-bis-(tetrahydropyranyloxy)phenylacetic acid
(4)
Compound 3 (1 g, 2.74 mmol) was dissolved in 20 mL of THF/
water (1 : 1) mixture at 0 C, followed by the addition of LiOH
(3.2 mmol), and allowed to react for 1 h. When the TLC indicated the absence of starting material, the pH of the reaction
mixture was adjusted to 4–5 using 5% HCl, extracted with ethyl
acetate, and evaporated to give 0.9 g of 4 (98%) as a mixture of
diastereoisomers, which were used in the next step without
further purication.
Synthesis of 3-(3,4-dihydroxyphenyl)-N-(1,10-phenanthrolin-5yl)propanamide (6)
5-Amino-1,10-phenanthroline (0.5 g, 2.5 mmol, 1 eq.) was dissolved in distilled DCM (50 mL) at 0 C. Compound 4 (0.8 g,
2.5 mmol, 1 eq.) was added, followed by EDCI (0.95 g, 5 mmol, 2
eq.) and, nally, 4-dimethylaminopyridine (DMAP) (0.3 g,
2.3 mmol, 1 eq.). Aer complete addition of reagents, the
reaction mixture was stirred at 0 C for a further 1 h and allowed
to complete for 24 h. Finally, the solvent was removed under
reduced pressure, and the red colour residue was dissolved in
EtOAc. The organic layer was washed with NaHCO3 (saturated)
solution and dried over Na2SO4. The organic layer was evaporated and puried by column chromatography to get compound
5 (0.9 g, 75%). Compound 5 was dissolved (0.50 g, 0.94 mmol) in
EtOH (10 mL), and PPTS was added (4 mg, 0.002 mmol). The
resulting mixture was stirred at 55 C for 6 h. Aer removal of
EtOH under vacuum, the residue was washed with EtOAc and
ether to give a pure white solid compound 6 (0.32 g, 94%). 1H
NMR (400 MHz, DMSO) d 9.12 (dd, J ¼ 4.2, 1.5 Hz, 1H), 9.05 (dd,
J ¼ 4.3, 1.7 Hz, 1H), 8.46 (dd, J ¼ 8.2, 1.7 Hz, 1H), 8.34 (dd, J ¼
8.4, 1.6 Hz, 1H), 8.11 (s, 1H), 7.76 (m, 2H), 6.67 (m, 2H), 6.53 (d, J
¼ 8.0 Hz, 1H), 2.83 (d, J ¼ 6.3 Hz, 2H), 2.77 (d, J ¼ 5.9 Hz, 2H).
13
C NMR (101 MHz, DMSO) d 171.71, 149.75, 149.36, 145.76,
145.22, 137.42, 135.75, 131.77, 129.94, 127.47, 124.78, 123.51,
122.74, 120.36, 118.87, 115.94, 115.53, 37.98, 30.56. HRMS:
calcd for C21H17N3O3: 359.3780 found [M + 1]+: 360.1609.
Synthesis of 4-(2-(1,10-phenanthroline-5-carboxamido)ethyl)1,2-phenylene diacetate (9)
4-(2-Aminoethyl)-1,2-phenylene diacetate (0.7 g, 3.12 mmol, 1
eq.) was dissolved in distilled DCM (30 mL) at 0 C. Compound
7 (1.28 g, 4.68 mmol, 1.5 eq.) was added, followed by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDCI) (0.95 g, 5 mmol,
2 eq.), HOBt (0.42 g, 3.12 mmol, 1 eq.), and, nally, Et3N (0.63 g,
6.3 mmol, 2 eq.). Aer complete addition of reagents, the
reaction mixture was stirred at 0 C for a further 1 h and allowed
to complete for 24 h. Completion of reaction was monitored by
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TLC using MeOH/DCM (10 : 90). The solvent was removed
under reduced pressure, and the red colour residue was dissolved in EtOAc. The organic layer was washed with NaHCO3
(saturated) solution and dried over Na2SO4. The organic layer
was evaporated and puried by column chromatography to get
compound 9 (0.5 g, 62%). 1H NMR (400 MHz, CDCl3) d 9.01 (d, J
¼ 3.0 Hz, 2H), 8.49 (dd, J ¼ 8.4, 1.5 Hz, 1H), 8.08 (dd, J ¼ 8.1,
1.4 Hz, 1H), 7.57 (s, 1H), 7.50 (ddd, J ¼ 10.3, 8.3, 4.3 Hz, 2H),
7.20 (s, 1H), 7.12 (m, 3H), 6.75 (s, 1H), 3.81 (dd, J ¼ 12.7, 6.4 Hz,
2H), 3.03 (t, J ¼ 6.6 Hz, 2H), 2.25 (s, 3H), 2.22 (s, 3H). 13C NMR
(101 MHz, CDCl3) d 168.45, 168.40, 168.03, 151.25, 150.35,
142.13, 140.77, 137.77, 136.76, 134.41, 132.87, 127.03, 126.93,
125.72, 125.57, 124.10, 123.69, 123.49, 123.42, 40.86, 34.84,
20.75, 20.71. HRMS: calcd for C25H21N3O5Na: 466.1373 found
[M + Na]+: 466.1352.
Materials and cell lines
All chemicals were purchased from Sigma-Aldrich and were
used as received. Lipofectamine™ 3000 reagent was purchased
from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS)
and Dulbecco's Modied Eagle's Medium (DMEM) were
purchased from Gibco BRL (Gaithersburg, MD, USA).
Cell viability assay
A549 cells were seeded at the density of 5000 cells per well in 96
well plates and incubated for 12 h. Complexes dissolved in
DMSO were added to cells at indicated concentrations for 48 h,
respectively. Then 10 mL of 5 mg mL1 MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent
were added to each well. Aer 4 h incubation in the dark, 100 mL
of DMSO were added to each well, and the intensity of absorbance was determined by a SpectraMax M5 microplate reader at
a wavelength of 570 nm.
Cell imaging
A549 cells were seeded into a glass-bottomed dish (35 mm dish
with 20 mm well). Aer 24 h, the cells were incubated with
complexes for indicated time periods or concentrations and
then washed with phosphate-buffered saline three times. The
luminescence imaging of complexes in cells was carried out by
a Leica TCS SP8 confocal laser scanning microscope system.
The excitation wavelength was 488 nm.
Immunoblotting
A549 cells were harvested in lysis buffer aer knockdown
treatment, and the protein concentration was determined by
using the BCA assay. Total proteins were separated on SDSpolyacrylamide gel electrophoresis and then transferred onto
polyvinylidene diuoride membranes (Millipore). Aer 1 h
incubation with blocking buffer at room temperature,
membranes were incubated with the primary antibodies at 4 C
overnight and the secondary antibodies for 1 h incubation at
room temperature. The protein bands were then stained by ECL
Western Blotting Detection Reagent (GE Healthcare) and visualized using the ChemiDoc™ MP Imaging System.
1124 | Chem. Sci., 2018, 9, 1119–1125
Edge Article
Dopamine D1/D2 receptor knockdown assay
A549 cells were seeded in 6 well plates at about 80% conuence
in DMEM for 12 h. Lipofectamine™ 3000 reagent and siRNA
were gently mixed in FBS-free DMEM medium. Aer 15 min
incubation at room temperature, 500 mL of siRNA-lipid complex
were directly added to cells in 1.5 mL DMEM culture medium.
Then, A549 cells were incubated at 37 C in a CO2 incubator for
48 h before use.
Photobleaching assay and FLIM imaging
A549 cells were seeded into a confocal glass-bottomed dish
(35 mm dish with 20 mm well) and incubated at 37 C for 12 h.
Subsequently, complex 13 (30 mM) was added and the wells were
further incubated for 1 h. Before cell imaging, cells were prexed with 4% paraformaldehyde for 15 min, followed by
washing three times with phosphate-buffered saline. DAPI
staining solution was added and the wells were incubated for
3 min. Aer washing in phosphate-buffered saline, uorescence
imaging was carried out with continuous excitation (l ¼ 405
nm) for 700 s using a Leica TCS SP8 confocal laser scanning
microscope. For FLIM imaging, complex 13 (60 mM) was added
and the wells were incubated for 1 h. The cells were pre-xed
with 4% paraformaldehyde for 15 min, followed by washing
three times with phosphate-buffered saline. DAPI staining
solution or buffer was added and the wells were further incubated for 3 min, followed by imaging using a Leica TCS SP5
confocal laser scanning microscope with a 40 objective lens.
An MP laser with 800 nm two-photon excitation wavelength and
80 MHz repetition rate were used for excitation. The luminescence signals were collected in the range of 550–650 nm. Photoluminescence lifetime images with 512 512 pixels were
acquired with scan speed of 400 Hz. The images were recorded
aer an excitation pulse without any time delay. Finally, the
FLIM data were analyzed using a pixel-based tting soware
(SPCImage, Becker & Hickl). An incomplete decay model in
SPCImage soware was employed for the calculation of lifetime
for complex 13.
Conclusions
Four iridium(III) complexes (11–14) bearing dopamine or 3-(3,4dihydroxyphenyl)propanoic acid as dopamine agonists were
synthesized. Among the synthesized complexes, complexes 11
and 13 displayed superior photophysical characteristics and
high stability in living cells. Moreover, D1R/D2R siRNA knockdown and dopamine competition experiments suggested that
complexes 11 and 13 could selectively bind to dopamine
receptors (D1R/D2R) in living cells. Finally, the photostable
complex 13 could also be used to monitor the internalization of
dopamine receptors (D1R/D2R) in living cells, and its application for long-lived luminescence imaging even in the presence
of endogenous uorescence background signal was also
demonstrated using FLIM. We envisage that these complexes
could serve as useful scaffolds for the development of luminescent dopamine receptors cell imaging probes.
This journal is © The Royal Society of Chemistry 2018
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Conflicts of interest
There are no conicts to declare.
Open Access Article. Published on 19 December 2017. Downloaded on 5/2/2026 2:50:53 AM.
This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.
Acknowledgements
This work is supported by Hong Kong Baptist University (FRG2/
15-16/002), the Health and Medical Research Fund (HMRF/
14130522 and HMRF/14150561), the Research Grants Council
(HKBU/12301115, HKBU/204612, HKBU/201913 and CityU/
11228316), the National Natural Science Foundation of China
(21575121, 21628502 and 21775131), the Guangdong Province
Natural Science Foundation (2015A030313816), Innovation and
Technology Fund (ITS/260/16FX), the Hong Kong Baptist
University Century Club Sponsorship Scheme 2017, the Interdisciplinary Research Matching Scheme (RC-IRMS/15-16/03),
the Science and Technology Development Fund, Macao SAR
(077/2016/A2), and the University of Macau (MYRG2015-00137ICMS-QRCM, MYRG2016-00151-ICMS-QRCM, MRG044/LCH/
2015/ICMS).
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