Cell imaging of dopamine receptor using agonist labeling iridium(III) complex

Article abstract of DOI:10.1039/c7sc04798c

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. Compl

Full text of DOI:10.1039/c7sc04798c

Chemical
Science
View Article Online
View Journal
EDGE ARTICLE
Cell imaging of dopamine receptor using agonist
labeling iridium(III) complex†
Cite this: DOI: 10.1039/c7sc04798c
a
b
a
b
Kasipandi Vellaisamy,‡ Guodong Li,‡ Chung-Nga Ko,‡ Hai-Jing Zhong,
c
c
d
a
Sarwat Fatima, Hiu-Yee Kwan, Chun-Yuen Wong,
Wai-Jing Kwong,*
ef
Chung-Hang Leung *b and Dik-Lung Ma
a
Weihong Tan,
*
*
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
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/
D2R) in living cells. To the best of our knowledge, complex 13 is the first metal-based compound that
has been used to monitor intracellular dopamine receptors in living cells.
Received 6th November 2017
Accepted 9th December 2017
DOI: 10.1039/c7sc04798c
rsc.li/chemical-science
3
important for cancer cell proliferation. Recently, Basu and
Introduction
coworkers have reported that a dopamine 2 receptor (D2R)
4
Lung cancer is a leading cause of death in both developed and agonist is able to inhibit angiogenesis. Studies with a D2R-
1
developing countries. Non-small cell lung cancer (NSCLC) is knockout mouse model have indicated the molecular mecha-
the most common subtype of lung cancer, with a 5 year survival nism through which D2R/VEGFR-2 crosstalk can mediate the
2
rate less than 15%. Angiogenesis, mediated by vascular endo- phosphorylation of VEGFR-2. Dopamine receptors also serve as
5
thelial growth factor (VEGF) and the VEGF receptor (VEGFR), is good targets for breast and colon cancers.
Early-stage identication of cancer is a high priority in
5,6
research. Therefore, the development of methods for the
a
Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, detection of cancer biomarkers in living systems has attracted
7–9
China. E-mail: edmondma@hkbu.edu.hk; dkwong@hkbu.edu.hk
b
tremendous interest.
Studies have shown that dopamine
State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese
receptor (D1R/D2R) expression is higher in NCI-H69 NSCLC
Medical Sciences, University of Macau, Macau, China. E-mail: duncanleung@umac.
mo
10
cells. Previously, the expression of the dopamine (D1/D2)
receptor in these cells has been monitored using an iodo-
c
School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong,
11
sulpride isotope probe which is subjected to biases caused by
China
d
1
2–14
the incubation of isotope. Fluorescent probes
and biotin
Department of Biology and Chemistry, City University of Hong Kong, Kowloon Tong,
15
Hong Kong, China
e
derivatives of dopamine agonists have also been developed as
Department of Chemistry, Department of Physiology and Functional Genomics, Center probes of D1R and D2R, whereas nitrobenzoxadiazole (NBD) and
for Research at the Bio/Nano Interface, Shands Cancer Center, UF Genetics Institute,

uorescein-coupled dopamine agonists have been applied in the
uorescent labelling of D1R and D2R sites in monkey pituitary
McKnight Brain Institute, University of Florida, Gainesville, USA. E-mail: tan@

chem.u.edu
f
gland. However, these probes are generally limited by their short
luminescence lifetimes (1–10 ns) and the phenomenon of self-
quenching, photobleaching and uorescence resonance energy
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
†
1
‡
Electronic supplementary information (ESI) available. See DOI: transfer (FRET). Although radiolabelled probes for dopamine
0.1039/c7sc04798c
123
receptors include S-labeled cRNA probes and [ I] N
3
-NAP have
These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 2017
Chem. Sci.

View Article Online
Chemical Science
Edge Article
16–21
been extensively reported,
great caution is required when dopamine receptors (D1R and D2R). In our designed strategy,
handling these radioactive materials. To the best of our knowl- the dopamine agonists were conjugated to N^N ligands 6 and 9.
edge, metal complex probes capable of identifying dopamine To synthesize ligand 6, compound 1 was rst converted to the
receptors (D1R and D2R) in living cells have not yet been corresponding methyl ester (2) in acidic conditions using MeOH
23–25
developed. In particular, the long luminescence lifetime, large as solvent (Scheme S1†).
Compound 2 was protected as the
Stokes shi, high luminescence quantum yield and high pho- tetrahydropyranyl ethers (THP) using pyridinium p-toluenesul-
tostability properties of iridium(III) complexes render them an fonate (PPTS) in dry DCM to generate compound 3 in 62%
30,31
26
excellent alternative agent for dopamine tracking.
yield. Subsequently, the THP-protected methyl ester (3) was
In this study, four cyclometalated iridium(III) complexes 11– selectively hydrolyzed using LiOH to furnish the corresponding
4 with general structure [Ir(N–C) (N–N)](PF ) (where N–N ¼ 3- THP-protected acid (4) aer acidic workup. Compound 4 was
1
2
6
(
3,4-dihydroxyphenylagonistsagonists)-N-(1,10-phenanthrolin- then coupled with 1,10-phenanthrolin-5-amine using standard
5-yl) propanamide (6) or 4-(2-(1,10-phenanthroline-5- 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) coupling
carboxamido)ethyl)-1,2-phenylene diacetate (9) and N–C ¼ 2- to generate compound 5. Finally, THP deprotection with a cata-
phenylpyridine (ppy), 2-(2,4-diuorophenyl)pyridine (dfppy), or lytic amount of PPTS in EtOH produced ligand 6 with a yield of
2
-phenylquinoline (pq)) were designed and synthesised 94%. Ligand 6 was characterized by NMR spectroscopy and
Scheme 1). As ligands 6 and 9 are derived from dopamine HRMS (Fig. S1 and S2†).
agonists, we hypothesized that the conjugated complexes would Ligand 9 was generated in 62% yield from an acetylated
(
be able to effectively recognize dopamine receptors (D2R/D1R). dopamine derivative (8) and 1,10-phenanthroline-5-carboxylic
Notably, complexes 11 and 13, showed superior cell imaging acid (7) using EDCI coupling (Scheme S2†). Ligand 9 was
characteristics, high stability and low cytotoxicity (>100 mM) in characterized by NMR spectroscopy and HRMS (Fig. S9 and
A549 lung cancer cells. siRNA knockdown and dopamine S10†).
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
Iridium(III) complexes synthesis
properties including long luminescence lifetimes, high photo- Complexes 11–14 were synthesised in good yields (85–91%) by
stability and high luminescence quantum yield. Since most of reacting two equivalents of N^N ligands (6 or 9) and the corre-
the background uorescence in cell medium has a lumines- sponding cyclometalated iridium(III) dimers in CH Cl /CH OH
2
2
3
cence lifetime of less than 3 ns, the relatively longer lumines- (1 : 1, v/v), followed by chloride anion exchange with NH PF .
4
6
cence lifetime for complex 13 should enable the temporal Complexes 11–14 were puried by silica gel column chroma-
1
13
separation of the probe signal from the intense background tography and characterized by H, C NMR spectroscopy and
signal by uorescence lifetime microscopy. To the best of our MALDI-HRMS. The photophysical properties of complexes 11–
knowledge, this is the rst application of iridium(III) complexes 14, including their luminescence quantum yields, emission
for imaging D2R/D1R within living cells.
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
Results and discussions
Synthesis of N^N ligands 6 and 9
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
We envisaged that conjugating dopamine agonists, such as
dopamine or 3-(3,4-dihydroxyphenyl)propanoic acid (1), to an
iridium(III) scaffold could generate effective probes for
22
13 exhibited an intense absorption at 255 and 280 nm and
a moderate peak at 331 and 446 nm in CH Cl . The bands are
2
2
assigned to the promotion of electrons based on the ligand-
centered (p–p*) transition and the metal-to-ligand charge
transfer (MLCT) transition respectively. Furthermore,
complexes 13 and 14 were both stable in a DMSO-d and D O
6
2
ꢀ
mixture (9 : 1) at 25 C for seven days, as revealed by NMR
spectroscopy, indicating that the acetyl groups of 14 are suffi-
ciently 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
Scheme 1 Iridium(III) dopamine complexes as D1R and D2R probes.
Chem. Sci.
This journal is © The Royal Society of Chemistry 2017

View Article Online
Edge Article
Chemical Science
from background uorescence by the use of time-resolved complex concentration (Fig. S18–S20†). In contrast, minimal
emission spectroscopy (TRES). luminescence was observed with complex 12, even when the
To validate this hypothesis, coumarin 460 (Cm-460) or thi- concentration of the complex was increased to 60 mM
oavin S (THS) was employed as model matrix interferents. In (Fig. S17†). We presume that complexes 11, 13 and 14 could
contrast to the iridium(III) complexes, organic uorophores interact strongly with the dopamine receptor (D1R/D2R) via
typically show nanosecond luminescence lifetimes. When the their dopamine agonist moieties, thereby leading to an
luminescence spectra were recorded directly aer the excitation enhanced luminescence of the cell.
pulse without any delay (lexc ¼ 355 nm), Cm-460 exhibited
a strong emission peak at 455 nm while THS exhibited
Validation of dopamine receptor binding in A549 cells
a moderate peak at 540 nm. Therefore, the peak of complex 13
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.
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 aer the excitation pulse, the short-lived
27–29
As shown by Western blotting experiments, the levels of both
D1R (Fig. 3a) and D2R (Fig. 3b) were reduced signicantly 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 signicantly, indicating that
the emission enhancement of the complexes required the pres-
ence 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 nonspecic 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

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 dopa-
mine receptor expression, using the MTT (3-(4,5-dimethylthiazol-2-
yl)-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 phos-
phate 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
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 interac-
tion between complexes and dopamine receptors (D1R/D2R) in
living cells, presumably via competitive binding to dopamine
receptors. Overall, complex 13 showed the highest lumines-
cence 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 Fig. 2 Luminescence and bright-field images of A549 cells stained
peak of complex 13 became more evident when delay ¼ 333 ns.
with 30 mM of complexes 11–14 for 1 h. Scale bar ¼ 15 mm.
This journal is © The Royal Society of Chemistry 2017
Chem. Sci.

View Article Online
Chemical Science
Edge Article
Fig. 3 (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
Fig. 5 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
(
30 mM) for 1 h. Scale bar ¼ 15 mm.
(green, 430 to 480 nm) and 2 (red, 550 to 650 nm).
within 60 min (Fig. 6). However, at 180 min, the luminescence
of cell boundaries was signicantly 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 conrmed by
assessing the luminescence in D1R/D2R knockdown A549 cells
aer 180 min incubation, which showed a decrease in the
luminescence of the cytoplasm as expected (Fig. S22†). Collec-
tively, 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.
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
Lifetime imaging
0
13 in paraformaldehyde-xed A549 cells. 4 ,6-Diamidino-2-
phenylindole (DAPI), an organic and commercial dye for Considering that the long luminescence lifetime of transition
staining nuclei in cells, was used as a benchmark for photo- metal complexes could overcome issues of an endogenous
stability. Aer continuous excitation at 405 nm for 700 s, the uorescent background signal, 13-stained A549 cells were visu-
mean luminescence intensity of complex 13 (550–650 nm, alized with uorescence lifetime imaging microscopy (FLIM) to
region 2) only decreased by 4.9%. Whereas the luminescence of demonstrate the merits of 13 in bioimaging. Most of the back-
DAPI (430–480 nm, region 1) decreased by 39.6% (Fig. 5). This ground uorescence has a luminescence lifetime of less than 3
result demonstrates that 13 exhibits higher photostability ns, which is in contrast to the long luminescence lifetimes of
compared to DAPI, indicating that 13 could be employed as iridium(III) complexes. This large difference enables the
a potential bioimaging luminescent probe for continuous temporal separation of the probe uorescence signal from the
tracking studies over a long period of time.
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 internali-
zation 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
1
80 min) were monitored by using uorescent microscopy. The
Fig. 6 A549 cells were stained by complex 13 for 0, 10, 30, 60 and
80 min. The white arrow indicates the punctuated pattern. Scale bar
results showed that luminescence of the cells increased over
1
time and was predominantly localized in the cell boundaries ¼ 15 mm.
Chem. Sci.
This journal is © The Royal Society of Chemistry 2017

View Article Online
Edge Article
Chemical Science
with 4.5 mL (5.1 mmol) of THP in 5 mL of DCM. The mixture
became homogeneous aer 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 (Na SO ), and evaporated to give 1.8 g of 3 (98%) as
2 4
a mixture of diastereoisomers, which were used in the next step
without further purication.
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
Synthesis of 3,4-bis-(tetrahydropyranyloxy)phenylacetic acid
(4)
focused onto the sample with a 40ꢁ objective lens for two-photon Compound 3 (1 g, 2.74 mmol) was dissolved in 20 mL of THF/
excitation. The luminescence signals were collected with the range of
50–650 nm.
ꢀ
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 indi-
5
(
cated 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 purication.
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 aer 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 lumines-
cence lifetime in the cell nucleus (orange) in which its signal
decay in 2.1 ns aer the pulsed excitation (Fig. 7b). To validate
the potential of 13 in bioimaging, FLIM imaging of A549 cells co-
incubated with 13 and DAPI was also performed. A short exci-
tation pulse with 80 MHz repetition rate excites all chromo-
phores 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.
Synthesis of 3-(3,4-dihydroxyphenyl)-N-(1,10-phenanthrolin-5-
yl)propanamide (6)
5
-Amino-1,10-phenanthroline (0.5 g, 2.5 mmol, 1 eq.) was dis-
ꢀ
solved 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.). Aer 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 NaHCO
solution and dried over Na SO . The organic layer was evapo-
rated and puried by column chromatography to get compound
(0.9 g, 75%). Compound 5 was dissolved (0.50 g, 0.94 mmol) in
3
(saturated)
2
4
5
EtOH (10 mL), and PPTS was added (4 mg, 0.002 mmol). The
ꢀ
resulting mixture was stirred at 55 C for 6 h. Aer removal of
EtOH under vacuum, the residue was washed with EtOAc and
1
ether to give a pure white solid compound 6 (0.32 g, 94%). H
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).
Experimental
Synthesis of 3,4-dihydroxyphenylacetic acid methyl ester (2)
1
1
3
C NMR (101 MHz, DMSO) d 171.71, 149.75, 149.36, 145.76,
A solution of 1 g (0.0054 mol) of 3,4-dihydroxyphenylacetic acid
1
1
45.22, 137.42, 135.75, 131.77, 129.94, 127.47, 124.78, 123.51,
(1) in 50 mL of MeOH containing 1 mL of concentrated sul-
22.74, 120.36, 118.87, 115.94, 115.53, 37.98, 30.56. HRMS:
phuric acid (H SO ) was reuxed overnight. The completion of
+
2
4
calcd for C21
H
17
N
3
O
3
: 359.3780 found [M + 1] : 360.1609.
reaction was monitored by TLC. Aer completion, the MeOH
was evaporated, and the residue was dissolved in 50 mL of
Synthesis of 4-(2-(1,10-phenanthroline-5-carboxamido)ethyl)-
EtOAc. The ethyl acetate layer was washed with dilute NaHCO
3
,
1,2-phenylene diacetate (9)
water, and brine, followed by drying with Na SO . The organic
2
4
4
-(2-Aminoethyl)-1,2-phenylene diacetate (0.7 g, 3.12 mmol, 1
layer was evaporated to give 1.2 g of the ester 2 (97%) that was
used in the next step without further purication.
ꢀ
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-
(
2
6
3-dimethylaminopropyl)carbodiimide (EDCI) (0.95 g, 5 mmol,
Synthesis of 3,4-bis-(tetrahydropyranyloxy)phenylacetic acid
methyl ester (3)
eq.), HOBt (0.42 g, 3.12 mmol, 1 eq.), and, nally, Et N (0.63 g,
3
.3 mmol, 2 eq.). Aer complete addition of reagents, the
ꢀ
A solution of 1 g (5.1 mmol) of 2 and 120 mg (0.102 mmol) of reaction mixture was stirred at 0 C for a further 1 h and allowed
pyridinium p-tosylate in 30 mL of DCM was treated dropwise to complete for 24 h. Completion of reaction was monitored by
This journal is © The Royal Society of Chemistry 2017
Chem. Sci.

View Article Online
Chemical Science
Edge Article
TLC using MeOH/DCM (10 : 90). The solvent was removed Dopamine D1/D2 receptor knockdown assay
under reduced pressure, and the red colour residue was dis-
A549 cells were seeded in 6 well plates at about 80% conuence
in DMEM for 12 h. Lipofectamine™ 3000 reagent and siRNA
were gently mixed in FBS-free DMEM medium. Aer 15 min
incubation at room temperature, 500 mL of siRNA-lipid complex
solved in EtOAc. The organic layer was washed with NaHCO3
saturated) solution and dried over Na SO . The organic layer
(
2
4
was evaporated and puried by column chromatography to get
1
3
compound 9 (0.5 g, 62%). H NMR (400 MHz, CDCl ) d 9.01 (d, J
were directly added to cells in 1.5 mL DMEM culture medium.
¼
3.0 Hz, 2H), 8.49 (dd, J ¼ 8.4, 1.5 Hz, 1H), 8.08 (dd, J ¼ 8.1,
ꢀ
Then, A549 cells were incubated at 37 C in a CO
2
incubator for
1
7
2
.4 Hz, 1H), 7.57 (s, 1H), 7.50 (ddd, J ¼ 10.3, 8.3, 4.3 Hz, 2H),
.20 (s, 1H), 7.12 (m, 3H), 6.75 (s, 1H), 3.81 (dd, J ¼ 12.7, 6.4 Hz,
48 h before use.
1
3
H), 3.03 (t, J ¼ 6.6 Hz, 2H), 2.25 (s, 3H), 2.22 (s, 3H). C NMR
(
101 MHz, CDCl ) d 168.45, 168.40, 168.03, 151.25, 150.35, Photobleaching assay and FLIM imaging
3
1
1
2
42.13, 140.77, 137.77, 136.76, 134.41, 132.87, 127.03, 126.93,
25.72, 125.57, 124.10, 123.69, 123.49, 123.42, 40.86, 34.84,
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.
21 3 5
0.75, 20.71. HRMS: calcd for C25H N O Na: 466.1373 found
Subsequently, complex 13 (30 mM) was added and the wells were
further incubated for 1 h. Before cell imaging, cells were pre-
+
[M + Na] : 466.1352.

xed with 4% paraformaldehyde for 15 min, followed by
Materials and cell lines
washing three times with phosphate-buffered saline. DAPI
All chemicals were purchased from Sigma-Aldrich and were staining solution was added and the wells were incubated for
used as received. Lipofectamine™ 3000 reagent was purchased 3 min. Aer washing in phosphate-buffered saline, uorescence
from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS) imaging was carried out with continuous excitation (l ¼ 405
and Dulbecco's Modied Eagle's Medium (DMEM) were nm) for 700 s using a Leica TCS SP8 confocal laser scanning
purchased from Gibco BRL (Gaithersburg, MD, USA).
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 incu-
bated for 3 min, followed by imaging using a Leica TCS SP5
confocal laser scanning microscope with a 40ꢁ objective lens.
An MP laser with 800 nm two-photon excitation wavelength and
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,
ꢂ1
respectively. Then 10 mL of
5
mg mL
MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent
were added to each well. Aer 4 h incubation in the dark, 100 mL
of DMSO were added to each well, and the intensity of absor-
bance was determined by a SpectraMax M5 microplate reader at
a wavelength of 570 nm.
80 MHz repetition rate were used for excitation. The lumines-
cence signals were collected in the range of 550–650 nm. Pho-
toluminescence lifetime images with 512 ꢁ 512 pixels were
acquired with scan speed of 400 Hz. The images were recorded
aer an excitation pulse without any time delay. Finally, the
FLIM data were analyzed using a pixel-based tting soware
Cell imaging
(SPCImage, Becker & Hickl). An incomplete decay model in
A549 cells were seeded into a glass-bottomed dish (35 mm dish SPCImage soware was employed for the calculation of lifetime
with 20 mm well). Aer 24 h, the cells were incubated with for complex 13.
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
Conclusions
a Leica TCS SP8 confocal laser scanning microscope system.
Four iridium(III) complexes (11–14) bearing dopamine or 3-(3,4-
The excitation wavelength was 488 nm.
dihydroxyphenyl)propanoic acid as dopamine agonists were
synthesized. Among the synthesized complexes, complexes 11
and 13 displayed superior photophysical characteristics and
Immunoblotting
A549 cells were harvested in lysis buffer aer knockdown high stability in living cells. Moreover, D1R/D2R siRNA knock-
treatment, and the protein concentration was determined by down and dopamine competition experiments suggested that
using the BCA assay. Total proteins were separated on SDS- complexes 11 and 13 could selectively bind to dopamine
polyacrylamide gel electrophoresis and then transferred onto receptors (D1R/D2R) in living cells. Finally, the photostable
polyvinylidene diuoride membranes (Millipore). Aer 1 h complex 13 could also be used to monitor the internalization of
incubation with blocking buffer at room temperature, dopamine receptors (D1R/D2R) in living cells, and its applica-
ꢀ
membranes were incubated with the primary antibodies at 4 C tion for long-lived luminescence imaging even in the presence
overnight and the secondary antibodies for 1 h incubation at of endogenous uorescence background signal was also
room temperature. The protein bands were then stained by ECL demonstrated using FLIM. We envisage that these complexes
Western Blotting Detection Reagent (GE Healthcare) and visu- could serve as useful scaffolds for the development of lumi-
alized using the ChemiDoc™ MP Imaging System.
nescent dopamine receptors cell imaging probes.
Chem. Sci.
This journal is © The Royal Society of Chemistry 2017

View Article Online
Edge Article
Chemical Science
1
3 S. L. Vincent, Y. Khan and F. M. Benes, Synapse, 1995, 19,
Conflicts of interest
112.
There are no conicts to declare.
14 V. Bakthavachalam, N. Baindur, B. K. Madras and
J. L. Neumeyer, J. Med. Chem., 1991, 34, 3235–3241.
1
5 B. K. Madras, D. Caneld, C. Pfaelzer, F. Vittimberga,
M. Diglia, N. Aronin, V. Bakthavachalam, N. Baindur and
J. L. Neumeyer, Mol. Pharmacol., 1990, 37, 833–839.
Acknowledgements
This work is supported by Hong Kong Baptist University (FRG2/
1
1
5-16/002), the Health and Medical Research Fund (HMRF/ 16 M. A. Ariano, F. J. Monsma, A. C. Barton, H. C. Kang,
4130522 and HMRF/14150561), the Research Grants Council
R. P. Haugland and D. R. Sibley, Proc. Natl. Acad. Sci. U. S.
A., 1989, 86, 8570–8574.
(HKBU/12301115, HKBU/204612, HKBU/201913 and CityU/
1
1228316), the National Natural Science Foundation of China 17 C. Le Moine and B. Bloch, J. Comp. Neurol., 1995, 355, 418–
426.
Natural Science Foundation (2015A030313816), Innovation and 18 N. Amlaiky and M. G. Caron, J. Biol. Chem., 1985, 260, 1983–
Technology Fund (ITS/260/16FX), the Hong Kong Baptist
1986.
University Century Club Sponsorship Scheme 2017, the Inter- 19 M. M. Kong, A. Hasbi, M. Mattocks, T. Fan, B. F. O'Dowd and
disciplinary Research Matching Scheme (RC-IRMS/15-16/03),
S. R. George, Mol. Pharmacol., 2007, 72, 1157–1170.
the Science and Technology Development Fund, Macao SAR 20 M. Hakim, Y. Y. Broza, O. Barash, N. Peled, M. Phillips,
077/2016/A2), and the University of Macau (MYRG2015-00137-
A. Amann and H. Haick, Chem. Rev., 2012, 112, 5949–5966.
ICMS-QRCM, MYRG2016-00151-ICMS-QRCM, MRG044/LCH/ 21 A. B. Chinen, C. M. Guan, J. R. Ferrer, S. N. Barnaby,
015/ICMS).
T. J. Merkel and C. A. Mirkin, Chem. Rev., 2015, 115,
0530–10574.
(21575121, 21628502 and 21775131), the Guangdong Province
(
2
1
2
2 M. Y. S. Kalani, N. Vaidehi, S. E. Hall, R. J. Trabanino,
P. L. Freddolino, M. A. Kalani, W. B. Floriano,
V. W. T. Kam and W. A. Goddard, Proc. Natl. Acad. Sci. U.
S. A., 2004, 101, 3815–3820.
Notes and references
1
2
3
A. Jemal, F. Bray, M. M. Center, J. Ferlay, E. Ward and
D. Forman, Ca-Cancer J. Clin., 2011, 61, 69–90.
K. Cetin, D. S. Ettinger, Y.-j. Hei and C. D. O'Malley, Clin. 23 J. Narayanan, Y. Hayakawa, J. Fan and K. L. Kirk, Bioorg.
Epidemiol., 2011, 3, 13.
Chem., 2003, 31, 191–197.
S. Basu, C. Sarkar, D. Chakroborty, J. Nagy, R. B. Mitra, 24 N. M. Gueddouda, M. R. Hurtado, S. Moreau, L. Ronga,
P. S. Dasgupta and D. Mukhopadhyay, Cancer Res., 2004,
R. N. Das, S. Savrimoutou, S. Rubio, A. Marchand,
O. Mendoza, M. Marchivie, L. Elmi, A. Chansavang,
V. Desplat, V. Gabelica, A. Bourdoncle, J.-L. Mergny and
J. Guillon, ChemMedChem, 2017, 12, 146.
64, 5551–5555.
4
L. H. Hoeppner, Y. Wang, A. Sharma, N. Javeed, V. P. Van
Keulen, E. Wang, P. Yang, A. C. Roden, T. Peikert and
J. R. Molina, Mol. Oncol., 2015, 9, 270–281.
2
5 A. Bedrat, L. Lacroix and J.-L. Mergny, Nucleic Acids Res.,
5
6
J. Sheng, J. Gan and Z. Huang, Med. Res. Rev., 2013, 33, 1119–
2016, 44, 1746–1759.
26 M. Miyashita, A. Yoshikoshi and P. A. Grieco, J. Org. Chem.,
1977, 42, 3772–3774.
27 J.-j. Xu, S.-y. Wang, Y. Chen, G.-p. Chen, Z.-q. Li, X.-y. Shao,
L. Li, W. Lu and T.-y. Zhou, Acta Pharmacol. Sin., 2014, 35,
1173.
X.-B. Zhang, Z. Wang, H. Xing, Y. Xiang and Y. Lu, Anal.
Chem., 2010, 82, 5005–5011.
S. K. Arya and S. Bhansali, Chem. Rev., 2011, 111, 6783–6809.
D. Feng, Y. Song, W. Shi, X. Li and H. Ma, Anal. Chem., 2013,
7
8
889–898.
8
5, 6530–6535.
Z. Li, X. Gao, W. Shi, X. Li and H. Ma, Chem. Commun., 2013,
9, 5859–5861.
2
8 M. Moreno-Smith, C. Lu, M. M. Shahzad, G. N. A. Pena,
J. K. Allen, R. L. Stone, L. S. Mangala, H. D. Han, H. S. Kim
and D. Farley, Clin. Cancer Res., 2011, 17, 3649–3659.
9
4
1
0 D. Ferone, M. Arvigo, C. Semino, P. Jaquet, A. Saveanu, 29 J. Gan, J. Sheng and Z. Huang, Sci. China: Chem., 2011, 54, 3–
J. E. Taylor, J.-P. Moreau, M. D. Culler, M. Albertelli and
23.
F. Minuto, Am. J. Physiol.: Endocrinol. Metab., 2005, 289, 30 C. Liu, C. Yang, L. Lu, W. Wang, W. Tan, C.-H. Leung and
E1044–E1050.
D.-L. Ma, Chem. Commun., 2017, 53, 2822.
1
1
1 S. E. Senogles, Anti-Cancer Drugs, 2007, 18, 801–807.
2 F. J. Monsma, A. C. Barton, H. C. Kang, D. L. Brassard,
R. P. Haugland and D. R. Sibley, J. Neurochem., 1989, 52,
3
1 L.-J. Liu, W. Wang, S.-Y. Huang, Y. Hong, G. Li, S. Lin, J. Tian,
Z. Cai, H.-M. D. Wang and D.-L. Ma, Chem. Sci., 2017, 8,
4756.
1641.
This journal is © The Royal Society of Chemistry 2017
Chem. Sci.