Dual-Emissive Cyclometalated Iridium(III) Polypyridine Complexes as Ratiometric Biological Probes and Organelle-Selective Bioimaging Reagents

Article abstract of DOI:10.1021/acs.inorgchem.5b00944

In this Article, we present a series of cyclometalated iridium(III) polypyridine complexes of the formula [Ir(N^C)₂(N^N)](PF₆) that showed dual emission under ambient conditions. The structures of the cyclometalating and diimine liga

Full text of DOI:10.1021/acs.inorgchem.5b00944

Article
pubs.acs.org/IC
Dual-Emissive Cyclometalated Iridium(III) Polypyridine Complexes as
Ratiometric Biological Probes and Organelle-Selective Bioimaging
Reagents
Kenneth Yin Zhang,*,† Hua-Wei Liu,‡ Man-Chung Tang,‡ Alex Wing-Tat Choi,‡ Nianyong Zhu,§
Xi-Guang Wei,‡ Kai-Chung Lau,‡ and Kenneth Kam-Wing Lo*,‡,∥
†Key Laboratory for Organic Electronics and Information Displays & Institute of Advanced Materials (IAM), Jiangsu National
Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts & Telecommunications, 9 Wenyuan
Road, Nanjing 210023, P. R. China
‡Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, P. R. China
§Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, P. R. China
∥State Key Laboratory of Millimeters Waves, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, P. R. China
S
* Supporting Information
ABSTRACT: In this Article, we present a series of cyclo-
metalated iridium(III) polypyridine complexes of the formula
[Ir(N^C)2(N^N)](PF6) that showed dual emission under
ambient conditions. The structures of the cyclometalating and
diimine ligands were changed systematically to investigate the
effects of the substituents on the dual-emission properties of
the complexes. On the basis of the photophysical data, the
high-energy (HE) and low-energy (LE) emission features of
the complexes were assigned to triplet intraligand (3IL) and
triplet charge-transfer (3CT) excited states, respectively. Time-
dependent density functional theory (TD-DFT) calculations
supported these assignments and indicated that the dual
emission resulted from the interruption of the communication
between the higher-lying 3IL and the lower-lying 3CT states by a triplet amine-to-ligand charge-transfer (3NLCT) state. Also, the
avidin-binding properties of the biotin complexes were studied by emission titrations, and the results showed that the dual-
emissive complexes can be utilized as ratiometric probes for avidin. Additionally, all the complexes exhibited efficient cellular
uptake by live HeLa cells. The MTT and Annexin V assays confirmed that no cell death and early apoptosis occurred during the
cell imaging experiments. Interestingly, laser-scanning confocal microscopy revealed that the complexes were selectively localized
on the cell membrane, mitochondria, or both, depending on the nature of the substituents of the ligands. The results of this work
will contribute to the future development of dual-emissive transition metal complexes as ratiometric probes and organelle-
selective bioimaging reagents.
of the two emission bands will enable self-calibration of the
fluorescence signals and hence more accurate quantification of
the analyte. The current design of dual-emissive probes relies
on covalent connection or hybridization of two fluorescent
compounds.32−36 Although it is a straightforward strategy, there
are some limitations: for example, (1) a complicated synthetic
process is required to put the two units together, (2) the
resultant probes usually have a large molecular size, which
reduces their cellular uptake efficiency, and (3) possible
electron- and/or energy-transfer between the two fluorescent
units may lower the accuracy of the sensing. Thus, simple dual-
emissive compounds with single-wavelength excitation are
INTRODUCTION
■
Due to the high sensitivity and short response time of
fluorescence sensing, there has been much interest in using
fluorescent materials including organic dyes,1−6 transition metal
complexes,5−10 lanthanide chelates,5,6,11 and inorganic nano-
particles12,13 as probes for environmental physical parameters
and chemical analytes. Fluorescent probes have been applied to
visualize live cells13−20 and measure intracellular temperature,21
oxygen content,22 pH,23 and the concentration of metal
cations24−27 and various endogenous analytes28−31 by laser-
scanning confocal microscopy. However, the results of
fluorescence intensity-based sensing may be easily affected by
the inherent variability in incident laser power and intracellular
concentration of the probe itself. An alternative approach
involving dual-emissive probes that display a change in the ratio
Received: April 27, 2015
© XXXX American Chemical Society
A
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more attractive candidates because these potential problems
can be avoided. Although Kasha’s rule states that fluorescence
only occurs from the lowest-energy excited state,37 transition
metal complexes that simultaneously display dual emission
from two excited states under ambient conditions have been
reported.38−40 For example, charge-neutral iridium(III) com-
plexes exhibiting dual emission have been used as a ratiometric
sensor for the Cu2+ ion.40 Since cationic complexes enter live
cells more readily than neutral ones,41−44 we anticipate that
cationic dual-emissive systems will afford intracellular ratio-
metric probes with high cellular uptake efficiency.
Chart 1. Structures of the Complexes
Previously, we have discovered a cationic cyclometalated
iridium(III) polypyridine complex with an amine on the
cyclometalating ligand and an amide on the diimine ligand
[Ir(ppy-C4)2(bpy-Et)](PF6) (Hppy-C4 = 2-(4-((N-n-butyl)-
aminomethyl)phenyl)pyridine, bpy-Et = 4-(N-(ethyl)-
aminocarbonyl)-4′-methyl-2,2′-bipyridine) that displays inter-
esting dual emission under ambient conditions.45 Remarkably,
the high-energy (HE) and low-energy (LE) emission features,
and hence the overall emission profile, depend strongly on the
polarity of the local environment of the complex. The complex
has also been derivatized to afford homogeneous sensors for
avidin, estrogen receptor α, and lipid-binding proteins. To
rationalize the roles of the amine and ligand structures in the
dual-emission properties of these complexes, and to understand
the effects of the ligands on their cellular uptake and
intracellular localization properties, we have launched a
program to design new dual-emissive cyclometalated iridium-
(III) polypyridine complexes and examine their photophysical
and biological properties. Herein we report the synthesis and
characterization of a family of structurally related complexes
[Ir(N^C)2(N^N)](PF6) (HN^C = Hppy-C4, 2-((4′-((N-n-
butyl)aminomethyl)-1,1′-biphenyl)-4-yl)pyridine (Hpppy-C4);
N^N = bpy-Et, 3,4,7,8-tetramethyl-1,10-phenanthroline (Me4-
phen), 4,4′-dimethyl-2,2′-bipyridine (Me2-bpy), 1,10-phenan-
throline (phen), 4,7-diphenyl-1,10-phenanthroline (Ph2-phen))
(Chart 1). Also, complexes without the amine groups
[Ir(pppy)2(N^N)](PF6) (Hpppy = 2-((1,1′-biphenyl)-4-yl)-
pyridine, N^N = bpy-Et, Me4-phen, Me2-bpy, phen, Ph2-phen)
were synthesized for comparison studies. Additionally, time-
dependent density functional theory (TD-DFT) calculations
were performed on [Ir(ppy)2(bpy-Et)](PF6) (Hppy = 2-
phenylpyridine) and [Ir(ppy-C4)2(bpy-Et)](PF6) to under-
stand their excited-state nature. With an aim to evaluate the
potential application of these complexes as ratiometric probes,
we incorporated biotin into the dual-emissive system, affording
the complexes [Ir(N^C)2(bpy-biotin)](PF6) (HN^C = Hppy-
C4, Hpppy-C4, Hpppy) (Chart 1) as luminescent probes for
avidin. Furthermore, the cellular uptake efficiency and live cell
staining properties of these complexes were studied using
human cervix epithelioid carcinoma (HeLa) cells as a model
cell line. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
tetrazolium bromide) and Annexin V assays were performed to
determine the optimal conditions for live cell imaging, under
which images of good quality can be obtained with minimum
cell death. Furthermore, the intracellular localization of all the
complexes was investigated by laser-scanning confocal micros-
copy, and the specific organelle staining was also confirmed by
co-staining experiments.
ppy-C4 ligand yielding the pppy-C4 ligand to study the effects
of the cyclometalating ligands. The importance of the amine
group was investigated using the pppy ligand as an amine-free
analogue for pppy-C4. Also, five diimine ligands were selected
to elucidate the role of the π*(N^N) orbitals on the dual-
emission properties of the complexes. The cyclometalating
ligands 2-((4′-formyl-1,1′-biphenyl)-4-yl)pyridine (Hpppy-
CHO) and Hpppy were synthesized by the Suzuki coupling
of 2-(4-bromophenyl)pyridine with 4-formylphenylboronic
acid and phenylboronic acid, respectively, using Pd(PPh3)2Cl2
as the catalyst (Scheme 1).46 The cyclometalated iridium(III)
polypyridine complexes [Ir(N^C)2(N^N)](PF6) (HN^C = 4-
(2-pyridyl)benzaldehyde (Hppy-CHO), Hpppy-CHO, Hpppy)
were obtained from the reaction of the dichloro-bridged dimer
[Ir2(N^C)4Cl2] with the diimine ligand N^N in refluxing
CH2Cl2/MeOH. During the preparation of the aldehyde
complexes [Ir(pppy-CHO)2(N^N)](PF6), we found that the
aldehyde groups reacted with MeOH in the solvent mixture
and formed stable acetals. To avoid this, a mixture of MeOH/
CH2Cl2/H2O (1:3:1, v/v) was used as the solvent for the
reaction. The amine-containing complexes [Ir(ppy-
C4)2(N^N)](PF6) and [Ir(pppy-C4)2(N^N)](PF6) were
synthesized from the reductive amination of their respective
aldehyde precursor complexes [Ir(ppy-CHO)2(N^N)](PF6)
and [Ir(pppy-CHO)2(N^N)](PF6) with n-butylamine. All the
1
complexes were characterized by H NMR spectroscopy, IR
spectroscopy, positive-ion ESI-MS, elemental microanalysis,
UV−vis absorption spectroscopy, and cyclic voltammetry.
The complexes exhibited good solubility in common organic
solvents and aqueous MeOH or DMSO solutions except
[Ir(pppy-C4)2(bpy-biotin)](PF6), which was almost insoluble
in CH2Cl2, and [Ir(pppy)2(N^N)](PF6) (N^N = Me4-phen,
Me2-bpy, phen, Ph2-phen), which were sparingly soluble in
aqueous buffers containing as much as 50% MeOH or DMSO.
RESULTS AND DISCUSSION
■
Synthesis and Characterization. Regarding the design of
our ligands, an additional phenyl ring was incorporated into the
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Scheme 1. Synthetic Procedure of the Cyclometalating Ligands Hpppy-CHO and Hpppy
Single crystals of [Ir(ppy-C4)2(Me2-bpy)](PF6) suitable for X-
ray crystallographic studies were obtained by layering
petroleum ether (40−60 °C) over a concentrated acetone
solution of the complex. The perspective view of complex
cation [Ir(ppy-C4)2(Me2-bpy)]+ with the atomic numbering
scheme is depicted in Figure 1. Selected bond lengths and bond
Information Table S2. All the complexes showed intense spin-
allowed IL (π → π*) (N^N and N^C) absorption bands and
1
shoulders in the UV region and less intense absorption features
related to spin-allowed metal-to-ligand charge-transfer
(1MLCT) (dπ(Ir) → π*(N^N and N^C)) transitions in the
visible region. The pppy-C4 complexes revealed stronger
absorption at ca. 250−325 nm than their pppy counterparts,
probably due to additional singlet amine-to-ligand charge-
transfer (1NLCT) (amine → π*(N^N and N^C)) transitions
(Supporting Information Table S2 and Figure S1). The
electrochemical properties of the iridium(III) complexes were
studied by cyclic voltammetry, and the electrochemical data are
listed in Supporting Information Table S3. The amine-
containing ppy-C4 and pppy-C4 complexes exhibited two
oxidation processes: an irreversible wave at +0.87 to +1.15 V
and a quasireversible couple at +1.24 to +1.46 V versus SCE,
which were assigned to the oxidation of the secondary amine
groups and the iridium center, respectively. The amine-free
pppy complexes only showed one reversible iridium(IV/III)
oxidation couple at +1.22 to +1.27 V. The first reduction
couples of the diimine ligands of all the complexes were
reversible in nature and appeared at ca. −1.23 to −1.63 V
versus SCE.
Emission Properties. Upon photoexcitation, all the
complexes exhibited intense and long-lived green to orange
luminescence in degassed fluid solutions under ambient
conditions. The photophysical data of the complexes are
summarized in Table 1. The emission properties of these
complexes are highly dependent on both cyclometalating and
diimine ligands. First, the effects of the cyclometalating ligands
on the emission properties were investigated using the
complexes [Ir(N^C)2(bpy-Et)](PF6) (N^C = ppy-C4, pppy-
C4, ppy, pppy) as examples. Interestingly, the amine-containing
ppy-C4 and pppy-C4 complexes showed well-resolved dual
emission that was composed of a vibronically structured high-
energy (HE) emission feature in blue-green region and a
structureless low-energy (LE) emission band in the yellow to
orange-red region (Figure 2a,b, Table 1). The emission colors
of these two complexes depended on the polarity of the
solvents. In less polar CH2Cl2, the HE emission intensity was
Figure 1. Perspective view of the complex cation [Ir(ppy-C4)2(Me2-
bpy)]+ with the atomic numbering scheme. Thermal ellipsoids are
shown at the 30% probability level. Hydrogen atoms are omitted for
clarity.
angles are listed in Supporting Information Table S1. Similar to
related bis(cyclometalated) iridium(III) polypyridine com-
plexes,18 the iridium(III) center of [Ir(ppy-C4)2(Me2-bpy)]+
adopted a distorted octahedral geometry, and the trans angles at
the metal center ranged from 171.85(17)° to 174.2(2)°. The
metal−carbon bonds around the iridium(III) center were in a
cis orientation, and the bond lengths were 2.022(5) Å. As
expected, the trans-influence of the carbon donors rendered
longer Ir−N bond lengths for the Me2-bpy ligand (2.159(4) Å)
than the ppy-C4 ligands (2.057(5) Å). The bite angles of the
ppy-C4 ligands (80.6(2)°) were larger than that of the Me2-bpy
ligand (76.0(2)°).
Electronic Absorption and Electrochemical Proper-
ties. The electronic absorption spectral data of the iridium(III)
complexes in selected solvents at 298 K are listed in Supporting
C
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Table 1. Photophysical Data of the Complexes in Deaerated Solutions
medium
medium
complex
(T/K)
λem/nm
τo/μs
Φem
0.11
complex
(T/K)
λem/nm
τo/μs
Φem
[Ir(ppy-C4)2(bpy-Et)] CH2Cl2
498 sh, 521
1.06
CH3CN
(298)
524 sh, 556
1.58
0.23
(PF6)
(298)
a
593 (max)
0.28
1.43
buffer
523 (max), 557,
619 sh
2.97
1.17
1.11
1.44
1.12
0.91
2.03
8.62
7.31
5.61
1.22
1.12
0.47
1.19
1.06
0.36
1.07
0.96
0.39
1.92
0.12
0.45
0.23
0.09
0.50
0.23
0.08
0.46
(298)
CH3CN
(298)
495, 523 sh
0.065
[Ir(pppy-C4)2(phen)]
(PF6)
CH2Cl2
(298)
561
612 sh
0.14
2.48
a
CH3CN
(298)
567
buffer
500, 525 sh
0.17
0.18
(298)
a
buffer
523 sh, 558
567
[Ir(pppy-C4)2(bpy-
Et)](PF6)
CH2Cl2
(298)
531, 576 sh
4.37
(298)
[Ir(pppy-C4)2(Ph2-
phen)](PF6)
CH2Cl2
(298)
602 (max)
0.55
4.73
CH3CN
(298)
528, 573 sh
0.079
0.14
CH3CN
(298)
577
604 (max)
0.30
6.52
a
buffer
575
a
buffer
528 (max), 563
(298)
(298)
[Ir(pppy)2(Me4-phen)] CH2Cl2
523 (max), 551,
603 sh
606 sh
609
0.052
0.40
(PF6)
(298)
[Ir(ppy)2(bpy-Et)]
(PF6)
CH2Cl2
(298)
0.13
0.058
0.0069
0.13
0.069
c
CH3CN
(298)
521 (max), 551,
601 sh
0.41
a
c
CH3CN
(298)
613
0.23
0.078
0.46
0.27
0.041
2.56
buffer
516 (max), 551,
601 sh
(298)
b
buffer
626
[Ir(pppy)2(Me2-bpy)]
(PF6)
CH2Cl2
(298)
522 sh, 555
526 sh, 560
525 sh, 554
564
0.44
(298)
[Ir(pppy)2(bpy-Et)]
(PF6)
CH2Cl2
(298)
598
CH3CN
(298)
0.28
a
c
CH3CN
(298)
602
buffer
(298)
a
buffer
608
[Ir(pppy)2(phen)]
(PF6)
CH2Cl2
(298)
0.42
(298)
[Ir(ppy-C4)2(Me4-
phen)](PF6)
CH2Cl2
(298)
482 sh
0.47
CH3CN
(298)
569
0.28
a
c
518
1.82
2.49
buffer
562
(298)
CH3CN
(298)
482 sh
0.43
[Ir(pppy)2(Ph2-phen)] CH2Cl2
571
0.51
(PF6)
(298)
538
1.58
4.45
a
CH3CN
(298)
578
0.45
buffer
482 (max), 512,
556 sh
0.46
0.39
(298)
a
c
buffer
562
[Ir(ppy-C4)2(Me2-
bpy)](PF6)
CH2Cl2
(298)
487 sh
1.98
(298)
[Ir(ppy-C4)2(bpy-
biotin)](PF6)
CH2Cl2
(298)
494 (max), 523
0.24
0.11
559
0.73
1.46
CH3CN
(298)
484 sh
0.23
0.15
574
0.57
2.06
CH3CN
(298)
492, 518 sh
565
0.39
2.41
a
buffer
490, 518 (max)
601 sh
0.34
2.61
(298)
a
buffer
492, 517 sh
0.13
0.15
576 sh
577
0.30
0.84
(298)
[Ir(ppy-C4)2(phen)]
(PF6)
CH2Cl2
(298)
0.41
0.25
0.10
[Ir(pppy-C4)2(bpy-
biotin)](PF6)
CH2Cl2
(298)
526 sh
4.57
CH3CN
(298)
584
0.51
2.92
589
0.44
5.48
a
MeOH
(298)
528, 574 sh
0.051
0.10
0.17
buffer
480 sh
(298)
602 (max)
0.15
7.03
566
579
0.35
0.93
a
buffer
528 (max), 567
[Ir(ppy-C4)2(Ph2-
phen)](PF6)
CH2Cl2
(298)
0.40
0.26
0.12
0.48
0.42
0.40
0.44
(298)
612 sh
590
0.065
0.50
CH3CN
(298)
580
577
0.53
0.37
1.02
6.80
10.00
1.33
[Ir(pppy)2(bpy-
biotin)](PF6)
CH2Cl2
(298)
a
buffer
(298)
CH3CN
(298)
601
611
0.28
0.074
[Ir(pppy-C4)2(Me4-
phen)](PF6)
CH2Cl2
(298)
522 (max), 552,
606 sh
b
c
buffer
0.051
(298)
CH3CN
(298)
520 (max), 550,
604 sh
a
b
a
buffer
520 (max), 552,
602 sh
50 mM potassium phosphate buffer pH 7.4/DMSO (7:3, v/v). 50
c
(298)
mM potassium phosphate buffer pH 7.4/MeOH (3:7, v/v). The
quantum yield cannot be determined accurately due to insufficient
solubility of the complex.
[Ir(pppy-C4)2(Me2-
bpy)](PF6)
CH2Cl2
(298)
524 sh, 553
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profile of the complex was independent of the pH. Thus, the
dual-emission properties of the complex should not result from
the different protonated situations of the amino groups. The
broad emission bands of the two amine-free ppy and pppy
complexes occurred at a similar wavelength to the LE emission
of the dual-emissive complexes, and their origins were assigned
to a 3CT state. This assignment is supported by the fact that the
pppy complex (598 nm) emitted at a shorter wavelength than
the ppy complex (609 nm) in CH2Cl2 (Table 1), as a result of
the electron-withdrawing property of the phenyl substituent of
the cyclometalating ligand. Interestingly, the 3CT emission
band of the pppy-C4 complex occurred at a longer wavelength
(602 nm) than that of the ppy-C4 complex (593 nm) in
CH2Cl2 (Table 1), probably due to the presence of the
electron-rich amino groups. The strong electron-donating
ability of the amino groups may neutralize and even reverse
the electron-withdrawing effect of the phenyl group.
To study the effects of diimine ligands, we selected four
diimine ligands (Me4-phen, Me2-bpy, phen, Ph2-phen) of
different π* energy levels. Among the ppy-C4 complexes,
complexes with the Me4-phen and Me2-bpy ligands (which are
of higher-lying π* energy levels), i.e., [Ir(ppy-C4)2(N^N)]-
(PF6) (N^N = Me4-phen, Me2-bpy), displayed dual emission,
which was similar to that of the bpy-Et complex (Figure 2e,f
and Table 1). Changing the diimine ligands did not influence
significantly the HE emission energy and lifetime, whereas the
LE emission energy of the Me4-phen complex was higher than
that of the Me2-bpy complex, which is in accordance with the π-
accepting properties of the diimine ligands. These are
Figure 2. Emission spectra of [Ir(N^C)2(bpy-Et)](PF6) (N^N = ppy-
C4 (a), pppy-C4 (b), ppy (c), pppy (d)), [Ir(ppy-C4)2(N^N)](PF6)
(N^N = Me4-phen (e), Me2-bpy (f)), and [Ir(ppy-C4)2(bpy-
biotin)](PF6) (g) in deaerated CH2Cl2 (red) and potassium
phosphate buffer (blue) at 298 K. (h) Emission spectra of [Ir(pppy-
C4)2(bpy-biotin)](PF6) in deaerated CH3CN (red) and potassium
phosphate buffer (blue) at 298 K.
3
consistent with the assignment of the HE emission to the IL
(π → π*) (ppy-C4) excited state and the LE emission to the
3CT state associated with the π*(N^N) orbital. Upon lowering
the π* energy level of the diimine ligand, the dual emission
feature became less noticeable; for example, the phen complex
[Ir(ppy-C4)2(phen)](PF6) only showed a broad LE band in
CH2Cl2 and CH3CN, and an additional weak HE shoulder also
appeared at ca. 480 nm (το = 2.92 μs) in aqueous buffer (Table
1 and Supporting Information Figure S3). The Ph2-phen
complex [Ir(ppy-C4)2(Ph2-phen)](PF6) did not show dual
emission in fluid solutions at all and only exhibited an LE band
at ca. 577−580 nm (Table 1 and Supporting Information
Figure S4).
The emission properties of the four pppy-C4 complexes
[Ir(pppy-C4)2(N^N)](PF6) (N^N = Me4-phen, Me2-bpy,
phen, Ph2-phen) were similar to those of their corresponding
ppy-C4 counterparts (Table 1). For the dual-emissive Me4-
phen and Me2-bpy complexes, the additional phenyl ring in the
cyclometalating ligand lowered the emission energy of the HE
bands, which was also observed in the case of the bpy-Et
complexes. As a result, the HE and LE emission bands of these
two complexes were not well-resolved (Supporting Information
Figures S5 and S6). Similar to their ppy-C4 counterparts, the
dual emission of the phen complex [Ir(pppy-C4)2(phen)](PF6)
was only observed in aqueous solution (Supporting Informa-
tion Figure S7), and the Ph2-phen complex [Ir(pppy-C4)2(Ph2-
phen)](PF6) exhibited only a broad emission band (Supporting
Information Figure S8) in all the solvents we used. As expected,
the amine-free pppy complexes [Ir(pppy)2(N^N)](PF6) (N^N
= Me4-phen, Me2-bpy, phen, Ph2-phen) displayed a single
emission band in fluid solutions (Table 1), which was assigned
to a mixed 3MLCT (dπ(Ir) → π*(N^N)) and 3LLCT
(π(pppy) → π*(N^N)) excited state. The Me4-phen and
Me2-bpy complexes showed structured emission bands,
lower than that of the LE band, and the emission was orange in
color. In more polar CH3CN, the LE emission intensity was
reduced. In aqueous buffer, the emission spectra were
dominated by the HE band, and the LE emission was
completely embedded or appeared as an unresolved shoulder,
giving green and yellow emission. The lifetimes of both
emission bands were in the microsecond or submicrosecond
time scales, indicative of triplet-state nature. Additionally, upon
increasing the polarity of the solvent, the lifetimes of the HE
emission were extended whereas those of the LE emission were
shortened. With the change in the cyclometalating ligand from
ppy-C4 to pppy-C4, the HE emission was shifted to a longer
wavelength by ca. 30 nm with increased lifetimes, whereas the
low-energy emission wavelength was less affected (Figure 2a,b).
On the basis of these photophysical data, we assigned the HE
3
band of the complexes to a triplet intraligand IL (π → π*)
(N^C) excited state and the LE feature to a triplet charge-
transfer (3CT) excited state of mixed 3MLCT (dπ(Ir) →
3
π*(bpy-Et)) and LLCT (π(N^C) → π*(bpy-Et)) character.
Additionally, the dual-emission properties were absent in the
amine-free ppy and pppy complexes, which only displayed a
broad emission band in fluid solutions (Figure 2c,d), indicative
of the crucial role of the amine groups in the dual-emissive
complexes. Thus, the possible pH-dependence of the emission
of [Ir(ppy-C4)2(bpy-Et)](PF6) was studied. We found that the
emission intensity of the complex was strongly dependent on
the pH, with a maximum intensity at ca. pH 5 (Supporting
Information Figure S2). Both increasing and decreasing the pH
value led to emission quenching. Importantly, the emission
E
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Figure 3. Molecular orbitals (isosurface plots) of [Ir(N^C)2(bpy-Et)](PF6) (N^C = ppy, ppy-C4).
Table 2. Calculated Triplet Transitions and the Frontier Orbitals of [Ir(N^C)2(bpy-Et)](PF6) (N^C = ppy, ppy-C4)
complex
state
energy/eV (nm)
molecular orbital
main transition character
MLbpy‑EtCT + LppyLbpy‑EtCT
[Ir(ppy)2(bpy-Et)](PF6)
T1
T2
2.16 (574.9)
2.64 (469.6)
HOMO → LUMO (100%)
HOMO − 2 → LUMO (65%)
HOMO − 4 → LUMO (13.9%)
HOMO → LUMO + 2 (56.6%)
HOMO − 1 → LUMO + 3 (17.3%)
HOMO − 1 → LUMO (47.6%)
HOMO − 3 → LUMO (34.9%)
HOMO → LUMO + 3 (62.2%)
HOMO − 1 → LUMO + 2 (26.8%)
HOMO → LUMO (83.4%)
MLbpy‑EtCT + LppyLbpy‑EtCT
T3
T4
T5
T1
T2
T3
T4
T5
T6
T7
2.69 (460.6)
2.70 (459.7)
2.73 (454.0)
2.08 (596.3)
2.50 (495.0)
2.53 (489.8)
2.61 (475.1)
2.65 (468.3)
2.65 (467.5)
2.68 (462.1)
MLppyCT + ILppy
MLbpy‑EtCT + LppyLbpy‑EtCT
MLppyCT + ILppy
[Ir(ppy-C4)2(bpy-Et)](PF6)
MLbpy‑EtCT + LppyLbpy‑EtCT + NamineLbpy‑EtCT
HOMO − 2 → LUMO (16.6%)
HOMO − 1 → LUMO (67.7%)
HOMO − 2 → LUMO (22.3%)
HOMO − 2 → LUMO (57.4%)
HOMO − 1 → LUMO (32.3%)
HOMO − 6 → LUMO (30.4%)
HOMO − 3 → LUMO (24.6%)
HOMO − 3 → LUMO (27.8%)
HOMO − 5 → LUMO (27.3%)
HOMO → LUMO + 2 (56.8%)
HOMO − 3 → LUMO + 3 (16.7%)
HOMO → LUMO + 3 (53.3%)
HOMO − 3 → LUMO + 2 (29.4%)
N
N
amineLbpy‑EtCT
amineLbpy‑EtCT
MLbpy‑EtCT + LppyLbpy‑EtCT
MLbpy‑EtCT + LppyLbpy‑EtCT + MLppyCT
MLppyCT + ILppy
MLppyCT + ILppy
3
suggesting that their emissive states may also possess some IL
(π → π*) (N^N) character.
Theoretical Calculations. To gain more insight into the
nature of the excited states, TD-DFT calculations have been
performed on [Ir(N^C)2(bpy-Et)](PF6) (N^C = ppy, ppy-
C4). The molecular orbitals (isosurface plots) of both
complexes are shown in Figure 3 and Supporting Information
Figure S8. The triplet transitions and frontier orbitals are listed
in Table 2. The HOMO and the nearby occupied MO of both
complexes were mainly localized at the iridium(III) center and
the cyclometalating ligands, which is commonly observed in
bis-cyclometalated iridium(III) diimine complexes.47−50 Im-
portantly, the secondary amines in [Ir(ppy-C4)2(bpy-Et)](PF6)
were also involved in these orbitals, especially in HOMO − 1
and HOMO − 2 (Figure 3). The LUMO and LUMO + 1 of
both complexes were primarily on the bipyridine ligand
including the substituted amide group, whereas LUMO + 2
and LUMO + 3 were mainly localized on the phenylpyridine
ligands (Supporting Information Figure S9). The calculated
excited states of both complexes were generally similar (Table
2); for example, the lowest-energy triplet excited states T1 of
[Ir(ppy)2(bpy-Et)](PF6) and [Ir(ppy-C4)2(bpy-Et)](PF6) oc-
curred at 2.16 and 2.08 eV (574.9 and 596.3 nm), respectively,
and were mainly composed of MLbpy‑EtCT and LppyLbpy‑EtCT
To develop luminescent bioprobes with dual-emissive
properties, we incorporated a biotin moiety into the diimine
ligand and prepared the complexes [Ir(N^C)2(bpy-biotin)]-
(PF6) (N^C = ppy-C4, pppy-C4, pppy). As expected, the
amine-containing ppy-C4 and pppy-C4 complexes showed dual
emission in fluid solutions (Table 1 and Figure 2g,h), and the
amine-free pppy complex only displayed a single broad
emission band. Since the addition of a biotin moiety does
not significantly affect the electronic structure of the bipyridine
unit, the emission behavior of these biotin complexes was very
similar to that of the bpy-Et complexes (Table 1).
Taken together, we can conclude that only the amine-
containing complexes exhibited dual emission. The HE IL (π
→ π*) (N^C) state was more sensitive to the modification of
the cyclometalating ligand. Also, changing the diimine ligands
3
3
can tune the energy of the LE CT state. Interestingly, when
the π* levels of the diimine ligands are too low in energy, single
LE 3CT emission bands were observed as a consequence of the
improved communication between the HE and the LE states.
F
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transitions, with a small extent of NamineLbpy‑EtCT character in
the ppy-C4 complex. Thus, these states of highly mixed nature
are best described as CT states. The second lowest-energy
avidin. Biotin binds to the glycoprotein avidin with an
extremely high affinity, and the biotin−avidin system has
been widely utilized as a powerful tool in bioanalytical
applications.51 The 4′-hydroxyazobenzene-2-carboxylic acid
(HABA) assay, which is based on the competition between
biotin and HABA on binding to avidin,52 confirmed that the
biotin moiety in the complexes [Ir(N^C)2(bpy-biotin)](PF6)
(N^C = ppy-C4, pppy-C4, pppy) maintained avidin-binding
affinity. Addition of the biotin complexes to a mixture of avidin
and HABA led to a decrease in absorbance at 500 nm,
indicating that the biotin moieties of the complexes can
displace the avidin-bound HABA molecules (Supporting
Information Figure S10). Also, the avidin-binding properties
of the complexes were studied by emission titrations. The dual-
emissive complexes [Ir(N^C)2(bpy-biotin)](PF6) (N^C = ppy-
C4, pppy-C4) showed a decrease in the HE band intensity and
an increase in LE band intensity in aerated buffer, resulting in
clear isoemissive points at 574 and 529 nm, respectively (Figure
5a,b). The ratiometric response became more significant when
3
state of [Ir(ppy)2(bpy-Et)](PF6) degenerated into T2−T5
(2.64−2.73 eV, 469.6−454.0 nm), between which the energy
difference was as small as 0.09 eV (Table 2). The ppy-C4
complex [Ir(ppy-C4)2(bpy-Et)](PF6) also showed very similar
degenerated states T4−T7, composed of transitions of basically
the same origins, at 2.61−2.68 eV (475.1−462.1 nm) (Table
2). Although these states are admixtures of MLbpy‑EtCT,
MLppyCT, LppyLbpy‑EtCT, and ILppy transitions, the IL character
should be highlighted due to the distinctive photophysical
properties (long lifetime and structural features) of the HE
band. The most significant difference between the two
complexes is that [Ir(ppy-C4)2(bpy-Et)](PF6) exhibited an
additional NamineLbpy‑EtCT transition (T2 and T3) occurring at
2.50−2.53 eV (495.0−489.8 nm), which originates from
HOMO − 1 and HOMO − 2 to LUMO (Table 2). Since
these states are absent in the ppy complex, they should play a
crucial role in the interesting dual-emission properties of the
amine-containing ppy-C4 complexes. On the basis of the above
experimental and theoretical information, we propose orbital
energy diagrams for the two complexes (Figure 4). On the basis
Figure 4. Proposed orbital energy diagrams of [Ir(N^C)2(bpy-
Et)](PF6) (N^C = ppy, ppy-C4).
of their excited-state nature, the degenerated frontier states and
the lowest-energy triplet excited states are denoted as TIL and
TCT, respectively. The excited ppy complex [Ir(ppy)2(bpy-
Et)](PF6) undergoes efficient internal conversion from the TIL
to the lowest-lying emissive TCT state, leading to the exclusive
Figure 5. Emission spectral traces of [Ir(N^C)2(bpy-biotin)](PF6)
(N^C = ppy-C4 (a), pppy-C4 (b)) (10 μM) in aerated potassium
phosphate buffer (50 mM, pH 7.4)/CH3OH (9:1, v/v) at 298 K upon
addition of avidin. (c) Emission spectra of [Ir(pppy-C4)2(bpy-
biotin)](PF6) (10 μM) in the absence (blue) and presence (red) of
avidin (2.5 μM) in degassed potassium phosphate buffer (50 mM, pH
7.4)/CH3OH (9:1, v/v) at 298 K.
3
LE emission with predominant CT character (Figure 4). In
contrast, in the case of the ppy-C4 complex [Ir(ppy-C4)2(bpy-
Et)](PF6), the additional TNLCT state interrupts the commu-
nication between the two states TIL and TCT. Also, it is likely
that the additional energy barrier between the TIL and TNLCT
states limits efficient internal conversion, resulting in dual
emission from both triplet excited states. We believe that the
existence of the TNLCT state is essential to the dual-emissive
properties of these complexes.
Utilization of Dual-Emissive Complexes as Ratiomet-
ric Probes. During the investigation of the emission properties
of these iridium(III) complexes, we found that the complexes
exhibited ratiometric emissive response toward the polarity of
solvents under deaerated conditions (Table 1 and Figure 2). To
extend the applications of these complexes as ratiometric
probes for other analytes, biotin was incorporated into the dual-
emissive system to afford potential ratiometric probes for
the titration was performed in deaerated solutions because the
HE emission was more intense in the absence of oxygen
(Figure 5c). In view of the solvent-dependent emission of the
ppy-C4 and pppy-C4 biotin complexes, we attributed the
changes of emission profile of both complexes to the increased
hydrophobicity of the local environment of the complexes upon
binding to avidin.45 For comparison, [Ir(pppy)2(bpy-biotin)]-
(PF6) only displayed a broad band ca. 599 nm in aqueous
buffer, and showed emission enhancement without a shift in
wavelength in response to avidin binding (Supporting
G
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Information Figure S11). Similar observations have been
commonly observed in luminescent transition metal biotin
complexes developed in this laboratory.53−55 It is noteworthy
that the possibility of using these dual-emissive complexes as
bioprobes is basically unlimited. We believe that the
incorporation of other biological substrates or molecular/ion
recognition units into this system will lead to new ratiometric
probes that will display pronounced changes in their emission
profile and spectral characteristics upon interaction with the
protein hosts or analyte molecules/ions.45
= pppy-C4, pppy; N^N = Me4-phen, Me2-bpy, phen, Ph2-
phen), which showed relatively high uptake efficiency, exhibited
intense emission from the peripheral cell membrane. As an
example, the confocal microscopy image of the cells treated
with [Ir(pppy)2(Me4-phen)](PF6) is shown in Figure 6a. Since
Cellular Uptake Efficiency and Cytotoxicity. The
cellular uptake efficiency of all the complexes was analyzed by
ICP-MS. Generally speaking, it is well-known that more
lipophilic complexes usually exhibit a higher diffusion rate
through the cell membrane.44 In this work, an average HeLa
cell incubated with the complexes (2 μM, 2 h, 37 °C) contained
0.54−5.82 fmol of iridium (Table 3). It is reasonable that the
Figure 6. Confocal microscopy images of HeLa cells pretreated with
[Ir(pppy)2(Me4-phen)](PF6) (10 μM) (a), [Ir(ppy-C4)2(bpy-
biotin)](PF6) (10 μM) (b), and no complex (c) at 37 °C for 12 h
and then incubated with the Annexin V, Alexa Fluor 647 conjugate (50
μL/mL) at 25 °C for 15 min.
Table 3. Numbers of Moles of Iridium Associated with an
Average HeLa Cell upon Incubation with the Iridium(III)
Complexes (2 μM) at 37 °C for 2 h As Determined by ICP-
MS
Annexin V only stains cells that express phosphatidylserine on
the cell surface, which is a characteristic of apoptosis,57 the
results indicate that these complexes caused apoptotic cell
death. Interestingly, the cells treated with the other complexes
including the ppy-C4, bpy-Et, and bpy-biotin complexes (10
μM, 12 h, 37 °C) showed much weaker emission (Figure 6b),
which was similar to the untreated cells (Figure 6c), indicating
that these complexes did not cause apoptotic cell death under
these conditions.
Intracellular Localization Studies. The intracellular
localization of all the complexes was investigated using laser-
scanning confocal microscopy. It is important and necessary to
employ a suitable condition under which the complexes give
good-quality images with minimum cell death. Thus, we
incubated HeLa cells with the complexes at a concentration of
0.5−5 μM for 1−6 h and traced the possible early apoptosis
using the Annexin V assay. Either lowering the complex
concentration or shortening the incubation duration signifi-
cantly reduced the peripheral membrane staining by the
Annexin V-Alexa Fluor conjugate (Supporting Information
Figure S12). When HeLa cells were incubated with [Ir-
(pppy)2(Me4-phen)](PF6) at a concentration lower than 3 μM
for less than 3 h, the confocal microscopy images (λex = 633
nm) displayed similar emission intensity compared to those of
the untreated cells, indicating that no apoptosis occurred. Thus,
we selected a complex concentration of 2 μM and an
incubation period of 2 h for our imaging studies.
HeLa cells treated with the complexes at 37 °C for 2 h
revealed intense emission. Interestingly, the intracellular
localization of these complexes was very different (Figure 7).
On the basis of the observations, the complexes were put
into three groups. Group I includes the most lipophilic
complexes such as the pppy complexes [Ir(pppy)2(N^N)]-
(PF6) (N^N = Me4-phen, Me2-bpy, phen, Ph2-phen, bpy-Et,
bpy-biotin) and the Ph2-phen complexes [Ir(N^C)2(Ph2-
phen)](PF6) (N^C = ppy-C4, pppy-C4). Similar to most
iridium(III) complexes, they were readily internalized and
localized in the perinuclear region of cells. Group II includes
complexes of medium lipophilicity, including [Ir(N^C)2-
(N^N)](PF6) (N^C = ppy-C4, pppy-C4; N^N = Me2-bpy,
phen, Me4-phen). Cells incubated with these complexes
displayed two sharp luminescent rings; the interior one was
around the nuclei while the exterior one surrounded the whole
cell. Group III includes the least lipophilic n-butylaminomethyl
complex
N^N
Ir/fmol
[Ir(ppy-C4)2(N^N)](PF6)
bpy-Et
0.59 0.05
0.54 0.03
0.66 0.09
0.72 0.10
0.74 0.10
0.77 0.12
1.34 0.19
1.04 0.15
5.24 0.43
5.20 0.47
4.91 0.38
5.60 0.55
3.91 0.40
2.99 0.23
5.55 0.62
5.51 0.49
5.44 0.50
5.82 0.73
bpy-biotin
Me4-phen
Me2-bpy
phen
Ph2-phen
bpy-Et
[Ir(pppy-C4)2(N^N)](PF6)
[Ir(pppy)2(N^N)](PF6)
bpy-biotin
Me4-phen
Me2-bpy
phen
Ph2-phen
bpy-Et
bpy-biotin
Me4-phen
Me2-bpy
phen
Ph2-phen
less lipophilic ppy-C4 complexes exhibited lower cellular uptake
efficiency (0.54−0.77 fmol) than their pppy-C4 (1.04−5.60
fmol) and pppy (2.99−5.82 fmol) counterparts. Also, the pppy-
C4 complexes, especially those with the bpy-Et and bpy-biotin
ligands, showed lower uptake efficiency than their pppy
counterparts, which is apparently a result of the polar amine
groups in the n-butylaminomethyl substituents. Additionally,
the complexes with the polar biotin unit [Ir(N^C)2(bpy-
biotin)](PF6) (N^C = ppy-C4, pppy-C4, pppy) showed the
lowest uptake efficiency among all the complexes.
The cytotoxic activity of the complexes was evaluated by the
MTT assay.56 HeLa cells treated with the complexes (10 μM)
for 12 h at 37 °C maintained >98% viability. Despite this
cellular viability, the possibility of early apoptosis induced by
the presence of the complexes cannot be excluded; this was
examined using the Annexin V assay.57 HeLa cells were
pretreated with the complexes (10 μM, 12 h, 37 °C) and then
incubated with the Annexin V-AlexaFluor 647 conjugate. Upon
excitation at 633 nm (where the complexes do not absorb), the
cells treated with the complexes [Ir(N^C)2(N^N)](PF6) (N^C
H
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occurred at the interior luminescent ring. The Group III
complex [Ir(ppy-C4)2(bpy-biotin)](PF6) was hardly co-local-
ized with MitoTracker (CC = 2.9%, Figure 8c). All these
confocal microscopy images can be interpreted as follows: the
most lipophilic pppy and Ph2-phen complexes exhibited the
most efficient internalization and subsequent localization at the
mitochondria. However, the amine-containing ppy-C4 and
pppy-C4 complexes were localized on the cell membrane or
migrate into mitochondria, depending on their diimine ligands:
(1) those complexes with the polar amide-substituted
bipyridine ligands almost resided exclusively on the cell
membrane; (2) those with the less polar ligands Me2-bpy,
phen, and Me4-phen stained both the cell membrane and the
mitochondria, giving rise to two luminescent rings. These
results clearly indicate the importance of the lipophilicity of the
complexes on their cellular staining properties. Interestingly,
this structure-dependent intracellular distribution was only
restricted to live cells since paraformaldehyde-fixed cells treated
with the complexes showed indistinguishable staining behavior
(Supporting Information Figure S14). Basically, the fixed cells
displayed intense emission evenly in the cytoplasm and less
intense emission in the nuclei. Thus, the membrane-staining
properties of Groups II and III complexes were a consequence
of limited uptake and suppression of further internalization by
live cells.
Figure 7. Confocal microscopy images of HeLa cells incubated with 2
μM of [Ir(N^C)2(N^N)](PF6) (N^C = ppy-C4, pppy-C4, pppy; N^N
= Me4-phen, Me2-bpy, phen, Ph2-phen, bpy-Et, bpy-biotin) at 37 °C
for 2 h. The emission at 570 50 nm was collected.
complexes with the polar amide-substituted bipyridine ligands
bpy-Et and bpy-biotin [Ir(N^C)2(N^N)](PF6) (N^C = ppy-
C4, pppy-C4; N^N = bpy-Et, bpy-biotin). HeLa cells stained by
these complexes revealed intense emission on their cell surface,
while the cytoplasm was much less emissive.
To determine the intracellular localization of these
complexes, we co-stained live HeLa cells with the complexes
and the fluorescent dye MitoTracker Deep Red FM. The
Group I complex [Ir(pppy)2(Ph2-phen)](PF6) was significantly
co-localized with MitoTracker, resulting in a co-localization
coefficient (CC) of 97%, which confirmed that this complex
was localized in the mitochondria of the cells (Figure 8a). High-
CONCLUSION
■
In this work, we systematically varied the cyclometalating and
diimine ligands to investigate the effects of the structures of the
iridium(III) polypyridine complexes [Ir(N^C)2(N^N)](PF6)
on their emission properties. Remarkably, complexes with a
secondary amine on the cyclometalating ligands ppy-C4 and
pppy-C4 showed interesting dual emission. Photophysical and
TD-DFT calculations revealed that such a property resulted
from the interruption of the internal conversion from the 3IL to
3
the CT state by an additional NLCT state. As a consequence,
the emission profile of the complexes strongly depended on
their coordinating ligands and their local environment. We
demonstrated the application of the dual-emissive biotin
complexes as ratiometric sensors for avidin. Additionally, all
the complexes exhibited efficient uptake by live HeLa cells.
Laser-scanning confocal microscopy revealed that the com-
plexes were localized on the cell membrane, mitochondria, or
both, depending on the nature of the substituents of the
ligands. The results of this work can contribute to the future
development of dual-emissive transition metal complexes as
ratiometric probes and organelle-selective bioimaging reagents.
Figure 8. Confocal microscopy images of HeLa cells co-stained by the
complexes and fluorescent dyes. Co-localization coefficients (CCs) are
indicated in the overlaid images. The emission at 570 50 and 670
20 nm was collected for the complexes and fluorescent dyes,
respectively.
EXPERIMENTAL SECTION
■
Materials and Synthesis. All solvents were of analytical reagent
grade and purified according to standard procedures.58 All buffer
components were of biological grade and used as received. IrCl3·
3H2O, Hppy-CHO, diethylamine, triethylamine, n-butylamine,
triphenylphosphine, and Pd(PPh3)2Cl2 were purchased from Aldrich.
N-Boc-1,6-diaminohexane hydrochloride was purchased from Interna-
tional Laboratory. Palladium(II) chloride, copper(I) iodide, DCC,
NHS, Na2CO3, 4-bromoanilane, pyridine, NaNO2, 4-formylphenylbor-
onic acid, phenylboronic acid, phen, Me4-phen, Ph2-phen, MgSO4,
NaBH4, ferrocene, KPF6, and cisplatin were purchased from Acros.
Me2-bpy was obtained from BDH. HABA and MTT were purchased
from Sigma. Avidin was purchased from Calbiochem. The diimine
ligands bpy-Et and bpy-biotin, and [Ir(ppy-C4)2(N^N)](PF6), were
prepared according to reported procedures.45 TBAP was obtained
from Aldrich and was recrystallized from hot ethanol and dried in
quality mitochondria staining images can be obtained using a
diluted solution of the complex and shorter incubation
duration; for example, cells treated with [Ir(pppy)2(Me4-
phen)](PF6) at 0.3 μM for 30 min showed specific
mitochondrial staining with a fairly high CC of 88% with
MitoTracker (Supporting Information Figure S13). The Group
II complex [Ir(pppy-C4)2(phen)](PF6) was partially co-
localized with MitoTracker, revealing a CC of 78% (Figure
8b). It can be seen in the overlaid image that co-staining
I
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vacuo at 110 °C before use. HeLa cells were obtained from American
Type Culture Collection. DMEM, FBS, PBS, trypsin-EDTA,
penicillin/streptomycin, MitoTracker Deep Red FM, and Annexin V,
Alexa Fluor 647 conjugate were purchased from Invitrogen. The
growth medium for cell culture contained DMEM with 10% FBS and
1% penicillin/streptomycin.
(100 μL) and incubated at 37 °C under a 5% CO2 atmosphere for 24
h. The iridium(III) complexes (10 μM) were then added to the wells
in a mixture of growth medium/DMSO (99:1, v/v). Wells containing
growth medium without cells were used as blank controls. The
microplate was incubated at 37 °C under a 5% CO2 atmosphere for 12
h. Then, 10 μL of MTT in PBS (5 mg mL−1) was added to each well.
The microplate was incubated at 37 °C under a 5% CO2 atmosphere
for another 4 h. Solubilization solution (100 μL) containing 10% SDS
in 2-propanol/0.04 M HCl (1:1, v/v) was then added to each well, and
the microplate was further incubated for 24 h. The absorbance of the
solutions at 570 nm was measured with a SPECTRAmax 340
microplate reader (Molecular Devices Corp., Sunnyvale, CA). The cell
viability of the complexes were calculated.
Annexin V Assay. The cell death process has been investigated
using the Annexin V assay. HeLa cells were grown on a sterile glass
coverslip in a 35 mm tissue culture dish and incubated at 37 °C under
a 5% CO2 atmosphere for 48 h. The culture medium was removed,
and the cells were incubated with the complex in medium/DMSO
(99:1, v/v). After that, the medium was removed, and the cell layer
was washed gently with cold PBS (1 mL × 3). The cells were then
incubated with the Annexin V, Alexa Fluor 647 conjugate (5 μL/100
μL) in the annexin-binding buffer (10 mM of HEPES, 140 mM of
NaCl, and 2.5 mM of CaCl2 pH 7.4) at room temperature for 15 min.
After washing with the annexin-binding buffer (1 mL × 3), the
coverslip was mounted onto slides for measurements. Imaging was
performed using a confocal microscope (Leica TCS SPE) with an
excitation wavelength at 633 nm. The emission at 670 20 nm was
collected.
Confocal Microscopy. HeLa cells were grown on a sterile glass
coverslip in a 35 mm tissue culture dish and incubated at 37 °C under
a 5% CO2 atmosphere for 48 h. The culture medium was removed and
replaced with medium/DMSO (99:1, v/v) containing the complex (2
μM). After incubation for 2 h, the medium was removed, and the cell
layer was washed gently with PBS (1 mL × 3). The coverslip was
mounted onto slides for measurements. Imaging was performed using
a confocal microscope (Leica TCS SPE) with an excitation wavelength
at 405 nm. The emission at 570 50 nm was collected. In the co-
localization experiments with MitoTracker, after being treated with
complexes, the HeLa cells were incubated with MitoTracker Deep Red
FM (100 nM) for 20 min and then washed with PBS (1 mL × 3). The
co-localization coefficient was determined by ImageJ (Version
1.4.3.67).
Crystal Structure Determination of [Ir(ppy-C4)2(Me2-bpy)]-
(PF6). Single crystals of the complex suitable for X-ray crystallographic
studies were obtained by slow diffusion of petroleum ether (40−60
°C) vapor into a concentrated acetone solution of the complex. A
crystal of dimensions 0.2 × 0.1 × 0.1 mm3 mounted in a glass capillary
was used for data collection at 28 °C on a MAR diffractometer with a
300 mm image plate detector using graphite monochromatized Mo Kα
radiation (λ = 0.710 73 Å). Data collection was made with 1.5°
oscillation steps of φ, 10 min exposure time, and a scanner distance at
120 mm. A total of 130 images were collected. The images were
interpreted and intensity integrated using the program DENZO.59 The
structure was solved by direct methods employing the program SIR-
9760 on a PC. Iridium, phosphorus, and many non-hydrogen atoms
were located according to the direct methods and the successive least-
squares Fourier cycles. Positions of other non-hydrogen atoms were
found after successful refinement by full-matrix least-squares using the
program SHELXL-97.61 One crystallographic asymmetric unit consists
of half of a formula unit. In the final stage of least-squares refinement,
all non-hydrogen atoms were refined anisotropically. Hydrogen atoms
were generated by SHELXL-97. The positions of the hydrogen atoms
were calculated on the basis of the riding mode with thermal
parameters equal to 1.2 times that of the associated carbon atoms, and
participated in the calculation of final R-indices. Crystal data and a
summary of data collection and refinement details are given in
Supporting Information Table S4.
Physical Measurements and Instrumentation. Equipment and
procedures for the characterization and electrochemical and photo-
physical measurements have been described previously.62 The details
have been included in the Supporting Information. Luminescence
quantum yields were measured by the optically dilute method63 using
an aerated aqueous solution of [Ru(bpy)3]Cl2 (Φ = 0.028) as the
standard solution.64
Computational Details. The ground-state (S0) structures of
[Ir(N^C)2(bpy-Et)](PF6) (N^C = ppy, ppy-C4) have been optimized
using density functional theory (DFT) with the B3LYP65,66 hybrid
functional. The LANL2DZ basis sets67,68 are used for iridium and all
other atoms. The quasirelativistic pseudopotential with 17 valence
electrons for Ir atom is employed. This pseudopotential has been
shown to give good agreements with experimental measurements.47−50
Vibrational frequency calculations have been performed to verify the
minimum nature of the optimized structures. On the basis of
optimized geometries of the S0 state, time-dependent (TD)-DFT
calculations using the B3LYP functional and the same basis sets are
carried out to obtain the vertical excitation energies of various triplet
transitions.69−71 The solvent effect of CH2Cl2 is taken into account by
the polarized continuum model (PCM).72,73 All calculations are done
with the Gaussian 09 package of programs.74
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedure, characterization data, and details of
physical measurements and instrumentation; Table S1
summarizing the selected bond lengths (Å) and bond angles
(deg) for [Ir(ppy-C4)2(Me2-bpy)](PF6); Tables S2 and S3
summarizing the electronic absorption spectral data and
electrochemical data, respectively; Table S4 summarizing the
data collection and refinement details in the crystal structure
determination; Figure S1 showing electronic absorption
spectra; Figure S2 depicts the pH-dependent emission spectra
of [Ir(ppy-C4)2(bpy-Et)](PF6); Figures S3−S8 displaying the
emission spectra; Figure S9 indicating molecular orbitals
obtained from TD-DFT calculations; Figure S10 showing
results of HABA assays; Figure S11 displaying emission spectral
traces of [Ir(pppy)2(bpy-biotin)](PF6) upon addition of avidin;
Figures S12−S14 showing confocal images; and the CIF file for
[Ir(ppy-C4)2(Me2-bpy)](PF6) (CCDC 1058968). The Sup-
porting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.inorgchem.5b00944.
Emission Titrations. In a typical experiment, aliquots (5 μL) of
avidin (150 μM) in phosphate buffer (50 mM, pH 7.4) were added
cumulatively to the iridium biotin complex (10 μM) in a mixture of
the same buffer and MeOH (9:1, v/v) at 1 min intervals. The solution
was excited at 350 nm, and the emission spectra were measured.
ICP-MS. HeLa cells were grown in a 60 mm tissue culture dish and
incubated at 37 °C under a 5% CO2 atmosphere for 48 h. The culture
medium was removed and replaced with medium/DMSO (99:1, v/v)
containing the complex (2 μM). After incubation for 2 h, the medium
was removed, and the cell layer was washed gently with PBS (1 mL ×
3). After that, the cell layer was trypsinized, digested in 65% HNO3 (2
mL) at 70 °C for 2 h, and then diluted in Milli-Q water to the final
volume of 10 mL for ICP-MS (PerkinElmer SCIEX, ELAN DRC Plus)
analysis.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: iamyzhang@njupt.edu.cn.
■
MTT Assay. The cytotoxic effect of the complexes toward HeLa
cells was studied using the MTT assay. HeLa cells were seeded in a 96-
well flat-bottomed microplate (10 000 cells/well) in growth medium
J
DOI: 10.1021/acs.inorgchem.5b00944
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Inorganic Chemistry
Article
(31) Choi, A. W.-T.; Yim, V. M.-W.; Liu, H.-W.; Lo, K. K.-W.
Chem.Eur. J. 2014, 20, 9633−9642.
*E-mail: bhkenlo@cityu.edu.hk.
Notes
The authors declare no competing financial interest.
(32) Yoshihara, T.; Yamaguchi, Y.; Hosaka, M.; Takeuchi, T.; Tobita,
S. Angew. Chem., Int. Ed. 2012, 51, 4148−4151.
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ACKNOWLEDGMENTS
■
The work described in this paper was supported by Nanjing
University of Posts and Telecommunications (NY213097) and
City University of Hong Kong (Project No. 9667090 and
7004210). K.K.-W.L. is grateful to the Croucher Foundation for
the award of a Senior Research Fellowship.
(34) Huang, C.; Jia, T.; Tang, M.; Yin, Q.; Zhu, W.; Zhang, C.; Yang,
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