Hydroxyl and amino functionalized cyclometalated Ir(III) complexes: Synthesis, characterization and cytotoxicity studies

Article abstract of DOI:10.1016/j.jorganchem.2015.05.035

A series of Ir(III) complexes (?N)₂Ir(N N) (N N are 4,4′-dihydroxy-2,2′-bipyridine and 4,4′-diamino-2,2′-bipyridine, and ?N are phenylpyridine, benzo[h]quinolone, and 2-phenylquinoline) were synthesized and characterized. Two of the complexes were structurally characterized via X-ray crystallography. The photophysical and photochemical properties of these complexes were studied. Preliminary studies of their applications on pH sensing, and cell imaging were also performed.

Full text of DOI:10.1016/j.jorganchem.2015.05.035

Accepted Manuscript
Hydroxyl and amino functionalized cyclometalated Ir(III) complexes: synthesis,
characterization and cytotoxicity studies
Zhaozhen Wu, Juanjuan Mu, Qiong Wang, Xing Chen, Lasse Jensen, Changqing Yi,
Mei-Jin Li
PII:
S0022-328X(15)30005-X
10.1016/j.jorganchem.2015.05.035
JOM 19076
DOI:
Reference:
To appear in: Journal of Organometallic Chemistry
Received Date: 23 September 2014
Revised Date: 12 May 2015
Accepted Date: 23 May 2015
Please cite this article as: Z. Wu, J. Mu, Q. Wang, X. Chen, L. Jensen, C. Yi M.-J. Li Hydroxyl and amino
functionalized cyclometalated Ir(III) complexes: synthesis, characterization and cytotoxicity studies,
Journal of Organometallic Chemistry (2015), doi: 10.1016/j.jorganchem.2015.05.035.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Hydroxyl and amino functionalized cyclometalated Ir(III) complexes: synthesis,
characterization and cytotoxicity studies
a
a
a
c
c
b,*
a,*
Zhaozhen Wu, Juanjuan Mu, Qiong Wang, Xing Chen, Lasse Jensen, Changqing Yi, Mei-Jin Li
a
b
c
Key Laboratory of Analysis and Detection Technology for Food Safety, Ministry of Education, Department of Chemistry, Fuzhou University, Fuzhou
3
50108, China
Key Laboratory of Sensing Technology and Biomedical Instruments (Guangdong Province), School of Engineering, Sun Yat-Sen University, Guangzhou
10006, P.R.China
Department of Chemistry, The Pennsylvania State University, 104 Chemistry Building, University Park, 16802, United States.
5
Abstract
A series of Ir(III) complexes (C^N) Ir(N^N) (N^N are 4,4’-dihydroxy-2,2’-bipyridine and 4,4’-diamino-2,2’-bipyridine, and C^N are
2
phenylpyridine, benzo[h]quinolone, and 2-phenylquinoline) were synthesized and characterized. Two of the complexes were structurally
characterized via X-ray crystallography. The photophysical and photochemical properties of these complexes were studied. Preliminary
studies of their applications on pH sensing, and cell imaging were also performed.
Keywords: Iridium(III) complex / Synthesis / pH sensing / Cytotoxicity
orange-yellow solids. The complexes were easily purified by
crystallization to provide pure crystals. The identities of the
complexes were confirmed by NMR spectroscopy, elemental
1. Introduction
analysis, and ESI mass spectrometry.
Ir(III) complexes as luminescent materials have drawn
OH
considerable research attention because of their relatively long
excited-state lifetime, high photoluminescence efficiency, and
+
OH
N
N
N
N
N
C
C
N
N
[
1]
excellent color tuning. In particular, tuning of the luminescence
strength and wavelength of the complexes has been extensively
N
N
N
N
N
OH
[
2]
N2
CH Cl
CH OH
OH
studied. The advantages of iridium(III) complexes make them
potentially useful in applications such as photosensitizers,
C
C
Cl
Cl
C
C
bpyOH
2
+
+
2
2
NH
2
3
NH
2
2
[
3]
C
C
N
N
molecular sensing, photocatalysis and organic light-emitting
[
4]
N
N
diodes (OLEDs). Metal complexes with simple ligands show
various electronic properties depending on the proton transfer
NH2
[
5]
reaction of the ligand. Ligands such as 2,2'-bipyridinyl-4,4'-
NH
bpyNH
2
high luminescence
N^N
[
5a,6]
[7]
diol,
1,10-phenanthroline-4,7-diol, and amino terpyridine
[
8]
complex
C^N
(
tpy-φ-NH2) have been used in pH sensing studies due to their
1
2
3
4
5
6
ppy
pq
bzq
ppy
pq
bpy(OH)
bpy(OH)
2
2
remarkable deprotonation ability. Based on the acid-base
equilibrium of the phenolic hydroxyl and amino groups of the N^N
ligands, the amino or hydroxyl functionalized Ir(III) complexes
were expected to be potential pH sensors. In this paper, we report
the synthesis, and characterization of a series of Ir(III) complexes
containing 2,2'-bipyridinyl-4,4'-diol or 2,2'-bipyridinyl-4,4'-
diamine. Preliminary studies of their applications on pH sensing,
and cell imaging were also performed.
N
C
N
N
N
=
bpy(OH)
2 2
bpy(NH )
2
ppy
pq
bzq
bpy(NH2)2
bpy(NH2)2
bzq
Scheme 1. Synthesis of the iridium(III) complexes.
2
.2. Crystal structure determination
The solid-state structures of 2 and 5 were determined by X-ray
crystallography and their ORTEP illustrations are shown in Fig. 1.
Selected bond distances and angles are listed in Table 1. The Ir(III)
center adopts a distorted-octahedral geometry, as indicated by the
crystal structures, with angles subtended by the N atoms of the
N^N ligand at the Ir(III) center of 75.17° and 74.4° for complexes
2 and 5, respectively. These angles are significantly smaller than
the ideal angle of 90° adopted in octahedral geometry. Ir(III) is
coordinated by two cyclometalated ligands pq and the dihydroxyl
or amino functionalized bipyridine moiety. Selected bond lengths
and angles for the molecules of complexes 2 and 5 are listed in
Table 2. The bond distances of Ir-C(pq) were 1.993(6) and
2
.Results and discussion
2
.1. Synthesis and characterization
,4’-Dihydroxy-2,2’-dipyridine (bpy(OH) ) and 4,4’-diamine-
4
2
2
,2’-dipyridine (bpy(NH ) ) were synthesized according to the
2 2
methods in the literature with slight modifications. Dichloro-
bridged dimers [Ir(C^N) -µ-Cl] were conveniently prepared from
a reaction of the appropriate ligand and IrCl ·xH O.
functionalized pyridine ligand was then reacted with [Ir(C^N) -µ-
2
2
[
9]
The
3
2
2
Cl] to provide the target complexes 1 to 6 (Scheme 1) as yellow or
2
2
.003(5) Å for complex 2, and 1.999(7) and 2.002(8) Å for
complex 5. The bond distances of Ir-N(pq) were 2.093(5) and
2.113(5) Å for complex 2, and 2.060(6) and 2.089(6) Å for
complex 5, similar to those found in cyclometalated Ir(III)
_
___________
*
Corresponding authors: Fax: (+)86 591 22866135 (M. J. Li)
E-mail addresses: mjli@fzu.edu.cn (M. J. Li), yichq@mail.sysu.edu.cn
C. Q. Yi)
[10]
complexes. All atoms of the modified bpy ligand, including the
(
1

ACCEPTED MANUSCRIPT
hydroxyl or amino moieties, are essentially coplanar, as indicated
by the crystal structures.
Table 2. Photophysical data of the new Ir(III) complexes.
4
3
-
λ
ab/nm (ε/10 dm mol
Complex
Solvent
1
-1
λ
em/nm (τ
o
/µs)
φ
em
cm )
DMSO
271(2.39), 370(0.38)
491 (0.18)
489
0.15
3
CH OH
1
CHCl
glass
3
489
474
DMSO
CH OH
272(3.75),338(1.63),
445(0.32)
560 (0.24)
558
0.29
0.03
0.16
0.23
0.03
3
2
3
4
5
6
CHCl
glass
3
561
547
DMSO
CH3OH
CHCl3
glass
273(4.00),331(1.47),
420(0.48)
530 (0.47)
523
(2)
(5)
521
Fig. 1. ORTEP diagram of the complexes 2 and 5 with atom labeling
scheme showing 30% thermal ellipsoids and the H atoms removed for
clarity.
500
DMSO
270(5.77),350(0.98),
420(0.26)
490 (0.13)
489
CH OH
3
Table 1. Selected bond distances (Å) and angles (°) of complexes 2 and 5.
CHCl
glass
3
488
476
Ir(1)-C(25)
Ir(1)-C(40)
Ir(1)-N(3)
1.993(6)
2.003(5)
2.093(5)
2.113(5)
2.143(4)
2.160(5)
1.344(7)
1.335(7)
80.4(2)
Ir(1)-C(16)
Ir(1)-C(1)
1.999(7)
2.002(8)
2.060(6)
2.089(6)
2.162(6)
2.167(6)
1.365(9)
1.364(9)
80.6(3)
DMSO
CH OH
272(1.60),340(0.56),
448(0.21)
566 (0.24)
563
3
Ir(1)-N(1)
CHCl
glass
3
561
Ir(1)-N(4)
Ir(1)-N(2)
552
Ir(1)-N(1)
Ir(1)-N(4)
DMSO
CH OH
275(8.25),344(2.92),
425(0.34)
530 (0.27)
523
Ir(1)-N(2)
Ir(1)-N(3)
3
O(1)-C(3)
N(5)-C(33)
N6-C(38)
CHCl
glass
3
521
O(2)-C(8)
503
C(25)-Ir(1)-N(3)
C(40)-Ir(1)-N(4)
N(1)-Ir(1)-N(2)
C(1)-Ir(1)-N(1)
C(16)-Ir(1)-N(2)
N(4)-Ir(1)-N(3)
The low-temperature photoluminescence spectra of all
complexes in ethanol-methanol (4:1, v:v) glass were studied and
shown in Fig. 2b. A blue-shift of emission maxima and vibronic
structures in the emission bands from fluid solution (MeOH) at
room temperature to rigid matrix at 77 K were observed. The blue
80.4(2)
80.1(3)
75.17(18)
74.4(2)
2
.3. Absorption and photoluminescence properties of the Ir(III)
-
1
complexes
shifts are about 350, 600 and 880 cm for pq (2 and 5), ppy (1 and
3) and bzq (3 and 6) complexes, respectively. Compared with the
emission maxima at room temperature, a small blue-shift of the
emission maxima of 2 and 5 at 77 K could be observed, which
shows the excited-state properties of pq complexes are different
from those of ppy and bzq complexes. The photoluminescence
spectra of all complexes in different solvents were also investigated
and the data are summarized in Table 2. The small dependence on
the solvent polarity was observed, especially for pq complexes.
This finding reflects that the emission of pq complexes originates
Electronic absorption data of the Ir(III) complexes were
collected in DMSO (Table 3). The electronic absorption spectra of
these complexes were mainly dominated by extremely high-energy
absorption bands at ca. 270 to 370 nm with molar extinction
coefficients in the order of 104 dm mol cm and comparatively
less intense low-energy absorption bands at ca. 420 nm to 448 nm.
With reference to previous spectroscopic studies on the related
3
−1
−1
[
11]
cyclometalated Ir(III) diimine systems,
absorption bands with
4
3
-1
-1
extinction coefficients in the order of 101 dm mol cm were
ascribed to spin-allowed π-π* intraligand ( IL) transitions of the
cyclometalating ligands or substituted bpy ligands. The less-intense
low-energy absorption bands at ca. 420 nm to 448 nm likely
originated from dπ→π∗ (ligand) spin-allowed metal-to-ligand
3
from an excited state of predominantly IL (π→π*) (pq) character
[
14]
with some contribution of charge-transfer character. While for
[
14,15]
ppy and bzq complexes, according to previous report,
the
more sensitivity to the temperature of photoluminescence spectra
shows the excited states for these complexes are mainly attributed
1
charge-transfer ( MLCT) transitions, similar to those observed in
previous studies of related Ir(III) diimine complexes.
[
12]
3
3
to the MLCT states and the contribution from LC is relatively
[
16]
The complexes were found to emit strongly at room
temperature upon excitation with both UV and visible light in
DMSO solution with emission maxima ranging from 490 to 566
nm. Images of their luminescence under a UV lamp of 365 nm are
shown in Fig. 2a, where the emission light is observably tuned
from green to yellow. With reference to previous spectroscopic
studies on other related cyclometalated Ir(III) diimine
small. Gudel et al. have reported the strong mixing between the
MLCT and LC excited states in the similar cationic iridium
complex [Ir(ppy)
3
+
bpy] .
2
b
1
2
3
4
5
6
a
1.0
0.8
0.6
0.4
1
2
3
4
5
6
[
11a,11b,12a,12b,13]
systems,
the
strong emission was tentatively
assigned to a spin-forbidden dπ(Ir)→π*(ligand) metal-to-ligand
charge-transfer ( MLCT) excited state, probably with some mixing
of
Photophysical data of the complexes in DMSO at room
temperature are presented in Table 2. The emission lifetimes of
complexes 1, 2, 3, 4, 5, and 6 are 180, 240, 470, 130, 240, and 270
ns, respectively, and their emission quantum yields in aerated
DMSO are 15.0%, 29.0%, 3.0%, 16.0%, 23.0%, and 3.0%,
respectively.
3
0
0
.2
.0
3
a
spin-forbidden π→π* intraligand ( IL) character.
450 500 550 600 650 700
Wavelengh / nm
450
500
550
600
650
700
Wavelength / nm
Fig. 2. Emission spectra and luminescent photograph of the Ir(III)
complexes in DMSO (a); Emission spectra of the complexes in
EtOH/MeOH (4:1, v/v) at 77 K (b).
2
.4. Photophysical properties of the complexes in acid/base
solutions
2

ACCEPTED MANUSCRIPT
The complexes 1 and 4 were chosen for the preliminary studies
of the effect of acid and base on the photophysical properties. No
obvious changes were observed in the UV-vis spectra obtained
upon addition of an aqueous solution of HCl to complexes 1 and 4
1
0
.0
.8
1
2
4
6
7
8
1
0.6
0.4
0.2
in DMSO/H O (1:1, v/v). Upon addition of an aqueous solution of
2
NaOH to the dihydroxyl complex 1, the absorption intensity at ca.
0
.0
2
71 nm increased obviously. No obvious changes were observed in
0
2
4
6
8 10 12 14
pH
0.5
the UV-vis spectra obtained upon addition of NaOH to complex 4.
Fig. 3 shows luminescent changes of Ir(III) complex 1 upon
addition of HCl and NaOH solutions. Upon addition of HCl
11.5
2.5
1
solution to a DMSO/H O (1:1, v/v) solution of Ir(III) complex 1,
2
the luminescence of the complex decreased in intensity at 492 nm
with a reduction of ca. 99% in strongly acidic solution (pH<2), and
a weak band appeared at around 550 nm. The lifetime is about
4
00
450
500
550
600
650
700
Wavelength / nm
0
.11µs in the acidic solution (pH=3.0). The luminescence intensity
of complex 1 was quenched upon addition of HCl solution, which
Fig. 4. Changes in the luminescence of Ir(III) complex 4 (6 µM) in a
mixture solution of DMSO/H O (1:1, v/v) upon addition of various
amounts of NaOH or HCl. Inset shows the changes in luminescence vs. pH.
may be ascribed to protonation of the dihydroxy ligand
2
[
17]
bpy(OH)2.
While the emission intensity of complex 1 was
strongly enhanced over a range of pH from nearly neutral to highly
alkaline with a small red shift of ca. 9 nm (Fig. 3) as a result of the
strong influence of the two OH groups in the 4,4’-dihydroxyl-2,2’-
2.5. The reversibility of the complex in acid/base solutions
[
18]
bipyridine ligand of these complexes.
In the basic solution
Fig. 5 illustrates the reversibility of complex 1 upon alternate
addition of NaOH and HCl solution (pH 4.5 to pH 9.5) for six
cycles. The luminescence intensity was enhanced after addition of
NaOH, and the luminescence was turned off after addition of HCl,
as shown in Fig. 5. The emission was then turned on again in basic
solution. This on-off process was reversible, and the complex was
extremely stable in acid/base solutions. Such results further
demonstrate that the change in luminescence could be attributed to
the electron-donating effect brought by the protonation and
deprotonation of the dihydroxyl group. It is difficult to estimate the
pKa of the ground state of the complexes, because of the tiny
changes in the electronic absorption spectra in acidic/basic
solutions. But multiple protonation-deprotonation equilibriums of
complex 1 are clearly evident in Fig.3, the plots of the relative
emission intensity vs. pH have two inflection points near pH 4.5
and 10.5, owing to the stepwise deprotonation of two hydroxyl
groups.
(pH=12), the lifetime is about 0.61 µs, which is longer than that in
the acidic solution, probaly due to the changes of the excited state.
2
6
4
6
7
8
4
2
9
1
1
.5
0.5
1.5
0
2
4
6
8
10 12
pH
4
50
500
550
600
650
Wavelength / nm
The reversibility of complex 4 upon alternate addition of NaOH
and HCl was also studied. The luminescence intensity of a solution
of complex 4 was turned off upon addition of NaOH solution and
recovered upon addition of HCl solution. The process was
reversible and the complex remained stable after several cycles.
These results indicate that amino functionalized Ir(III) complexes
are fully and reversibly pH-controlled “off-on-off” luminescent
signaling systems. From pH 4 to pH 11, the luminescence was
almost constant. At around pH 0 to pH 4 and pH 11 to pH 14, the
luminescence of the complexes was gradually switched off.
Fig. 3. Changes in the luminescence of Ir(III) complex 1 (6 µM) in a
solution of DMSO and H O (1:1, v/v) upon addition of various amounts of
2
HCl or NaOH. Inset shows the changes in luminescence vs. pH.
Upon addition of HCl (0.1 M) to a solution of complex 4 (6
µM), the pH ranged from approximately 7 to 2 and the
luminescence intensity was quenched with a reduction of about
9
5% (Fig. 4). The electron-donating NH2 group becomes an
+
electron-withdrawing (NH ) group upon protonation, which
3
quenches the emission intensity of the complexes because of the
1
+ NaOH
[
19]
electron transfer effect.
Interestingly, upon addition of NaOH,
the luminescence of complex 4 was also quenched with a reduction
of approximately 96% (Fig. 4). These results differ from those
observed in complex 1. The emission intensity of the amino
functionalized Ir(III) complexes decreased in basic solution,
probably due to the partial quinoid character resulted from
delocalization of the negative charge on the aniline anion at high
[
18,19]
pH.
To differentiate the same emission intensity at different
1
+ HCl
pHs, the emission lifetimes of complex 4 at pH=3 and 11 were
investigated, and the lifetime is determined to be 0.60 and 0.35 µs,
1
2
3
4
5
6
times
respectively The results suggested that the energy gap might be
.
affected by the pH, leading to the defferent lifetime in the acidic or
basic solution.
Fig. 5. Luminescent changes of Ir(III) complex 1 (6 µM) in a mixture
solution of DMSO/H
HCl for six cycles.
2
O (1:1, v/v) upon alternate addition of NaOH and
3

ACCEPTED MANUSCRIPT
subsequent optical property investigations. As show in Table S2,
2
.6. Cytotoxicity and imaging studies
the TDDFT calculations predict that two absorption bands are
dominant in the high energy region with the wavelength from 400
to 200 nm, which well agree with the experimental results. The
excitations are ascribed to metal-to-ligand charge transfer
(MLCT) as shown in Fig. S3, which schematically depicts the
molecular orbitals involved in the electron transitions.
Cell viability upon treatment with the new Ir(III) complexes was
determined by testing mitochondrial enzyme functions according to
the
colorimetric
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) method. Fig. 6 presents the
viability of HepG2 cells after incubation with complexes 1 to 6 for
2
4 h in DMSO/PBS. Approximately >70% of the HepG2 cells
remained alive after incubation for 24 h with complexes 2, 4, and 6
at concentrations lower than 25 µM, which reveals that the
toxicities of these complexes are acceptable. By contrast, high
toxicity to HepG2 cells was found with complexes 1 and 5 even at
extremely low concentrations. In particular, complex 3 showed a
significantly positive effect on cell proliferation without an obvious
dose-dependent tendency. It is a usual phenomena for some
complexes that show proliferative effects at low concentrations
As proposed by previous works, the emission energies of the
iridium(III) complexes calculated by TDDFT from the S
0
[
24]
geometries are sufficient to reproduce the experimental results.
We computed the S -T vertical transition energies based on the
optimized geometry excluding the solvent effect in
0
1
S
0
consideration of the computational cost. The spin-orbit coupling
leads to the spin-forbidden transition allowed, as the triplet
manifold mixes with higher-energy singlets. The S -T transition
0 1
[
20]
while showing cytotoxicity toward cells at high concentrations.
energies with non-neglected oscillator strength are shown in
Table S3. The theoretically predicted emission energies deviates
from the experimental values. It may arise from the instinct
defect of B3LYP functional for the lack of long-range correction
and the absence of solvent effects. The frontier molecular orbitals
The exact molecular mechanism of cytotoxicity induced by Ir(III)
complexes is still unclear. In general, the higher lipophilicity would
result in the higher cytotoxicity of the complexes. It has been
reported that the strong DNA binding,
complexes,
[
21]
the dissociation of the
[
22]
[23]
or generation of intracellular ROS
should be
make the dominant contributions to the S -T transition. As
0 1
responsible for the cytotoxicity induced by metal complexes.
shown in Fig 7 (Fig. S4) and Table 4, the HOMOs are featured by
the d orbital of iridium(III) in combination with orbital
delocalized on the conjugated (C^N) ligands, and the LUMOs
delocalized on the (N^N) ligands. It is evident that the singlet-
triplet vertical transition has the mixed intraligand π−π* and
MLCT character.
The interaction of living cells with emissive iridiumIII)
complexes 1 and 4 was also investigated by fluorescence
microscopy. HepG2 cells were incubated with each complex in
DMSO/PBS (pH 7.4, 99:1, v/v) for 1h at 37℃. Fig S2(Supporting
information) shows the images of HepG2 cells incubated with the
complex 1 at 50 µM and complex 4 at a concentration of 25 µM.
Bright-field measurements after treatment with the Ir(III)
complexes showed that the cells were visible throughout the
imaging experiments. Interestingly, the complex 4 displayed
intense intracellular luminescence, and the luminescence and
bright-field images showed that the luminescence was evident in
cytoplasm, not in the nucleus and membrane; whereas the complex
We further investigated the pH-dependent photophysical
properties of complexes 1 and 4. Two extreme cases were
considered: the totally protonated species and alkalized species.
The T geometries of two protonated species and two alkalized
1
species were optimized, and the energy difference between S0
and T is calculated based on the optimized T geometry. For the
1
1
protonated complexes 1 and 4, the energy gap is only 0.05 and
.14 eV, respectively. For the alkalized complex 1 whose energy
0
gap is 2.10 eV matching the experimental value measured in high
pH solution. The excitation is assigned to π−π* character (Table
1
displayed weak or scattered luminescence in membrane and dead
cell. These observations suggest that the complex 1 was hardly
internalized into the living cells, and the complex 4 did better. The
results were basically coincided with those of MTT cytotoxicity
studies. It could be concluded that the hydrophilic and steric
hindrance of the complexes collectively decided on the abilities of
passing cell membrane.
S4). While for the alkalized complex 4, the T is more stable than
1
S0. That means the phosphorescence signal is beyond the
measured wavelength. Moreover, according to the Einstein theory
of spontaneous emission, the radiative rate constant is
proportional to the emission energy and transition dipole moment.
As the nearly identical transition dipole moment for the Ir(III)
complex in different pH solution, the tiny energy gap between S0
and T1 leads to the fast radiative rate. Accordingly, the
nonradiative rate becomes enhanced and the nonradiative
deactivation channel is facilitated.
2
2
1
1
50
00
50
00
5
μ M
25μ
0μ
75μ
M
M
M
5
1
00μ M
5
0
0
Fig .7. Calculated HOMOs and LUMOs involved in the singlet-
triplet electron transitions of complexes 1 and 4
1
2
3
4
5
6
Fig. 6. Viability of HepG2 cells after incubation with complexes 1
to 6 in DMSO/PBS (pH 7.4, 99:1, v/v) for 24 h.
3. Conclusions
A series of cyclometalated Ir(III) diimine complexes were
successfully synthesized and characterized. The luminescence of
the complexes containing hydroxyl groups could be switched
off/on upon addition of acid and base, whereas the luminescence of
the complexes functionalized with amino groups could be switched
off/on/off upon addition of acid and base as a result of the
protonation or deprotonation of amino and hydroxyl groups. The
2
.7 Calculations
Compared with the geometry parameters selected from the crystal
structures, the equilibrium geometries optimized at B3LYP/6-
3
1G*/LANL2DZ level provide the reliable descriptions in theory
(Table S1). Therefore, we used the optimized S geometry for the
0
4

ACCEPTED MANUSCRIPT
results show that the complexes could be potentially used for the
(m ,4H), 7.64 (m, 2H) , 7.49 (t, 4H, J = 7.6 Hz), 7.13 (t, 2H, J = 7.6
Hz), 6.94 (m, 2H), 6.19 ( d, 2H,J = 7.2 Hz). Positive Q-TOF
HRMS: m/z found 737.2121 (calcd 737.1529) for C H IrN O
luminescence pH sensors. The cytotoxicity studies demonstrated
that the toxicities of the complexes 2, 4 and 6 were acceptable, and
they have promising potential as specific cell-imaging agents.
36
24
4
2
+
({M-Cl} ). Anal. Calcd (%) for C36
H
24ClIrN
.66, N 6.78. Found：C 52.23, H 3.45, N 6.78. IR(KBr) vmax/cm
414, 2366, 1618, 1495, 1448, 1330, 1029, 835, 720, 620. The
O ·H O: C 52.33, H
4 2 2
-
1
3
3
4. Experimental Section
13
signal was very weak in CNMR spectrum due to the poor
solubility of the complex.
4
.1. Reagents
4
.3.4. [Ir(ppy) [bpy(NH ) ]]Cl (4)
2 2 2
1
The product was isolated as a yellow solid with 70% yield. H
All reagents were used as received from commercial sources.
NMR(400 MHz, d -DMSO), δ (ppm): 8.22 (d, 2H, J = 8.2 Hz),
6
Solvents were purified to deoxygenization by dry nitrogen. Other
solid or liquid reagents received from commercial sources were
used without further purification. 4,4’-Diamine-2,2’-dipyridine was
7
5
.92 (t, 2H, J = 7.1 Hz ), 7.85 (d, 2H, J = 7.1 Hz), 7.72 (d, 2H, J =
.2 Hz), 7.32 (s ,2H), 7.24 (t, 2H, J = 6.6 Hz) , 7.17 (d, 2H, J = 6.4
Hz), 7.06 (s, 4H), 6.96 (t, 2H, J = 7.4 Hz ), 6.83 (t, 2H, J = 7.4 Hz),
.57 ( d, 2H, J = 6.4Hz), 6.17(d, 2H, J = 7.4 Hz). Positive Q-TOF
HRMS: m/z found 687.1949 (calcd 687.1848) for C H IrN ({M-
[
25]
synthesized based on the method provided in the literature. The
6
ligand 4,4’-dihydroxy-2,2’-dipyridine (bpy(OH) ) was synthesized
2
3
2
26
6
[
26]
according to the literature with slight modifications.
Cyclometalated Ir(III) chloride-bridged dimer [(C^N) IrCl] was
+
Cl} ). Anal. Calcd (%) for C H ClIrN ·2H O: C 50.69, H 3.99, N
1.08. Found：C 50.99, H 4.04, N 10.78. CNMR(d -DMSO), δ
32
26
6
2
13
2
2
1
6
[
1a,27]
synthesized according to the literature.
(ppm): δ 107.6, 111.9, 120.1, 121.9, 124.0, 125.3, 130.4, 131.7,
1
38.5, 144.5, 148.8, 149.9, 153.1, 156.0, 156.5, 167.8. IR(KBr)
4
.2. Physical Measurements and Instrumentation
-1
vmax/cm 3414, 1622, 1503, 1474, 1265, 1029, 844, 759.
.3.5. [Ir(pq) [bpy(NH ) ]]Cl (5)
Physical measurements and instrumentation, cell culture and
4
[
28]
2
2 2
cell viability assay were the same to the previous report.
.3. Synthesis
A solution of [(C^N) IrCl] (20 mg, 0.018 mmol) in oxygen-
1
The product was isolated as red crystals with 99% yield. H
4
NMR (400 MHz, CD OD ), δ (ppm): 8.36 (m, 4H), 8.10 (d, 2H, J =
3
2
2
7
(
2
.7 Hz ), 7.86 (d, 2H, J = 7.2 Hz), 7.70 (d, 2H, J = 9.0 Hz), 7.49
d ,2H, J = 6.4 Hz), 7.44 (t, 2H, J = 7.4 Hz) , 7.12 (m, 4H), 6.99 (s,
H), 6.75 (t, 2H, J = 7.0 Hz ), 6.49 (m, 4H). 4.57 (b, 4H). Positive
free CH Cl /CH OH (10 mL; 1:1 v/v) was stirred under nitrogen,
after which an excess of bpy(OH) or bpy(NH ) (0.041 mmol) was
added to it. The mixture was heated to reflux for 12 h to provide a
fluorescent solution. Subsequently, the solvent was removed under
reduced pressure. The residue was purified by column
chromatography on silica gel.
.3.1. [Ir(ppy) [bpy(OH) ]]Cl (1)
2 2
The residue was purified by column chromatography on silica
gel using a mixture of CH Cl and methanol (20:1, v/v) as the
eluent to afford the pure compound as a yellow solid with 75%
yield. H NMR(400 MHz, d -DMSO), δ (ppm): 8.20 (d, 2H, J =
.2 Hz), 7.90 (t, 2H, J = 8.2 Hz ), 7.84 ( d, 2H, J = 7.8 Hz), 7.71
d ,2H, J = 5.8 Hz), 7.38 (s, 2H) , 7.20 (m, 4H), 6.94 (t, 2H, J = 7.5
Hz ), 6.82 (t, 2H, J = 7.4 Hz), 6.49 ( b, 2H), 6.18 (d, 2H, J = 7.4
Hz). Positive Q-TOF HRMS: m/z found 689.1609 (calcd 689.1529)
for C H IrN O
2
C H ClIrN O ·2H O: C 50.55, H 3.71, N 7.37. Found: C 50.65,
H 3.62, N 7.60. CNMR(d -DMSO), δ (ppm): 112.6, 116.2, 120.3,
22.3, 124.2, 125.4, 130.5, 131.6, 138.9, 144.4, 149.1, 151.0, 151.8,
2
2
3
2
2 2
Q-TOF HRMS: m/z found 787.2262 (calcd 787.2161) for
C H IrN ({M-Cl} ). Anal. Calcd (%) for C H ClIrN ·4H O: C
+
40
30
6
40 30
6
2
5
3.71, H 4.28, N 9.50. Found ： C 53.80, H 4.10, N 9.85.
13
CNMR(d -DMSO), δ (ppm): δ 106.5, 110.9, 117.1, 121.8, 125.5,
6
4
1
1
1
26.1, 126.6, 127.8, 128.6, 129.8, 130.1, 134.3, 139.3, 146.2, 146.3,
-1
47.8, 153.2, 156.1, 156.4, 170.4. IR(KBr) vmax/cm 3416, 2360,
2
2
624, 1542, 1512, 1449, 1402, 1333, 1265, 1005, 826, 764.
4
.3.6 [Ir(bzq) [bpy(NH ) ]]Cl (6)
2 2 2
1
6
1
The product was isolated as an orange solid with 90% yield. H
NMR(400 MHz, d -DMSO) , δ(ppm): 8.55 (d, 2H, J = 7.1 Hz),
8
J = 8.0 Hz), 7.35 (d, 2H, J = 2.3 Hz) , 7.11 (m, 8H), 6.49 (m, 2H),
6
7
8
(
6
.14 (d, 2H, J = 5.4 Hz ), 7.89 (m, 4H), 7.68 (m, 2H), 7.47 (d ,2H,
.18 (d, 2H, J = 7.1 Hz). Positive Q-TOF HRMS: m/z found
+
({M-Cl} ). Anal. Calcd (%) for
+
32
24
4
35.1951 (calcd 735.1848) for C H IrN ({M-Cl} ). Anal. Calcd
36
26
6
32
24
4
2
2
(%) for C H ClIrN ·4H O: C 51.33, H 4.07, N 9.98. Found：C
13
36 26 6 2
6
13
5
1
1
2
1.88, H 4.01, N 9.93. CNMR(d -DMSO), δ (ppm): 107.7, 112.0,
6
1
1
1
20.0, 123.0, 124.5, 127.0, 128.9, 129.9, 130.0, 134.1, 137.4, 141.1,
-
1
57.4, 167.0, 167.6. IR(KBr) vmax/cm 3423, 1616, 1553, 1471,
268, 1024, 829, 764.
4
-1
48.5, 149.5, 149.8, 156.4, 156.5, 157.4. IR(KBr) vmax/cm 3417,
363, 1624, 1503, 1444, 1330, 1095, 829, 724.
.3.2. [Ir(pq) [bpy(OH) ]]Cl (2)
2 2
4
.4. Crystal Structure Determination
Single crystals of complexes 2 and 5 were obtained via the liquid
The product was recrystallized from dichloromethane-hexane
phase diffusion method by slow diffusion of n-hexane into
dichloromethane/methanol (20/1, v/v) solutions of complexes 2
and 5. Crystallographic data collections for complexes 2 and 5
were carried out on a beam line 3W1A at the Beijing Synchrotron
Radiation Facility with a mounted MarCCD-165 detector using
synchrotron radiation (λ = 0.7501 Å). Data reduction and
numerical absorption correction were applied using HKL2000
1
to provide red crystals with 99% yield. H NMR(400 MHz, d -
6
DMSO) , δ (ppm): 12.0 (b, 2H) 8.53 (s, 4H), 8.24 (d, 2H, J = 7.6
Hz ), 7.95 ( d, 2H, J = 7.2 Hz), 7.77 (d ,2H, J = 5.6 Hz), 7.56 (s,
H) , 7.42 (m, 4H,), 7.13 (m, 4H), 7.00 (d, 2H, J = 6.4 Hz), 6.77 ( t,
2
2
H, J = 7.0 Hz), 6.39 (d, 2H, J =7.5 Hz). Positive Q-TOF HRMS:
+
m/z found 789.1944 (calcd 789.1842) for C H IrN O ({M-Cl} ).
40
28
4
2
Anal. Calcd (%) for C H ClIrN O ·4H O: C53.59, H 4.05, N 6.25.
4
0
28
4
2
2
[29]
software.
1
3
Found: C 53.45, H 3.88, N 5.99. CNMR(d -DMSO), δ (ppm):
6
The structures were solved by direct methods, and all of the
1
1
10.6, 114.8, 117.3, 122.2, 125.0, 126.8, 127.3, 128.1, 128.8, 130.2,
30.3, 134.2, 139.6, 146.0, 147.2, 148.3, 151.8, 157.4, 166.9, 170.5.
2
non-hydrogen atoms were refined anisotropically on F by the full-
matrix least squares technique using the SHELXH-97
-
1
IR(KBr) vmax/cm 3411, 2371, 1615, 1560, 1516, 1451, 1399, 1337,
260, 1016, 826, 761.
[30]
crystallographic software package.
The asymmetric unit of
1
-
complex 5 contains four Ir(III) complex molecules and four Cl
ions. The remainder of the unit is occupied by extreme electron
density, which could be identified as free solvent molecules. Given
that these guest solvent molecules in the crystal are too many and
impossible to refine via conventional discrete-atom models, the
4
.3.3. [Ir(bzq) [bpy(OH) ]]Cl (3)
2 2
1
The product was isolated as an orange solid with 90% yield. H
NMR(400 MHz, d -DMSO) , δ(ppm): 12.0 （b, 2H）, 8.56(d, 2H,
6
J = 7.1Hz), 8.12 (d, 2H, J =5.4 Hz ), 8.02 ( d, 2H, J=2.5 Hz), 7.9
[
31]
SQUEEZE
subroutine of the PLATON software suite was
5

ACCEPTED MANUSCRIPT
[36]
applied to remove the scattering from the highly disordered solvent
molecules, and sets of solvent-free diffraction intensities were
produced. SQUEEZE analysis showed that the void space was
occupied by 49 electrons per formula, corresponding to 4 water
corrected functional, LC-PBEh,
considering the excitations
featured by the metal-to-ligand charge transfer. The solvent effects
were taken into account by using the COSMO salvation model
in the excitation energy calculations. All these calculations were
[
37]
[
38]
performed with NWChem 6.1.1.
transition energies, SOC-TDDFT
For the S0-T1 vertical
molecules
per
formula
unit.
The
final
formula
[
39]
[40]
and pSOC-TDDFT
(
C H N Ir)Cl·(H O) was calculated from the SQUEEZE results
and elemental analysis. The crystal parameters are listed in Table 3.
40
30
6
2
4
[41]
calculations with the ZORA Hamiltonian were carried out with
[
42]
ADF.
The B3LYP functional was applied with all electron
[
43]
Slater-type orbital basis sets,
TZP for iridium and DZ for the
Table 3. Crystallographic data for complexes 2 and 5.
rest. 20 singlet and triplet excitations were investigated in SR-
TDDFT calculations and the lowest 4 spin-mixed excitations were
studied in the SOC-TDDFT calculations. HOMO and LUMO
energy levels (eV) are showed in table 4.
Complex
2
5
Empirical
40 4 2 2
C H28ClIrN O ·H O
C40H30ClIrN6
formula
·CH
2
Cl ·0.5C H
2
6 14
Table 4. HOMO and LUMO energy levels (eV) and compositions (%) of
iridium(III) complexes.
Formula weight
970.34
P21/n
822.35
Space group
P2(1)/c
HOMO
C^N
LUMO
C^N N^N
Complex
E
H
(eV)
L
E (eV)
a/Å
b/Å
c/Å
α (°)
β(°)
γ(°)
V/Å
Z
14.482(3)
17.694(4)
16.564(3)
90
19.246(2)
23.9560(10)
33.275(2)
Ir
N^N
Ir
1
2
3
4
5
6
-8.25
-8.19
-8.16
-7.95
-7.92
-7.88
-5.48
-5.32
-5.47
-4.99
-4.79
-4.98
45.18 50.88 3.94 2.00 1.74 96.26
45.97 50.64 3.39 1.86 3.60 94.54
35.96 60.42 3.62 2.06 1.66 96.28
46.07 49.49 4.44 1.78 9.11 89.11
46.79 49.58 3.63 1.97 7.59 90.44
37.25 59.12 3.63 1.84 1.75 96.41
90
109.04(3)
90
104.554(5)
90
Supporting Information
4012.2(14)
4
14849.4(19)
16
The supporting information includes the synthetic procedure
for the ligands, MS spectra of the Ir(III) complexes and X-ray
crystallographic data in CIF format.
−
3
ρcalcd/mg cm
1.606
1.471
3.704
−
1
µ/mm
3.573
Radiation, λ/Å
0.75010
0.75010
Acknowledgments
T/K
203(2)
194(2)
This work was supported by the National Scientific Foundation of
China (No. 21171038 and No. 31100723), the Program for
Changjiang Scholars and Innovative Research Team in University
a
R1(F
o
)
0.0814
0.2757
1.144
0.0494
0.0866
1.372
2
o
b
wR (F
2
)
(No. IRT1116), and the Key Laboratory Funding Scheme
GOF
a
Guangzhou Municipal Government (2060204). The Beijing
Institute of Research provided access to the equipment used for
crystal structural determination.
b
2
2
2
2 2 1/2
R1=∑| F
o
-F
c
o o c o
|/∑F wR2=∑[w(F -F ) ]/∑[w(F ) ] .
4
.5 Cytotoxicity and imaging studies
_
___________
MTT assay was used to determine viability of HepG2 cells upon
treatment with Ir(III) complexes, as described in detail
elsewhere.
plates at the density of 4 × 10 cells per well and incubated 3 days.
After the treatment with Ir(III) complexes for 24h, the plates were
washed twice with culture medium, and then MTT was added and
incubated for another 4h. Cells without treatment of Ir(III)
complexes were used as control. The relative cytotoxicity was
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microscope.HepG2 cells in growth medium (1×10 cells mL )
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.6 Computational details
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(
The singlet ground state (S0) and first triplet state (T1) were
optimized by density functional theory. The exchange-correlation
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functional was chosen as B3LYP
with the LANL2DZ basis
for the rest. The singlet-singlet
vertical transition energies were calculated by the long-range
[
34]
[35]
3276；
set
for iridium and 6-31G*
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7

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Jacobsen, L. Jensen, J. W. Kaminski, G. van Kessel, F. Kootstra, A.
Kovalenko, M. V. Krykunov, E. van Lenthe, D. A. McCormack, A.
Michalak, M. Mitoraj, S. M. Morton, J. Neugebauer, V. P. Nicu, L.
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Philipsen, D. Post, C. C. Pye, W. Ravenek, J. I. Rodríguez, P. Ros, P.
R. T. Schipper, G. Schreckenbach, J. S. Seldenthuis, M. Seth, J. G.
Snijders, M. Solà, M. Swart, D. Swerhone, G. te Velde, P. Vernooijs,
L. Versluis, L. Visscher, O. Visser, F. Wang, T. A. Wesolowski, E. M.
van Wezenbeek, G. Wiesenekker, S. K. Wolff, T. K. Woo and A. L.
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1
156.
Received: ((will be filled in by the editorial staff))
Published online: ((will be filled in by the editorial staff))
8

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Entry for the Table of Contents (Please choose one layout)
Layout 1:
Key Topic
A series of Ir(III) complexes with
amino or hydroxyl groups were
Zhaozhen Wu, Juanjuan Mu, Qiong
Wang, Xing Chen, Lasse Jensen,
Changqing Yi, Mei-Jin Li … Page
1
synthesized and characterized. Two of
the complexes were structurally
characterized via X-ray crystallography.
The photophysical and photochemical
properties of these complexes were
studied. Preliminary studies of their
applications on pH sensing, and cell
imaging were also performed.
2
3
4
5
6
*
*
No. – Page No.
Hydroxyl and Amino Functionalized
Cyclometalated Ir(III) Complexes:
450
500
550
600
650
700
Synthesis,
Characterization
and
Wavelengh / nm
Cytotoxicity Studies
+
+
OH
NH
2
N
Ir
C
N
Ir
C
N
N
N
C
N
N
N
Keywords: Iridium(III) complex /
C
OH
NH
2
Synthesis /pH sensing / Cytotoxicity
9

ACCEPTED MANUSCRIPT
Highlights
ò
ò
ò
ò
ò
A series of Ir(III) complexes were successfully synthesized and characterized
Two of the complexes were structurally characterized via X-ray crystallography
The emission of the complexes could be switched off or on in acid or base solution
The complexes could be potentially used for the luminescence pH sensors
Some of the complexes have promising potential as specific cell-imaging agents

ACCEPTED MANUSCRIPT
A series of Ir(III) complexes with amino or hydroxyl groups were synthesized and characterized. Two of the complexes
were structurally characterized via X-ray crystallography. The photophysical and photochemical properties of these
complexes were studied. Preliminary studies of their applications on pH sensing, and cell imaging were also performed.

ACCEPTED MANUSCRIPT
Supporting Information
Hydroxyl and amino functionalized cyclometalated
Ir(III) complexes: synthesis, characterization and
cytotoxicity studies
a
a
a
c
c
b,*
Zhaozhen Wu, Juanjuan Mu, Qiong Wang, Xing Chen, Lasse Jensen, Changqing Yi,
a,*
Mei-Jin Li
a
b
c
Key Laboratory of Analysis and Detection Technology for Food Safety, Ministry of Education,
Department of Chemistry, Fuzhou University, Fuzhou 350108, China
Key Laboratory of Sensing Technology and Biomedical Instruments (Guangdong Province), School of
Engineering, Sun Yat-Sen University, Guangzhou 510006, P.R.China
Department of Chemistry, The Pennsylvania State University, 104 Chemistry Building, University
Park, 16802, United States.
Content
I. General Information
II. The synthetic procedure for the ligands
III. MS spectra of the ligand and Ir(III) complexes
IV. Imaging studies
IV. Calculations
1

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I. General Information
All reagents were used as received of commercial sources. Solvents were purified to
deoxygenization by dry nitrogen. Other solid or liquid reagents received from
commercial source were used without any further purification. 2-Phenylpyridine (ppy),
2
-phenylquinoline (pq), benzo [h]quinoline (bzq) were all purchased from Alfa Aesar
Reagent Co. Ltd. Iridium(III) chloride hydrate was obtained from Aladdin chemistry Co.
Ltd. 4,4’-Dimethoxy-2,2’-bipyridine was obtained from Sigma-Aldrich Co. LLC. All of
these reagents were used without any further purification.
1
H NMR spectra were recorded on a Bruker Avance III (400 MHz) spectrometer
using deuteriated solvents at room temperature with chemical shifts reported relative to
tetramethylsilane(Me4Si). Positive Q-TOF mass spectra (MS) were recorded on an
Agilent 6520 accurate mass spectrometer..
II. The synthetic procedure for the ligands
Synthesis of 4,4’-dihydroxy-2,2’-dipyridine (bpy(NH
2
2
) ).
[
1]
4,4’-Diamine-2,2’-dipyridine was synthesized by the literature method.
2
,2’-Bipyridine-N,N-dioxide
N
O
O
N
2,2’-Bipyridine-N,N-dioxide was prepared by heating 2,2’-bipyridine (1.0 g.), glacial
acetic acid (7.5 ml.), and 30% hydrogen peroxide (1.3 ml.) together at 70-80℃ for 3 hr.
An additional 0.9 ml. of 30% hydrogen peroxide was added and the temperature
2

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maintained at 70-80℃ for a further 19 hr. On addition of acetone, 2,2’-bipyridine
N,N’-dioxide precipitated. The material was recrystallized from hot water by the addition
of a large excess of acetone. Yield: 867mg of fine white needles (72%).
2
,2’-Bipyridine N,N-dioxide
O N
2
O
N
N
O
O N
2
3
.6mL of concentrated sulfuric acid was added to 750mg of 2,2’-Bipyridine-N,N-dioxide.
The mixture was first cooled in ice, before addition of 1.25mL of fuming nitric acid
CAUTION). The resulting solution was held at reflux (95–100 ℃) for 20 h before
(
cooling to room temperature. The acidic mixture was then poured onto ice (-40 ℃),
prepared by the addition of an excessive amount of liquid nitrogen onto 20mL of water
with constant stirring. After continued stirring, a bright yellow precipitate formed, to
which a further measure of liquid nitrogen was added. The solution was subsequently
filtered and the yellow precipitate collected. The solid was washed successively with
water and allowed to air dry. Yield: 920mg of yellow powder (83%).
5
,6-diamino-1,10-phenanthrolin
H N
2
N
N
H N
2
A mixture of 350mg of 2,2’-Bipyridine N,N-dioxide and 630 mg Pd/C (5%) in 38mL of
ethanol was purged with N gas. The suspension was then heated to reflux under nitrogen
2
and, after the complex was completely dissolved, 2.7mL of hydrazine hydrate in 10mL
ethanol was added dropwise over a period of 20min. The resulting solution was held at
reflux for 15 h. When completed, the mixture was immediately filtered under reduced
pressure, and washed with boiling ethanol. After removal of the solvent, the yellow
3

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precipitate was ground in 20mL of water and left at 2℃ overnight. The white solid that
separated was vacuum filtered, washed with cold water and dried at 50℃. Yield: 195 mg
of white powder (83%).
Synthesis of 4,4’-dihydroxy-2,2’-dipyridine (bpy(OH) ). The ligand was synthesized
2
[2]
according to the literature with slight modifications. HBr solution (41 wt%, 0.554 mL,
2
4
.77 mmol) was added to a mixture of glacial acetic acid (13.85 mL) and
,4’-dimethoxy-2,2’-bipyridine (60 mg, 0.277 mmol); the resulting mixture was
subsequently refluxed overnight. After cooling to room temperature, the solvent was
removed under reduced pressure. The residue was dissolved in distilled water and
neutralized to pH 6–7 by addition of aqueous ammonium hydroxide to provide a white
precipitate with 64% (33 mg) yield.
Synthesis of [(C^N) IrCl] . Cyclometalated Ir(III) chloride-bridged dimer [(C^N) IrCl]
2
2
2
2
[3]
3 2
was synthesized according to the literature. A mixture of IrCl ·3H O and an excess of
the desired C^N ligand, ppy, pq, or bzq, in oxygen-free 2-ethoxyethanol/H O (3:1, v/v)
2
was heated to reflux for 24 h in the dark under nitrogen to provide a crude
chloride-bridged dimer. After cooling to room temperature, the precipitate was filtered
off and washed with ethanol followed by an equal amount of acetone. The solid was
dissolved and isolated to afford a colored powder.
4

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Ⅲ
. MS spectra of the Ir(III) complexes
OH
N
Ir
N
N
N
OH
OH
OH
N
N
N
Ir
N
OH
OH
N
N
Ir
N
N
5

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NH
2
N
Ir
N
N
N
NH
2
NH
2
N
N
N
Ir
N
NH
2
NH
2
N
N
Ir
N
N
NH
2
Fig.S1 MS spectra of the Ir(III) complexes
6

ACCEPTED MANUSCRIPT
IV. Imaging studies
a
b
c
1
4
Fig. S2. Images of fluorescence microscopy of HpeG2 cells in the presence of Ir
complexes 1 and 4: (a) Brightfield images; (b) luminescence imaging of HepG2 cells
incubated solely with 50 μM complex 1 (or 25μM 4) in DMSO/PBS(pH 7.4, 99:1,v/v); (c)
Merged image of (a) and (b).
V. Calculations
Table S1. Selected geometry parameters from optimized geometries compared with the experimental
data.
Geometry
parameters
2.010
2.007
Geometry
parameters
2.010
2.008
2.137
1
Complex 2
Error (%)
Complex 5
Error (%)
Ir(1)-C(25)
Ir(1)-C(40)
Ir(1)-N(3)
Ir(1)-N(4)
Ir(1)-N(1)
Ir(1)-N(2)
O(1)-C(3)
O(2)-C(8)
0.85
0.20
2.11
1.31
5.68
4.34
0.15
0.82
1.01
0.88
2.97
Ir(1)-C(16)
Ir(1)-C(1)
Ir(1)-N(1)
Ir(1)-N(2)
Ir(1)-N(4)
0.65
0.30
3.60
2.43
4.55
3.82
0.29
0.22
1.13
0.50
1.92
2.138
2.141
2.272
2.258
1.346
1.346
79.6
2.141
2.265
2.253
1.361
1.361
79.7
Ir(1)-N(3)
N(5)-C(33)
N(6)-C(38)
C(1)-Ir(1)-N(1)
C(16)-Ir(1)-N(2)
N(4)-Ir(1)-N(3)
C(25)-Ir(1)-N(3)
C(40)-Ir(1)-N(4)
N(1)-Ir(1)-N(2)
79.7
73.0
79.7
73.0
1
Error is defined as |p -pexpt/pexpt*100|
cal
7

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Table S2. Calculated singlet-singlet vertical transition energies (E) and wavelengths() with the large
oscillator strengths (f) incorporated with transition assignment for the interested Ir(III) complexes in
DMSO.
Complex
E(eV),(nm)
f
Transition
138-140
134-140
Assignment
MLCT
3.70, 334.7
4.81, 257.9
3.34, 371.6
0.091
1
MLCT
MLCT
MLCT
0.232
0.108
1
38-142
164-165
2
3
4
5
1
1
61-166
62-166
MLCT
MLCT
MLCT
4
3
.28, 289.4
.66, 339.1
0.150
0.080
150-152
1
1
47-153
48-152
MLCT
MLCT
MLCT
4
.48, 276.9
.68, 337.7
0.268
0.090
3
138-140
1
1
35-141
34-140
MLCT
MLCT
MLCT
4
.79, 258.8
.30, 375.3
0.223
0.101
3
164-165
1
1
62-166
60-166
MLCT
MLCT
MLCT
4
3
.28, 289.8
.62, 342.4
0.147
0.078
150-151
6
1
1
48-152
50-154
MLCT
MLCT
4
.41, 281.0
0.194
Table S3. Calculated singlet-triplet vertical transition energies (E) and wavelengths() with the
oscillator strengths (f).
Complex
E(eV),(nm)
2.04, 606.5
2.15,576.5
2.02,612.3
2.23,555.1
2.34,529.3
2.23,556.9
f
1
2
3
4
5
6
0.0008
0.0005
0.0011
0.0012
0.0005
0.0019
8

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complex 1
1
34
138
140
142
complex 2
1
61
162
164
165
166
complex 3
1
47
148
150
152
153
complex 4
1
34
135
138
140
141
complex 5
1
60
162
164
165
166
complex 6
9

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1
48
150
151
152
154
Fig. S3. Molecular orbitals involved in the singlet-singlet electron transitions with pronouced
transition dipole moments.
Fig. S4. Calculated HOMOs and LUMOs involved in the singlet-triplet electron transitions.
1
0

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N
C
N
=
OH2+
OH2+
O-
charge
N
N
N
N
N
N
N
C
C
N
N
Ir
+
2
OH
O-
OH
N
N
N
1-a
NH3+
1-b
1-c
=
NH-
2-
NH2
N
N
N
N
N
N
N
N
N
NH3+
NH3+
2-
NH-
N
4
-a
4-b
4-c
4-d
Scheme S1. Protonated and deprotonated forms of Iridium(III) complexes 1 and 4.
Table S4. Energy gap between the triplet and singlet states and compositions of iridium(III)complexes
in HOMO and LUMO
HOMO
C^N
61.46
55.82
4.00
60.63
55.24
3.82
LUMO
C^N
0.09
79.34
9.08
0.31
74.79
46.44
9.08
Complex ∆E(eV)
Ir
N^N
5.35
4.66
89.95
5.19
4.64
Ir
N^N
99.60
14.0
88.98
98.97
19.27
49.90
88.98
1-a
-b
0.04
-1.20
2.10
0.14
1.00
1.59
-1.90
33.19
39.52
6.05
34.18
40.12
3.83
0.31
6.61
1.94
0.72
5.94
3.66
1.94
1
1
4
-c
-a
4
4
4
-b
-c
-d
92.35
89.96
6.04
4.00
Reference
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1
1