A Convenient Approach To Synthesize o-Carborane-Functionalized Phosphorescent Iridium(III) Complexes for Endocellular Hypoxia Imaging

Article abstract of DOI:10.1002/chem.201603340

The structure–property relationship of carborane-modified iridium(III) complexes was investigated. Firstly, an efficient approach for the synthesis of o-carborane-containing pyridine ligands a–f in high yields was developed by utilizing stable and cheap B₁₀H₁₀(Et₄N)₂as the starting material. By using these ligands, iridium(III) complexes I–VII were efficiently prepared. In combination with DFT calculations, the photophysical and electrochemical properties of these complexes were studied. The hydrophilic nido-o-carborane-based iridium(III) complex VII showed the highest phosphorescence efficiency (abs.?_P=0.48) among known water-soluble homoleptic cyclometalated iridium(III) complexes and long emission lifetime (τ=1.24 μs) in aqueous solution. Both of them are sensitive to O₂, and thus endocellular hypoxia imaging of complex VII was realized by time-resolved luminescence imaging (TRLI). This is the first example of applying TRLI in endocellular oxygen detection with a water-soluble nido-carborane functionalized iridium(III) complex.

Full text of DOI:10.1002/chem.201603340

DOI: 10.1002/chem.201603340
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Imaging Agents |Hot Paper|
A Convenient Approach to Synthesize o-Carborane-Functionalized
Phosphorescent Iridium(III) Complexes for Endocellular Hypoxia
Imaging
Xiang Li,[a] Xiao Tong,[b] Hong Yan,*[a] Changsheng Lu,*[a] Qiang Zhao,*[b] and Wei Huang[b]
Abstract: The structure–property relationship of carborane-
modified iridium(III) complexes was investigated. Firstly, an
efficient approach for the synthesis of o-carborane-contain-
ing pyridine ligands a–f in high yields was developed by uti-
lizing stable and cheap B10H10(Et4N)2 as the starting material.
By using these ligands, iridium(III) complexes I–VII were effi-
ciently prepared. In combination with DFT calculations, the
photophysical and electrochemical properties of these com-
plexes were studied. The hydrophilic nido-o-carborane-based
iridium(III) complex VII showed the highest phosphores-
cence efficiency (abs. ꢀP =0.48) among known water-soluble
homoleptic cyclometalated iridium(III) complexes and long
emission lifetime (t=1.24 ms) in aqueous solution. Both of
them are sensitive to O2, and thus endocellular hypoxia
imaging of complex VII was realized by time-resolved lumi-
nescence imaging (TRLI). This is the first example of applying
TRLI in endocellular oxygen detection with a water-soluble
nido-carborane functionalized iridium(III) complex.
Introduction
tively reduce self-quenching phenomena. The anion nido-
C2B9H12À, which is easy to obtain by removal of one boron
vertex from the carborane icosahedron, can be used as a hydro-
philic building motif to construct water-soluble luminescent
During the last decade, considerable progress has been ach-
ieved in the design and synthesis of phosphorescent transition
metal complexes (PTMCs),[1] especially iridium(III) complexes
owing to their excellent optical properties such as high quan-
tum efficiency, tunable emission, long phosphorescence life-
time, and high photo- and thermostabilities.[2] Phosphorescent
iridium(III) complexes have exhibited promising applications in
organic light-emitting diodes,[3] light-emitting electrochemical
cells,[4] information recording and security protection,[5] chemo-
sensors, and bioimaging.[6,7] Since the photophysical properties
of iridium(III) complexes are strongly dependent on their li-
gands, it is crucial to design novel ligands to efficiently tune
the optical properties of these complexes.
complexes.[9] Until now, some examples containing carboranyl
_
substituents on the phenyl ring of HCN ligands have been re-
ported,[9a,b,10] but incorporation of a carborane motif on the
_
pyridine ring of HCN ligands still remains much rarer,[11] owing
to the difficult synthesis. The structure–activity relationships of
carborane-modified iridium(III) complexes still deserve further
investigation. To do so, the development of efficient and envi-
ronmentally benign synthetic routes to novel o-carborane-sub-
_
stituted HCN ligands are needed.
In this work, an efficient synthetic method starting from
cheap B10H10(Et4N)2 to synthesize o-carborane-containing pyri-
dine derivatives in high yields was developed. Thus, a variety
Recently, carboranyl substituents were demonstrated to be
effective in tuning the phosphorescence of metal complexes.[8]
Icosahedral carboranes, especially 1,2-C2B10H12 (o-carborane),
show strong electron-withdrawing ability. In addition, the
bulky cage can provide steric hindrance to suppress intermo-
lecular interactions between phosphors and therefore effec-
of ligands were conveniently synthesized by introduction of
_
carboranyl substituents into pyridine rings of the HCN ligands.
The corresponding iridium(III) complexes I–VI were synthe-
sized, and the luminescence properties of these new com-
plexes investigated. Owing to the water solubility of nido-
C2B9H12À, a hydrophilic nido-o-carborane-based homoleptic cy-
clometalated iridium(III) complex VII was prepared and applied
as an excellent oxygen-sensitive phosphor for endocellular O2
sensing and hypoxia imaging.
[a] X. Li, Prof. Dr. H. Yan, Prof. Dr. C. Lu
State Key Laboratory of Coordination Chemistry
School of Chemistry and Chemical Engineering
Nanjing University, Nanjing 210023 (P.R. China)
E-mail: hyan1965@nju.edu.cn
luchsh@nju.edu.cn
Results and Discussion
[b] X. Tong, Prof. Dr. Q. Zhao, Prof. Dr. W. Huang
Key Laboratory for Organic Electronics and Information Displays and
Institute of Advanced Materials, Nanjing University of Posts and
Telecommunications, Nanjing 210023 (P.R. China)
E-mail: iamqzhao@njupt.edu.cn
Synthesis
Carborane-modified compounds are generally synthesized
from B10H14 (Method 1 in Scheme 1) or carborane isomers
(Method 2 in Scheme 1). In Method 1, the reaction is conduct-
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201603340.
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ed in one pot by treating B10H14 with an alkyne in the presence
of a Lewis base;[12] however, B10H14 is hypertoxic. Method 2
uses expensive carborane isomers as precursors, is limited to
expensive iodopyridines, and gives low yields even with pro-
longed reaction times. Besides, the functionalization of carbon
atoms of carboranes with pyridine rings has proved to be
quite difficult. An alternative method is to start from commer-
cially available B10H10(Et4N)2, which is cheap, stable, and non-
toxic. It can be readily converted to the precursor
B10H12(Et2S)2,[12] which is reactive, but easy to handle and suita-
ble for synthesis of o-carborane–pyridine derivatives by reac-
tion with pyridine-bearing alkynes (Scheme 1).[12,13a–f] On the
basis of this modified synthetic approach, three new o-carbor-
_
ane-functionalized HCN ligands (a–c in Figure 1 and Scheme 2)
were prepared in high yields and with low costs.
_
Scheme 2. Synthetic routes to cyclometalated HCN ligands. i) Trimethylsilyl-
acetylene, [Pd(PPh3)2Cl2], CuI, triethylamine/toluene (1:1), overnight, RT. ii)
Methanol, K2CO3, 4 h, RT. iii) Toluene, reflux 72 h, methanol. iv) THF, CH3I, lithi-
um diisopropylamide, 2 h, RT. v) [Pd(PPh3)4], K2CO3, THF, reflux, overnight.
vi) [Pd(PPh3)4], K2CO3, THF/H2O (1:1), 608C, overnight.
Scheme 1. Synthetic routes to carborane-containing pyridine derivatives.
Figure 1. closo-Carborane-functionalized ligands and the corresponding iridium(III) complexes.
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As o-carborane is electron-deficient, the C-carboranyl group
can serve as an electron acceptor. Thus, we combined the elec-
tron-withdrawing nature of o-carborane with the commonly
used electron donor diphenylamine (DPA) to rationally design
a donor–acceptor (D–A) system. From that point of view, o-car-
borane-modified D–A structures attached to an electron-defi-
cient pyridine moiety are assumed to be able to enhance
charge transfer from D to A. More importantly, these com-
pounds can also function as cyclometalating ligands for syn-
thesis of iridium(III)-centered phosphors.[13g,h] Therefore, cyclo-
metalated ligands containing a D–A structure (d–f in Figure 1)
were also prepared according to the modified method
(Scheme 2). The corresponding iridium(III) complexes I–V were
synthesized (Scheme 3) and their photophysical properties
were examined. To develop water-soluble phosphorescent iri-
dium(III) complexes for bioimaging, homoleptic complexes VI
and VII were prepared as well. All the new ligands and iridiu-
m(III) complexes were fully characterized by 1H, 13C, and
11B NMR spectroscopy, mass spectrometry, and elemental anal-
ysis. X-ray single-crystal structures further confirmed the con-
figuration of ligands b and e, and complexes II and VI.
Figure 2. Crystal structures of ligands b and e and complexes II and VI. (H
atoms and solvent molecules omitted for clarity).
phosphorescent emission of iridium(III) complexes in solution
and in the solid state. The crystal structure of complex VI
(Figure 2) reveals that the three o-carborane cages are on the
same side. Complex VI adopts a distorted octahedral structure
with cis-cyclometalated nitrogen atoms, which is a facial
isomer, similar to other reported homoleptic cyclometalated iri-
dium(III) complexes.[9a]
Photophysical properties
_
Scheme 3. Synthetic route to [Ir(CN)2(acac)] complexes. vii) IrCl3·3H2O, 2-
Photophysical properties of ligands
ethoxyethanol/H2O (3:1), reflux 24 h. viii) Acetonitrile, Na2CO3, pentane-2,4-
dione, 808C, 24 h.
The introduction of o-carborane has no significant effect on
the fluorescence of ligands a–c (Supporting Information, Fig-
ure S1). For ligands d–f, which feature a D–A structure, the
solid-state fluorescence is significantly different. Emissions of d,
e, and f show bathochromic shifts compared with control com-
pound m (Supporting Information, Figure S2). According to
time-dependent (TD) DFT calculations, the energy gap (from
the S1 excited state) is decreased from 3.19 eV for m to 1.41 eV
for d, 1.55 eV for e, and 1.50 eV for f (Supporting Information,
Figure S8). The TD-DFT calculations also show that the o-car-
borane units are hardly involved in the HOMOs of a–c, and
completely uninvolved in those of d–f. The o-carborane units
participate in the LUMOs of all ligands (Supporting Information
Figure S8). In the cases of ligands d, e, and f, the excited states
are mainly attributed to charge transfer, whereas some fraction
of p–p* transitions contribute to those of ligands a, b, and c.
Thus, o-carborane units play different roles in the two kinds of
ligands, and fluorescence intensity has been tuned accordingly
by means of different substituents (Supporting Information,
Figure S2). The intensity magnitude follows the order e (CH3)>
f (Ph)>d (H) (Supporting Information, Table S2) in comparison
to the control compound m. A CH3 group can suppress rota-
tion of the CÀC bond of carborane unit more efficiently (ꢀFL =
Structural analysis
To determine the solid-state structures of the ligands and com-
plexes, single crystals suitable for X-ray diffraction analysis
were grown from solutions in hexane and dichloromethane by
slow evaporation. The structures of carborane-based ligands
b and e and iridium(III) complexes II and VI were solved
(Figure 2).
In the unit cells of ligands b and e, the phenyl and pyridine
rings are not in the same plane. There is a twist angle, and the
o-carborane cage is positioned on the periphery. In iridium(III)
complex II, two cyclometalated ligands b (5-car-ppy) adopt
a trans configuration of the pyridine rings, which is similar to
that of other reported heteroleptic iridium(III) complexes.[10b]
The bond lengths and angles around the iridium(III) center are
also in normal ranges, similar to the reported ones.[10] Due to
the bulkiness of o-carborane, no intermolecular p–p interac-
tions were observed in the packing model of complex II
(Figure 2). The advantage of the bulky carborane cage in inhib-
iting intermolecular interactions is prominent in tuning the
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0.60 for ligand e) than a Ph ring (ꢀFL =0.10 for ligand f) or H
atom (ꢀFL =0.06 for ligand d). The different substituents at the
_
o-carboranyl units in the D–A-structured HCN ligands do not
change the excited states, but can suppress the rotation of the
CÀC bond, which leads to enhanced solid state emission.[14]
Photophysical properties of iridium(III) complexes
UV/Vis absorption and photoluminescence (PL) measurements
on the two series of iridium(III) complexes were performed in
degassed acetonitrile at room temperature (5.0ꢁ10À5 m, lex =
365 nm for PL). The absorption spectra of complexes I, II, and
III are quite similar, and exhibit a bathochromic shift of about
10 nm compared with model complex 1 (Figure 3). Incorpora-
tion of o-carborane at the pyridine ring results in a narrowed
bandgap between HOMO and LUMO levels compared with
that in complex 1, which is in accordance with the calculated
spectra (Supporting Information, Figure S3). The UV spectrum
of complex 2 shows strong absorption around 380 nm, which
is quite different from those of complexes I–III and 1. This is
ascribed to the charge transfer occurring in complex 2. Similar
absorptions were also observed in the UV spectra of IV and V,
which show bathochromic shifts of 18 and 26 nm, respectively
(Table 1). Thus the o-carboranyl unit has enhanced the elec-
tron-accepting ability of the acceptor in the D–A-structured iri-
dium(III) complexes, similar to previously reported com-
plexes.[15]
Figure 3. Absorption spectra of complexes I, II, III, IV, and V and model com-
plexes 1 and 2 in degassed CH3CN (5.0ꢁ10À5 m) at room temperature.
the control complex 1, complex IV exhibits a blueshift of
15 nm, which indicates that the D–A ligand structure can in-
crease the energy gap of iridium(III) complexes. Comparison of
1 with V reveals an 8 nm blueshift in emission, from 530 to
522 nm. Thus, both calculated and experimental results have
demonstrated that incorporating a carboranyl unit on the pyri-
_
dine ring of CN ligands can tune the energy gap between
HOMO and LUMO. In the cases of complexes with D–A struc-
tures, the energy gaps can be increased to a slightly greater
extent than those without D–A structures. Complex III is com-
pletely quenched in CH3CN solution. In general, the quantum
yields (FP) are low for all the newly synthesized iridium(III)
complexes (Table 1).
As for the emission properties, the HOMO and LUMO levels
of complex IV are lowered compared with those of 2. This indi-
cates that the introduction of carborane to the pyridine ring
can lower both the HOMO and LUMO energy levels and has
more impact on the former than on the later. The net increase
in the HOMO–LUMO bandgap of 0.05 eV is responsible for the
blueshift from 526 to 515 nm (Table 1). For complex V, the
HOMO level is decreased by 0.10 eV and the LUMO level is de-
creased by 0.08 eV compared with complex 2. This leads to an
overall increase in the bandgap of 0.02 eV, which is responsible
for a blueshift of 4 nm (from 526 to 522 nm; Figure 4 and
Table 1). The energy gaps increased by 0.04–0.05 eV, from
2.34 eV (in complex 1) to 2.38 eV (in complex I) and 2.39 eV (in
complex II), corresponding to blueshifts of 8 and 12 nm com-
pared with control compound 1. For complexes I and IV, the
energy gaps between HOMO and LUMO are 2.38 and 2.41 eV,
which result in blueshifts from 522 to 515 nm. Compared with
To explore the nature of the emission, quantum chemical
calculations were carried out on all the iridium(III) complexes
(Figure 5). TD-DFT calculations revealed that the HOMOs are
largely located on the iridium(III) centers and pyridine rings of
the cyclometalated ligands, whereas the LUMOs are located on
_
the whole CN ligands and partially on the carborane units. Evi-
dently, the o-carboranyl units are involved in excited states,
and the CÀC bonds of carboranes directly participate in the
LUMOs. The variable CÀC bond of carborane can dissipate
energy, which might be responsible for the observed emission
quenching (Table 1).[16]
Table 1. Photophysical and electrochemical data of iridium(III) complexes.
[a]
[a]
[b]
ox
onset
[c]
labs [nm] (lge)
lem [nm]
t [ns][b]
FPL
E
[eV]
Eg [eV]
HOMO/LUMO [eV][b]
V
IV
2
1
I
261 (4.7), 382 (4.6), 420 (4.7), 481 (4.2)
262 (4.3), 350 (4.1), 412 (4.2), 493 (4.3)
296 (4.6), 376 (4.6), 394 (4.5), 458 (3.5)
260 (4.5), 339 (4.0), 398 (3.9), 463 (3.6)
275 (4.7), 350 (4.1), 396 (3.9) 480 (3.5)
275 (4.7), 356 (4.0), 398 (3.8), 484 (3.7)
276 (4.6), 358 (3.9), 423 (3.7), 496 (3.6)
522.0
515.0
526.0
529.5
522.0
518.0
0
159.4
339.7
339.9
817.8
112.1
157.1
26.4
0.01
0.01
0.04
0.11
0.02
0.01
0.01
0.639
0.628
0.539
0.43
0.649
0.681
0.645
2.375
2.408
2.357
2.342
2.375
2.394
0
À5.44/À3.06
À5.43/À3.02
À5.34/À2.98
À5.23/À2.89
À5.45/À3.07
À5.48/À3.09
À5.45/À3.05
II
III
[a] In CH3CN. [b] Data in degassed solvents at 298 K. [c] HOMO [eV]=Àe(Eoonxset +4.8), Eg =1240/l, LUMO [eV]=Eg +HOMO.
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Figure 6. Cyclic voltammograms of iridium(III) complexes at a scan rate of
100 mVsÀ1
.
complexes containing embedded DPA groups have two oxida-
tion waves on each curve due to the electrochemically active
DPA group (Figure 6). The oxidation potentials (IV>2) clearly
indicate that o-carborane-modified D–A structures can lower
the HOMO energy level. Therefore, the energy gap in IV is in-
creased, in accordance with the blueshifted emissions
(Figure 3).
Figure 4. Emission spectra of o-carborane-functionalized complexes and
model iridium(III) complexes 1 and 2 in CH3CN (5.0ꢁ10À5 m, lex =365 nm).
According to cyclic voltammetry studies, energy gaps in all
the o-carborane-incorporated complexes have been increased,
in accordance with the calculated results. This suggests that
modification with o-carborane can increase the oxidation po-
tentials and lower the HOMO energy levels of iridium(III) com-
plexes, owing to stabilization of the HOMO levels by the induc-
tive electron-withdrawing effect.[10a,b,16e]
Bioimaging
Hypoxia, that is, an insufficient supply of oxygen, is an impor-
tant symptom of conditions such as inflammatory diseases,
tumors, and cardiac ischemia.[17] Hence, monitoring oxygen
levels in complicated biological systems is of great importance
for diagnosis and therapy evaluation. Recently, PTMCs have
been used for hypoxia imaging in living cells.[18] The bright
emissive phosphorescence of PTMCs is sensitive to oxygen
concentration and can be quenched by energy transfer be-
tween the long-lived triplet excited state of PTMCs and the
triplet ground state of oxygen. However, most of the hypoxia
probes are hydrophobic and irrelevant to bioimaging.[19] There-
fore, development of high-performance hydrophilic phosphor-
escent probes for endocellular O2 detection is still a challenge.
o-Carborane can be converted to the anionic form nido-
C2B9H12À, which is suitable for the preparation of water-soluble
PTMCs.[9a]
Figure 5. Calculated HOMO and LUMO orbitals at excited state (T1) for all
the iridium(III) complexes (marked in black). The experimental energy levels
are marked in blue.
Cyclic voltammetry
Electrochemical studies showed that all the iridium(III) com-
plexes exhibit reversible waves with potentials in the range of
0.4–1.3 V, which are attributed to iridium-centered oxidation/
reduction (Figure 6). For complexes I, II, and III, the oxidation
potentials are positively shifted compared with that of model
complex 1. This indicates that incorporation of o-carboranyl
units can lower the HOMO energy level and increase the oxida-
tion potentials, which is in accordance with the experimental
emissions.
Experimentally, all the newly synthesized heteroleptic cyclo-
metalated iridium(III) complexes I–V exhibit almost phosphor-
escent silence (Table 1). Thus, we shifted to homoleptic cyclo-
metalated complexes. According to our previous report,[9a] an
efficient synthetic route to nido-carborane-functionalized iridiu-
m(III) complexes starts from heteroleptic cyclometalated closo-
carborane functionalized iridium(III) complexes. In this work,
the stable intermediate iridium(III) complex VI was prepared,
and then selective removal of one boron vertex from the o-car-
This result is probably ascribable to stabilization of the
HOMO level by the inductive electron-withdrawing effect of
o-carborane, as observed in a previous report[16e] (Table 1). In
addition, substituents at the C atom of o-carboranyl can fur-
ther fine-tune the energy levels, and indeed brings about oxi-
dation-potential shifts in the order of III<I<II. The order of
observed potential shifts is consistent with that of the elec-
tron-withdrawing abilities of the substituted o-carboranes. The
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boranyl unit under basic conditions (Scheme 4) gave rise to
the homoleptic cyclometalated nido-carborane-functionalized
complex VII in high yield. Its photophysical parameters in dif-
ferent degassed solvents were measured (Supporting Informa-
tion, Table S3 and Figure S4). This complex exhibits high phos-
phorescent efficiency in H2O (abs. ꢀP =0.48) and long emission
lifetime (t=1.24 ms; Figure 7 and Supporting Information,
Table S3). Its Stern–Volmer plot under excitation at 365 nm re-
vealed a good linear relationship between I0/I and [O2] (Sup-
porting Information, Figure S5), that is, the photophysics of
this complex is ratiometric to O2 concentrations in aqueous so-
lution. This may be useful to detect endocellular O2 (Figure 8).
Figure 8. Emission spectra of VII in aqueous solution (2.5ꢁ10À5 m,
lex =365 nm) at O2 concentrations from 0 to 100%; inset: luminescence
image.
bright-field and confocal luminescence images demonstrated
that the luminescence appeared in cytoplasm over nucleus
and membrane (Figure 9A, images c, f, and i). The images are
much brighter under the conditions of 2.5% O2 compared to
21% O2. This implies that VII may be used for monitoring en-
docellular oxygen concentration.
Scheme 4. Synthetic routes and chemical structures of homoleptic cyclome-
talated iridium(III) complexes VI and VII. i) Glycerol, 2008C for 48 h; ii) KOH,
EtOH, reflux for 48 h.
TRLI, including phosphorescence lifetime imaging microsco-
py (PLIM) and time-gated luminescence imaging (TGLI), can ef-
fectively eliminate the interference of short-lived autofluores-
cence from long-lived phosphorescence. Hence PTMCs are
suitable for applications in TRLI because of their long emission
lifetimes. To date, reports on the application of TRLI in endo-
cellular hypoxia imaging are rare. Owing to the long phosphor-
escence lifetime and high quantum efficiency of complex VII,
which are ratiometric to oxygen levels, phosphorescence life-
time imaging experiments on VII were performed with 21, 5,
and 2.5% O2 (Figure 9B). Emission lifetimes of t=90 ns under
21% O2 and t=360 ns under 5% O2 were observed. In particu-
lar, if the O2 content was further reduced to 2.5%, high-quality
signals with longer emission lifetime (t=450 ns) were ob-
tained with obvious color change from blue to orange. There-
fore, endocellular phosphorescence lifetime imaging of com-
plex VII is feasible in living cells. Thus, this complex is a promis-
ing probe for hypoxia imaging by using both emission intensi-
ty and lifetime as read-out signals.
Figure 7. Absorption and PL spectrum of VII in aqueous solution
(lex =365 nm); inset: luminescence image.
To further investigate the emission lifetime of phosphores-
cence signals in bioimaging, complex VII was tested by TGLI
experiments. TGLI can selectively collect long-lived phosphor-
escence signals by exerting a delay time. In doing so, phos-
phorescence intensity images at different time periods can be
collected. From the images of HeLa cells under 21, 5, and 2.5%
O2, evident phosphorescence was obtained by collecting sig-
nals at the time ranges of 0–1000, 250–1000, and 500–1000 ns,
which confirmed that complex VII is efficiently suitable for ap-
plication in TRLI to remove short-lived fluorescence interfer-
ence in bioimaging (Figure 9C).
First, the cytotoxicity of complex VII towards HeLa cancer
cells was evaluated by MTT assay. After treatment of HeLa cells
with different concentrations of VII for 48 h, the cell viabilities
were estimated to be greater than 80% even at 100 mm, and
no cytotoxicity was evident at 10 mm (Supporting Information,
Figure S6). Therefore, it is compatible with live-cell imaging.
Then, complex VII was used to detect endocellular oxygen
levels in HeLa cells, which were cultured under 21, 5, and 2.5%
O2 atmosphere for 24 h at 378C with a concentration of 10 mm.
Figure 9A shows phosphorescence images of living cells in the
wavelength range from 450 to 550 nm, which show that VII
was efficiently taken up by HeLa cells. The overlays of both
The phosphorescence collected in the same time ranges is
evidently weaker under 21% O2 than that under 2.5% O2.
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Figure 9. A) Phosphorescence images (a, d, and g), bright-field images (b, e, and h), and overlay images (c, f, and i). B) Phosphorescence-lifetime images of
HeLa cells incubated with VII (10 mm) in phosphate-buffered saline at 378C at 21 (j), 5 (k), and 2.5% O2 concentration (l) on excitation at 405 nm. C) TGLI
images of HeLa cells incubated with VII under 21, 5, and 2.5% O2 with different delay times at lex =405 nm (o–w). All images were taken at room tempera-
ture.
thesized according to literature procedures[21] (see Supporting In-
Therefore, detection of O2 concentration with complex VII is
practical by TGLI. To the best of our knowledge, this is the first
formation). NMR spectra (1H, 13C, and 11B) were recorded with
Bruker DRX-500 and DRX-400 spectrometers at ambient tempera-
example of applying TGLI in endocellular oxygen detection by
ture. EI-MS (Micromass GC-TOF, 70 eV) was used for all ligands.
using a water-soluble nido-carborane PTMCs.
Mass spectra of the complexes were recorded with a Bruker Dal-
tonics Autoflex IITM MALDI-TOF MS spectrometer, and ESI-MS (LCQ
Fleet, Thermo Fisher Scientific) was used to detect ionic com-
pounds. Elemental analyses for C, H, and N were performed with
a Vario MICRO elemental analyzer. IR spectra were recorded with
a Nicolet NEXUS870 FTIR spectrometer. UV/Vis absorption spectra
Conclusions
_
Two series of o-carborane functionalized HCN ligands have
been conveniently prepared in high yields by using low-toxici-
were recorded with a Shimadzu UV-2550 spectrophotometer. FL
ty, cheap, and commercially available B10H10(Et4N)2 as starting
and PL spectra were recorded with a Hitachi F-4600 fluorescence
material. From these ligands, iridium(III) complexes I–VII were
synthesized, and their structure–activity relationship was inves-
spectrophotometer. PL lifetime measurements were performed by
employing an Edinburgh FLS 920 spectrophotometer equipped
tigated. Introduction of o-carborane can increase oxidation po-
tentials and lower HOMO energy levels of the iridium(III) com-
plexes owing to stabilization of HOMO by the inductively elec-
tron withdrawing carborane cage. DFT calculations showed
that the o-carboranyl unit is involved in the excited states in all
the complexes, and the CÀC bond of carborane participates in
the LUMOs. The nontoxic and water-soluble nido-carborane-
functionalized complex VII has long emission lifetime and high
phosphorescence efficiency and has been successfully applied
in cell hypoxia imaging.
with laser impulse fluorometer of picosecond time resolution. Ab-
solute quantum yields were measured by using an integrating
sphere. X-ray diffraction data were collected with a Bruker Smart
CCD Apex DUO diffractometer with graphite-monochromated
MoKa radiation (l=0.71073 ꢂ) in w–2q scan mode.
Quantum yield determination
Absolute quantum yields of all the iridium(III) complexes in CH3CN
or in H2O and the ligands in the solid state were measured by em-
ploying an integrating sphere. Lifetime studies were carried out
with an Edinburgh FL 920 photocounting system with a hydrogen-
filled lamp as the excitation source.
Experimental Section
General experimental methods
Electrochemical experiments
All the synthetic steps were carried out under an inert argon at-
mosphere by using standard Schlenk and glovebox techniques.
Commercial reagents were used without any further purification.
THF and toluene were distilled from sodium/benzophenone. Ace-
tonitrile and EtOH were distilled from CaH2. Compounds 1a,[20a]
1b,[20b] 1c,[20c] 11 d,[20d] 11 e,[20b] and 11 f[20c] were synthesized ac-
cording to literature procedures.[20] The intermediate compound
B10H12(Et2S)2 was synthesized by a modified method according to
literature reports. [{(Rppy)2Ir(m-Cl)}2] (R=o-carboranyl) was also syn-
Cyclic voltammetry was carried out with an IM6ex (Zahner) by
using three-electrode cell assemblies. All measurements were car-
ried out in a one-compartment cell under argon, equipped with
a glassy-carbon working electrode, a platinum wire counter elec-
trode, and a Ag/Ag+ reference electrode at a scan rate of
100 mVsÀ1. The supporting electrolyte was a 0.10 molLÀ1 acetoni-
trile solution of tetrabutylammonium hexafluorophosphate
(Bu4NPF6). Each oxidation potential was calibrated with ferrocene
as reference.
Chem. Eur. J. 2016, 22, 1 – 10
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Quantum chemical calculations
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calculations. Electronic transition energies including electron corre-
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B3LYP functional (TD-B3LYP). The LANL2DZ basis set was used to
treat iridium atoms, and the 6-31G(d) basis set for all other atoms.
All calculations were performed with Gaussian 09.
Confocal luminescence imaging and phosphorescence life-
time imaging
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Confocal luminescence imaging was carried out with an Olympus
IX81 laser scanning confocal microscope equipped with a 40ꢁ im-
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tion of HeLa liver cancer cells incubated with complex VII at
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PLIM imaging was performed with an Olympus IX81 laser scanning
confocal microscope. The phosphorescence signal was detected by
the confocal microscope, and correlative calculation of the data
was performed by professional software provided by PicoQuant
Company. Light from the pulse diode laser head (PicoQuant, PDL
800-D) with excitation wavelength of 405 nm and frequency of
0.5 MHz was focused on the sample with a 40ꢁ/NA 0.95 objective
lens for single-photon excitation. The emitted phosphorescence
signal was collected at 450–550 nm.
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Financial supports from the National Natural Science Founda-
tion of China (21271102, 21472086, and 21531004), the Major
State Basic Research Development Program of China
(2013CB922100), and high-performance computational center
of Nanjing University are acknowledged.
Keywords: carboranes
·
imaging agents
·
iridium
·
phosphorescence · structure–property relationships
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Received: July 14, 2016
Published online on && &&, 0000
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FULL PAPER
A leading light: A hydrophilic nido-o-
carborane-based homoleptic cyclometa-
lated iridium(III) complex was prepared
which showed high phosphorescence
efficiency (abs. ꢀP =0.48 in H2O). It was
applied in oxygen detection as well as
for revealing intracellular hypoxia by
phosphorescence lifetime imaging mi-
croscopy (PLIM) and time-gated lumi-
nescence imaging (TGLI; see figure).
&
Imaging Agents
X. Li, X. Tong, H. Yan,* C. Lu,* Q. Zhao,*
W. Huang
&& – &&
A Convenient Approach to Synthesize
o-Carborane-Functionalized
Phosphorescent Iridium(III) Complexes
for Endocellular Hypoxia Imaging
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Chem. Eur. J. 2016, 22, 1 – 10
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ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!