Mitochondria-specific oxygen probe based on iridium complexes bearing triphenylphosphonium cation

Article abstract of DOI:10.1246/cl.2012.262

Organelle-selective oxygen probe BTP-Mito was designed and synthesized to selectively target mitochondria. BTP-Mito, which is an iridium complex bearing a triphenylphosphonium cation, exhibited selective mitochondria localization in HeLa cells. The phosphorescence of BTP-Mito was significantly quenched by molecular oxygen in living cells, demonstrating that BTP-Mito can be used as mitochondria-specific oxygen sensor.

Full text of DOI:10.1246/cl.2012.262

doi:10.1246/cl.2012.262
262
Published on the web February 25, 2012
Mitochondria-specific Oxygen Probe Based on Iridium Complexes
Bearing Triphenylphosphonium Cation
Tokiko Murase, Toshitada Yoshihara, and Seiji Tobita*
Department of Chemistry and Chemical Biology, Gunma University, Kiryu, Gunma 376-8515
(Received December 21, 2011; CL-111126; E-mail: tobita@gunma-u.ac.jp)
Organelle-selective oxygen probe BTP-Mito was designed
properties of BTP by introducing an appropriate substituent
into the ancillary ligand.
and synthesized to selectively target mitochondria. BTP-Mito,
which is an iridium complex bearing a triphenylphosphonium
cation, exhibited selective mitochondria localization in HeLa
cells. The phosphorescence of BTP-Mito was significantly
quenched by molecular oxygen in living cells, demonstrating
that BTP-Mito can be used as mitochondria-specific oxygen
sensor.
Herein, we report a newly developed mitochondria-specific
oxygen probe (BTP-Mito).8 As shown in Figure 1, BTP-Mito
has a structure in which BTP is conjugated to a delocalized
lipophilic cation (triphenylphosphonium ion; TPP+). In bio-
logical cells the delocalized lipophilic cations are known to
preferentially accumulate into mitochondria in response to
negative inside transmembrane potentials.4
Figure 2 illustrates the absorption and phosphorescence
spectra of BTP and BTP-Mito in deaerated tetrahydrofuran
(THF). BTP and BTP-Mito exhibit the S1 ← S0 absorption
bands with their maxima at 486 and 480 nm, respectively, and
show intense phosphorescence from a ligand-centered ³³*
excited state with significant admixtures of metal-to-ligand
charge-transfer (MLCT) character.5 On the longer-wavelength
tail of the intense S1 ← S0 absorption band, a weak absorption
band due to the electronic transition to the lowest excited triplet
state from the ground state is superimposed. It is noted from
Figure 2 that the shape and position of the phosphorescence
spectra of BTP are only slightly affected by introducing TPP+
into the ancillary ligand.
The phosphorescence quantum yield (Φp) and lifetime (¸p)
of BTP and BTP-Mito were measured in aerated and deaeraed
THF solutions (Table 1).6 From Stern-Volmer analyses of the
phosphorescene quenching due to oxygen, the quenching rate
constants (kq) for BTP and BTP-Mito were determined to be
6.3 © 104 and 5.3 © 104 mmHg¹1 s¹1, respectively.8 It is con-
firmed from Table 1 that BTP-Mito retains the phosphorescence
Oxygen is one of the key metabolites in aerobic systems.1
In the normal physiological state, the majority of cellular oxygen
consumption occurs in mitochondria where oxygen functions as
the terminal electron acceptor for the electron-transport chain
during aerobic respiration. Oxygen deprivation (hypoxia) is
connected with various diseases and occurs in tumor micro-
environments.2 The oxygen level in mitochondria is therefore
one of the central parameters in many physiological, patholog-
ical, and therapeutic processes.
We recently demonstrated that a phosphorescent iridium(III)
complex
acetylacetonatobis[2-(2¤-benzothienyl)pyridinato-
κN,κC3¤]iridium(III) (BTP, Figure 1) can be used as an optical
probe for visualizing the oxygen levels in biological cells
and tissues.3 Under deaerated conditions BTP dissolved in
n-hexane exhibited red phosphorescence with a moderately
long emission lifetime (6.3 ¯s) and a high quantum yield (0.31).
The phosphorescence of BTP was significantly quenched by
dissolved oxygen in solution. Similar quenching by oxygen
has also been observed for living cells, where BTP showed
efficient access to the endoplasmic reticulum (ER) in the
cytoplasm.
× 20
(a) BTP
1
2
1
A great advantage of the iridium complexes lies in their
facility in chemical modifications of the ligands. The ligand
molecules of BTP consist of two benzothienylpyridinato groups
and an acetylacetone as an ancillary ligand. The spectral
properties of BTP are determined almost exclusively by the
benzothienylpyridinato groups. On the other hand, the acetyl-
acetone moiety scarcely influences the spectral properties of the
complex. We can, therefore, improve the physicochemical
Ar–saturated
0.5
aerated
0
2
0
1
× 20
(b) BTP Mito
(a)
(b)
0.5
0
1
0
N
O
O
Ir
S
N
O
O
Ir
400
600
800
O
S
2
HN
Br-
Wavelength / nm
P+
2
Figure 2. Absorption (black and blue lines) and phospho-
rescence (red lines) spectra of (a) BTP and (b) BTP-Mito in THF
at room temperature. The phosphorescence spectra were taken
under Ar-saturated and aerated conditions.
Figure 1. Molecular structures of (a) BTP and (b) BTP-Mito.
Chem. Lett. 2012, 41, 262-263
© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

263
Table 1. Photophysical parameters of BTP and BTP-Mito in THF at room temperature
Φp
¸p/¯s
Deaerated
¾
kq
Compound
¹1
¹1
/dm3 mol¹1 cm
/104 mmHg¹1 s
Aerated
Deaerated
Aerated
BTP
BTP-Mito
6700a
6200b
0.0047
0.0056
0.30
0.29
0.093
0.110
5.7
5.5
6.3
5.3
b
aMolar absorption coefficient at 486 nm. At 480 nm.
(a) BTP-Mito (b) Mitotracker (c) merge
(a) 20% O2
(b) 2.5% O2
20 μm
20 μm
(d) BTP-Mito
(e) ER tracker
(f) merge
Figure 4. BTP-Mito imaging of HeLa cell line. The HeLa
cells were placed at (a) 20% O2 and (b) 2.5% O2.
lifetime measurements have been reported by using Pt (or Pd)
porphyrins or Ru complexes as phosphorescent oxygen probes.7
BTP is the first iridium complex-based oxygen sensor for
biological cells,3 and BTP-Mito will become the first mitochon-
dria-specific iridium complex sensor which can be used for
lifetime-based oxygen measurements.
20 μm
Figure 3. Subcellular localization of BTP-Mito in HeLa cells.
BTP-Mito was added to the medium at a final concentration of
500 nM, and HeLa cells were incubated for 5 h at 2.5% O2.
Organelle markers used were Mitotracker and ER tracker which
selectively stain mitochondria and ER.
This work was financially supported by a Grant-in-Aid
(Development of Advanced Measurement and Analysis
Systems) from the Japan Science and Technology Agency.
characteristics of BTP, and both compounds show significant
response to the variation of the oxygen concentration in solution.
We then verified the intracellular localization of BTP-Mito
in living cells by morphologic examination using HeLa cells.8
Cellular BTP-Mito localization seemed to be limited to the
cytoplasm and not to the nucleus (Figure 3). We examined color
merging using green-colored mitochondria-specific probe Mito-
tracker and ER-specific probe ER tracker. We observed a merged
yellow color between BTP-Mito and Mitotracker (Figure 3c).
However, no green-colored ER tracker merged with BTP-Mito
(Figure 3f). These observations suggest that BTP-Mito exhibits
selective mitochondria localization in the cytoplasm. This is in
remarkable contrast to efficient ER localization of BTP in HeLa
cells.3
References and Notes
1
J. Aragonés, P. Fraisl, M. Baes, P. Carmeliet, Cell Metab. 2009,
9, 11.
2
C. Brahimi-Horn, E. Berra, J. Pouysségur, Trends Cell Biol.
2001, 11, S32; M. Höckel, P. Vaupel, J. Natl. Cancer Inst. 2001,
93, 266.
3
4
S. Zhang, M. Hosaka, T. Yoshihara, K. Negishi, Y. Iida, S.
Tobita, T. Takeuchi, Cancer Res. 2010, 70, 4490; T. Yoshihara,
Y. Karasawa, S. Zhang, M. Hosaka, T. Takeuchi, Y. Iida, K.
Endo, T. Imamura, S. Tobita, Proc. SPIE 2009, 7190, 71900X.
L. F. Yousif, K. M. Stewart, S. O. Kelley, ChemBioChem 2009,
10, 1939; L. F. Yousif, K. M. Stewart, K. L. Horton, S. O. Kelley,
ChemBioChem 2009, 10, 2081; A. T. Hoye, J. E. Davoren,
P. Wipf, M. P. Fink, V. E. Kagan, Acc. Chem. Res. 2008, 41, 87;
J. S. Modica-Napolitano, J. R. Aprille, Adv. Drug Delivery Rev.
2001, 49, 63.
Since we could confirm selective mitochondria localization
of BTP-Mito in HeLa cells, we examined oxygen response of
the BTP-Mito phosphorescence in living cells. HeLa cells were
cultured under 20% or 2.5% O2 concentrations for 24 h at 37 °C.
BTP-Mito was added to the medium at a final concentration
of 500 nM and cells were incubated for 4 h, and then the
luminescence images were taken under 20% or 2.5% O2
concentrations (Figure 4). In normoxia (20% O2), the cell line
did not exhibit notable phosphorescence of BTP-Mito. In
contrast, it emitted bright phosphorescence in a 2.5% O2 culture.
The optical properties of BTP-Mito in solutions and living
cells demonstrate that BTP-Mito can be applied to real-time
monitoring of oxygen levels of mitochondria in living cells.
Recently, quantification of cellular oxygen levels based on
5
6
S. Lamansky, P. Djurovich, D. Murphy, F. Abdel-Razzaq, H.-E.
Lee, C. Adachi, P. E. Burrows, S. R. Forrest, M. E. Thompson,
J. Am. Chem. Soc. 2001, 123, 4304.
K. Suzuki, A. Kobayashi, S. Kaneko, K. Takehira, T. Yoshihara,
H. Ishida, Y. Shiina, S. Oishi, S. Tobita, Phys. Chem. Chem.
Phys. 2009, 11, 9850; H. Ishida, S. Tobita, Y. Hasegawa, R.
Katoh, K. Nozaki, Coord. Chem. Rev. 2010, 254, 2449.
D. Sud, M.-A. Mycek, J. Biomed. Opt. 2009, 14, 020506; A.
Fercher, G. V. Ponomarev, D. Yashunski, D. Papkovsky, Anal.
Bioanal. Chem. 2010, 396, 1793; O. S. Finikova, A. Y. Lebedev,
A. Aprelev, T. Troxler, F. Gao, C. Garnacho, S. Muro, R. M.
Hochstrasser, S. A. Vinogradov, ChemPhysChem 2008, 9, 1673.
Supporting Information is available electronically on the CSJ-
Journal Web site, http://www.csj.jp/journals/chem-lett/index.
html.
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Chem. Lett. 2012, 41, 262-263
© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/