Photochemical properties of red-emitting tris(cyclometalated) iridium(III) complexes having basic and nitro groups and application to ph sensing and photoinduced cell death

Article abstract of DOI:10.1021/acs.inorgchem.5b00369

Cyclometalated iridium(III) complexes, because of their photophysical properties, have the potential for use as luminescent probes for cellular imaging. We previously reported on a pH-activatable iridium complex that contains three N,N-diethylamino groups

Full text of DOI:10.1021/acs.inorgchem.5b00369

Article
pubs.acs.org/IC
Photochemical Properties of Red-Emitting Tris(cyclometalated)
Iridium(III) Complexes Having Basic and Nitro Groups and
Application to pH Sensing and Photoinduced Cell Death
Aya Kando,† Yosuke Hisamatsu,† Hiroki Ohwada,† Taiki Itoh,† Shinsuke Moromizato,† Masahiro Kohno,‡
and Shin Aoki*,†
†Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
‡Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama,
Kanagawa 226-8503, Japan
S
* Supporting Information
ABSTRACT: Cyclometalated iridium(III) complexes, because of their photophysical properties, have the potential for use as
luminescent probes for cellular imaging. We previously reported on a pH-activatable iridium complex that contains three
N,N-diethylamino groups, namely, fac-Ir(deatpy)3 5, which was synthesized via a regioselective aromatic substitution reaction at
the 5′-position with tolylpyridine groups of fac-Ir(tpy)3 2. It was found that 5 shows a considerable enhancement in emission
intensity in the pH range from neutral to slightly acidic (pH 6.5−7.4) in aqueous solution and selectively stains lysosome in
HeLa-S3 cells, due to the protonation of the diethylamino groups. In addition, 5 functions as a pH-dependent singlet oxygen
(1O2) generator and induces necrosis-like cell death. However, observing the green emission of 5 is often hampered by
autofluorescence emanating from nearby tissues. To overcome this problem, we designed and synthesized a series of new
pH-activatable Ir(III) complexes that contain diethylamino, guanidyl, and iminoimidazolidinyl groups on the mpiq ligand of
Ir(mpiq)3 7 and the tfpiq ligand of Ir(tfpiq)3 8, which exhibit a red emission, namely, Ir(deampiq)3 13, Ir(gmpiq)3 14,
Ir(imzmpiq)3 15, and Ir(imztfpiq)3 16. The emission intensity of these Ir complexes is enhanced substantially by protonation of
their basic groups, and they induce the necrosis-like cell death of HeLa-S3 cells by photoirradiation at 465 nm. A strong orange-red
emission of Ir(mpiq-NO2)3 9 and Ir(tfpiq-NO2)3 10 is also reported.
compared to fluorescent molecules (τ on the order of nano-
seconds).4 These attractive properties make them promising and
important candidates, not only for use in organic light emitting
diodes (OLEDs) as phosphorescent emitters5 but also for
biological applications such as chemosensors,6 cellular imaging
probes,7 in vivo tumor imaging,8 and photosensitizers for the
production of singlet oxygen (1O2).9 Although some pH-respon-
sive Ir complexes have been reported so far,10 applications of
them to cellular imaging remain limited.10c,g,h
INTRODUCTION
■
Intracellular pH plays a key role in various biological processes, and
pH-activatable probes1 can be useful tools for monitoring intracellular
pH and cancer tissues, the pH of which is slightly acidic in comparison
with that of normal tissues.2 A number of fluorescent pH probes have
been reported, and some of them are now commercially available
(e.g., LysoSensor, Blue DND-167, and SNARF-5F).1 As representa-
tive examples, Urano and co-workers demonstrated highly specific in
vivo cancer visualization by using pH-activatable BODIPY dyes
bearing a cancer-targeting monoclonal antibody.3
Meanwhile, cyclometalated iridium(III) complexes have
received considerable attention due to their high phosphorescent
quantum yields, excellent color-tuning capability, larger Stokes’
shifts, and relatively longer lifetimes (τon the order of microseconds)
We recently reported on the regioselective halogenation, nitra-
tion, and formylation of phenylpyridine-type tris(cyclometalated)
Received: February 17, 2015
© XXXX American Chemical Society
A
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15 (Ir(imzmpiq)3, imzmpiq = 1-(5′-iminoimidazolidinyl-4′-
methylphenyl)isoquinoline) and 16 (Ir(imztfpiq)3, imztfpiq =
1-(5′-iminoimidazolidinyl-4′-trifluoromethylphenyl)isoquinoline).
Prior to the synthesis, the pKa values of these derivatives were
predicted based on the pKa values of the corresponding aniline
derivatives using the SciFinder database. It was revealed that the
emission of 11 and 13 changes at a pH below 6, which is lower
than that of Ir(deatpy)3 5, and that the pKa values of 14 and 15
(ca. 8−9) are higher than that of 5. Finally, we succeed in the
synthesis of 16 Ir(imztfpiq)3, which has a pKa value of 7, by
the introduction of three iminoimidazolidinyl groups to the
trifluoromethyl analogue 8. We report herein on the synthesis
and pH-dependent photophysical behaviors of these Ir
complexes, the generation of singlet oxygen (1O2), and the
induction of cell death by photoirradiation with visible light.
We also report on the strong orange-red emission of 9 and 10,
which are nitro derivatives of 7 and 8.
Chart 1
EXPERIMENTAL SECTION
■
General Information. IrCl3·3H2O and ethyl iodide were purchased
from Kanto Chemical Co. 10% Pd/C, 1,3-diphenylisobenzofuran
(DPBF), and carbonyl cyanide 3-chlorophenylhydrazone (CCCP)
were purchased from Sigma-Aldrich. Imidazolidine-2-thione, NaN3,
propidium iodide (PI), and organic solvent for spectroscopic analyses
were purchased from Wako Chemicals Co., Ltd. MEM (Gibco
Minimum Essential Medium), MitoTracker Green FM, and Lyso-
Tracker Green DND-26 were purchased from Invitrogen. All aqueous
solutions were prepared using deionized water. All synthetic procedures
were performed under an atmosphere of argon. Negligible hazard exists
in all synthetic procedures. Melting points were measured on a Yanaco
MP-33 Micro melting point apparatus and are uncorrected. IR spectra
were recorded on a PerkinElmer FTIR-Spectrum 100 (ATR). 1H NMR
(300 MHz) spectra were recorded on a JEOL Always 300 spectrometer.
Electrospray ionization (ESI) mass spectra (MS) were recorded on
Varian 910-MS. Thin-layer (TLC) and silica gel column chromatog-
raphies were performed using Merck 5554 (silica gel) TLC plates and
Fuji Silysia Chemical FL-100D, respectively. Alumina (Al2O3) column
chromatography was performed using Aluminum oxide 90 active neutral
(Merck). Gel permeation chromatography (GPC) experiments were
performed using a system consisting of a PONM P-50 (Japan Analytical
Industry Co., Ltd.), a UV−vis detector S-3740 (Soma Japan), a Manual
Sample Injector 7725i (Rheodyne, USA) and a MDL-101 1 pen
recorder (Japan Analytical Industry Co., Ltd.), equipped with two GPC
columns, Jaigel-1H and jaigel-2 (Japan Analytical Industry Co., Ltd.,
20ϕ × 600 mm, Nos. A605201 and A605214). For measurement of
luminescence spectra in aqueous solutions at given pH values, buffer
solutions (CAPS, pH 11.0 and 10.0; CHES, pH 9.0; EPPS, pH 8.0;
HEPES, pH 7.4 and 7.0; MES, pH 6.0, 5.0, and 4.0), citric acid−sodium
citrate buffer (pH 3) and HCl−KCl buffer (pH 2.5, 2, and 1.0) were
used. The Good’s buffer reagents (Dojindo) were obtained from
commercial sources: 2-morpholinoethanesulfonic acid (MES, pKa =
4.8), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES,
pKa = 7.5), 3-[4-(2-hydroxy-ethyl)-1-piperazinyl]propanesulfonic acid
(EPPS, pKa = 8.0), 2-(cyclohexylamino)ethanesulfonic acid (CHES,
pKa = 9.5), 3-cyclohexylamino-1-propanesulfonic acid (CAPS, pKa =
10.4). Emission lifetimes were determined using a TSP-1000 (Unisoku
Co., Ltd.). Inductively coupled plasma atomic emission spectrometry
(ICP−AES) measurements were performed on a Shimadzu ICPE-9000.
Density functional theory (DFT) calculations were also performed using
the Gaussian09 program (B3LYP, the LanL2DZ basis set for a Iratom and
the 6-31G basis set for H, C, F, O, and N atoms).14,15 Microwave reactions
were conducted using the Discover Bench Mate microwave apparatus and
the corresponding vials. The reaction was performed in glass vials
(capacity 10 mL) sealed with a septum, under magnetic stirring. The
temperature of the reaction mixture was monitored using a calibrated
infrared temperature control mounted under the reaction vial.
Ir complexes (e.g., fac-Ir(ppy)3 1 (ppy = 2-phenylpyridine), fac-
Ir(tpy)3 2 (tpy = 2-(4′-tolyl)pyridine), and 3 (mppy = 2-(4′-
methoxylphenyl)pyridine); see Chart 1) at the 5′-position (R2 at
the p-position with respect to the C−Ir bond) of the phenyl rings
and their subsequent conversion to amino, cyano, and sulfonyl
Ir complexes that exhibit a blue-red-color emission.11,12 Among
these analogues, fac-Ir(atpy)3 4 (atpy = 2-(5′-amino-4′-tolyl)-
pyridine) containing three amino groups was found to show
a weak red emission at ca. 600 nm in the neutral−alkaline pH
range and a green emission (at ca. 530 nm) at pH < 5, due to the
protonation of its amino groups (Chart 1).11a The tris-
(diethylamino) derivative Ir(deatpy)3 5 (deatpy = 2-(5′-N,N-
diethylamino-4′-tolyl)pyridine) functions as pH-activatable
probe that exhibits a considerable change in emission intensity
between neutral and slightly acidic pH (pH 6.5−7.4).11c On
the other hand, the emission characteristics of Ir(4Pyppy)3 6
(4Pyppy = 2-(5′-(p-pyridyl)phenyl)pyridine), which contain
pyridyl groups at the 5′-position of the 2-phenylpyridine (ppy)
ligand, are opposite to that for 4.11d Namely, the green-colored
emission (at ca. 500 nm) of 6 changes to red (at ca. 590 nm)
when the three pyridyl groups are protonated.11d The results of a
cellular imaging study of HeLa-S3 cells indicate that 5 could be
useful for the selective staining of lysosomes, which are acidic
organelles that are located in cells, and that 6 could be used to
stain mitochondria, whose pH is ca. 7.5. Also note that 5 and 6
are able to induce cell death when photoirradiated with UV
light.11c,d However, the actual visualization of their green emis-
sion is often hampered by autofluorescence from nearby living
cells and tissues, and the use of less toxic visible light would be
preferable for the photochemical induction of cell death.
To overcome these problems, we designed and synthesized the
amino, diethylamino, guanidyl, and iminoimidazolidinyl deriv-
atives of fac-Ir(mpiq)3 713 (mpiq = 1-(4′-methylphenyl)isoquinoline)
and fac-Ir(tfpiq)3 8 (tfpiq = 1-(4′-trifluoromethylphenyl)-
isoquinoline) that exhibit a red emission (at ca. 600 nm). The
examples include 11 (Ir(ampiq)3, ampiq = 1-(5′-amino-4′-
methylphenyl)isoquinoline), 12 (Ir(atfpiq)3, atfpiq = 1-(5′-amino-
4′-trifluoromethylphenyl)isoquinoline), 13 (Ir(deampiq)3, deampiq =
1-(5′-diethylamino-4′-methylphenyl)isoquinoline), 14 (Ir-
(gmpiq)3, gmpiq = 1-(5′-guanidyl-4′-methylphenyl)isoquinoline),
Improved Method for the Synthesis of fac-Tris[1-(4′-
methylphenyl)isoquinoline]iridium(III) (Ir(mpiq)3) (7).13 A solution
B
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of 17 (1.00 g, 4.56 mmol), Na2SO4 (648 mg, 4.56 mmol), and IrCl3·
3H2O (53.6 mg, 0.15 mmol) in dioxane/H2O (1/1, 8 mL) was added to
a microwave vial. The reactor was subjected to microwave irradiation at
150 °C for 50 min. After adding 30 mL of water, the solution was
extracted with CHCl3 (30 mL × 3). The combined organic layer was
concentrated under reduced pressure, to which Na2SO4 (648 mg,
4.56 mmol) and dioxane/H2O (1/1, 8 mL) were added, and the
resulting mixture was microwave-irradiated at 150 °C to 50 min. After
this procedure was repeated once more, the mixture was concentrated
under reduced pressure. After adding 20 mL of water, the solution was
extracted three times with CHCl3 (30 mL). The combined organic layer
was dried over Na2SO4, filtered, and concentrated under reduced
pressure, and the resulting residue was purified by silica gel column
chromatography (hexanes/CHCl3 = 2:1) and recrystallization from
hexanes/CH2Cl2 to afford 7 as a red powder (63.8 mg, 50% yield).
1-(4′-Trifluoromethylphenyl)isoquinoline (18). 4-(Trifluoromethyl)-
phenylboronic acid (678 mg, 3.57 mmol) and Pd(PPh3)4 (100 mg,
86.5 μmol) were added to a solution of 1-chloroisoquinoline (292 mg,
1.78 mmol) in 20 mL of toluene/EtOH/2 N Na2CO3 aq (2/1/1), and
the solution was degassed by bubbling with argon gas for 15 min. The
reaction mixture was heated at 70 °C for 3 h. After the mixture cooled to
room temperature, the insoluble materials were removed by filtration.
After adding 15 mL of H2O to the filtrate, the solution was extracted with
AcOEt (30 mL × 3). The combined organic layer was dried over
Na2SO4, filtered, and concentrated under reduced pressure, and the
resulting residue was purified by silica gel column chromatography
(hexanes/AcOEt, 50:1) and recrystallization from hexanes/AcOEt
to afford 18 as a white powder (486 mg, 99% yield). mp > 300 °C. IR
(ATR): ν = 1618, 1556, 1405, 1320, 1158, 1105, 1059, 1017, 831, 757,
702, 680, 609 cm−1. 1H NMR (300 MHz, CDCl3/TMS): δ = 8.63 (d, J =
5.4 Hz, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.87−7.77
(m, 4H), 7.77−7.67 (m, 2H), 7.58 (dd, J = 8.3, 1.5 Hz, 1H) ppm. ESI-
MS (m/z): Calcd for C16H11F3N (M+H)+: 274.0838. Found: 274.0837.
Anal. Calcd for C16H10F3N: C, 70.33; H, 3.69; N, 5.13. Found: C, 70.35;
H, 3.31; N, 4.99.
fac-Tris[1-(4′-trifluoromethylphenyl)isoquinoline]iridium(III)
(Ir(tfpiq)3) (8). A mixture of 18 (300 mg, 1.10 mmol), Na2SO4 (150 mg,
1.07 mmol), and IrCl3·3H2O (12.9 mg, 36.6 μmol) in 5 mL of dioxane/
H2O (1/1) was added to a microwave vial. The reactor was subjected to
microwave irradiation at 150 °C for 50 min. After it cooled to room
temperature, the mixture was concentrated under reduced pressure,
30 mL of water was added, and the resulting solution was extracted with
CHCl3 (30 mL × 3). The combined organic layer was concentrated
under reduced pressure, to which Na2SO4 (150 mg, 1.07 mmol) and
5 mL of dioxane/H2O (1/1) were added, and the resulting mixture was
subjected to microwave irradiation at 150 °C to 50 min. After adding
20 mL of water, the solution was extracted with CHCl3 (30 mL × 3).
The combined organic layer was dried over Na2SO4, filtered, and
concentrated under reduced pressure, and the resulting residue was
purified by silica gel column chromatography (hexanes/CHCl3, 3:4)
and recrystallization from hexanes/CH2Cl2 to afford 8 as a red powder
(22.2 mg, 60% yield). mp > 300 °C. IR (ATR): ν = 3061, 1558, 1448,
1380, 1318, 1269, 1159, 1111, 904, 815, 731, 707, 677, 646, 572 cm−1.
1H NMR (300 MHz, CDCl3/TMS): δ = 8.98−8.84 (m, 3H), 8.23 (d, J =
CDCl3/TMS): δ = 9.03 (s, 3H), 8.95 (d, J = 8.4 Hz, 3H), 7.86−7.75 (m,
9H), 7.36 (d, J = 6.2 Hz, 3H), 7.31 (d, J = 6.2 Hz, 3H), 6.87 (s, 3H), 2.45
(s, 9H) ppm. ESI-MS (m/z): Calcd for C48H33(191Ir)N6 (M+):
980.2062. Found: 980.2061. Anal. Calcd for C48H33IrN6O6: C, 58.71;
H, 3.39; N, 8.56. Found: C, 58.77; H, 3.16; N, 8.47%.
fac-Tris[1-(5′-nitro-4′-trifluoromethylphenyl)isoquinoline]-
iridium(III) (Ir(tfpiq-NO2)3) (10). Cu(NO3)2·3H2O (243 mg,
241 μmol) was added to a solution of 8 (174 mg, 723 μmol) in Ac2O
(10 mL), and the reaction solution was stirred at room temperature for
24 h, after which 20 mL of water was added; the solution was then
extracted with CHCl3 (30 mL × 3). The combined organic layer was
dried over Na2SO4, filtered, and concentrated under reduced pres-
sure, and the resulting residue was purified by silica gel column
chromatography (hexanes/CHCl3 = 3:2) and recrystallization from
hexanes/CH2Cl2 to afford 10 as an orange powder (250 mg, 91% yield).
mp > 300 °C. IR (ATR): ν = 1592, 1521, 1460, 1336, 1283, 1144, 1060,
1006, 908, 821, 732, 688, 647, 593 cm−1. 1H NMR (300 MHz, CDCl3/
TMS): δ = 8.86 (d, J = 6.1 Hz, 6H), 7.99−7.79 (m, 9H), 7.48 (d, J = 6.2
Hz, 3H), 7.40 (d, J = 6.2 Hz, 3H), 7.26 (s, 3H) ppm. ESI-MS (m/z):
Calcd for C48H24F9(191Ir)N6O6 (M+): 1142.1214. Found: 1142.1203.
Anal. Calcd for C48H24F9IrN6O6·H2O: C, 49.62; H, 2.26; N, 7.23.
Found: C, 49.81; H, 2.00; N, 7.25%.
fac-Tris[1-(5′-amino-4′-methylphenyl)isoquinoline]iridium-
(III) (Ir(ampiq)3) (11). SnCl2·2H2O (293 mg, 1.30 mmol) and NaBH4
(49.2 mg, 1.30 mmol) were added to a solution of 9 (82.7 mg,
84.2 μmol) in EtOH (3 mL), and the reaction mixture was heated at
reflux for 20 min. NaBH4 (49.2 mg, 1.30 mmol) was added, and the
reaction mixture was refluxed for 20 min. After the mixture cooled to
room temperature, the insoluble materials were removed by filtration.
After 15 mL of H2O was added to the filtrate, the solution was extracted
with CHCl3 (20 mL × 3). The combined organic layer was dried over
Na2SO4, filtered, and concentrated under reduced pressure, and the
resulting residue was purified by silica gel column chromatography
(CHCl3/MeOH = 30:1 to 10:1) and recrystallization from hexanes/
CH2Cl2 to afford 11 (24.8 mg, 33% yield) as a dark red-brown powder.
mp > 300 °C. IR (ATR): ν = 3341, 2923, 2853, 1615, 1591, 1535, 1500,
1438, 1402, 1347, 1297, 1266, 1122, 810, 750, 735, 639, 558, 418 cm−1.
1H NMR (300 MHz, CDCl3/TMS): δ = 8.92 (m, 3H), 7.64−7.57 (m,
12H), 7.26 (m, 3H), 6.97 (d, J = 6.2 Hz, 3H), 6.71 (s, 3H), 2.02 (s, 9H)
ppm. ESI-MS (m/z): Calcd for C48H39(191Ir)N6 (M+): 890.2837,
Found: 890.2835. Anal. Calcd for C48H39IrN6·0.5CH2Cl2·H2O: C,
61.15; H, 4.44; N, 8.82. Found: C, 61.46; H, 4.22; N, 8.80%.
fac-Tris[1-(5′-amino-4′-trifluoromethylphenyl)isoquinoline]-
iridium(III) (Ir(atfpiq)3) (12). SnCl2·2H2O (91.4 mg, 405 μmol) and
NaBH4 (15.5 mg, 410 μmol) were added to a solution of 10 (32.0 mg,
280 μmol) in EtOH (4 mL), and the reaction mixture was heated at
reflux for 20 min. NaBH4 (15.5 mg, 410 μmol) was added, and the
reaction mixture was then refluxed for 20 min. After the mixture cooled
to room temperature, the insoluble materials were removed by filtration.
After adding 15 mL of H2O to the filtrate, the solution was extracted with
CHCl3 (20 mL × 3). The combined organic layer was dried over
Na2SO4, filtered, and concentrated under reduced pressure, and the
resulting residue was purified by silica gel column chromatography
(CHCl3/MeOH = 50:1 to 10:1) and recrystallization from hexanes/
CH2Cl2 to afford 12 (11.4 mg, 39% yield) as a dark red-brown powder.
mp > 300 °C. IR (ATR): ν = 3376, 1621, 1540, 1408, 1255, 1138, 1096,
1052, 1019, 896, 817, 743, 701, 546 cm−1. 1H NMR (300 MHz, CDCl3/
TMS): δ = 8.94−8.83 (m, 3H), 7.78−7.74 (m, 3H), 7.71−7.67 (m, 9H),
7.30 (d, J = 6.3 Hz, 3H), 6.93 (d, J = 6.3 Hz, 3H), 6.95 (s, 3H) ppm.
ESI-MS (m/z): Calcd for C48H30F9(191Ir)N6 (M+): 1052.1989, Found:
1052.1987. Anal. Calcd for C48H30F9IrN6·1/4H2O: C, 54.47; H, 2.90; N,
7.94. Found: C, 54.86; H, 2.75; N, 7.54%.
8.4 Hz, 3H), 7.80 (m, 3H), 7.75−7.65 (m, 6H), 7.33 (d, J = 6.2 Hz, 3H),
7.29−7.14 (m, 6H), 7.07 (s, 3H) ppm. ESI-MS (m/z): Calcd for
C48H27F9(191Ir)N3 (M+): 1007.1662. Found: 1007.1647. Anal. Calcd for
C48H27F9IrN3·0.25CH2Cl2: C, 56.25; H, 2.69; N, 4.08. Found: C, 56.65;
H, 2.35; N, 4.24%.
fac-Tris[1-(5′-nitro-4′-methylphenyl)isoquinoline]iridium(III)
(Ir(mpiq−NO2)3) (9). Cu(NO3)2·3H2O (128 mg, 530 μmol) was added
to a solution of 7 (300 mg, 354 μmol) in Ac2O (10 mL), and the reaction
solution was stirred at room temperature for 24 h. After 20 mL of water
was added, the resulting solution was extracted with CHCl3 (30 mL × 3).
The combined organic layer was dried over Na2SO4, filtered, and con-
centrated under reduced pressure, and the resulting residue was purified
by silica gel column chromatography (hexanes/CHCl3 = 3:2) and
recrystallization from hexanes/CH2Cl2 to afford 9 as an orange powder
(336 mg, 96% yield). mp > 300 °C. IR (ATR): ν = 2925, 2854, 1590,
1539, 1493, 1319, 1294, 1256, 921, 823, 758 cm−1. 1H NMR (300 MHz,
fac-Tris[1-(5′-diethylamino-4′-methylphenyl)isoquinoline]-
iridium(III) (Ir(deampiq)3) (13). Ethyl iodide (1.90 g, 12.2 mmol) and
Cs2CO3 (397 mg, 1.22 mmol) were added to a solution of 11 (30.2 mg,
33.9 μmol) in MeCN (2.0 mL). The reaction mixture was stirred at
room temparature for 12 h, and the insoluble materials were removed by
filtration. The filtrate was washed with CHCl3, concentrated under the
reduced pressure, and diluted with CHCl3. The resulting CHCl3
solution was washed with 1N NaOH (aq), and the combined organic
C
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layer was dried over Na2SO4, filtered, and concentrated under reduced
pressure. The resulting residue was purified by NH silica gel column
chromatography (hexanes/CHCl3 = 2:1), and the eluted solution
containing 13 was combined, washed with 1N NaOH (aq), and
(s, 4H) ppm. ESI-MS (m/z): Calcd for C19H19N2O4S (M+H)+:
371.1060. Found: 371.1069. Anal. Calcd for C19H18N2O4S: C, 61.61; H,
4.90; N, 7.56. Found: C, 61.79; H, 4.75; N,7.70%.
fac-Tris[1-(5′-bis-Cbz-iminoimidazolidinyl-4′-methylphenyl)-
isoquinoline]iridium(III) (Ir(Cbz-imzmpiq)3) (22). The synthesis
of this compound was performed using the method reported for the
iminoimidazolidyl compounds.19 Triethylamine (42.9 mg, 425 μmol)
and HgCl2 (28.8 mg, 106 μmol) was added to solution of 11 (31.6 mg,
35.4 μmol) and 20 (41.3 mg, 112 μmol) in dry DMF (2.0 mL) at 0 °C.
The resulting brown suspention was stirred at 0 °C for 1.5 h, and the
mixture was diluted with CHCl3 and filtered. The filtrate was washed
with water (30 mL) and extracted with CHCl3 (30 mL × 3). The organic
layer was washed with water, dried over Na2SO4, and filtered.
Evaporation of the filtrate gave a crude product that was purified by
silica gel column chromatography (CHCl3) followed by GPC (CHCl3)
and concentrated. The resuluting material was recrystallized from
hexanes/CH2Cl2 to afford 22 as a red-brown powder (36.2 mg, 53%
from 11). mp > 300 °C. IR (ATR): ν = 1760, 1698, 1499, 1454, 1382,
1
concentrated (this manipulation is necessary to obtain clear H NMR
spectra). The resuluting material was recrystallization from hexanes/
CH2Cl2 to afford 13 as a dark red-brown powder (19.0 mg, 53% from
11). mp > 300 °C. IR (ATR): ν = 2966, 2803, 1581, 1498, 1437, 1378,
1343, 1258, 1107, 1084, 812, 739, 637, 565, 415 cm−1. 1H NMR
(300 MHz, CDCl3/TMS): δ = 8.84−8.74 (m, 3H), 7.86 (s, 3H), 7.72−
7.68 (m, 3H), 7.64−7.60 (m, 6H), 7.17 (d, J = 6.2 Hz, 3H), 7.03 (d, J =
6.2 Hz, 3H), 6.65 (s, 3H), 3.14−2.88 (m, 12H), 2.11 (s, 9H), 1.00 (t, J =
7.0 Hz, 18H) ppm. ESI-MS (m/z): Calcd for C60H64(191Ir)N6 (M+H)+:
1059.4793. Found: 1059.4794. Anal. Calcd for C60H63IrN6·H2O: C,
66.82; H, 6.08; N, 7.79. Found: C, 66.76; H, 5.85; N, 7.83%.
fac-Tris[1-(5′-bis-Cbz-guanidyl-4′-methylphenyl)isoquinoline]-
iridium(III) (Ir(Cbz-gmpiq)3) (21 (Cbz = carbobenzoxy)). The
synthesis of this compound was performed using the method reported
for the guanidyl compounds.16 Triethylamine (27.2 mg, 269 μmol) and
HgCl2 (20.0 mg, 73.7 μmol) were added to a solution of 11 (20.0 mg,
22.4 μmol) and 1917 (25.3 mg, 70.6 μmol) in dry dimethylformamide
(DMF, 2.0 mL) at 0 °C. The resulting brown suspention was stirred at
0 °C for 1 h, and the mixture was diluted with CHCl3 and filtered.
The filtrate was washed with water (30 mL) and dissolved in CHCl3
(30 mL × 3). The organic layer was washed with water, dried over
Na2SO4, and filterd. Evaporation of the solvent gave a crude product that
was purified by silica gel column chromatography (CHCl3) followed by
GPC (CHCl3). The resuluting material was recrystallized from hexanes/
CH2Cl2 to afford 21 as a red-brown powder (29.5 mg, 72% from 11).
mp > 300 °C. IR (ATR): ν = 1725, 1615, 1546, 1498, 1421, 1398, 1363,
1325, 1234, 1206, 1119, 1051, 908, 803, 734, 694, 633, 581, 558, 384 cm−1.
1H NMR (300 MHz, CDCl3/TMS): δ = 12.04 (s, 3H), 10.12 (s, 3H), 9.17
1
1295, 1207, 1146, 1106, 999, 813, 755, 737, 697, 400 cm−1. H NMR
(300 MHz, CDCl3/TMS): δ = 8.66 (d, J = 8.1 Hz, 3H), 7.55−7.51 (m,
6H), 7.45 (d, J = 7.2 Hz, 3H), 7.26−7.11 (m, 51H), 6.98 (d, J = 6.3 Hz,
3H), 3.92−3.81 (m, 6H), 3.78−3.68 (m, 6H), 1.89 (s, 9H) ppm. ESI-
MS (m/z): Calcd for C105H88(191Ir)N12O12(M+H)+: 1899.6245.
Found: 1899.6252. Anal. Calcd for C105H87IrN12O12·2H2O: C, 65.10;
H, 4.74; N, 8.84. Found: C, 64.92; H, 4.35; N, 8.58%.
fac-Tris[1-(5′-iminoimidazolidinyl-4′-methylphenyl)-
isoquinoline]iridium(III) (Ir(imzmpiq)3) (15). Compound 22
(17 mg, 8.9 μmol) was dissolved in THF/MeOH (1/1 v/v, 4 mL), and
13 mg of 10% Pd/C was added. The hydrogenation reaction was performed
at room temperature under H2 atmosphere for 3 h. The catalyst was removed
by filtration through a pad of diatomaceous earth, and the filtrate was
concentrated under reduced pressure. The resulting residue was purified by
Al2O3 column chromatography (CH2Cl2/MeOH) and recrystallization from
CH2Cl2/MeOH to afford 15 as a red-brown powder (8.0 mg, 82% from 22).
mp > 300 °C. IR (ATR): ν = 2919, 2851, 1652, 1586, 1542, 1499, 1436,
1395, 1346, 1260, 1085, 1031, 814, 736, 559 cm−1. 1H NMR (300 MHz,
CD3OD/TMS): δ = 8.91−8.88 (m, 3H), 8.07 (s, 3H), 7.92−7.89 (m, 3H),
7.79−7.76 (m, 6H), 7.36 (s, 6H), 6.92 (s, 3H), 3.72 (s, 12H) 2.07 (s, 9H)
ppm. ESI-MS (m/z): Calcd for C57H52(191Ir)N12(M+H)+: 1095.4038.
Found: 1095.4027. Anal. Calcd for C57H51IrN12·3CH2Cl2·H2O: C, 52.64; H,
4.34; N,12.28. Found: C, 52.93; H, 4.40; N, 12.58%.
fac-Tris[1-(5 ′-bis-Cbz-iminoimidazolidi nyl-4 ′-
trifluoromethylphenyl)isoquinoline]iridium(III) (Ir(Cbz-imztfpiq)3)
(23). Triethylamine (55.2 mg, 546 μmol) and HgCl2 (37.4 mg, 138 μmol)
were added to a solution of 12 (48.0 mg, 45.5 μmol) and 20 (53.2 mg,
144 μmol) in dry DMF (4.0 mL) at 0 °C. The reaction mixture was
stirred at 0 °C for 2 h, diluted with CHCl3, and filtered. The filtrate was
washed with water (30 mL) and extracted with CHCl3 (30 mL × 3). The
combined organic layar was washed with water, dried over Na2SO4,
filtered, and concentrated under reduced presure. The resulting crude
product was purified by silica gel column chromatography (hexanes/
CHCl3) followed by GPC (CHCl3) and concentrated. The resulting
material was recrystallized from hexanes/CH2Cl2 to afford 23 as a red-
brown powder (49.0 mg, 52% from 12). mp > 300 °C. IR (ATR): ν =
1763, 1693, 1379, 1290, 1208, 1140, 1116, 1056, 998, 905, 819, 730,
697 cm−1. 1H NMR (300 MHz, CDCl3/TMS): δ = 8.69−8.66 (m, 3H),
7.68 (d, J = 6.3 Hz, 3H), 7.62 (s, 3H), 7.57−7.54 (m, 3H),7.45 (s, 3H),
7.38−7.35 (m, 6H), 7.24−7.17 (m, 33H), 7.10−7.02 (m, 12H), 3.85−
3.76 (m, 12H) ppm. ESI-MS (m/z): Calcd for C105H79F9(191Ir)N12O12
(M+H)+: 2061.5397. Found: 2061.5429. Anal. Calcd for C105H78F9IrN12O12·
H2O: C, 60.60; H, 3.87; N, 8.08. Found: C, 60.35; H, 3.47; N, 8.11%.
fac-Tris[1-(5′-iminoimidazolidinyl-4′-trifluoromethylphenyl)-
isoquinoline]iridium(III) (Ir(imztfpiq)3) (16). Compound 23
(34.9 mg, 16.9 μmol) was dissolved in THF/MeOH (1/1 v/v, 5 mL),
and 25 mg of 10% Pd/C was added. The hydrogenation reaction was
performed in a Parr hydrogenation apparatus for 6 h. The catalyst was
removed by filtration though a pad of diatomaceous earth, and the
filtrate was concentrated under reduced pressure. The resulting residue
was purified by Al2O3 column chromatography (CH2Cl2/MeOH) and
recrystallization from CH2Cl2/MeOH to afford 16 as a red-brown
(d, J = 8.7 Hz, 3H), 8.57 (s, 3H), 7.59 (d, J = 8.4 Hz, 3H), 7.43−7.29 (m,
21H), 7.26 (s, 6H), 7.20 (d, J = 6.3 Hz, 3H), 7.06 (t, J = 7.5 Hz, 3H), 6.99
(d, J = 6.3 Hz, 3H), 6.82 (s, 3H), 5.20 (d, J = 8.7 Hz, 12H), 2.13 (s, 9H)
ppm. ESI-MS (m/z): Calcd for C99H82(191Ir)N12O12 (M+H)+: 1821.5776.
Found: 1821.5764. Anal. Calcd for C99H81IrN12O12·1.5H2O: C, 64.27; H,
4.58; N, 9.09. Found: C, 64.19; H, 4.22; N, 9.09%.
fac-Tris[1-(5′-guanidyl-4′-methylphenyl)isoquinoline]iridium(III)
(Ir(gmpiq)3) (14). Compound 21 (29.1 mg, 15.9 μmol) was dissolved in
tetrahydrofuran (THF)/MeOH (4/1 v/v, 10 mL), and 20 mg of 10%
Pd/C was added. The hydrogenation reaction was performed at room
temperature under H2 atmosphere for 8 h. The catalyst was removed by
filtration through a pad of diatomaceous earth, and the filtrate was con-
centrated under reduced pressure. The resulting residue was purified by
Al2O3 column chromatography (CH2Cl2 to MeOH) and recrystalliza-
tion from Et2O/CH2Cl2/MeOH to afford 14 as a red-brown powder
(14.0 mg, 86% from 21). mp > 300 °C. IR (ATR): ν = 3147, 1614, 1436,
1396, 1260, 1037, 879, 837, 816, 740, 633 cm−1. 1H NMR (300 MHz,
CD3OD/TMS): δ = 8.82−8.79 (m, 3H), 7.89 (s, 3H), 7.80−7.76 (m,
3H), 7.67−7.64 (m, 6H), 7.26−7.21 (m, 6H), 6.82 (s, 3H), 1.96 (s, 9H)
ppm. ESI-MS (m/z): Calcd for C51H45(191Ir)N12 (M+): 1016.3491.
Found: 1016.3491. Anal. Calcd for C51H45IrN12·5.5CH2Cl2·Et2O: C,
46.60; H,4.27; N, 10.78. Found: C, 46.74; H, 4.26; N, 10.99%.
1,3-Bis(Cbz)imidazolidine-2-thione (20). Compound 20 was
synthesized according to a reported method for the Cbz protection of
some amines.18 A mixture of imidazolidine-2-thione (1.00 g, 9.79 mmol)
and NaH (55%, 1.28 g, 29.4 mmol) in 20 mL of dry THF was stirred at
room temperature for 2 h. Benzyl chloroformate (502 mg, 29.4 mmol)
was then added dropwise at 0 °C, and the resulting mixture was stirred at
room temperature for 3 h and quenched by adding water to the reaction
mixture. The reaction mixture was extracted with AcOEt, and the
combined organic layer was washed with water twice, once with brine,
dried over Na2SO4, and filtered. Evaporation of the solvent gave a crude
product that was purified by silica gel column chromatography (CHCl3)
and concentrated. The resulting material was recrystallized from hexanes/
CH2Cl2 to afford 20 as a pale yellow crystal (2.17g, 5.85 mmol, 60% from
imidazolidine-2-thione). mp > 300 °C. IR (ATR): ν = 1756, 1455, 1379,
1
1251,1251, 1207, 1141, 987, 919, 751, 691, 608, 473 cm−1. H NMR
(300 MHz, CDCl3/TMS): δ = 7.44−7.34 (m, 10H), 5.30 (s, 4H), 4.00
D
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Chart 2
powder (19.4 mg, 91% from 23). mp > 300 °C. IR (ATR): ν = 2877,
H2SO4 was assumed to be 0.55 (excitation at 366 nm) and corrected by
using the standard Φ value of Ir(mpiq)3 (7) (Φ = 0.26).13 eq 1 was used
to calculate the emission quantum yields, in which Φs and Φr denote the
quantum yields of the sample and reference compound; ηs and ηr are the
refractive indexes of the solvents used for the measurements of the
sample and the reference (1.477 for dimethyl sulfoxide (DMSO) (ηs)
and 1.333 for H2O (ηr)); As and Ar are the absorbance of the sample and
the reference; and Is and Ir stand for the integrated areas under the
emission spectra of the sample and reference, respectively (all of the Ir
compounds were excited at 366 nm for luminescence measurements in
this study). For the determination of Φs in mixed-solvent systems, the
η values of the main solvents were used for the calculation.
1655, 1589, 1392, 1305, 1244, 1217, 1112, 1055, 901, 817, 749, 712,
1
670 cm−1. H NMR (300 MHz, CD3OD/TMS): δ = 8.93−8.89 (m,
3H), 8.02 (s, 3H), 7.95−7.90 (m, 3H), 7.82−7.78 (m, 6H), 7.45 (d, J =
6.0 Hz, 3H), 7.34 (d, J = 6.0 Hz, 3H), 7.17 (s, 3H), 3.54 (s, 12H) ppm.
ESI-MS (m/z): Calcd for C57H43F9(191Ir)N12 (M+H)+: 1257.3190.
Found: 1257.3117. Anal. Calcd for C57H42 F9IrN12·CH2Cl2·MeOH: C,
51.53; H, 3.52; N,12.22. Found: C, 51.73; H, 3.22; N, 12.00%.
Crystallographic Study of 9 (Ir(mpiq-NO2)3). A red block crystal
of 9 was obtained from the slow diffusion of Et2O into a CH2Cl2 solution
of 9. All measurements were performed using a Bruker APEX CCD area
detector using Mo Kα radiation at −170 °C. The structure was solved by
direct methods. All calculations were performed using the Crystal
Structure crystallographic software package except for refinements,
which were performed using SHELXL-97. Crystal data: C48H33IrN6O6,
Mr = 982.00, monoclinic, space group C/2c, T = 273 K, a = 19.902(3),
b = 29.796(4), c = 17.541(2) Å, α = 90, β = 95.096(2), γ = 90°, V =
10360(2) Å3, Z = 8, ρcalcd = 1.259 g·cm−3, 2θmax = 24.94°, R = 0.0573 (for
7237 reflections with I > 2σ(I)), Rw = 0.1935 (for 9017 reflections),
GOF = 1.076. Additional crystallographic information is available in the
Supporting Information.
Φ = Φ(η 2ArIs)/(η 2AsIr)
(1)
s
r
s
r
The luminescence lifetimes of sample solutions in degassed DMSO
at 25 °C were measured on a TSP1000-M-PL (Unisoku, Osaka, Japan)
instrument by using third harmonic generation (THG) (355 nm) of
Nd:YAG laser, Minilite I (Continuum, CA) as excitation source. The
signals were monitored with an R2949 photomultiplier. Data were
analyzed by using the nonlinear least-squares procedure.
Cell Culturing and Luminescence Imaging. HeLa-S3 cells,
provided by Dr. Tomoko Okada (National Institute of Advanced
Industrial Science and Technology), were grown in MEM supple-
mented with 10% fetal calf serum (FCS) under 5% CO2 at 37 °C. HeLa-
S3 cells (2.0 × 105 cells/mL) were plated in 1.0 mL of MEM on 35 × 10 mm
Greiner CELLview Petri Dish, Four Compartments (Greiner) under the
same conditions and allowed to adhere for 24 h. Subsequently, 0.5 mL of
MEM containing Ir complexes (50 μM) in MEM/DMSO (95/5, v/v) was
added, and the samples in MEM/DMSO (95/5, v/v) were incubated at
37 °C under 5% CO2 for 30 min. The culture medium was then removed
Measurements of UV−vis Absorption and Luminescence
Spectra. UV−vis spectra were recorded on a Jasco V-550 UV−vis
spectrophotometer and emission spectra were recorded on a Jasco FP-
6200 spectrofluorometer at 25 °C. Sample solutions in quartz cuvettes
equipped with Teflon septum screw caps were degassed by bubbling Ar
through the solution for 10 min prior to making the luminescence
measurements. The quantum yields for luminescence (Φ) were deter-
mined by comparison with the integrated corrected emission spectrum
of a quinine sulfate standard, whose emission quantum yield in 0.1 M
E
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A 100 μL aliquot of MEM was added, and the solution was then
subjected to photoirradiation at 465 nm (Twinlight 465, RELYON,
Japan; the number of photon per second was determined to be
1.3 × 10−4 mol/s by means of ferrioxalate actinometry)20 at 25 °C. After
photoirradiation, the solution was incubated for 2 h, and 3.0 μL of PI
solution (1 mM) was then added, followed by incubation for 30 min at
37 °C. The culture medium was then removed and washed twice with
PBS. Finally, 100 μL of MEM was added for observation (excitation at
540 nm for PI) on a BIOREVO BZ-9000 instrument (Keyence, Japan).
Singlet Oxygen Trapping Experiments in DMSO/H2O (3/2).11c,d
The solutions of Ir complexes (12 μM) in DMSO/H2O (1.3/1.2 v/v,
2.5 mL) were bubbled with O2 for 10 min, to which a solution of DPBF
(300 μM) in DMSO (500 μL) was added. The protonated Ir complexes
(10 μM) were prepared in situ upon the addition of 0.01 M HCl aq
(final concentration: 50 μM HCl). Measurement of 1O2 generation by
deprotonated and protonated forms of Ir complexes was performed
separately. The resulting solutions containing Ir complex (10 μM) and
DPBF (50 μM) in DMSO/H2O (3/2) were photoirradiated at 470 nm
(FP-6200, Jasco; the number of photon per second was determined to
be 9.7 × 10−7 mol/s by means of ferrioxalate actinometry)20 at 25 °C,
and changes in the UV−vis spectra of DPBF were recorded.
Table 1
entry
ligand
conditions
reflux, 7 d
yield
mer-7: trace
1
2
3
4
R3 = Me
R3 = Me
R3 = CF3
R3 = CF3
fac-7: 67%
fac-7: 50%
fac-8: 3%
microwave, 50 min, twice
reflux, 7 d
mer-7: trace
mer-8: 35%
mer-8: trace
microwave, 50 min, twice
fac-8: 60%
and washed twice with phosphate buffered saline (PBS). Finally, 0.5 mL
of MEM was added for luminescence imaging by confocal microscopy
(Leica TCS SP2, Leica) with an excitation wavelength at 350, 360, and
458 nm for Ir complexes and 488 nm for MitoTracker and LysoTracker.
The emission of Ir complexes was measured at 580−650 nm, and that of
MitoTracker and LysoTracker was measured at 490−550 nm using a
prism spectrometer.
Photoinduced Cell Death and Propidium Iodide Staining.
HeLa-S3 cells were incubated in MEM/DMSO (99/1, v/v) containing
the Ir complex (10 μM) for 30 min at 37 °C, and washed twice with PBS.
Electron Spin Resonance Analysis of Nitroxide Radical Generated
by Reaction of TPC with Singlet Oxygen in DMSO/H2O (3/2).11d
Solutions of Ir complexes (40 μM) and 2,2,5,5-tetramethyl-3-pyrroline-
3-carboxamide (TPC, 80 mM) in DMSO/H2O (3/2 v/v, 200 μL) were
prepared, and then the solutions were photoirradiated at 465 nm
(Twinlight 465, RELYON, Japan) at 25 °C. After the photoirradia-
tion, the samples were transferred to a quartz cell, and electron spin
resonance (ESR) spectra were recorded on an X-band ESR
Chart 3
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As shown in Figure 2a,b, the addition of H+ induced a
considerable blue shift in the UV−vis absorption spectra of 11
(ca. 90 nm) and 16 (ca. 50 nm), and almost the same behaviors
were observed for 12−15 (Figure S3 in Supporting Informa-
tion). Figure 2c,e displays that acid-free 11 and 16 had a very
weak emission and that the addition of HCl (in 1,4-dioxane)
induced a considerable enhancement in their emission at
spectrometer (JES-FA-100, JEOL, Tokyo, Japan). The measurement
conditions for ESR were as follows: field sweep, 330.50−340.50 mT;
field modulation frequency, 100 kHz; field modulation width, 0.05 mT;
amplitude, 80; sweep time, 2 min; time constant, 0.03 s; microwave
frequency, 9.420 GHz; microwave power, 4 mW. To calculate the spin
concentration of each nitroxide radical, 20 μM TEMPOL was used as a
standard sample, and the ESR spectrum of manganese (Mn2+), which
was located in the ESR cavity, was used as an internal standard. The spin
concentration was determined using Digital Data Processing (JEOL,
Tokyo, Japan).
RESULTS AND DISCUSSION
■
Synthesis of Ir(mpiq)3 Derivatives. Prior to the design of
new Ir(III) complexes, we predicted the pKa values of some
derivatives of 7 based on the known aniline derivatives using the
SciFinder database, as summarized in Figure S1 in Supporting
Information. We previously reported that the introduction of
N,N-diethylamino groups to the 5′ position of Ir(tpy)3 2 to
obtain Ir(deatpy)3 5, which has pKa value of ca. 7.11c Therefore,
we introduced N,N-diethylamino groups on Ir(mpiq)3 7 and
obtained 13 (Chart 2). Because the pKa value of 13 was lower
than predicted, as described below, we synthesized 14 and 15,
containing guanidyl and iminoimidazolidinyl groups, respectively.
In addition, 16 was synthesized by introducing a trifluoromethyl
group to permit the fine-tuning of the pKa value based on the
aforementioned prediction (Figure S1 in Supporting Information).
The fac-Ir(mpiq)3 7 was prepared as a racemic mixture of Δ
and Λ forms in 67% yield from IrCl3·3H2O and the mpiq ligand
(30 equiv) in dioxane/H2O under reflux for one week using our
previously reported procedure (entry 1 in Table 1).11a When we
applied this method to the preparation of 8, the facial form of 8
(fac-8) was obtained in only 3% yield, and major product was the
meridional form (mer-8, 35%, entry 3 in Table 1). Alternatively,
microwave irradiation resulted in a considerable improvement in
the chemical yield (60% in 100 min) of fac-8 (entry 4 in Table 1),
and 7 was obtained in moderate yield (50%) under the same
conditions (entry 2 in Table 1). Note that a smaller amount of
ligand (30 equiv against IrCl3) is required than that in the
reported procedure.21
Figure 1. (a) Top and (b) side views of X-ray structure of 9 with thermal
ellipsoids (50% probability).
The nitration of 7 and 8 was then performed using Cu(NO3)2
and Ac2O to give the tris(nitro) derivatives 9 and 10, and their
nitro groups were then reduced with SnCl2·2H2O and NaBH4
to give 11 and 12 (Chart 3).11a The tris(diethylamino) derivative
Ir(deampiq)3 13 was obtained by the ethylation of 11. The
reaction of 11 with the Cbz-protected S-methylthiourea 19 gave
21,16 the Cbz groups of which were removed by hydrogenation
to give Ir(gmpiq)3 14. For the synthesis of Ir(imzmpiq)3 15, we
treated 11 with 20 to produce 22, and its Cbz groups were
deprotected. Similarly, Ir(imztfpiq)3 16 was synthesized from 12
via 23.
The structure of 9 was confirmed by a X-ray single-crystal
structure analysis of fine red crystals obtained by recrystallization
from Et2O/CH2Cl2, as displayed in Figure 1a,b. Representative
parameters for the crystal structure of 9 are listed in Table S1 in
Supporting Information.22
Photophysical Properties of 11−16. UV−vis spectra of
the Ir(III) complexes 2 and 7−10 in DMSO at 25 °C are shown
in Figure S3 in Supporting Information. Because of the extension
of π-conjugation by the isoquinoline parts, the absorption spectra
of 7−10 are red-shifted considerably, in comparison with that of
Ir(tpy)3 2, which contains phenylpyridine units. Their emission
wavelength and luminescent quantum yields (Φ) (determined
based on the standard complex Ir(mpiq)3 7 (Φ = 0.26 in toluene)13
are summarized in Table 2.
Table 2. Photophysical Properties of 7−16 (10 μM) in
Degassed DMSO at 25 °C
a
λmax
b
complex
λmax (absorption)
(emission)
Φ
τ (μs)
7
295 nm, 327 nm, 423 nm
319 nm, 354 nm, 414 nm
316 nm, 347 nm, 394 nm
354 nm, 399 nm, 456 nm
289 nm, 334 nm, 501 nm
613 nm
600 nm
576 nm
585 nm
0.26
0.25
0.21
0.14
1.34
1.07
1.44
1.19
8
9
10
11
protonated·11 288 nm, 321 nm, 411 nm
12 325 nm, 383 nm, 493 nm
protonated·12 314 nm, 380 nm, 450 nm
13 292 nm, 329 nm, 451 nm
protonated·13 352 nm, 411 nm
602 nm
598 nm
593 nm
607 nm
608 nm
599 nm
0.09
<0.01
0.29
0.48
0.35
1.74
1.30
1.34
0.97
14
256 nm, 335 nm, 465 nm
protonated·14 298 nm, 354 nm, 419 nm
0.18
15
protonated·15 324 nm, 354 nm, 420 nm
16 388 nm, 466 nm
protonated·16 353 nm, 372 nm, 418 nm
288 nm, 336 nm, 475 nm
0.17
0.19
a
Emission spectra were measured by photoirradiation at 366 nm.
b
Ir(mpiq)3 7 (Φ = 0.26 in toluene)13 was used as the standard for the
phosphorescence quantum yield measurement.
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Figure 2. UV−vis spectra of 11 (a) and 16 (b) (10 μM) in DMSO at 25 °C before and after the addition of 1 M HCl in 1,4-dioxane. Change in emission
spectra of 11 (c) and 16 (e) (10 μM) in degassed DMSO at 25 °C (excitation at 366 nm) upon the repeated addition of acid (1 M HCl in 1,4-dioxane)
and base (1 M DBU in 1,4-dioxane). (i) 11 or 16 before addition of H+, (ii) (i) + HCl, (iii) (ii) + DBU, (iv) (iii) + HCl, (v) (iv) + DBU, and (vi) (v) +
HCl. Change in emission intensity of 11 at 602 nm (d) and 16 at 599 nm (f) caused by the repeated addition of HCl and DBU. [11] or [16] = 10 μM and
a.u. is in arbitrary units.
ca. 600 nm (red). When 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) was added to these solutions as a base, the red emission
was nearly completely quenched, and these processes proceeded
in a reversible manner, as plotted in Figure 2d,f (excitation
H
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spectra of deprotonated and protonated forms of 16 are
shown in Figure S4 in Supporting Information). Almost the
same behaviors were observed for 13, 14, and 15 (Figure S5 in
Supporting Information). Note that emission intensity of
protonated 11 (Φ = 0.09) and 12 (Φ < 0.01) are much weaker
than that of 13−16 (Table 2).
The pH-dependent emission spectra of 11 and 13−16
(1−100 μM) in DMSO/100 mM buffer (1/6) are shown
in Figure 3 and Figure S6 in Supporting Information. The
Figure 3. pH-Dependent change in emission intensity of 5 (1 μM) at
■
○
494 nm (×), 11 (100 μM) at 602 nm ( ), 13 (1 μM) at 590 nm ( ), 14
◇
△
(10 μM) at 600 nm ( ), 15 (10 μM) at 601 nm ( ) and 16 (10 μM) at
595 nm ( ), in degassed DMSO/100 mM buffer (1/6, v/v). This
●
relative emission intensity of 11 and 13 was calculated based on 1 at
pH 1, and that of 5, 14, 15, and 16 was calculated based on 1 at pH 4.
Excitation at 366 nm; a.u. is in arbitrary units.
enhancement in emission intensity (at 602 nm) for 11 was
observed at pH < 3, which is lower than that of 4 (pH 4−6)
(Figure S6a).11a Note that the pKa value of the excited state of 11
(estimated by the pH-dependent change in emission) is close to
that of its ground state (determined by UV−vis titration), as
previously reported for 4.11a,c It is assumed that the more extensive
π-conjugation of the mpiq ligand and the more hydrophobic
structure serve to decrease the pKa of 11 compared to 4, which
contains a tpy ligand. The introduction of diethylamino groups in
13 induced a shift in emission intensity threshold to ca. pH 4−5
from that of its original compound 11 (pH < 3) (Figure S6b). On
the other hand, the pKa values of guanidine and iminoimidazolidine
derivatives 14 and 15 were estimated to be >8, possibly due to the
strong basicity of these basic groups (Figure 3 and Figure S6c and
S6d in Supporting Information). Lastly, we found that the emission
for 16 containing iminoimidazolidine and trifluoromethyl groups
was enhanced at ∼pH 7−8 (Figure 3 and Figure S6e; the predicted
pKa values of the corresponding aniline derivatives are listed in
Figure S1 in Supporting Information and change in UV−vis
spectra of 16 at pH 4−11 is shown in Figure S7 in Supporting
Information).23 The photos in Figure 4 clearly show the pH-
dependent behaviors of Ir(tpy)3 derivatives 4 and 5 and
Ir(mpiq)3 derivatives 11, 13, 14, 15, and 16. Note that the
change in the emission of 5 and 16 occurs at almost the same pH
range (Figure 4b,g).
Figure 4. Photograph showing solutions of (a) 4 (100 μM), (b) 5
(1 μM), (c) 11 (50 μM), (d) 13 (1 μM), (d) 14 (10 μM), (f) 15
(10 μM), and (g) 16 (5 μM) in degassed DMSO/100 mM buffer (from
pH 4 to 10) at 25 °C. Excitation at 365 nm.
performed using the Gaussian09 program, and the results are
summarized in Figure S8 in Supporting Information with the
results for 2 and 5.14,15 The B3LYP hybrid functional was used,
together with the LanL2DZ basis set for the Ir atom and the
6-31G basis set for the H, C, F, N, and O atoms. The highest
occupied molecular orbital−lowest unoccupied molecular orbital
(HOMO−LUMO) shapes of the Ir(III) complexes 7−16 are
almost similar, as shown in Figure 5. The HOMO orbital consists
of phenyl(tolyl)-π and Ir-d orbitals, while the LUMO orbital is
mainly localized on the isoquinoline ring. Introduction of an
electron-withdrawing group (a nitro group in 9 and 10) stabilizes
Density Functioned Theory Calculations of 7−16.
Density functional theory (DFT) calculations of 7−16 were
I
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Figure 6. HOMO−LUMO energy gap of acid-free and protonated 11
(a) and 16 (b) calculated by DFT method using the B3LYP hybrid
functional together with the LanL2DZ basis set for Ir atom and the
6-31G basis set for H, C, F, O, and N atoms.
(Ir(mppy)3), although the emission of 24a and 24b is very
weak.25 The luminescence lifetimes of complexes 9 and 10 were
determined to be 1.44 and 1.19 μs, respectively, in DMSO at
25 °C (Table 2), supporting the conclusion that the emission
occurs via excited triplet states. Generally, a nitro group functions
as a strong quencher of luminescent dyes.26 We assume that 9
and 10 are rare examples of the nitrated tris-cyclometalated Ir
complexes that have strong emission properties.
To elucidate these phenomena, we performed DFT cal-
culations of 2, 24a, and 9, which afforded HOMO and LUMO
orbitals presented in Figure 8. Generally, the HOMO orbital of
tris(cyclometalated) Ir complexes such as 2 consists of phenyl-π
and Ir-d orbitals, and its LUMO orbital is mainly localized on the
pyridine ring (Figure 8a, left).4p As for 24a, its LUMO orbital is
localized on nitrophenyl parts (Figure 8b, left), implying that
photoinduced intramolecular electron transfer occurs from the Ir
center (HOMO) to the nitrophenyl units (LUMO), resulting
in the quenching of the emission.26c In contrast, the LUMO of 9
is mainly localized on the isoquinoline rings and not on the
nitrotolyl ring (Figure 8c, left). These data allowed us to
conclude that strong emission of 9 and 10 results from the
absence of LUMO on the nitrotolyl units of their ligand
moieties.26c
Live Cell Imaging of HeLa-S3 Cells Using 13−16 with
MitoTracker and LysoTracker. We previously reported that
5 is useful for the selective staining of lysosomes, an acidic
organelle in cells in response to its pH.11c These data prompted
us to perform luminescent imaging of living cells with the above-
synthesized Ir complexes. After incubating HeLa-S3 cells in
MEM/DMSO (pH 7.4, 95/5, v/v) with 13, 15, and 16 (50 μM)
for 30 min at 37 °C and washing the cells with PBS, they were
observed by confocal microscopy (Leica TCS SP2, Leica).
Since the pH of lysosomes is lower than 5.527 and the pH of
mitochondria is ∼7.5,28 costaining studies of these Ir complexes
Figure 5. HOMO and LUMO surfaces of 11 and protonated·11 (a) and
16 and protonated·16 (b) calculated by DFT method using the B3LYP
hybrid functional together with the LanL2DZ basis set for Ir atom and
the 6-31G basis set for H, C, F, O, and N atoms.
both the HOMO and LUMO energy levels, resulting in an
increase in the HOMO−LUMO energy gap of 9 (Eg = 3.27 eV)
and 10 (Eg = 3.21 eV). This result may explain the blue shift in
the emission wavelength (ca. 15−40 nm) of nitro derivatives 9
and 10, as described below. On the other hand, an electron-
donating group (amino group) induces destabilization of HOMO
energy level to decrease in energy gap of 11 (Eg = 2.45 eV), in
comparison with that of 7 (Eg = 2.92 eV) (Figure S8 in Supporting
Information).
The HOMO and LUMO energy levels of the protonated and
deprotonated forms of 11 and 16 are shown in Figure 6, and the
results indicate that the HOMO−LUMO energy gaps are en-
hanced upon protonation of their amino groups. Similar behaviors
were observed for our previous compounds, namely, 411a and 5.11c
Photophysical Properties of Nitro Derivatives 9 and
10. UV−vis absorption and emission spectra of 9 and 10 in
degassed DMSO at 25 °C are shown in Figure 7 with those for 7
and 8 (excitation at 366 nm). Note that the tris(nitro) derivatives
9 and 10 exhibit a rather strong orange-red color emission at
576 nm (Φ = 0.21) and 585 nm (Φ = 0.14), respectively, which
are ca. 20 nm shorter than the corresponding values for 7 and 8
(Figure 7b,c). This is in agreement with the values for 24a11a and
24b24 (Chart 4), the trinitro derivatives of 2 (Ir(tpy)3) and 3
J
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Figure 8. HOMOs and LUMOs of 2 (a), 24a (b), and 9 (c), calculated
by DFT method using the B3LYP hybrid functional together with the
LanL2DZ basis set for Ir atom and the 6-31G basis set for H, C, O, and N
atoms.
Figure 9 displays a typical bright field image, a luminescent
image of Ir complexes, a luminescent image of MitoTracker
Green or LysoTracker Green, and a merged image. For example,
Figures 9a−d and 9e−h show the results for the costaining of
HeLa-S3 cells with 13 and MitoTracker and LysoTracker,
respectively. The cytotoxicity of these Ir complexes was very low
at 50 μM (data not shown). The emission area of 13 and
MitoTracker and their merged image (Figure 9a−d) indicate that
13 is overlapped with MitoTracker to a negligible extent. In
addition, the intracellurar emission intensity of 13 is low probably
due to its low pKa value. On the other hand, the luminescent image
with 13 and LysoTracker are largely overlapped (Figure 9e−h).
The luminescent images of 14 or 15 are overlapped with both
LysoTracker and MitoTracker, possibly due to their higher pKa
values (Figure 9i−p; the luminescent images with 14 are shown in
Figure S9 in Supporting Information). The results of costaining
studies of 16 and LysoTracker (Figure 9q−x) indicate that the
emission area of 16 and LysoTacker are overlapped to a greater
extent than those with Mitotracker. We therefore conclude that 16
is the optimal probe for the staining of lysosomes among red Ir
complexes synthesized in this work.
Figure 7. UV−vis spectra of 2 (plain curve), 24a (plain and dashed
curve), 7 (bold curve), and 9 (bold and dashed curve) (a). Emission
spectra of 7 (plain curve) and 9 (plain and dashed curve) (b) and 8
(plain curve) and 10 (plain and dashed curve) (c) in DMSO at 25 °C.
a.u. is in arbitrary units, and the intensity of emission was normalized
with the absorbance of each compound at 366 nm, which was used for
the excitation of the Ir complexes. [Ir complex] = 10 μM.
Chart 4
It has been reported that CCCP inhibits oxidative
phosphorylation,29 decreases ATP production, and lowers the
metabolic rate, resulting in the suppression of active transport.
NaN3 is also known to function as a metabolic inhibitor.30 As
shown in Figure S10 in Supporting Information, the cellular
uptake of 15 and 16 was not inhibited by CCCP (40 μM) nor
NaN3 (5 mM) at 37 °C for 30 min in MEM/DMSO (95/5, v/v),
indicating that 15 and 16 are taken up by cells via a passive
transport mechanism. Although the intracellular concentrations
of 13−16 in HeLa-S3 cells were measured by ICP-AES accord-
ing to our previous method,11c these values were too low
(<0.1 fmol/cell) to permit their detection.
with a lysosomal dye (LysoTracker) and a mitochondrial dye
(MitoTracker) were conducted.
K
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Figure 9. Luminescence microscope images (Leica TCS SP2, Leica) of HeLa-S3 cells stained with Ir complexes (50 μM), MitoTracker Green (100 nM),
or LysoTracker Green (500 nM) in MEM/DMSO (95/5, v/v) at 37 °C for 30 min. (a, e, i, m, q, and u) brightfield images of HeLa-S3, (b, f) emission
images of 13, (j, n) emission images of 15, (r, v) emission images of 16, (c, k, and s) emission images of MitoTracker Green, (g, o, and w) emission
images of LysoTracker Green, (d) overlay image of (a−c), (h) overlay image of (e−g), (l) overlay image of (i−k), (p) overlay image of (m−o), (t)
overlay image of (q−s), (x) overlay image of (u−w). Excitation at 350, 360, and 458 nm for Ir complexes and excitation at 488 nm for MitoTracker
Green and LysoTracker Green.
Generation of Singlet Oxygen by the Photoirradiation
of Ir Complexes. Ir complexes are known to function as photo-
sensitizers for the production of singlet oxygen (1O2) from triplet
oxygen (3O2) by photoirradiation.9 We previously reported that the
pH-dependent generation of 1O2 by photoirradiation of the Ir(III)
complexes 5 and 6 and their application for inducing necrosis-like
cell death of HeLa-S3 cells.11c,d These results allowed us to check
the generation of O2 by the Ir complexes 13, 14, 15, and 16
(10 μM) in DMSO/H2O (3/2, v/v) upon photoirradiation at 470 nm,
which is less toxic to living cells than UV light. The decomposition
of DPBF was monitored by the change in the UV−vis spectra, for
which the absorbance at 415 nm decreases in the case of a reaction
with 1O2.31 As shown in Figure 10, the protonated forms of 13, 14,
15, and 16 (prepared in situ by addition of 0.01 M HCl aq)
generate 1O2 more efficiently than their acid-free forms. Since these
Ir complexes have an absorbance at longer wavelengths than that
of 5 (Figure 2b in the text and Figure S3d−f in Supporting
1
1
Information), the higher efficiency in O2 generation of 13−16
than those of 5 and protonated 5 can be explained by the fact that
the absorbance of 13−16 at 470 nm is stronger that for 5.32
L
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Figure 10. Photooxidation of DPBF (50 μM) caused of photo-
irradiation of Ir complexes in O2-aerated DMSO/H2O (3/2 v/v), (a)
□
□
◇
blank in DMSO/H2O (×), (b) 14 ( ), (c) 15 (gray ), (d) 5 ( ), (e)
◆
○
●
△
16 ( ), (f) 13 ( ), (g) protonated 5 ( ), (h) protonated 13 ( ), (i)
■
○
protonated 15 (gray ), (j) protonated 14 ( ) and (k) protonated 16
▲
(
) (excitation at 470 nm; FP-6200, JASCO). [5], [13], [14], [15], or
[16] = 10 μM. The protonated forms of Ir complexes were prepared
in situ upon the addition of 0.01 M HCl aq. (final concentration:
50 μM HCl).
1
The generation of O2 by the photoirradiation of acid-free 5
and 13 was examined using the ESR analysis of nitroxide radicals
generated through the oxidation of the sterically hindered
amines, 2,2,6,6-tetramethyl-4-piperidinol (4-hydroxy-TEMP)
and TPC.11d,33 As shown in Figure S12 in Supporting Information,
the intensity of the ESR signal of the nitroxide radical of TPC
generated by acid-free 5 and 13 is increased. In these experiments,
the activity of the protonated forms of 5 and 13 was not determined,
1
because TPC is protonated and is essentially unreactive for O2
under acidic conditions. Thus, we conclude that the photo-
irradiation of 5 and 13 facilitates the generation of 1O2 and that their
protonated forms generate 1O2 more effectively than their acid-
free forms (Figure 10 in the text and Figure S11 in Supporting
Information).
Induction of Cell Death of HeLa-S3 Cells upon
Photoirradiation in the Presence of Ir Complexes. We
examined the photoinduced cell death of HeLa-S3 cells upon
photoirradiation at 465 nm (Twinlight 465, Relyon, Japan) in the
presence of the complexes 5, 13, 14, 15, and 16 by staining with
PI, which exhibits strong red emission by intercalating with DNA
in the nucleus because the nuclear membrane is damaged on
necrotic cell death. Figure 11 shows a bright field image, a PI
emission image, and an overlay image of the bright field and PI
emission images in the absence and presence of Ir complexes
after irradiation at 465 nm for 30 min. Negligible cell death was
observed with photoirradiation in the absence of Ir complexes
(Figure 11a−c) and no photoirradiation in the presence of
16 (Figure 11s−u and Figure 12). As shown in PI emission
images obtained after the photoirradiation with Ir complexes
(Figure 11e,h,k,n,q), a strong red emission from PI was observed,
indicating that necrosis-like cell death was induced by photo-
irradiation of the Ir complexes (note that PI is excited at 540 nm,
which scarcely excites Ir complexes). The lower cytotoxicity of
13 than that of 5 can be attributed to the lower intracellular
concentration of its protonated form due to the lower pKa value
of diethylamino groups of 13 (see also Figure 12).
Figure 11. Luminescence microscope images (Biorevo BZ-9000,
Keyence) of HeLa-S3 cells irradiated at 465 nm (Twinlight 465,
Relyon) for 30 min with (a−c) only DMSO, (d−f) 5, (g−i) 13, (j−l) 14,
(m−o) 15, and (p−r) 16 or (s−u) incubated for 30 min with 16 (no
irradiation). After photoirradiation, the cells were incubated for 2 h
again, and stained with PI (30 μM) for 30 min. Excitation at 540 nm to
observe PI. (a, d, g, j, m, p, and s) brightfield of HeLa-S3, (b, e, h, k, n, q,
and t) emission images of PI, (c) overlay of (a, b), (f) overlay of (d, e),
(j) overlay of (g, h), (l) overlay of (j, k), (o) overlay of (m, n), (r) overlay
of (p, q), (u) overlay of (s, t).
We also examined the inhibition of the induction of cell death
by NaN3, an inhibitor of 1O2 generation.9h As shown in Figure 13,
M
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14−16 are able to generate singlet oxygen (1O2) from triplet
oxygen (3O2) more efficiently than 5 and 13 upon photo-
irradiation at 465 nm and induce the necrosis-like cell death of
HeLa-S3 cells. Luminescence imaging studies of such complexes at
acidic pH and the photoinduced cell death of cancer tissues using
these Ir complexes are currently under way. It is of interest to note
that the nitrated Ir complexes 9 and 10 exhibit a strong emission at
576−585 nm (an orange-red color), while the corresponding
Ir(tpy)3 and Ir(mppy)3 counterparts, 24a and 24b, emit very
weakly.
The results presented herein provide useful information for
the future design and synthesis of new pH-activatable Ir com-
plexes and their applications to photocatalytic reactions,
photodynamic therapy, biological chemistry, medicinal chem-
istry, and related fields.
Figure 12. Change in cell viability of HeLa-S3 cells monitored by PI
staining upon photoirradiation at 465 nm (Twinlight 465, Relyon) in
ASSOCIATED CONTENT
* Supporting Information
■
●
○
the absence (×) and presence of 5 (10 μM) ( ), 13 (10 μM) ( ), 14
S
□
◆
△
(10 μM) ( ), 15 (10 μM) ( ), and 16 (10 μM) ( ) in MEM/DMSO
(99/1). Cell viability = (number of cells that were not stained by
PI/number of all cells) × 100.
Calculated pKa values of aniline derivatives, parameters for X-ray
crystal structure analysis, crystal structure of 9, UV−vis and
excitation spectra, acid-induced changes in emission spectra,
pH-dependent change in emission spectra, DFT-calculated
HOMO−LUMO energy gap of Ir complexes, luminescence
images of stained cells under microscope, photooxidation of
DPBF, comparative photooxidation of TPC. The Supporting
Information is available free of charge on the ACS Publications
website at DOI: 10.1021/acs.inorgchem.5b00369. CCDC
1048292 contains the supplementary crystallographic data for
the paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.
uk/data_request/cif.
AUTHOR INFORMATION
Corresponding Author
*E-mail: shinaoki@rs.noda.tus.ac.jp.
■
Notes
The authors declare no competing financial interest.
Figure 13. Cell death of HeLa-S3 cells upon photoirradiation at 465 nm
(Twinlight 465, Relyon) with 16 (10 μM) in the absence (faded bar)
and presence (filled bar) of NaN3 (500 μM) in MEM/DMSO (99/1 v/v,
monitored by PI staining). Cell death of HeLa-S3 cells (%) = (number
of cells which were stained by PI/number of all cells) × 100.
ACKNOWLEDGMENTS
■
This work was supported by grants-in-aid from the Ministry of
Education, Culture, Sports, Science and Technology (MEXT) of
Japan (Nos. 22890200, 24890256, and 26860016 for Y.H. and
Nos. 19659026, 22659055, 24659085, and 24640156 for S.A.),
the Uehara Memorial Foundation for Y.H., and High-Tech
Research Center Project for Private Universities (matching fund
subsidy from MEXT). We appreciate Prof. H. Kawai (Faculty of
Science, Tokyo Univ. of Science) for the measurement of
phosphorescence quantum yield of Ir(III) complexes. We thank
Prof. R. Abe and Dr. T. Suzuki (Research Institute for Biomedical
Sciences, Tokyo Univ. of Science) for the helpful discussion and
the preparation for the HeLa-S3 cells. We appreciate Mrs. F.
Hasegawa (Faculty of Pharmaceutical Sciences, Tokyo Univ. of
Science) for the measurement of mass spectra of Ir complexes
and Ms. T. Matsuo (Reserch Institute of Sciences and
Technology, Tokyo Univ. of Science) for the X-ray single-crystal
structure analysis. We also appreciate Dr. K. Yoza (Bruker AXS)
for helpful suggestions for the X-ray crystal structure analysis.
NaN3 inhibits the photoinduced cell death by 16. Considering
the fact that NaN3 has little effect on the cellular uptake of Ir
complexes (Figure S10 in Supporting Information), these
1
findings suggest that cell death is triggered by O2, which is
generated by the excitation of the Ir complexes.
CONCLUSIONS
■
In summary, we report on the synthesis, pH-dependent photo-
chemical properties, cellular imaging, and photosensitizing
activity of a series of new pH-activatable Ir(III) complexes 13−
16, based on the predicted pKa values of the corresponding
aniline derivatives. These Ir complexes show a red emission, and
their emission intensity is enhanced to a considerable extent
upon the protonation of their basic groups in aqueous solution.
It was found that the emission of 13 is nearly nonexistent at
pH > 5, and a red emission is observed at pH < 5. In contrast, the
guanidine and iminoimidazolidine derivatives 14 and 15 exhibit a
strong emission at pH < 9, and the emission intensity of 16
having three CF3 groups at the 4′ position of the phenyl-
isoquinoline ligand is increased at pH < 8. Moreover, Ir complexes
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P
DOI: 10.1021/acs.inorgchem.5b00369
Inorg. Chem. XXXX, XXX, XXX−XXX