Phosphorescence imaging of living cells with amino acid-functionalized tris(2-phenylpyridine)iridium(III) complexes

Article abstract of DOI:10.1021/ic201860s

A series of nine luminescent cyclometalated octahedral iridium(III) tris(2-phenylpyridine) complexes has been synthesized, functionalized with three different amino acids (glycine, alanine, and lysine), on one, two, or all three of the phenylpyridine ligands. All starting complexes and final compounds have been fully analyzed by one-dimensional (1D) and twodimensional (2D) NMR spectroscopy, and photophysical data have been obtained for all the mono-, bis-, and tri- substituted iridium(III) complexes. Cellular uptake and localization have been studied with flow cytometry and confocal microscopy, respectively. Confocal experiments demonstrate that all nine substituted iridium(III) complexes show variable uptake in the tumor cells. The monosubstituted iridium(III) complexes give the highest cellular uptake, and the series substituted with lysines shows the highest toxicity. This systematic study of amino acid-functionalized Ir(ppy)₃ complexes provides guidelines for further functionalization and possible implementation of luminescent iridium complexes, for example, in (automated) peptide synthesis or biomarker specific targeting.

Full text of DOI:10.1021/ic201860s

Article
pubs.acs.org/IC
Phosphorescence Imaging of Living Cells with Amino Acid-
Functionalized Tris(2-phenylpyridine)iridium(III) Complexes
Peter Steunenberg,† Albert Ruggi,† Nynke S. van den Berg,‡ Tessa Buckle,‡ Joeri Kuil,‡
Fijs W.B. van Leeuwen,‡ and Aldrik H. Velders*,†,§
†Supramolecular Chemistry and Technology, MESA+ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500 AE,
Enschede, The Netherlands
‡Division of Diagnostic Oncology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands;
Interventional Molecular Imaging Section, Department of Radiology, Leiden University Medical Center, Leiden, The Netherlands
§BioNanoTechnology group, Laboratory of Physical Chemistry and Colloid Science Wageningen University, Dreijenplein 6, 6703
HB, Wageningen, The Netherlands
S
* Supporting Information
ABSTRACT: A series of nine luminescent cyclometalated
octahedral iridium(III) tris(2-phenylpyridine) complexes has been
synthesized, functionalized with three different amino acids
(glycine, alanine, and lysine), on one, two, or all three of the
phenylpyridine ligands. All starting complexes and final compounds
have been fully analyzed by one-dimensional (1D) and two-
dimensional (2D) NMR spectroscopy, and photophysical data have
been obtained for all the mono-, bis-, and tri- substituted
iridium(III) complexes. Cellular uptake and localization have
been studied with flow cytometry and confocal microscopy,
respectively. Confocal experiments demonstrate that all nine substituted iridium(III) complexes show variable uptake in the
tumor cells. The monosubstituted iridium(III) complexes give the highest cellular uptake, and the series substituted with lysines
shows the highest toxicity. This systematic study of amino acid-functionalized Ir(ppy)3 complexes provides guidelines for further
functionalization and possible implementation of luminescent iridium complexes, for example, in (automated) peptide synthesis
or biomarker specific targeting.
for diagnostic imaging have been performed by Lo et al.,15 and
INTRODUCTION
■
Li et al.,16 using cationic iridium(III) complexes with neutral
Luminescent complexes with lanthanide ions or late first-row
transition metal ions, for example, zinc or copper,1,2 have been
bidentate, for example, 2,2′-bipyridine (bpy), ligands. Although
these complexes show good cellular uptake,17 low toxicity,
applied in optical imaging already for a while, but only more
recently d6 transition metal complexes have proven their
reduced photobleaching, and exclusive staining of the
potential as luminophores in fluorescence cell microscopy.3
cytoplasm, these cationic dyes still suffer from a significantly
lower quantum yield compared with the neutral fully
cyclometalated complexes: 0.4 for Ir(ppy)3 vs 0.06 for
[(ppy)2Ir(bpy)](PF6) (in degassed dichloromethane solu-
tions).18 Some cationic iridium(III) complexes show even
lower quantum yields (CH3CN 0.017, MeOH 0.0064 and PBS/
MeOH 9:1 even 0.0021); the values obtained for neutral
complexes show at least a 10-fold improvement compared to
those values.19
Here we report on the cellular uptake and localization of nine
neutral iridium(III) complexes with fully cyclometalated neutral
tris(2-phenylpyridine) cores. The archetypical octahedral
Ir(ppy)3 itself is poorly soluble in aqueous media,20 and Li et
al. briefly described the troublesome cellular uptake of neutral
iridium(III) complexes.21 However, upon introduction of one,
Luminescence based imaging is one of the most convenient
ways to study cells as it visualizes morphological details with
subcellular resolution which cannot be achieved by other
imaging modalities.4−6 Organic fluorophores still constitute the
class of the most used fluorescent probes,7 but they have
limitations such as easy photobleaching, small Stokes shifts, and
the same (short) fluorescent lifetime as the autofluorescence
observed in organisms/tissues.8 Iridium(III) complexes exhibit
a high luminescent quantum yield (0.5−1.0 in most degassed
organic solvents and still about 0.1 in aqueous solutions), have
a tunable luminescent color (from blue to red), and relatively
long lifetimes, which may yield favorable properties in imaging
applications.9 These properties have resulted in their utilization
in organic light-emiting diodes,10 phosphorescent chemo-
sensing systems,11 light-emiting electrochemical cells,12 lumi-
nescence sensitizers,13 and pressure sensitive paints (aerody-
namic applications).14 First studies on applications of iridium
Received: August 25, 2011
Published: February 3, 2012
© 2012 American Chemical Society
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Article
brine (50 mL), and dried over MgSO4. Upon evaporation of the
solvent the compound was obtained in a 90% yield (1.8 g, 9 mmol) as
a white solid. H NMR in accordance with literature.
two, or three ppy ligands conjugated to three different amino
acids, an improved water solubility is observed; the quantum
yields of the luminescence (0.1−0.15) further allows good
conditions for comparison of their cellular uptake, distribution,
and (bio)imaging properties. The use of cationic iridium(III)
complexes, as described before,15,16 is well established, but the
higher stability of neutral iridium(III) complexes, allowing a
wider range of synthetic modifications, together with the small
improvements of the photophysical properties, can already
mean a major advantage. The cellular uptake is significantly
influenced by the number and type of substituents, yielding
initial guidelines for the design of new biolabels.
24,25
1
fac-(ppy)2(mppy)Ir (4) and fac-(ppy)(mppy)2Ir (5). [(ppy)2Ir-
(μ-Cl)2Ir(ppy)2] (1) (200 mg, 0.2 mmol), mppy (2) (200 mg, 0.9
mmol), AgCF3SO3 (100 mg, 0.4 mmol), and toluene (15 mL) were
placed in a reaction vessel and degassed. The reaction mixture was
heated at 120 °C while under continuous stirring for 5 h. The resulting
dark orange-red solution was cooled to room temperature and directly
transferred to a silica column. The product was eluted with CH2Cl2.
The monosubstituted product 4 (45 mg, 0.06 mmol, 15%) was found
in the first fractions, the bis-substituted product 5 (65 mg, 0.09 mmol,
22%) in the middle fractions.26 fac-(ppy)2(mppy)Ir (4): H NMR
1
(DMSO) δ 3.72 (s, 3H, MeO), 6.6−6.8 (m, 6H, H-Ph6, H-Ph5, H-
Ph6′,H-Ph5′), 6.8−6.9 (m, 3H, H-Ph4, H-Ph4′), 7.13, 7.19 (2t, 2H, J =
6.5, H-Py5′), 7.50, 7.55 (2 d, 2H, J = 6.5, H-Py6′), 7.79 (d, 2H, J = 8.0,
H-Ph3′), 7.8−7.9 (m, 3H, H-Ph3, H-Py4′), 7.99 (s, 1H, J = 2.0, H-
Py6), 8.17 (m, 3H, H-Py3′, H-Py4), 8.26 (d, 1H, J = 8.0, H-Py3), 13C
NMR (DMSO) δ 52.6, 118.9, 119.7, 120.4, 123.4, 124.2, 124.8, 126.3,
129.5, 130.6, 136.6, 137.5, 137.9, 142.9, 144.2, 147.6, 148.5, 160.3,
164.0, 164.9, 165.8, 170.1, IR (neat) 3035 (w), 1711 (s), 1598 (s),
1578 (s), 1471 (s), 1290 (m), 1126 (m), 747 (vs), 734 (vs) cm−1,
MALDI-TOF MS (dithranol) [M]+ Calcd. for: 713, Found: 713,
HRMS (ESI-MS) Calcd. for: C35H27IrN3O2 [M]+, 713.1656 Found:
713.1598. fac-(ppy)(mppy)2Ir (5): 1H NMR (DMSO) δ 3.72, 3.74 (2
s, 6H, MeO), 6.6−6.8 (m, 9H, H-Ph6,5,4,6′,5′,4′), 7.17 (t, 1H, J = 6.0,
H-Py5′), 7.60 (d, 1H, J = 6.0, H-Py6′), 7.80 (d, 1H, J = 8.0, H-Ph3′),
7.88 (m, 3H, H-Ph3, H-Py4′), 7.97, 8.00 (2 d, 2H, J = 2.0, H-Py6),
8.24 (m, 3H, H-Py3′, H-Py4), 8.26, 8.31 (2 d, 2H, J = 8.0, H-Py3), 13C
NMR (DMSO) δ 52.3, 118.9, 119.8, 120.2, 123.1, 124.1, 124.6, 126.4,
129.6, 136.5, 137.4, 137.8, 142.8, 144.3, 147.9, 148.5, 160.5, 163.9,
165.5, 165.7, 169.7, IR (neat) 3044 (w), 1715 (s), 1544 (s), 1288 (m),
1252 (m), 1124 (m), 743 (vs), 730 (vs) cm−1, MALDI-TOF MS
(dithranol) m/z [M+H]+ Calcd. for: 772, Found: 772, HRMS (ESI-
MS), Calcd. for: C37H28IrN3O4 [M]+ 771.1709, Found: 771.1707.
Tetrakis(2-phenyl-5-methoxycarbonyl-pyridine-C2,N′)(μ-
dichloro)diiridium(III) (6). Iridium(III) trichloride hydrate (400 mg,
1.1 mmol) was combined with mppy (2) (400 mg, 1.2 mmol),
dissolved in 2-ethoxyethanol (30 mL), and refluxed for 24 h (the
solution was purged with argon and stirred in the dark). The reaction
was cooled to room temperature, diluted with 30 mL water, and the
orange precipitate (550 mg, 0.8 mmol, 74%) was collected on a glass
EXPERIMENTAL SECTION
■
1H NMR (600 MHz) and 13C NMR (151 MHz) data were obtained
using a Bruker Avance II NMR-spectrometer. All spectra are
referenced to residual proton solvent signal. Abbreviations used
include singlet (s), doublet (d), doublet of doublets (dd), triplet (t),
and unresolved multiplet (m). 13C NMR assignments were made by
analyzing the HC-HMBC, HC-HMQC, and HH−COSY spectra.
Mass spectral data were obtained using an Applied Biosystems
Voyager DE-RP MALDI-TOF Spectrometer. HRMS analyses were
obtained using a Waters/Micromass LCT mass spectrometer.
Emission measurements were acquired using a Perkin-Elmer
Fluorescence spectrometer. IR-spectra were recorded using a Thermo
Scientific Nicolet TM 6700 FT-IR spectrometer equipped with a
Smart Orbit diamond ATR accessory. Abbreviations used include (w)
weak, (m) medium, (s) strong, and (vs) very strong. UV−vis spectra
were measured on a Perkin-Elmer Lambda 850 UV−vis spectropho-
tometer. Steady-state luminescence spectra were measured using an
Edinburgh FS900 fluorospectrometer. A 450 W xenon arc lamp was
used as excitation source. Luminescence quantum yields at room
temperature (Φ and Φair) were evaluated by comparing wavelength-
integrated intensities (I and IR) of isoabsorptive optically diluted
solutions (Abs <0.1) with reference to [Ru(bpy)3]Cl2 (ΦR = 0.028 in
air-equilibrated water) standard and by using the eq 1
2
n I
Φ = Φ
R
2
n I
R R
(1)
1
filter frit. H NMR (CD2Cl2) δ 3.86 (s, 12H, MeO), 5.87 (d, 4H, J =
where n and nR are the refractive index of the sample and reference
solvent, respectively.22
8.0, H-Ph6), 6.71 (t, 4H, J = 8.0, H-Ph5), 6.91 (t, 4H, J = 8.0, H-Ph4),
7.69 (d, 4H, J = 8.0, H-Ph3), 8.03 (d, 4H, J = 9.0, H-Py3), 8.33 (d, 4H,
J = 9.0, H-Py4), 9.86 (s, 4H, H-Py6), 13C NMR (DMSO) δ 52.9 (C-
MeO), 118.2 (C-Py3), 122.4 (C-Ph4), 124.3 (C-Ph3), 125.6 (C-Py5),
130.4 (C-Ph5), 130.5 (C-Ph6), 138.6 (C-Py4), 142.7 (C-Ph2), 147.1
(C-Ph1), 152.7 (C-Py6), 162.5 (C−COOMe), 172.1 (C-Py2), IR
(neat) 2996 (w), 1714 (s), 1604 (s), 1582 (s), 1285 (m), 1254 (m),
1124 (m), 796 (vs) cm−1, MALDI-TOF MS (dithranol) [M-Cl]+
Calcd. for: 1268, Found: 1268, HRMS (ESI-MS) Calcd. for:
C52H40Cl2Ir2N4O8 [M]+ 1304.1482, Found: 1304.1459.
fac-(mppy)3Ir (7). [(mppy)2Ir(μ-Cl)2Ir(mppy)2] (6) (250 mg, 0.2
mmol), mppy (2) (200 mg, 0.9 mmol), AgCF3SO3 (100 mg, 0.4
mmol), and toluene (15 mL) were placed in a reaction vessel and
degassed. The reaction mixture was heated at 120 °C while under
continuous stirring for 5 h. The resulting dark orange-red solution was
cooled to room temperature and directly transferred to a silica column.
The product was eluted with CH2Cl2/Aceton 8/2. fac-(mppy)3Ir was
obtained in a yield of 45% (150 mg, 0.09 mmol). 1H NMR (DMSO) δ
3.72 (s, 9H, MeO), 6.59 (d, 3H, J = 7.5, H-Ph6), 6.70 (t, 3H, J = 7.5,
H-Ph5), 6.80 (t, 3H, J = 7.5, H-Ph4), 7.80 (d, 3H, J = 7.5, H-Ph3),
7.90 (d, 3H, J = 2.0, H-Py6), 8.15 (dd, 3H, J = 9.0, J = 2.0, H-Py4),
8.22 (d, 3H, J = 9.0, H-Py3), 13C NMR (DMSO) δ 52.9 (OMe), 118.9
(C-Py4), 121.0 (C-Ph4), 124.1 (C-Py5), 126.9 (C-Py6), 131.2 (C-
Ph5), 136.9 (C-Ph6), 138.3 (C-Py3), 142.9 (C-Ph2), 148.5 (C-Py6),
161.9 (C-Ph1), 164.1 (C-PyCOOMe), 170.0 (C-Py2), IR (neat) 3036
(w), 2950 (w), 1720 (s), 1598 (s), 1580 (s), 1430 (m), 1284 (m),
1250 (m), 1125 (m), 747 (vs) cm−1, MALDI-TOF MS (dithranol):
Materials and Reagents. IrCl3·4H2O and 2-phenylpyridine were
purchased from ABCR. Phenylboronic acid, 6-bromo-pyridine-3-
carboxylic acid, and the amino acids (glycine, alanine, and lysine)
were acquired from Sigma Aldrich, and tetrakis(triphenylphosphino)-
palladium from Acros. All other reagents and solvents were purchased
from VWR. All commercial materials were used without further
purification. The dimeric complex [(ppy)2Ir(μ-Cl)2Ir(ppy)2] (1) was
synthesized as previously reported.23
Methyl 2-Phenyl-5-carboxypyridine (mppy) (2). A mixture of
phenylboronic acid (1.6 g, 14 mmol), 6-bromo-pyridine-3-carboxylic
acid (2.0 g 10 mmol), tetrahydrofuran (THF, 50 mL), water (50 mL),
and Na2CO3 (1M, 50 mL) was stirred under argon; then
tetrakis(triphenylphosphino)palladium (250 mg, 0.25 mmol) was
added in one portion. The yellow solution was stirred for 24 h at 100
°C. Afterward the THF was removed, and the remaining aqueous
phase was extracted with dichloromethane (3 times 50 mL). The
aqueous phase was then acidified by conc. HCl and 2-phenyl-5-
carboxypyridine precipitated. The compound was isolated by filtration
and dried under vacuum over P2O5. 2-Phenyl-5-carboxypyridine was
obtained in 1.8 g (90%) as a white solid. This product was used in the
next step without further purification. 2-Phenyl-5-carboxypyridine (2
g, 10 mmol) was refluxed overnight in MeOH (70 mL) and H2SO4 (4
mL). After evaporation of the solvent, water (100 mL) was added, and
the mixture was neutralized with saturated NaHCO3 solution. The
aqueous phase was then extracted with diethylether (2 times 100 mL).
The combined organic fractions were washed with water (100 mL),
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m/z [M]+ Calcd. for: 829, Found: 829 HRMS (ESI-MS), Calcd. for:
C39H31IrN3O6 [M+H]+ 830.1844, Found: 830.1797.
Scheme 1. Synthesis of Amino Acid Conjugated Complexes
11−19
fac-(ppy)2(cppy)Ir (8). The obtained red solid mononuclear
iridium complexes 4 (general synthetic route for 9 and 10 as well)
(0.1 mmol) from the previous step were dissolved in THF/water (20/
20 mL), and LiOH (30 mg) was added. The reaction mixture was
heated until 45 °C and stirred at this temperature overnight.
Afterwards, the reaction mixture was allowed to cool to room
temperature, and THF was removed under reduced pressure. The
remaining aqueous solution was acidified by 2 N HCl until
precipitation of the product. The red precipitate was isolated by
filtration, washed with 20 mL of water and dried. fac-(ppy)2(cppy)Ir
1
was obtained in quantitative yield. H NMR (DMSO) δ 6.6−6.8 (m,
6H, H-Ph6, H-Ph6′, H-Ph5, H-Ph5′), 6.8−6.9 (m, 3H, H-Ph4, H-
Ph4′), 7.14, 7.20 (2 t, 2H, J = 6.5 H-Py5′), 7.50, 7.55 (2d, 2H, J = 6.5,
H-Py6′), 7.80 (d, 2H, J = 8.0, H-Ph3′), 7.8−7.9 (m, 3H, H-Ph3, H-
Py4′), 7.99 (d, 1H, J = 2.0, H-Py6), 8.15 (m, 3H, H-Py3′, H-Py4), 8.25
(d, 1H, J = 8.0, H-Py3), 13C NMR (DMSO) δ 119.3, 119.4, 120.4,
123.6, 124.7, 126.2, 129.6, 130.5, 136.9, 137.5, 138.1, 143.1, 144.1,
147.4, 149.0, 160.5, 163.8, 166.0, 170.1, IR (neat) 3292 (w), 3036 (w),
1693 (s), 1598 (s), 1259 (m), 750 (vs) cm−1, MALDI-TOF MS
(dithranol) [M]+ Calcd. for: 699, Found: 699, HRMS (ESI-MS)
+
Calcd. for: C34H24IrN3O2, [M] 669.1498, Found: 669.1449.
fac-(ppy)(cppy)2Ir (9). This complex was prepared according to
the procedure for the synthesis of complex 8 using complex 5 (instead
1
of complex 4) as the reactant. H NMR (DMSO) δ 6.6−6.8 (m, 9H,
H-Ph6,6′,5,5′,4,4′), 7.13 (t, 1H, J = 6.0, H-Py5′), 7.54 (d, 1H, J = 6.0,
H-Py6′), 7.75 (d, 1H, J = 8.0, H-Ph3′), 7.81 (m, 3H, H-Ph3, H-Py4′),
7.94, 7.99 (2 d, 2H, J = 2.0, H-Py6), 8.22 (m, 3H, H-Py3′, H-Py4),
8.26, 8.31 (2 d, 2H, J = 8.0, H-Py3), 13C NMR (DMSO) δ 119.1,
119.6, 120.4, 123.4, 124.7, 125.5, 126.1, 129.6, 130.9, 136.8, 137.7,
138.2, 143.2, 144.4, 147.8, 148.7, 159.2, 162.9, 163.1, 165.6, 169.3, IR
(neat) 3035 (w), 1694 (s), 1579 (s), 1574 (s), 1379 (m), 1223 (m),
749 (vs), 729 (vs) cm−1, MALDI-TOF MS (dithranol): m/z [M]+
Calcd. for: 743, Found 743, HRMS (ESI-MS) Calcd. for:
CH2-Gly), 6.6−6.8 (m, 6H, H-Ph6,6′,5,5′), 6.8−6.9 (m, 3H, H-Ph4,4′),
7.15 (t, 2H, J = 6.0, H-Py5′), 7.50, 7.53 (2 d, 2H, J = 6.0, H-Py6′), 7.78
(2d, 2H, J = 8.0, H-Ph3′), 7.80 (m, 2H, H-Py4′), 7.85 (d, 1H, J = 8.0,
H-Ph3), 8.04 (s, 1H, H-Py6), 8.16 (2 d, 2H, J = 9.0, H-Py3′), 8.25 (m,
2H, H-Py3,Py4), 9.04 (t, 1H, NH), IR (neat) 3322 (w), 2927 (w),
2849 (w), 1624 (s), 1537 (s), 1309 (m), 1228 (m), 1087 (m), 749
(vs) cm−1, 13C NMR (DMSO) δ 40.9, 118.4, 119.3, 120.1, 123.1,
124.4, 125.6,127.4, 129.3, 130.6, 134.3, 136.8, 137.6, 143.2, 144.0,
147.4, 148.8, 160.8, 163.0, 164.2, 165.9, 166.1, 168.7, 171.8, MALDI-
TOF MS (dithranol) Calcd. for: m/z [M]+ 756, Found 756, HRMS
(ESI-MS) Calcd. for: C36H27IrN4O3 [M]+ 756.1712, Found: 756.1667,
UV−vis: λabs 382 nm, λabs 483 nm, λem = 579 nm, ε382 = 9,096 M−1
cm−1 ε483 = 1,1871 M−1 cm−1, Φ 0.12 (in DMSO).
+
C35H24IrN3O4 [M] 743.1398, Found: 743.1423.
fac-(cppy)3Ir (10). This complex was prepared according to the
procedure for the synthesis of complex 8 using complex 7 (instead of
complex 4) as the reactant. 1H NMR (DMSO) δ 6.59 (d, 3H, J = 7.5,
H-Ph6), 6.70 (t, 3H, J = 7.5, H-Ph5), 6.80 (t, 3H, J = 7.5, H-Ph4), 7.80
(d, 3H, J = 7.5, H-Ph3), 7.90 (d, 3H, J = 2.0, H-Py6), 8.15 (dd, 3H, J =
9.0, J = 2.0, H-Py4), 8.22 (d, 3H, J= 9.0, H-Py3) 13C NMR (DMSO) δ
119.0 (C-Py4), 120.9 (C-Ph4), 124.3 (C-Py5), 126.4 (C-Py6), 131.1
(C-Ph5), 136.8, (C-Ph6) 138.5 (C-Py3), 143.5 (C-Ph2), 148.9 (C-
Py6), 162.1 (C-Ph1), 164.5 (C-PyCOOMe), 169.5 (C-Py2), IR (neat)
3365 (w), 1682 (s), 1597 (s), 1580 (s), 1403 (m), 1226 (m), 749 (vs),
723 (vs) cm−1, MALDI-TOF MS (dithranol): m/z [M]+ Calcd. for:
787, Found: 787 HRMS (ESI-MS), Calcd. for: C36H24IrN3O6 [M+H]
+, 787.1294 Found: 787.1265.
fac-(ppy)(gly-ppy)2Ir (12). Reactants: glycine methyl ester
1
hydrochloride and complex 9 H NMR (DMSO) δ 3.83 (dd, 4H, J
= 3.8, CH2-Gly), 6.6−6.8 (m, 6H, H-Ph6,6′,5,5′), 6.8−6.9 (m, 3H, H-
Ph4,4′), 7.16 (t, 1H, J = 6.0, H-Py5′), 7.58 (d, 1H, J = 6.0, H-Py6′),
7.77 (d, 1H, J = 8.0, H-Ph3′), 7.83 (t, 1H, J= 6.0, H-Py4′), 7.86 (dd,
2H, J = 8.0, H-Ph3), 8.02 (s, 2H, H-Py6), 8.19 (d, 1H, J = 6.0, H-
Py3′), 8.26 (d, 2H, J = 9.0, H-Py4), 8.28 (dd, 2H, J = 9.0, J = 2.0, H-
Py3), 9.14 (m, 2H, NH), 13C NMR (DMSO) δ 41.3, 118.5, 119.4,
120.5, 123.9, 124.7, 125.9, 120.9, 135.6, 136.6, 138.2, 143.4, 144.4,
147.9, 148.2, 160.0, 162.6, 165.8, 168.1, 171.4, IR (neat) 3322 (w),
2927 (w), 2849 (w), 1624 (s), 1537 (s), 1309 (m), 1228 (m), 1087
(m), 749 (vs) cm−1, MALDI-TOF MS (dithranol) m/z [M]+ Calcd.
for: 857, Found: 857, HRMS (ESI-MS) Calcd. for: C39H31IrN5O6 [M
+H]+ 858.1906, Found: 858.1947, UV−vis: λabs 401 nm, λabs 482 nm,
λem = 576 nm, ε401 = 7,402 M−1 cm−1 ε482 = 1,858 M−1 cm−1 Φ 0.13
(in DMSO).
General Procedure for Amino Acid Coupling. The general
synthetic route for amino acid conjugated 2-phenylpyridine mono-
nuclear iridium(III) complex conjugated to amino acids is described in
Scheme 1. Briefly, 1 equiv of the mononuclear iridium(III) complex
(0.06 mmol) was dissolved in dimethylformamide (DMF, 10 mL). To
this solution was added DCC (0.18 mmol, i.e., 3 equiv per free acid),
HOBt (0.18 mmol or 3 equiv per free acid), amino acid methyl ester
(HCl-salt, 0.018 mmol, i.e., 3 equiv per free acid), and DIPEA (0.1
mL), and the reaction mixture was stirred overnight at room
temperature. The reaction mixture was concentrated under vacuum,
and the crude iridium complex-conjugates were purified by column
chromatography (CH2Cl2:Aceton 4:1). The so-obtained red solid was
dissolved in THF/water (20/20 mL), and LiOH (30 mg per free acid)
was added. The reaction mixture was heated until 45 °C and stirred at
this temperature overnight. The reaction mixture was cooled to room
temperature and acidified to pH 1 by 1.0 M HCl until precipitation of
the product. The red precipitate was isolated by filtration, washed with
20 mL water and dried. The amino acid conjugated complexes were
obtained as a single product in near quantitative yield.
fac-(gly-ppy)3Ir (13). Reactants: glycine methyl ester hydro-
1
chloride and complex 10 H NMR (DMSO) δ 3.82 (m, 6H, CH2-
Gly), 6.60 (d, 3H, J = 7.5, H-Ph6), 6.72 (t, 3H, J = 7.5, H-Ph5), 6.84
(t, 3H, J = 7.5, H-Ph4), 7.88 (d, 3H, J = 7.5, H-Ph3), 8.05 (s, 3H, H-
Py6), 8.30 (m, 6H, H-Py3, H-Py4), 9.16 (t, 3H, J = 5.5, NH), 13C
NMR (DMSO) δ 41.2 (C-G1), 118.5 (C-Py4), 120.4 (C-Ph4), 125.7
(C-Ph3), 127.4 (C-Py5), 130.6 (C-Ph5), 135.4 (C-Py3), 136.7 (C-
fac-(ppy)2(gly-ppy)Ir (11). Reactants: glycine methyl ester
hydrochloride and complex 8 1H NMR (DMSO) δ 3.84 (m, 2H,
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Ph6), 143.0 (C-Ph2), 148.4 (C-Py6), 161.9 (C-Ph1), 163.7 (C-
PyCO), 168.5 (C-Py2), 171.6 (C-GCOOH), IR (neat) 3340 (w),
3040 (w), 2928 (w), 1720 (s), 1637 (s), 1594 (s), 1579 (s), 1473 (m),
1299 (m), 1207 (m), 746 (vs), 730 (vs) cm−1 MALDI-TOF MS
(dithranol) [M]+ Calcd. for: 959, Found: 959, HRMS (ESI-MS)
Calcd. for: C42H33IrN6O9 [M+H]+ 959.2020, Found: 959.2016, UV−
vis: λabs 405 nm, λabs 485 nm, λem = 564 nm, ε405 = 8,443 M−1 cm−1
ε485 = 2,013 M−1 cm−1 Φ 0.11 (in DMSO).
fac-(ppy)2(ala-ppy)Ir (14). Reactants: alanine methyl ester
hydrochloride and complex 8 1H NMR (DMSO) δ 1.30 (s, 3H,
CH-(CH3)-COOH) 4.24 (m, 1H, CH-(CH3)-Ala), 6.6−6.8 (m, 6H,
H-Ph6,6′,5,5′), 6.8−6.9 (m, 3H, H-Ph4,4′), 7.15 (2t, 2H, J = 6.0, H-
Py5′), 7.50, 7.53 (2 d, 2H, J = 6.0, H-Py6′), 7.75 (2d, 2H, J = 8.0, H-
Ph3′), 7.81 (m, 2H, H-Py4′), 7.87, (2d, 1H, J = 8.0, H-Ph3), 7.98, 8.00
(2d, 1H, J = 2.0, H-Py6), 8.15 (m, 2H, H-Py3′) 8.26 (dd, 1H, J = 9.0,
H-Py4), 8.31, 8.33 (m, 1H, J = 9.0, J = 2.0, H-Py3), 8.8 (m, 1H, NH),
13C NMR (DMSO) δ 17.5 48.8, 118.5, 119.5, 120.2, 123.4, 124.8,
125.8, 130.6, 135.6, 136.4, 138.1, 143.5, 144.5, 147.9, 159.8, 162.7,
163.4, 165.6, 168.5, 174.8, IR (neat) 3353 (w), 3034 (w), 2924 (w),
1716 (s), 1635 (s), 1597 (s), 1579 (s), 1472 (m), 1256 (m), 1030
(m), 750 (vs), 732 (vs) cm−1, MALDI-TOF MS (dithranol) m/z [M
+H]+ Calcd. for: 771, Found: 771, HRMS (ESI-MS) Calcd. for:
C37H29IrN4O3 [M]+ 770.1869, Found: 771.1795 UV−vis: λabs 382 nm,
λabs 484 nm, λem = 579 nm, ε382 = 7,152 M−1 cm−1 ε484 = 1,444 M−1
cm−1 Φ 0.11 (in DMSO).
atm. for 12 h. Afterward the resulting reaction mixture was filtered
through Celite 545. The solvent was evaporated to isolate the product
as an orange solid in near quantitative yield.
fac-(ppy)2(lys-ppy)Ir (17). Reactants: H2N-Lys(Cbz)-OMe hy-
1
drochloride and complex 8 H NMR (DMSO) δ 1.2−1.4 (m, 6H,
CH2-CH2-CH2-Lys), 2.73 (m, 2H, H2N−CH2-Lys), 3.60, 3.62 (2s, 3H,
MeO), 4.33, CH-Lys), 6.6−6.9 (m, 9H, H-Ph6,6′,5,5′,4,4′), 7.15 (m,
2H, H-Py4′), 7.53 (2 dd, 2H, J = 6.0, H-Py3′), 7.8−8.0 (m, 7H, NH2,
H-Ph3, 3′, H-Py5), 8.00 (2 s, 1H, H-Py6), 8.10 (m, 1H, H-Py6′), 8.26
(2 d, 1H, J = 9.0, H-Py4), 8.38, 8.44 (2 d, 1H, J = 9.0, H-Py3), 8.98 (br
t, 1H, NH), 13C NMR (DMSO) δ 22.5, 24.4, 29.6, 38.4, 53.1, 118.2,
119.4, 120.3, 123.3, 125.1, 125.9, 129.4, 130.5, 136.3, 138.2, 143.3,
144.2, 160.3, 162.9, 165.5, 168.6, 174.1, IR (neat) 3333 (w), 2928 (w),
1732 (s), 1644 (s), 1596 (s), 1579 (s), 1471 (m), 1299 (m), 1159
(m), 1030 (m), 750 (vs), 730 (vs) cm−1, MALDI-TOF MS (dithranol)
Calcd. for: [M+H]+ 842, Found: 842 HRMS (ESI-MS) Calcd. for:
C41H39IrN5O3 [M+H]+ 842.2685, Found: 842.2516 UV−vis: λabs 384
nm, λabs 484 nm, λem = 585 nm, ε384 = 10,653 M−1 cm−1 ε484 = 2,222
M−1 cm−1 Φ 0.13 (in DMSO).
fac-(ppy)(lys-ppy)2Ir (18). Reactants: H2N-Lys(Cbz)-OMe hy-
1
drochloride and complex 9 H NMR (DMSO) δ 1.2−1.4 (m, 12H,
CH2-CH2-CH2-Lys), 2.73 (m, 4H, H2N−CH2-Lys), 3.60, 3.62 (2 s,
6H, MeO), 4.33, CH-Lys), 6.6−6.9 (m, 9H, H-Ph6,6′,5,5′,4,4′), 7.15 (t,
2H, H-Py4′), 7.53 (m, 2H, H-Py3′), 7.8−8.0 (m, 10H, NH2, H-
Ph3,3′,H-Py5′,6), 8.10 (2 d, 2H, J = 9.0 H-Py6′), 8.26 (t, 1H, J = 9.0,
H-Py4), 8.38, 8.44 (2d, 1H, J = 9.0, H-Py3), 8.98 (br t, 1H, NH), 13C
NMR (DMSO) δ 22.3, 24.4, 29.7, 38.4, 52.6, 118.2, 119.7, 120.3,
123.3, 125.1, 125.9, 129.6, 130.6, 136.3, 137.9, 143.3, 144.2, 160.3,
163.1, 165.3, 168.6, 173.8, IR (neat) 3322 (w), 2926 (w), 1737 (s),
1625 (s), 1597 (s), 1473 (m), 1224 (m), 1030 (m), 749 (vs), 733 (vs)
cm−1, MALDI-TOF MS (dithranol) [M+2H]+ 1029, Found 1029,
HRMS (ESI-MS) C49H52IrN7O6 [M+H]+ Calcd. for: 1028.3690,
Found: 1028.3634 UV−vis: λabs 400 nm, λabs 485 nm, λem = 576 nm,
ε400 = 100,460 M−1 cm−1 ε485 = 2,892 M−1 cm−1 Φ 0.14 (in DMSO).
fac-(lys-ppy)3Ir (19). Reactants: H2N-Lys(Cbz)-OMe hydro-
fac-(ppy)(ala-ppy)2Ir (15). Reactants: alanine methyl ester
hydrochloride and complex 9 1H NMR (DMSO) δ 1.32 (s, 6H,
CH-(CH3)-COOH) 4.24 (m, 2H, CH-(CH3)-Ala), 6.6−6.8 (m, 6H,
H-Ph6,6′,5,5′), 6.8−6.9 (m, 3H, H-Ph4,4′), 7.15 (2t, 1H, J = 6.0, H-
Py5′), 7.55, 7.58 (2 d, 1H, J = 6.0, H-Py6′), 7.77 (d, 1H, J = 8.0, H-
Ph3′), 7.83 (m, 3H, H-Py4′, H-Ph3), 7.98 (m, 2H, H-Py6), 8.15 (m,
1H, H-Py3′) 8.26 (d, 2H, J = 9.0, H-Py4), 8.33 (m, 2H, H-Py3) 8.8
(m, 2H, NH), 13C NMR (DMSO) δ 17.4, 48.4, 118.5, 119.7, 120.2,
123.4, 124.8, 125.8, 130.6, 135.6, 136.6, 137.8, 143.5, 144.8, 147.9,
160.2, 162.7, 163.4, 165.6, 168.4, 174.8, IR (neat) 3334 (w), 2929 (w),
1725 (s), 1629 (s), 1580 (s), 1474 (m), 1255 (m), 1029 (m), 1012
(m), 750.6 (vs), 736 (vs) cm−1, MALDI-TOF MS (dithranol) [M]+
Calcd. for: 885, Found: 885 HRMS (ESI-MS) Calcd. for:
C41H35IrN5O6 [M+H]+ 886.2219, Found: 886.2182 UV−vis: λabs
1
chloride and complex 10 H NMR (DMSO) δ 1.2−1.4 (m, 18H,
CH2-CH2-CH2-Lys), 2.70 (m, 6H, H2N−CH2-Lys), 3.60 (s, 9H,
MeO), 4.20,(m, 3H, CH-Lys), 6.55, 6.58 (2d, 3H, J = 8.0, H-Ph6),
6.72 (dd, 3H, J = 8.0, H-Ph5) 6.85 (dd, 3H, J = 8.0, H-Ph4) 7.88 (m,
9H, NH2-Lys, H-Ph3), 8.07, 8.25 (2 s, 2H, H-Py6), 8.25, 8.32 (2 d,
3H, J = 9.0, H-Py4), 8.32, 8.47 (2 d, 3H, J = 9.0, H-Py3) 9.0−9.5 (m,
3H, NH), 13C NMR (DMSO) δ 22.1, 26.8, 30.6, 38.8, 52.2, 118.6,
120.5, 125.8, 127.6, 130.4, 136.2, 136.7, 143.6, 148.9, 162.5, 163.9,
168.7, 173.4, IR (neat) 3302 (w), 2927 (w), 2851 (w), 1732 (s), 1633
(s), 1597 (s), 1476 (m), 1225 (m), 1029 (m), 734 (vs) cm−1, MALDI-
TOF MS (dithranol) [M+2H]+ Calcd. for: 1215, Found 1215 HRMS
(ESI-MS) Calcd. for: C49H52IrN7O6 [M+H]+ 1214.4696, Found:
401 nm, λabs 483 nm, λem = 570 nm, ε401 = 6,554 M−1 cm−1 ε483
2,005 M−1 cm−1 Φ 0.12 (in DMSO).
=
fac-(ala-ppy)3Ir (16). Reactants: alanine methyl ester hydro-
1
chloride and complex 10 H NMR (DMSO) δ 1.30 (s, 9H, CH-
(CH3)-COOH), 4.24 (m, 3H, CH-(CH3)-Ala), 6.60 (dd, 3H, J = 6.0,
H-Ph6), 6.72 (dd, 3H, J = 6.0, H-Ph5), 6.84 (t, 3H, J = 6.0, H-Ph4),
7.88 (d, 3H, J = 8.0, H-Ph3), 8.02, 8.10 (2 d, 3H, J = 2.0, H-Py6), 8.30
(dd, 3H, J = 9.0, H-Py4), 8.35 (dd, 3H, J = 9.0, J = 2.0, H-Py3), 9.0 (m,
3H, NH), 13C NMR (DMSO) δ 16.7 (C-A3), 48.3 (C-A1), 118.5 (C-
Py3), 120.4 (C-Ph4), 125.7 (C-Ph3), 130.6 (C-Py5), 136.1 (C-Py4),
136.7 (C-Ph6), 143.0 (C-Ph2), 149.2 (C-Py6), 162.3 (C-Ph1), 163.7
(C-PyCO), 168.5 (C-Py2), 174.6 (C-ACOOH), IR (neat) 3323 (w),
2927 (w), 2844 (w), 1624 (s), 1573 (s), 1475 (m), 1448 (m), 1309
(m), 1242 (m), 1087 (m), 732 (vs) cm−1, MALDI-TOF MS
(dithranol) [M]+ Calcd. for: 1000, Found: 1000 HRMS (ESI-MS)
Calcd. for: C45H39IrN6O9 [M]+ 1000.2411, Found: 1000.2451 UV−
vis: λabs 402 nm, λabs 480 nm, λem = 567 nm, ε402 = 7,697 M−1 cm−1
ε480 = 2,369 M−1 cm−1 Φ 0.13 (in DMSO).
General Procedure for Hydrogenation/Cleavage of Cbz. The
general synthetic route for lysine functionalized iridium(III) complexes
is described in Scheme 1. Briefly, the mononuclear iridium(III)
complex (0.06 mmol) was dissolved in DMF (10 mL). To this
solution was added DCC (0.18 mmol or 3 equiv per free acid), HOBt
(0.18 mmol or 3 equiv per free acid), Cbz protected lysine (HCl-salt,
0.018 mmol or 3 equiv per free acid), and DIPEA (0.1 mL), and the
reaction mixture was stirred overnight at room temperature. The
reaction mixture was concentrated under vacuum, and the crude
iridium complex-conjugates were purified by column chromatography
(CH2Cl2:Aceton 4:1). Afterward the Cbz-protected iridium complex
(0.06 mmol) was dissolved in MeOH (25 mL) and 10% Pd/C (50
mg) was added, and the mixture stirred at room temperature under H2
1214.4962, UV−vis: λabs 402 nm, λabs 484 nm, λem = 567 nm, ε402
=
10,504 M−1 cm−1 ε484 = 3,157 M−1 cm−1Φ 0.15 (in DMSO).
Calculated log D (Clog D) Values. ChemDraw structures were
copied into the structure builder on the Web site http://intro.bio.umb.
edu/111-112/OLLM/111F98/newclogp.html. The calculated log of
the distribution coefficient (Clog D) at pH 7.4 was obtained via Tools,
Partitioning, logD for each complex.
Cell Culture. The murine breast cancer cell line 4T1 (kindly
provided by Dr. Olaf van Tellingen (NKI/AvL, Amsterdam, The
Netherlands) was maintained in Gibco’s minimum essential medium
(MEM) enriched with 10% fetal bovine serum, 5 mL Penicillin/
Streptomycin (10000 units/mL Penicillin/10000 μg/mL Streptomy-
cin), 5 mL L-glutamine, 5 mL nonessential amino acids, 5 mL sodium
pyruvate, and 5 mL MEM vitamin solution (all Life Technologies Inc.,
Breda, The Netherlands). Cells were kept under standard culture
conditions.
Flow Cytometry. Freshly cultured 4T1 cells were trypsinized,
washed with 0.1% bovine serum albumin in phosphate buffered saline
(0.1% BSA/PBS), and then incubated for 1 h at room temperature
with the synthesized compound 11−19 (0.1 μM) in 0.1% BSA/PBS.
Following incubation, cells were washed with 0.1% BSA/PBS, and 5
min prior to analysis, propidium iodide (PI; 1:10000; BD Biosciences)
was added to distinguish living from dead cells. After staining, cells
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were analyzed using a BD LSRII Special Order System (excitation 355
nm; emission filter 655/8 nm; BD Biosciences). Data was analyzed
using FACSDiva Software version 6.1.2 (BD Biosciences).
Scheme 2. Synthesis of Complexes 4 and 5
Confocal Microscopy Analysis. Prior to the start of each
experiment, 1.0 × 105 4T1 cells were seeded on coverslips (⌀ 24 mm;
Karl Heicht GmbH&Co, Sondheim, Germany). After 24−48 h (80%
confluency), the old medium was substituted with fresh culture
medium containing 10 μM complex in 0.3% DMSO/cell culture
medium and incubated for 1 h at 4 or 37 °C. To visualize the nucleus,
lysosomes or endosomes, cells were incubated for 10 min at 37 °C
with DAPI (1:1000; Roche, Woerden, The Netherlands), Transferrin
conjugate Alexa fluor 647 (1:1000; Invitrogen, Bleiswijk, The
Netherlands) or Lysotracker Yellow HCK-123 (1:1000; Invitrogen),
respectively. Following incubation, cells were placed on ice and washed
three times with icecold PBS. Then images were taken (magnification
630×) and analyzed using a Leica TSC-SP2-AOBS Live confocal
microscope (Leica Microsystems Heidelberg GmbH, Mannheim,
Germany) equipped with Leica confocal software (Leica Microsystems
Heidelberg GmbH). Luminescence emission of the iridium(III)
complexes 17, 18, and 19 was collected from 580 to 640 nm
(excitation at 405 nm), whereas the nucleus was visualized using DAPI
(emission collected from 445 to 485 nm; excitation at 405 nm).
Lysotracker was used to visualize the lysosomes (emission collected
from 510 to 550 nm; excitation at 488 nm) and endosomes could be
imaged using transferrin conjugate (emission collected from 650 to
680 nm: excitation at 633 nm).
[Ir(mppy)3] (7) the dichloro bridged dimer [(mppy)2Ir(μ-
Cl)2Ir(mppy)2] (6) was complexed with the mppy ligand 2 in
the presence of silver triflate, leading to the formation of the
mononuclear iridium complex fac-[Ir(mppy)3] (7) in 45% yield
(see Scheme 3).
Scheme 3. Synthesis of Complex 7
RESULTS AND DISCUSSION
■
Synthesis of Mononuclear Iridium Complexes. The
iridium(III) complexes 4, 5, and 7 were synthesized with the
ligand 2, mppy. The synthesis of 2 deviates from the synthetic
route described by Tanaka et al. as we performed a Suzuki
coupling with 6-bromo-pyridine-3-carboxylic acid instead of
methyl 6-chloro-pyridine-3-carboxylate.24 First, phenylboronic
acid is coupled to 6-bromo-pyridine-3-carboxylic acid in the
presence of [Pd(PPh3)4] and Na2CO3 (Suzuki coupling)
leading to the formation of 2-phenyl-5-carboxypyridine in a
90% yield.25 The so-obtained product was then esterified by
MeOH/H2SO4, the formed methyl ester was isolated in a yield
of 90%. The NMR-spectrum of the formed compound 2 was in
accordance to that previously reported.24
The reaction of the dichloro bridged dimer [(ppy)2Ir(μ-
Cl)2Ir(ppy)2] 1 with the mppy ligand 2 in the presence of silver
triflate26 resulted in a mixture of four mononuclear iridium(III)
complexes. This result implies that ligand exchange reactions
took place under these experimental conditions leading to the
formation of fac-[Ir(ppy)3] (3), the fac-[Ir(ppy)2mppy] (4),
and fac-[Ir(ppy)mppy2] (5). The fac-[Ir(ppy)mppy2] (5) is the
major product (20%), then fac-[Ir(ppy)2mppy] (4) (15%), and
finally fac-[Ir(ppy)3] (3) (5%, see Scheme 2). As described
recently,27 a small amount of fac-[Ir(mppy)3] (7) was also
formed, but even after prolonged reaction times the ratio of the
product did not change. TLC-monitoring during the reaction
reveiled that fac-[Ir(ppy)2mppy] (4) was formed first, then fac-
[Ir(ppy)mppy2] 5 and fac-[Ir(ppy)3] 3, and finally a small
amount of fac-[Ir(mppy)3] (7) could be detected in the
reaction mixture. Over time, the amount of fac-[Ir(ppy)mppy2]
(5) increased and that of fac-[Ir(ppy)2mppy] (4) decreased,
also indicating a form of ligand exchange/scrambling. It is also
possible to synthesize 4 and 5 upon reaction of the dichloro
bridged dimer [(mppy)2Ir(μ-Cl)2Ir(mppy)2] (6) with the ppy
ligand. In that case, after 6 h of reaction time, the major product
is also fac-[Ir(ppy)mppy2] (5) (25%), then fac-[Ir(ppy)2mppy]
(4) (10%), and finally fac-[Ir(ppy)3] (3) together with some
traces of fac-[Ir(mppy)3] (7). For the formation of fac-
Changing the symmetry of the complex going from the
simple spectrum of 3 with C3 symmetry, to the more complex
spectra of 4 and 5 with C1 symmetry, and finally back to
compound 7 with C3 symmetry immediately has its effect on
the complexity of the NMR-spectra (Figure 1). Most
characteristic is the dissapearance of Py5, observed in 3 at 7.1
ppm and corresponding to the three Py5 protons in the three
identical ppy ligands. In 4 two Py5 signals are observed,
corresponding to those of the two ppy ligands that now are
inequivalent because of the introduction of the mppy ligand on
the iridium and in which a methyl ester is present on the Py5
position. In 5 only one Py5 proton signal is observed,
corresponding to the one of the single ppy ligand present in
the complex, and in 7 all ppy ligands are substituted by mppy
ligands and no Py5 signal is observed. Similar trends in peak
shifts and change of peak pattern is observed for other protons.
The analysis of the NMR-spectra of the iridium(III) complexes
has also been used to determine the ligand configuration of the
complexes, either facial (fac) or meridonial (mer).28 A fac
isomer of a homoleptic complex would have all three phenyl
ligands on the same face of the molecule, trans to a pyridyl
nitrogen, with C3 symmetry. From the spectrum of compound
7 it can be concluded that the configuration is fac, because the
1D-NMR-spectrum gives evidence of the formation of a C3
symmetric complex, instead of the more complicated pattern
that would be obtained for a mer complex, with C1 symmetry.
Also it can be concluded that ligand scrambling has no
influence on the configuration, there all complexes obtained are
fac.
After formation of the iridium(III) complexes, the methyl
esters were hydrolyzed by LiOH in THF/water, acidification by
1 N HCl resulted in an orange precipitate.29 The amino acids
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Figure 2. HH−COSY spectrum (DMSO) of Ir(ppy)3 complex
conjugated with a glycine on each ppy ligand (13).
Figure 1. 1H NMR-Spectra (DMSO-d6) of mono-, bis- and tris(ester)
substituted Ir(ppy)3 complexes 4, 5, and 7 compared with fac-
[Ir(ppy)3] 3.
were coupled to the iridium(III) complexes by standard peptide
chemistry coupling procedures (DCC, HOBt, DIPEA, DMF),
and directly after purification, the methyl esters were cleaved
under the above-reported conditions. The amino acid
conjugated complexes were obtained with high purity in near
quantitative yields. As a starting-point for the synthesis of these
amino acid conjugated iridium(III) complexes, we started with
the simplest amino acid glycine; this was followed by alanine, a
chiral amino acid yielding diastereomeric complexes, as can be
observed by NMR-spectroscopy. Finally the complexes were
conjugated to lysine. In aqueous solution at neutral pH, lysine
substituents yield the iridium(III) complexes with a positive
charge, while glycine and alanine give negatively charged
molecules.
The iridium(III) complexes 3, 4, 5, 7, 13, 16, and 19 were
characterized using 1H and 13C homo- and heteronuclear NMR
experiments. The integration of the signals in the 1D spectra of
the complexes showed in all cases the expected number of
protons, but severe overlap of peaks in some case complicated
unambiguous assignement. The proton signals were assigned to
their position in the bicyclic system of the ligand by Correlation
Spectroscopy (COSY), Heteronuclear Multiple Quantum
Coherence (HC-HMQC), and Heteronuclear Multiple Bond
Correlation (HC-HMBC) spectra. HC-HMQC experiments
were used to assign the carbon signals to the corresponding
proton signals, while assignment of the quaternary carbons and
the bridgeheads of the phenyl-pyridine ligands was done by
HC-HMBC experiments. Typical and representative 2D NMR
spectra of the amino acid conjugated iridium(III) complex 13
are shown in Figures 2 and 3. From the COSY spectra of 13,
the Ph-6,5,4 and 3 protons at 6.60, 6.72, 6.84, and 7.88 ppm are
readily assigned, and likewise the pyridine 6 and overlapping 4
and 3 protons, at 8.05 and 8.30 ppm, respectively. Quarternary
carbons were assigned from HC-HMBC/HC-HMQC analyses
Figure 3. HMBC(blue)/HMQC(red)-spectrum (DMSO) of Ir(ppy)3
complex conjugated with a glycine on each ppy ligand (13).
(Figure 3): through correlation with the CH2 and NH from the
glycine the position of the carboxylic acid can be
unambiguously assigned at 171.6, and hence the amide at
163.7. Other quarternary carbons are found at 168.5 (Py-2),
143.0 (Ph-2), and 127.4 (Py-5) ppm.
In Figure 4, the NMR-spectra of the amino acid conjugated
iridium(III) complexes 13, 16, and 19 are shown together for
illustrating the spectral changes due to the formation of
diastereomers in the latter two complexes. Upon conjugating
the Ir(ppy)3 core, consisting of two enantiomeric forms Λ and
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Figure 5. Cellular uptake of complexes 11−19 (10 μM in PBS) in 4T1
cells after 1 h incubation.
Table 1. Calculated log D (Clog D) Values at pH 7.4
compound
Clog D
11
12
13
14
15
16
17
18
19
2.05
Figure 4. Upfield (aromatic) region of the 1H NMR spectra (DMSO)
of tris-substituted Ir(ppy)3 complexes conjugated with glycine (13),
alanine (16), and lysine (19). The doubling of peaks due to the
formations of diastereomers in 16 and 19 is most evident for the Ph3,
Ph6, and Py6 protons.
−3.03
−8.12
2.61
−1.92
−6.45
3.18
Δ,30 with a homochiral amino acid (L-alanine or L-lysine)
instead of an achiral L-glycine, the pattern of the peaks in the
spectrum changes dramatically, giving evidence for the two
stereoisomeric forms of the iridium(III)-complexes 16 and 19.
With respect to the spectrum of 13, the number of peaks for
the Py3, Py4, and Py6 protons is doubled, as well as the peaks
of Ph3, Ph4, Ph5, and Ph6. This is most evident for the Ph3
and Ph6 protons, which both show a doublet when conjugated
to glycine (13), but show both two doublets when conjugated
to lysine (19). The peak pattern of the Ph4 and Ph5 also
doubles, but the changes in chemical shift are small, and the
multiplets overlap severely. Also illustrative is the Py6 peak,
observed as a clear singlet for 13, but two singlets are observed
in the spectra of 16 and 19. For the lysine conjugated complex
19 the difference observed for the diastereotopic protons (up to
∼0.2 ppm) is significantly bigger than that observed for the
alanine conjugated iridium(III) complex 16, which can be
attributed to the larger functional group on the lysine and the
consequential greater steric constraint with the tris(ppy) unit.
The quantitative cellular uptake in 4T1 cells of the
iridium(III) complexes 11−19 was compared using flow
cytometry (Figure 5). Interestingly, the monosubstituted
complexes 11, 14, and 17, gave a remarkable 20-fold higher
cellular uptake than the corresponding bis- and tris- substituted
analogues (Figure 5). This difference is most likely because the
monosubstituted iridium(III) complexes are more hydro-
phobic, and this generally favors the uptake.31,32 To quantify
the hydrophobicity of complexes 11−19, log P or Clog D
values were determined. Because of poor solubility in both the
octanol and water layer (solutions stay turbid and the
complexes act as surfactants), the log P values could not be
measured and were therefore calculated at pH 7.4 (Table 1).
These values clearly illustrate that the monosubstituted
complexes are hydrophobic and the bis- and tri- substituted
complexes are hydrophilic, suggesting that hydrophobicity is
important for the cellular uptake of the dye. The Clog D values
also show that the lysine conjugated complexes are slightly
more hydrophobic than the glycine and alanine derivatives.
−0.76
−4.70
This difference could explain why lysine complexes 17−19
display relatively higher uptake with respect to the correspond-
ing alanine or glycine derivative. However, this relatively high
uptake could also be caused by the positive charge at the lysine
side chain, since positive charges commonly enhance uptake
(e.g., cell penetrating peptides). The amphiphilic character of
the iridium complexes, with a hydrophobic core and hydro-
philic substituents, makes it possible to tune the complexes with
concern to their lipophilicity, and therefore, cellular uptake.33
The monosubstituted iridium(III) complexes are significantly
more hydrophobic and possibly this favors the uptake.
Because of their relatively high uptake, the cellular
distribution of complex 17, 18, and 19 was studied in more
detail using confocal microscopy (Figure 6). After incubating
the cells with 10 μM 17−19 for 1 h at 4 and 37 °C, different
types and intensities of cell staining were observed.34 Upon
incubation of cells at 4 °C, cellular metabolism is shut down
and therefore, a limited amount of cellular dye uptake is
expected under these conditions (nonspecific uptake through
the membrane), whereas at 37 °C cellular metabolism is fully
functional and more specific dye uptake in the cell can be
expected, for example, via membrane vesicles. At 4 °C, slight
incorporation of complex 17 (Figure 6A) occurred; during the
imaging procedure the cells were maintained at 37 °C, which
resulted in stronger uptake of the dye. This uptake seems to
occur through the lysosomes as a similar staining pattern was
observed when 4T1 cells were incubated with Lysotracker
(Figure 6B). Very little to no staining was observed when 4T1
cells were incubated at 4 °C with complex 18 or 19,
respectively (Figure 6A). On the other hand, incubation of
4T1 cells with complex 17, 18, or 19 at 37 °C resulted in
significantly more intense staining. In contrast to complex 18
and 19, strong staining throughout the cells was observed for
complex 17 (Figure 6A), except in the nucleus (compared to
nucleus staining in Figure 6B). For complex 18 and 19, the
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Inorganic Chemistry
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From the quantum yield, shown in Table 2, it can also be
concluded that the fully cyclometalated iridium(III) complexes
maintain their relatively high luminescence in solution/cells.
Moreover, the quantum yield of the complexes lies within the
same range (0.11−0.15) and for this reason the cellular uptake
determined by flow cytometry of the different amino acid
conjugated iridium(III) complexes can be compared.
Flow cytometric analysis (common PI uptake assay; Figure
7) was used to to determine the influence of the different
Figure 6. Confocal microscopy images of the cellular distribution of
the various lysine substituted iridium(III) complexes. (A) Difference in
distribution of the number of lysine residues from left to right: mono-
(17), bis- (18), and tris- (19) substituted complexes. Cells were
incubated for 1 h at 4 °C (top row) or 37 °C (bottom row) with 10
μM complex in 0.3% DMSO/cell culture medium. Cell transmission
images were overlaid with the luminescence image. (B) Various cells
structures: nucleus (staining with DAPI), endosomes (staining with
transferrin conjugate), and lysosomes (staining with Lysotracker). Cell
transmission images were overlaid with the luminescence image.
Figure 7. Cell viability after treatment of 4T1 cells with complexes
11−19.
iridium(III) complexes have on the tumor cells. Only the lysine
functionalized complexes showed reduced cell viability.
Viability further decreased with the number of lysine
substituents from 71% and 70% to 39%, from the mono and
bis, respectively, to the tris functionalized complex. This
observation runs in parallel with the increasing positive charges
on complexes 17, 18, and 19.
staining patterns again suggested that the complexes incorpo-
rate through the lysosomes (Figure 6B). In vitro spectroscopic
measurements of the luminescence spectra all showed a
maximum emission around 580 nm (see Table 2), proving
Table 2. Photophysical Data of Complexes 11−19
CONCLUSION
■
compound
λabs/nm (103 M−1 cm−1
)
λem/nm λexc 480 nm
Φ
In conclusion, a series of nine iridium(III) complexes with the
archetypical parent Ir(ppy)3 as core unit, has been synthesized,
fully characterized by 1D and 2D NMR spectroscopy, and the
cellular uptake pattern has been investigated. The number
(mono-, bis-, tris-) and type (glycine, alanine, lysine) of amino
acids influence both the cellular uptake and, to a lesser extent,
intracellular localization of the complex. All three monosub-
stituted iridium(III) complexes gave a remarkably higher
cellular uptake than the bis- and tris- substituted complexes.
Iridium(III) complexes conjugated to lysine show in general a
higher uptake with respect to the correspondingly substituted
glycine and alanine derivatives. Moreover, the lysine derivatives
show an increased cellular toxicity. The high quantum yield of
the dyes, combined with their relative high cellular uptake,
makes the fully cyclometalated tris(ppy) iridium(III) complexes
a promising class of luminescent labels for (bio)imaging agents,
allowing finetuning of the properties of the complexes. Further
investigations are currently focused on integration of amino-
acid functionalized iridium(III) complexes in (automated)
peptide synthesis and biomarker specific targeting agents.
11
12
13
14
15
16
17
18
19
382(9,096), 483(1,187)
401(7,402), 482(1,858)
405(8,443), 485(2,013)
382(7,152), 484(1,444)
401(6,554), 483(2,005)
402(7,697), 480(2,369)
384(10,653), 484(2,222)
400(10,460), 485(2,892)
402(10,504), 484(3,157)
579
576
564
579
570
567
585
576
567
0.12
0.13
0.11
0.11
0.12
0.13
0.13
0.14
0.15
the intracellular luminescence was indeed caused by the
respective iridium(III) complexes. Similar to Fernandez-
Moreira et al.,35 under all conditions tested, no nuclear staining
was observed (Figure 6B). However, as shown in Supporting
Information, Figure S13, we found nuclear staining of complex
19 when we used a higher percentage (1%) of DMSO during
incubation, as observed by others.21,36 In the bright field images
of the stained cells a dark contrast was observed at locations
that had a high luminescence intensity, that is, the nucleoli.
This increase in light attenuation corroborates with the high
local concentration of iridium(III) complexes as observed from
the luminescence images. Reference staining for the nucleus,
endosomes, and lysosomes were obtained in separate cell-
culture samples of the same 4T1 cell line (Figure 6B).
ASSOCIATED CONTENT
■
S
* Supporting Information
The aromatic region of HH-COSY, HC-HMBC and HC-
HMQC spectra of compounds 3, 4, 5, and 7. Additional
2112
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Inorganic Chemistry
Article
(13) (a) Chen, F. F.; Bian, Z. Q.; Liu, Z. W.; Nie, D. B.; Chen, Z. Q.;
Huang, C. H. Inorg. Chem. 2008, 47, 2507−2513. (b) Chen, F. F.;
Bian, Z. Q.; Lou, B.; Ma, E.; Liu, Z. W.; Nie, D. B.; Chen, Z. Q.; Bian,
J.; Chen, Z. N.; Huang, C. H. Dalton Trans. 2008, 37, 5577−5578.
(14) DeRosa, M. C.; Hodgson, D. J.; Enright, G. D.; Dawson, B.;
Evans, C. E. B.; Crutchley, R. J. J. Am. Chem. Soc. 2004, 126, 7619−
7626.
confocal images of compound 19. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: a.h.velders@utwente.nl; Aldrik.Velders@wur.nl.
■
(15) Lo, K. K.-W.; P.-K. Lee, P.-K.; Lau, J. S.-Y. Organometallics 2008,
Notes
13, 2998−3006.
The authors declare no competing financial interest.
(16) Yu, M.; Zhao, Q.; Shi, L.; Li, F.; Zhao, Z.; Yang, H.; Yia, T.;
Huang, C. Chem. Commun. 2008, 44, 2115−2117.
ACKNOWLEDGMENTS
The authors would like to thank Tieme Stevens, Bianca
Snellink-Ruel, Frank van Diepen, and Anita Pfauth for their
■
(17) Zhao, Q.; Yu, M.; Shi, L.; Lui, S.; Li, C.; Shi, M.; Zhou, Z.;
Huang, C.; Li, H. Organometallics 2010, 29, 1085−1091.
(18) Kim, J. I.; Shin, I.-S.; Kim, H.; Lee, J.-K. J. Am. Chem. Soc. 2005,
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technical support.
(19) Zhang, K. Y.; Lo, K. K.-W. Inorg. Chem. 2009, 48, 6011−6025.
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