Increasing the triplet lifetime and extending the ground-state absorption of biscyclometalated Ir(III) complexes for reverse saturable absorption and photodynamic therapy applications

Article abstract of DOI:10.1039/c6dt02416e

The synthesis, photophysics, reverse saturable absorption, and photodynamic therapeutic effect of six cationic biscyclometalated Ir(iii) complexes (1-6) with extended π-conjugation on the diimine ligand and/or the cyclometalating ligands are reported in this paper. All complexes possess ligand-localized ¹π,π? absorption bands below 400 nm and charge-transfer absorption bands above 400 nm. They are all emissive in the 500-800 nm range in deoxygenated solutions at room temperature. All complexes exhibit strong and broad triplet excited-state absorption at 430-800 nm, and thus strong reverse saturable absorption for ns laser pulses at 532 nm. Complexes 1-4 are strong reverse saturable absorbers at 532 nm, while complex 6 could be a good candidate as a broadband reverse saturable absorber at 500-850 nm. The degree of π-conjugation of the diimine ligand mainly influences the ¹π,π? transitions in their UV-vis absorption spectra, while the degree of π-conjugation of the cyclometalating ligand primarily affects the nature and energies of the lowest singlet and emitting triplet excited states. However, the lowest-energy triplet excited states for complexes 3-6 that contain the same benzo[i]dipyrido[3,2-a:2′,3′-c]phenazine (dppn) diimine ligand but different cyclometalating ligands remain the same as the dppn ligand-localized ³π,π? state, which gives rise to the long-lived, strong excited-state absorption in the visible to the near-IR region. All of the complexes exhibit a photodynamic therapeutic effect upon visible or red light activation, with complex 6 possessing the largest phototherapeutic index reported to date (>400) for an Ir(iii) complex. Interactions with biological targets such as DNA suggest that a novel mechanism of action may be at play for the photosensitizing effect. These Ir(iii) complexes also produce strong intracellular luminescence that highlights their potential as theranostic agents.

Full text of DOI:10.1039/c6dt02416e

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DOI: 10.1039/C6DT02416E
Table of Contents Entry
Tuning the photophysics of cationic Ir(III) complexes via π-expansive ligands dramatically impacts their
applications as broadband reverse saturable absorbers and in photodynamic therapy and theranostic.
0.06
0.8
600
0.6
400
0.4
0.04
0.2
200
1E-5
1E-4
Incident Energy (J)
ε
ε
ε
ε
0.02
0.00
600
700
800
900
Wavelength (nm)
-0.02
-0.04
400
500
600
700
800
Wavelength (nm)

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DOI: 10.1039/C6DT02416E
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Increasing the triplet lifetime and extending the ground-state
absorption of cationic biscyclometalated Ir(III) complexes by
tuning ligand -conjugation for applications in reverse saturable
absorption and photodynamic therapy
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Chengzhe Wang,a Levi Lystrom,a Huimin Yin,b Marc Hetu,b Svetlana Kilina,a Sherri A. McFarland*b
www.rsc.org/
and Wenfang Sun*a
The synthesis, photophysics, reverse saturable absorption, and photodynamic therapeutic effect of six cationic
biscyclometalated Ir(III) complexes (1-6) with extended -conjugation on the diimine ligand and/or the cyclometalating
ligands are reported in this paper. All complexes possess ligand-localized 1,* absorption bands below 400 nm and
charge-transfer absorption bands above 400 nm. They are all emissive in the 500-800 nm range in deoxygenated solutions
at room temperature. All complexes exhibit strong and broad triplet excited-state absorption at 430-800 nm, and thus
strong reverse saturable absorption for ns laser pulses at 532 nm. Complexes 1-4 are strong reverse saturable absorbers at
532 nm, while complex 6 could be a good candidate as broadband reverse saturable absorber at 500-850 nm. The degree
of -conjugation of the diimine ligand mainly influences the 1,* transitions in their UV-vis absorption spectra, while the
degree of -conjugation of the cyclometalating ligand primarily affects the natures and energies of the lowest singlet and
emitting triplet excited states. However, the lowest-energy triplet excited states for complexes 3-6 that contain the same
benzo[i]dipyrido[3,2-a:2',3'-c]phenazine (dppn) diimine ligand but different cyclometalating ligands remain the same as
the dppn ligand-localized 3,* state, which gives rise to the long-lived, strong excited-state absorption in the visible to the
near-IR region. All of the complexes exhibit a photodynamic therapeutic effect upon visible or red light activation, with
complex 6 possessing the largest phototherapeutic index reported to date (>400). Interactions with biological targets such
as DNA suggest that a novel mechanism of action may be at play for the photosensitizing effect. These Ir(III) complexes
also produce strong intracellular luminescence that highlights their potential as theranostic agents.
(and other) favorable optical properties, such complexes are
rarely explored in context of reverse saturable absorption
(RSA),7 probably due in part to a lack of well-established
structure-property correlations with respect to organometallic
complexes and RSA.
RSA is a nonlinear optic (NLO) process whereby a
molecule absorbs more photons in its excited state than its
ground state.8 This NLO property can be used for optical
rectification,9 laser pulse compression and stabilization,10 and
optical switching.11 The effectiveness of RSA is mainly
governed by the ratio of absorption cross sections between the
ground and excited states.12 To produce efficient RSA with
Introduction
Biscyclometalated iridium (III) complexes bearing diimine
ligands have attracted much interest over the past two decades
because the spin-orbit coupling coefficient of iridium is among
the largest, which results in very efficient intersystem crossing
(ISC) in such systems.1 This heavy-atom induced singlet-triplet
mixing produces intense phosphorescence at room
temperature,2 which has been widely exploited for organic
light-emitting diodes (OLEDs),3 light-emitting electrochemical
cells (LEECs),4 biosensors,5 and chemosensors.6 Despite these
nanosecond laser pulses, a long-lived triplet excited state with
strong absorption is required.13 Transition-metal complexes
(Pt,14 Fe,15 Ru,16 Hg,14i,17 Au,14i,17 etc.) are attractive for this
purpose because the heavy-atom induced SOC increases the
quantum yield for triplet state formation. However, efficiently
populating the triplet excited state via one-photon absorption of
visible to near-IR (NIR) light (400 – 900 nm) while maintaining
a large triplet state absorption cross-section and long lifetime
remains a challenge.
a.Department of Chemistry and Biochemistry, North Dakota State University, Fargo,
ND 58108-6050, USA. E-mail: Wenfang.Sun@ndsu.edu
b.Department of Chemistry, Acadia University, 6 University Avenue, Wolfville, NS
B4P 2R6, Canada. E-mail: sherri.mcfarland@acadiau.ca
Electronic Supplementary Information (ESI) available: Experimental details, NTOs
for the high energy and intermediate energy absorption bands, comparison of
experimental and computational UV-vis absorption spectra of 1-6, excitation
spectra of 1-6, solvent-dependent UV-vis and emission spectra and emission
characteristics of 1-6, time-resolved ns transient absorption spectra of ligands and
complexes 1-6, in vitro dose-response curves for 1-3, confocal luminescence
images of SK-MEL-28 cells dosed with 1-3 in the dark or activated by visible light,
DNA photocleavage with 1-3 and visible light, See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 2016
Dalton Trans., 2016, 00, 1-11 | 1
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A long-lived triplet excited state, high triplet quantum complexes yield the highest light potencies and largest
View Article Online
DOI: 10.1039/C6DT02416E
yield, and a broad ground-state absorption in the NIR region are phototherapeutic margins to date.
also desirable features for biomedical applications such as
We have also previously demonstrated that the triplet state
photodynamic therapy (PDT).18 Briefly, PDT is a selective lifetimes of Ir(III) complexes containing -conjugated aromatic
means of destroying tumors and tumor vasculature by applying substituents appended to diimine ligands are drastically
a light trigger to activate an otherwise nontoxic compound lengthened ( = 11.3 s) in comparison to the analogous
called a photosensitizer (PS). The excited triplet state of the PS complexes without these substituents.23 As observed for the -
then reacts with ground state oxygen to produce cytotoxic expansive Ru(II) complexes, the long lifetimes were achieved
reactive oxygen species (ROS) through Type I (electron by extending the 휋-conjugation of certain coordinating ligands
3
3
transfer) or Type II (energy transfer) mechanisms. Singlet to lower the energy of the IL state below that of the MLCT.
oxygen that is generated via Type II mechanism has been Because the Ru(II) complexes bearing these highly -
implicated as the most important mediator of PDT’s anticancer conjugated ligands exhibit long-wavelength activation (>600
effects.19 Despite much promise, PDT is underutilized and is nm) and high singlet oxygen quantum yields,24,25 which are
not a mainstream approach for treating cancer, owing in part to attractive features for PDT, we were inspired to explore Ir(III)
the absence of PSs with the aforementioned properties.
It has been reported that strong SOC makes direct
complexes with similar properties as PSs for PDT.
We designed six Ir(III) cyclometalated complexes (Chart
absorption from the singlet ground state to the lowest triplet 1) to interrogate the effects of extending the
휋-conjugation of
excited state possible in some Ir(III) complexes.20 Such the diimine ligand (1-3) versus the cyclometalating ligand (4-6
)
transitions give rise to very weak but broad ground-state on the ground and triplet excited state absorption and the triplet
absorption of visible and NIR light. For example, Ir(III) lifetime. Their applications as reverse saturable absorbers and
complexes bearing 2,3-diphenylbenzo[g]quinoxaline (dpbq) PSs for PDT are reported herein.
cyclometalating ligands20d possess a weak but broad ground-
state absorption from 600-800 nm due to this direct absorption
from the singlet ground state to the triplet excited state. This
weak ground-state absorption provides another path for
populating the triplet excited state in addition to ISC from the
singlet excited state. On the other hand, this efficient ISC also
facilities the decay of the lowest triplet excited state, reducing
the triplet lifetime. Balancing these two effects is critical for
developing long-lived and broadband-absorbing (in both the
ground state and excited state) transition-metal complexes for
RSA and PDT applications.
It is well known that the triplet excited state lifetime is
mainly governed by the nature and energy of the lowest triplet
excited state (T1), which could be of metal-to-ligand charge
transfer (MLCT), metal-centered (MC), ligand-to-metal charge
transfer (LMCT), ligand-to-ligand charge transfer (LLCT),
intraligand charge transfer (ILCT), intraligand (IL or ,*), or
metal-to-metal charge transfer (MMCT) character, or mixtures
of such states. Previous studies involving Ru(II) complexes
have shown that increasing the degree of -conjugation of
certain diimine ligands can lengthen triplet lifetimes
dramatically. For example, the 750-ns triplet lifetime of
[Ru(bpy)2dppz]2+
dipyrido[3,2-a:2',3'-c]phenazine) is lengthened to 33
the extension of the -system by one fused benzene ring to
yield [Ru(bpy)2dppn]2+ (dppn = benzo[i]dipyrido[3,2-a:2',3'-
c]phenazine).21 This is made possible when the lowest energy
3IL state drops below 2.1 eV, the energy of the lowest MLCT
state in typical Ru(II) polypyridyl complexes. Similarly, our
own investigations with Ru(II) complexes bearing the 5-(pyren-
1′-ylethynyl)-1,10-phenanthroline (5-PEP) ligand have yielded
T1 lifetimes as long as 240 s for complexes of the type
[Ru(bpy)2(5-PEP)]2+ and 270 s for the homoleptic [Ru(5-
PEP)3]2+.22 In the same study, we highlighted the utility of such
long-lived 3IL states for PDT by demonstrating that these
(bpy=2,2’-bipyridine
and
dppz
=
±5 휇
s with
Chart 1 Structures of Ir(III) complexes 1-6
.
Results and discussion
Synthesis and characterization
3
The synthetic route for complexes 1–6 is shown in Scheme 1, and
the synthetic details and characterization data are provided in the
Experimental Section. All of the ligands were synthesized according
to the literature procedures.20d,26 The procedure reported by
Nonoyama was followed to convert the cyclometalating ligands into
the chloro-bridged dinuclear Ir(III) precursors.27 Reactions of the
dinuclear Ir(III) complexes with the corresponding diimine ligand in
2 | Dalton Trans., 2016, 00, 1-12
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the presence of AgSO3CF3 catalyst in mixed CH2Cl2/CH3OH coefficients in this region (~ 5 – 10  104 L/mol.cmV,ieTwaAbrtliecle1O)nalinree
solvent28 gave the desired mononuclear Ir(III) complexes 1-6. The consistent with 1,* transitions (referred to
DOI: 10.10a3s9/C16IDLT02w4h16eEn
1
intermediate compounds were confirmed by H NMR spectroscopy, incorporated into a metal complex) localized on the diimine or the
while the ligands and the Ir(III) complexes were characterized by 1H cyclometalating ligands. This assignment was supported by the
NMR, HRMS, and elemental analyses.
natural transition orbitals29 (NTOs, ESI Table S1) obtained from the
TDDFT calculations, which also indicated minor contributions from
charge transfer (CT) transitions in this energy range. Figure 1A
clearly shows that the most intense band observed for complexes 1-3
in this region shifted bathochromically in a systematic manner with
increasing -conjugation of the diimine ligand. In contrast, there was
no shift in the most intense UV band (~320 nm) for 4-6, but the
molar extinction coefficients were noticeably different. The NTOs
(ESI Table S1) revealed considerable contributions from the CT
transitions (i.e. 1MLCT, 1LLCT, and 1ILCT) in addition to the major
1
dppn-localized ,* transition for 5 and 6, which do not contribute
as much for
4 due to the smaller -conjugation of the
cyclometalating ligand in 4. The larger contributions from the CT
transitions decreased the intensity of these high-energy absorption
bands for 5 and 6 relative to that observed for 4.
Table 1 Photophysical data of complexes 1-6 in acetonitrile.
λabs / nm ( / 104 L.mol-1.
λem / nm (em
s; em
588 (2.98;
0.22)
/
λT1-Tn / nm (T / s;  T1-Tn
/ 104 L.mol-1.cm-1; T)
516 (3.19; 7.6; 0.25)
cm-1)
)
1
257 (7.19); 289 (5.65);
340 (2.01); 378 (1.36);
434 (0.7)
2
3
4
5
278 (6.86); 354 (2.16);
379 (1.61); 434 (0.58)
291 (6.84), 322 (9.21),
395 (1.68), 420 (1.79)
322 (9.93), 397 (1.66),
418 (1.87), 456 (0.65)
319 (8.21), 375 (3.49),
394 (3.19), 417 (2.12),
464 (0.87)
590 (0.40;
0.034)
591 (2.69;
0.026)
554 (2.52;
0.015)
629 (2.39;
0.083)
518 (0.43; 19.2; 0.052)
536 (35.7; 19.9; 0.12)
537 (36.9; 11.1; 0.23)
537 (39.6; 19.6; 0.069)
6
322 (9.29), 399 (2.51),
417 (2.57), 476 (0.92),
537 (0.39)
553 (0.06; -a), 537 (15.1; 31.8; 0.054)
774 (-;-)a
a Too weak to be measured.
The less intense bands in the range of 320-400 nm for
complexes 1-3 and 350-450 nm for 4-6 have mixed
1,*/1LLCT/1MLCT/1ILCT/1LMCT character in view of their
relatively large extinction coefficients (which are on the order of 2 –
4  104 L/mol.cm) and the NTOs shown in ESI Table S2. When the
-conjugation was increased on the diimine ligands, the intensities of
these absorption bands gradually increased from complex 1 to
complex 3. In line with this trend, the increased -conjugation on the
cyclometalating ligands also led to increased absorption for the band
near 420 nm for complexes 4-6. However, the intensity of the band
at ca. 400 nm for complexes 4-6 does not follow this trend, with
complex 5 possessing the strongest absorption and complex 4
showing the weakest absorption among these three complexes. This
can be explained by the different characters of contributing
transitions to this band (see the NTOs for S10 of complex 4, S9, S10,
and S18 of complex 5, and S15, S16, and S20 of complex 6 in Table
S2). As these NTOs indicate, the major contributing transitions to
this band in complex 4 are 1,*(dppn)/1MLCT; while in complex 5
are the 1,*(dppn)/1MLCT mixed with 1ILCT/1LLCT. The
additional contribution from the 1ILCT/1LLCT transitions in
Scheme 1 Synthetic routes for complexes 1-6.
UV-vis absorption
The UV-vis absorption of all of the ligands and complexes were
measured in acetonitrile solutions. Concentration dependence
experiments (210-6 mol/L to 110-4 mol/L solutions) indicated that
Beer’s law was obeyed, suggesting the absence of ground state
aggregation due to the octahedral geometry of the Ir(III) complexes.
All complexes showed intense and structured UV-vis
absorption at wavelengths below 350 nm (below 320 nm for
complexes 1-3) (Figure 1). The magnitudes of their molar extinction
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complex 5 makes this band stronger than that in complex 4. On the nature of this band in complex 6 accounts for the decreased intensity
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contrary, the charge transfer transitions (i.e. LLCT/1MLCT/1ILCT) of this band in comparison to the predominant ,*(dppn)/ MLCT
1
1
1
DOI: 10.1039/C6DT02416E
contribute predominantly to this band in complex 6, with minor band in complex 5 in the similar energy region.
1
contribution from the ,*(dpbq). The predominant charge transfer
1x105
600
A
1
2
3
C
4
5
6
2000
1000
0
8x104
6x104
4x104
400
200
0


500
600
700
800
900
600
700
800
900

Wavelength (nm)
Wavelength (nm)
2x104
0
1 Computed
2 Computed
3 Computed
4 Computed
5 Computed
6 Computed
D
B
200
300
400
500
600
700 300
400
500
600
700
800
Wavelength (nm)
Wavelength (nm)
Fig. 1 UV-Vis absorption spectra of complexes 1-6 in acetonitrile. Panels A and C: experimental spectra; Panels B and D: calculated spectra.
In addition to the aforementioned structured absorption bands, When the -conjugation on the diimine ligand was increased on
each complex exhibited broad and featureless absorption band(s) at going from complex 1 to complex 3, the energy of the diimine ligand
400-500 nm for complexes 1-3 and 450-600 nm for complexes 4-6. localized * orbital decreased (i.e. the LUMO energy decreased, see
Considering the moderate intensity ( ~ 103 – 104 L/mol.cm) and the the calculated ground-state energy diagram displayed in Fig. 2),
structureless features, we attribute these bands mainly to charge- while the (Ph)/d(Ir) based HOMO energy was unchanged, resulting
transfer transitions (1MLCT/1LLCT and/or 1ILCT), likely mixed in a decreased HOMO-LUMO gap. This systematic decrease in the
1
with some ,* character. This assignment was supported by the energy of S1 from 1 to 3 is evident as a red-shift in the lowest-energy
TDDFT calculations (see NTOs illustrated in Table 2 for these low- absorption bands. Although the nature of S1 differed between 4-6,
energy absorption bands). For the lowest-energy transitions in these there was also a systematic gradual decrease in the S1 state with
complexes (S1 state), the promoted electrons are all exclusively increased -conjugation on the cyclometalating ligands.
localized on the diimine ligand except for complex 6, in which the
A close examination of the UV-vis absorption spectra of these
electron is delocalized on the cyclometalating dpbq ligand and the d- complexes revealed a very weak tail beyond 500 nm for complexes
orbital of the Ir(III). While the holes are primarily on the phenyl 1-3 and past 600 nm for complexes 4-6. As reported for other Ir(III)
rings of the cyclometalating ligand and the d-orbital of the Ir(III) complexes,20 these bands are most likely due to direct S0-Tn
except for complex 4, which has the hole localized on part of the absorption via the spin-forbidden 3,*/3CT transition. Extending
dppn ligand. According to the electron and hole distributions, the the -conjugation on the cyclometalating ligand dramatically
nature of the S1 states for complexes 1-3 and 5 can be attributed to expanded this band into the red to near-IR region, especially for
1
1MLCT/1LLCT; while for complex 4 it has ,*/1ILCT character, complex 6 , which could potentially be exploited for both RSA and
and complex 6 has major 1ILCT/1MLCT character mixed with some PDT, as discussed in the introduction.
contributions from 1,*/1LMCT.
4 | Dalton Trans., 2016, 00, 1-12
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Table 2 Natural transition orbitals (NTOs) representing transitions contributing to the low-energy absorption bands of
complexes 1−6 in CH3CN.
View Article Online
DOI: 10.1039/C6DT02416E
Sn
Holes
Electrons
Sn
Holes
Electrons
1
4
S1
S1
464 nm
f = 0.006
507 nm
f = 0.021
S2
S3
436 nm
f = 0.112
437 nm
f = 0.008
S1
S1
2
5
490 nm
f = 0.002
512 nm
f = 0.011
S2
S2
449 nm
f = 0.009
506 nm
f = 0.019
S3
S3
435 nm
f = 0.107
494 nm
f = 0.055
S1
S1
3
6
533 nm
f = 0.001
541 nm
f = 0.050
S2
S2
504 nm
f = 0.021
510 nm
f = 0.016
S5
S6
435 nm
f = 0.109
470 nm
f = 0.049
1
2
3
4
5
6
-2
-3
-2
-3
d

*(piq)/ (Ir)

*(N^N)/

*(piq)
d


d
2
z
*(piq)/ (Ir)
*(dpp)/ (Ir)
*(N^N)


*(N^N)


*(N^N)
delocalized
d


(Ir)/ *(piq)
*(N^N)
d


d
*(piq)/ (Ir)
*(piq)/ (Ir)

d

*(dpp)/ (Ir)
*(N^N)


*(N^N)
*(N^N)/d (Ir)

*(N^N)
*(N^N)
*(N^N)
*(N^N)



*(N^N)
*(dpqx-N)/ (Ir)
d

*(dpqx)/d (Ir)



*(dpbq)/d(Ir)
*(dpbq)/d(Ir)

*(N^N)

*(N^N)

*(N^N)
*(N^N)


*(N^N)



(Ir)/ (Ph)
d (Ir)/ (Ph)
d (Ir)/ (Ph)
d
d
-6
-7
-6
-7

d
(Ir)/ (dpbq)
d(Ir)/ (dpqx)


d

(Ir)/ (Ph)

(N^N)

(N^N)
(dpbq-N)/d(Ir)
(dpbq)/ (Ir)

(dpp)
(N^N)



2
(piq)/ (Ir)
(Ir)/ (piq)

(piq)/d (Ir)
(piq)/ (Ir)

d

dz
dz
2
d


(Ir)/ (Ph)
d (Ir)/ (Ph)

dz
(dpp)/ (Ir)
d
(dpqx)/ (Ir)

d

(dpbq)

2
(Ir)/ (dpp)
d (Ir)/

(dpqx)


(Ir)/ (Ph)
d
(Ir)/ (Ph)
d


(dpqx)/d (Ir)
d (Ir)/ (Ph)
dz
dz
2

2

(Ir)/ (piq)
(Ir)/ (piq)

(dpp)
Fig. 2 Ground-state energy diagram for complexes 1-6 in CH3CN.
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DOI: 10.1039/C6DT02416E
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characteristics to the NTOs representing the 1MLCT/1LLCT
transitions, it is easy to understand the red-shifted emission of 5 with
respect to that of 4.
Photoluminescence
The room temperature emission from complexes 1-6 in different
solvents was studied. The observed luminescence was sensitive to
oxygen, with lifetimes in the range of hundreds of nanoseconds to
several s in deoxygenated solutions (except for complex 6, vide
infra). The magnitude of measured Stokes shifts (the difference
between the emission energy and the excitation energy) were in the
range of 5015-6946 cm-1. These facts suggested that the observed
emission in all cases was phosphorescence.
1
2
1.0
3
4
5
6
0.8
0.6
0.4
0.2
0.0
Fig. 3 illustrates the emission spectra of complexes 1-6 in
CH3CN. The emission lifetimes and quantum yields in CH3CN are
listed in Table 1. The emission spectra, lifetimes, and quantum
yields in other solvents are provided in ESI Fig. S3 and Table S4.
The emission energies and spectral features of complexes 1-3
resemble each other, with emission maxima near 590 nm and a
shoulder around 615 nm. The lifetimes in deoxygenated solutions of
1-3 are similar (~2.5-3.0 s in all solvents studied except that the
lifetime of 2 in CH3CN (400 ns) is much shorter). These features
indicate that the origin of the emitting states for these complexes
500
600
700
800
Wavelength (nm)
should be the same, i.e. likely the MLCT/3LLCT states with the
3
Fig.
3
Normalized emission spectra of complexes 1-6 in
deoxygenated acetonitrile. The excitation wavelength was 435 nm
for 1, 436 nm for 2, 419 nm for 3, 418 nm for 4, 466 nm for 5, and
436 nm for 6. The concentration used was 110-5 mol/L.
electrons presumably localized on the phenyl ring of the
cyclometalating ligand and the d-orbital of the Ir(III), and holes on
the dpq (dipyrido[3,2-d:2',3'-f]quinoxaline) part of the diimine
ligand. A lack of correlation between the emission energy and -
conjugation of the diimine ligand as well as emission assigned to the
3MLCT/3LLCT state have been reported for Ru(II) complexes with
dppz and dppn ligands.21
The emission of 6 is distinct from all of the other complexes.
This complex exhibited dual emission, with a broad high-energy
emission band at 553 nm and a low-energy emission band at 774 nm.
The excitation spectra monitored at the band maxima of these two
emission bands were different (see ESI Fig. S5), indicating the
different natures of the emitting states. Considering the similar
energy of the high-energy emission band of 6 to that of 4, we
attribute this emission originating from the 3MLCT/3LLCT states.
For the low-energy emission band at 774 nm, we ascribe it
For complexes 4-6, the emission features (such as energies,
lifetimes and shapes) are drastically different. When the -
conjugation of the cyclometalating ligand increased from complex 4
to 5, the emission energy significantly decreased (λem changed from
554 nm for 4 to 629 nm for 5). However, the emission lifetimes of 4
and 5 were on the same order of magnitude in all solvents studied. In
view of their structureless emission spectra, the thermally induced
Stokes shifts (see ESI Fig. S4), and their lifetimes, we tentatively
assign the emission from these two complexes to 3MLCT/3LLCT
states with the electrons presumably localized on the coordinating
phenyl ring of the cyclometalating dpp or dpqx ligand and the d-
orbital of the Ir(III), and holes on the dppn ligand. The red-shifted
emission of 5 could probably be attributed to the better electron
delocalization on the dppn ligand in 5 compared to that in 4, which
stabilized the dppn based holes in 5. Although the NTOs
corresponding to the triplet transitions contributing to the emitting
states of 1-6 were unavailable at this time, the 1MLCT/1LLCT
transitions in 4 (S3 state) and 5 (S1 state) (see the NTOs in Table 2)
clearly indicated the more electron density delocalization on the
dppn based electrons in 5. Assuming the NTOs representing the
triplet transitions contributing to the emission have the similar
3
predominantly to the dpbq localized ILCT/3,* emission. Such an
assignment is primarily based on the similar energy of this band to
the phosphorescence energy of the dpbq ligand (765 nm, see ESI
Fig. S6). The possibility of this low-energy emission band arising
from the trace amount of dpbq ligand or the [(dpbq)2IrCl]2 dimer
precusor has been excluded based on the following facts: First, TLC
analysis and elemental analysis results confirmed the 99.5% purity
of complex 6; secondly, the emission spectra obtained at a varity of
excitation wavelengths all had the same dual emission feature,
indicating the dual emission arising from a single species; thirdly, if
trace amount of dpbq ligand was present in the complex, its
fluorescence at 480 nm should be observed upon excitation at the
UV region, however, it was not detected; fourthly, even if trace
amount of [(dpbq)2IrCl]2 was present, its emission at 780 nm was
extremely weak, which could not give the detectable signals at 774
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3
nm. Therefore, we believe the dual emission truly emanating from lifetimes of 6 in comparison to those of 3-5, the ILCT states may
View Article Online
DOI: 10.1039/C6DT02416E
complex 6. It should be pointed out that although dual emission is an contribute to the transient signal of 6 as well. The presence of a non-
3
uncommon feature, it has been well documented in the literture for emissive low-energy, long-lived ,* state and an emissive short-
transition-metal complexes including Ir(III) and Ru(II) lived CT excited state has been previously reported for some
complexes.20a,30 The distinct nature of the emitting states for Ru(II)21,31 and Pt(II) complexes.32 This phenomenon suggests that
complexes 4-6 clearly reflects the impact of the extended - the 3CT state and 3,* state are not thermally equilibrated.
conjugation of the cyclometalating ligand on the emission, which Moreover, we speculate that the decay from the high-lying 3CT state
shifted the emitting state from 3MLCT/3LLCT in 4 and 5 to the dpbq via nonradiative process to the low-lying 3,* state is very
3
ligand-based ILCT state in 6. This effect was quite different from inefficient or completely blocked for reasons that are not well
that observed with extending -conjugation on the diimine ligand, understood, which makes the emission from the high-lying 3CT state
which essentially shows no impact on the emission energy of the possible.
Ir(III) complexes.
It is worth noting that although the nature of the emitting state
for complexes 3-6 varies, their lowest triplet excited states remain
centered on the extended -expansive dppn ligand. With the
Transient difference absorption
3
increased -conjugation on the C^N ligand, the dpbq-based ILCT
3
In order to further understand how the extended -conjugation on the
diimine and/or cyclometalating ligand influences the triplet excited-
state characteristics, nanosecond transient absorption (TA)
experiments were carried out to investigate the triplet excited-state
absorption and lifetime, as well as the triplet quantum yield. The TA
state could mix with the dppn ,* state due to a decreased energy
of the 3ILCT state that lies in proximity to the dppn 3,* state.
Reverse saturable absorption (RSA)
spectra of complexes 1-6 in deoxygenated acetonitrile at zero delay It is well known that RSA occurs when excited-state absorption is
after 355 nm excitation are illustrated in Fig. 4, and the time- stronger than ground state absorption. The TA spectra of complexes
resolved spectra for each complex are provided in ESI Fig. S8.
1-6 clearly demonstrates a strong positive absorption band in the
region of 450-800 nm, suggesting stronger excited-state absorption
compared to the ground-state absorption. Therefore, it is reasonable
0.08
to expect
a strong RSA in this spectral region. Nonlinear
1
2
transmission experiments were carried out to manifest the RSA at
532 nm using a 4.1-ns pulsed laser. The transmittance vs. incident
energy curves for complexes 1-6 are presented in Fig. 5.
0.06
0.04
0.02
0.00
-0.02
3
4
5
6
0.8
0.6
400
500
600
700
800
1
2
Wavelength (nm)
0.4
3
4
5
Fig. 4 Nanosecond transient absorption spectra of complexes 1-6 in
deoxygenated acetonitrile at zero delay after 355 nm excitation.
A355 = 0.4 in a 1-cm cuvette.
6
0.2
1E-5
1E-4
1E-3
The TA spectral features for complexes 1 and 2 were similar,
and their TA lifetimes agreed with their emissive lifetimes. These
characteristics suggest that the excited state giving rise to the
Incident Energy (J)
Fig. 5 Transmittance vs. incident energy curves of complexes 1-6 in
acetonitrile in a 2-mm cuvette for 4.1 ns laser pulses at 532 nm. The
linear transmission of the sample solutions were adjusted to 80% at
532 nm in the 2-mm cuvette. The radius of the beam waist at the
focal plane was approximately 96 m.
3
observed TA is also the emitting MLCT/3LLCT state. In contrast,
the TA spectra of 3-6 are similar to each other, but different from
those of 1 and 2. The TA lifetimes measured for 3-6 were much
longer than their respective emission lifetimes. In addition, the TA
spectral features of complexes 3-6 all resemble those of the dppn
ligand (see ESI Fig. S7) and the Ru(II) complex bearing the dppn
ligand.21 Thus, the triplet excited state giving rise to the observed TA
spectra for these four complexes should predominantly be the dppn
ligand-localized 3,* state. However, in view of the shorter
All complexes exhibited a moderate to strong transmission
decrease with increasing incident energy, a clear indication of RSA.
The degree of RSA for complexes 1-4 was similar, and was
comparable to the benchmark Ir(III) complexes with -conjugated
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aromatic substituents appended to diimine or cyclometalating
ligands.7d,20c,23,33 In contrast, RSA decreased in complexes 5 and 6 in
comparison to those of 1-4. Our previous studies on RSA materials
revealed that the key parameter in determining the degree of RSA is
the ratio of the excited-state absorption cross section to the ground-
state absorption cross section (ex/0). The 0 can be deduced from
the molar extinction coefficient at 532 nm from the UV-vis
absorption data, while the ex can be estimated from the OD values
at the TA band maximum and 532 nm, and the T1-Tn value at the TA
band maximum wavelength using the method described previously
by our group.34 The values obtained are presented in Table 3. Due to
very efficient heavy-atom induced ISC in the Ir(III) complexes, the
RSA of ns laser pulses could mainly arise from the triplet excited-
state absorption rather than the singlet excited-state absorption. Thus
the quantum yield of the triplet excited state should play a significant
role in determining the degree of RSA. Considering these factors, the
exT/0 ratios for complexes 1-6 roughly correlate to the observed
RSA trend for these complexes. With the increased -conjugation on
the cyclometalating ligand, the ground-state absorption at 532 nm
drastically increased in 5 and 6, which reduced the ex/0 ratio and
thus decreased the RSA at 532 nm. However, the broadened ground-
state absorption into the near-IR region would broaden the RSA
region for complex 6, making it a potential broadband RSA
complex.
given complex. In comparison to the relatively few PI values
View Article Online
reported for the other Ir(III) complexes,35f,35hDcOoI:m10p.l1e0x3e9s/C16-D6Ty0i2e4ld16eEd
the most potent photocytotoxicities and largest PIs reported to date
towards SK-MEL-28 and HL60 cell lines upon broad visible or red
light activation. It should be noted that previous reports of
photoactivatable Ir(III) complexes utilized UV or blue (425 nm)
light. Such high photon energy excitation would be expected to yield
even greater photobiological activity from the present series, but
these wavelengths are of less interest for practical application.
In general the Ir(III) complexes investigated were more potent
toward SK-MEL-28 melanoma cells than HL60 leukemia cells
regardless of whether a light trigger was applied, except for complex
6 which was more cytotoxic toward HL60 cells in the dark. Notably,
6 also produced the largest PI of the series in both cell lines (SK-
MEL-28, PI>400; HL60, PI>140) with submicromolar visible light
EC50 values (350-600 nM). The PDT effect observed for 6 was
attenuated approximately 10-fold with red light activation but was
still more than 40% larger than the best reported to date with UV
light activation towards HeLa cells35f (Fig. 6). Had the previous
reports used low-energy red light, this difference would have been
much larger. The large PDT effects of 6 upon both visible and red
light irradiation could partially be attributed to the broader and
stronger absorption in the visible and red regions in comparison to
the other complexes investigated in this work, which would populate
more PSs to the excited states under identical light fluence and thus
enhance the PDT effects. However, we demonstrated the feasibility
of utilizing the blue-green absorbing [Ru(bpy)2dppn]2+ complex for
effective red or NIR PDT even if the number of absorbed photons
was extremely low (  100 M-1cm-1) in the red-NIR region.25 The
efficient PDT at low photo absorption was attributed to the
extremely photosensitizing long-lived 3,* configuration. Taking
into account of this finding and the lack of correlation between the
light absorptivity and PIs for complexes 1-5, we speculate that the
strong PDT effect of 6 could be mainly ascribed to the extremely
high photosensitization efficiency of the 3,* configuration
although the strong and broad absorption in the visible to the NIR
region also contribute.
Table 3 Ground-state and excited-state absorption cross sections of
complexes 1-6 in acetonitrile.
1
2
3
4
5
6
0 / 10-18 cm2
ex / 10-18 cm2
1.6
270
173
43
1.6
710
432
22
3.9
780
197
24
2.5
380
151
35
8.4
730
87
6
15.7
130
8

ex/0
exT/0
0.4
Photodynamic therapy (PDT)
Reports on the use of Ir(III) complexes as PSs for PDT are rare.35
Due to the exceptionally long-lived triplet excited states of
complexes 3-6 measured by TA (as long as ~40 s), it was
reasonable to propose that these complexes could be good candidates
for PDT applications. We have previously shown that Ru(II)-based
metal-organic dyads possessing long-lived triplets contributed by -
expansive organic units with low-energy excited states (≤ 2.1 eV)
have proven to be highly effective as PDT agents.22,25,36 The
requirement appears to be a 3,* state that is in close energetic
Table 4 (Photo)cytotoxicity of complexes 1-6 towards SK-MEL-28
and HL60 cells.
Dark
Vis PDT
Red PDT
EC50 (M) EC50 (M)
0.400.07 0.0040.001
0.270.04 0.0030.001
1.530.03 0.0190.002
2.110.13 0.0290.003
1.370.07 0.0620.008
PI
EC50 (M)
PI
1
2
3
4
5
6
1
2
3
4
5
6
100 0.190.01
2.1
3
90
81
72
22
0.0340.002 7.9
proximity to the MLCT state, which lies at approximately 2.1 eV
SK-
0.250.03
1.830.08
0.650.07
6.1
1.2
2.1
32
for typical Ru(II) polypyridyl complexes. We hypothesize that slow
ISC from triplet excited states with substantial organic character
provides ample opportunity for bimolecular reactions with oxygen
and other quenchers.
To demonstrate that this concept could be operative in Ir(III)
systems, the photobiological activities of 1-6 were assessed in terms
of dark and light EC50 values and phototherapeutic indices (PIs)
using two cancer cell lines and two irradiation conditions (Table 4).
Briefly, EC50 refers to the effective concentration required to reduce
cell viability by 50%, and PI is the ratio of dark to light EC50 values.
Light treatments consisted of broadband visible or red light
irradiation (100 J/cm2) delivered 16 h after cells were dosed with a
MEL-28
14456.9
0.3540.066
407 4.450.08
0.43±0.03 0.0170.001
0.41±0.04 0.0100.001
1.57±0.05 0.060.01
4.51±0.13 0.0490.002
1.39±0.31 0.1170.006
83.9±1.40 0.5880.052
25
41
26
92
12
0.160.01
0.110.02
0.540.02
2.410.08
0.900.03
2.7
3.7
2.9
1.9
1.5
16
HL60
143 5.330.37
Of considerable importance for PDT applications in particular,
complex 6 was the only PS in the series that could be considered
8 | Dalton Trans., 2016, 00, 1-12
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nontoxic without a light trigger (dark EC50 >100 M), which was the but 6 was relocalized from nuclei to cytoplasm. WhileVtihewesAerticclheaOnnglienes
DOI: 10.1039/C6DT02416E
source of the large phototherapeutic margin. To the best of our are not conclusive of a particular type of cell death, they do serve to
knowledge, all other phototoxic Ir(III) complexes reported to date highlight the utility of such complexes as theranostic agents, namely
have considerable dark cytotoxicity. Typically these EC50 values are they integrate diagnostic capability with therapeutic capacity. Such
between 30 and 50 M,35f with some being as low as 8 M.35g platforms are of great interest in photomedicine.
Similar observations have been made for cyclometalated Ru(II)
complexes.37 Complexes 1 and 2 produced submicromolar toxicity
in the absence of any light trigger (Table 4 and ESI Fig. S9), and
complexes 3-5 displayed dark toxicity with EC50 values ranging
from 1 to 5 M. Despite PIs of up to 100, complexes 1-5 may be
better suited as traditional anticancer agents provided there is some
selectivity toward cancer cells over normal cells. For example,
complex 2 is 100-fold more potent than cisplatin toward melanoma
cells, and efforts are underway to probe for selectivity.
Fig. 7 Confocal luminescence images of SK-MEL-28 cells treated
with 50 M complex 4 (a) or 5 (b) and 6 (c) in the dark.
Fig. 6 In vitro dose-response curves for complexes 4 (a), 5 (b) and 6
(c) in SK-MEL-28 cells (left column) and HL60 cells (right column)
with visible light activation.
The emission observed from 1-6 could be used to track cellular
accumulation and distribution due to relatively bright intracellular
luminescence (Figs. 7-8 and ESI Figs. S10-S11). Laser scanning
confocal microscopy was used to detect the complexes in SK-MEL-
28 melanoma cells after a short incubation period (15 min). Cellular
uptake occurred both with and without a light trigger for all of the
complexes. However, the application of a light trigger influenced the
distribution of the PSs within the cell. For example, localization in
the membrane was evident for complexes 1-3 prior to light treatment
Fig. 8 Confocal luminescence images of SK-MEL-28 cells treated
(ESI Fig. S10). Application of a sublethal visible light treatment of
with 50 M complex 4 (a) or 5 (b) and 6 (c) with a visible light
50 J/cm2 caused the PSs to move from the cell membrane to the
treatment (50 J/cm2).
cytosol and mitochondria. Complexes 4-6 accumulated throughout
the cell without light activation, with some preference for the nuclei
of adherent cells and the cytosol of the suspension phenotype. Upon
Because some of the PSs appeared to accumulate in the nuclei
of cells, complexes 1-6 were probed for their abilities to interact with
light activation, complexes 4 and 5 caused a significant change in
plasmid DNA with and without a light treatment. Topological
cell shape such that it was difficult to compare localization trends,
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changes to plasmid DNA exposed to various exogenous agents and activated by light. Such a difference could also be applicable for
View Article Online
treatment conditions can be readily discerned by changes in interactions with potential non-genomic tarDgeOtIs: 1a0s.10w3e9l/lCa6sDTc0e2ll4u1l6aEr
electrophoretic mobility of the DNA through an agarose gel slab. uptake, efflux, metabolism, and localization. Typically singlet
Under the gel electrophoresis conditions employed (Fig. 9 and ESI oxygen generators that bind DNA well produce notable conversion
Fig. S12), the relative migration distances of plasmid DNA increase of Form I DNA to Form II in this gel mobility shift assay. The lack
in the order of aggregated (Form IV, induced aggregation or of significant Form II production coupled with clear evidence of
condensation), nicked circular (Form II, single-strand breaks), linear DNA interactions via aggregation points toward the involvement of
(Form III, two single-strand breaks in close proximity on opposite other reactive intermediates for cellular damage (unless DNA
strands or frank double-strand breaks), and supercoiled (Form I, no interactions suppress this sensitization pathway for some reason).
strand scission).
Plasmid pUC19 DNA (20 M nucleotide phosphates) was
dosed with complexes 1-6 at concentrations between 5 and 100 M
(PS-to-nucleotide ratios 0.25-5). The samples were either kept in the
dark or irradiated with visible light (14 J/cm2) and then
electrophoresed on 1% agarose gels in 1 TAE for 30 min. The
irradiation conditions used for the DNA experiments were
intentionally “softer” than those used for the cellular assays to ensure
that light did not damage the DNA in the absence of PS. Complexes
1-3 caused aggregation of plasmid DNA in a concentration-
dependent manner regardless of whether a light treatment was
applied (ESI Fig. S12, lanes 8-9). The major difference between the
dark and light treated samples was that photoactivation of complexes
1-3 produced a slight increase in Form II DNA concomitant with a
decrease in Form I with increasing concentration of PS in the order
of 3 > 2 > 1. As expected, the extent of PS-induced aggregation also
followed this order and was in agreement with what has been
observed with increasing -expansion, and thus intercalating power,
on diimine ligands in certain Ru(II) complexes. Complexes 1-3 did
not appear to interefere with the ability of ethidium bromide (EtBr)
to intercalate and stain the DNA.
When -conjugation was increased on the cyclometalating
ligand, PS-induced DNA aggregation also increased in the order of 6
> 5 > 4, with complexes 4 and 5 showing very little Form IV DNA
on the gel with or without a light trigger (Fig. 9). As observed for 1-
3, a slight increase in Form II over Form I occurred but was barely
detectable by eye. Unlike complexes 1-3, 6 showed a marked
difference in PS-induced DNA aggregation between the dark and
light-treated samples. Light activation of 6 produced 100% Form IV
DNA, while the dark treatment produced very little aggregated DNA
with no change in the relative amounts of Forms I and II. Complex 5
behaved differently than the rest of the photoactivated complexes in
Fig. 9 DNA photocleavage of pUC19 DNA (20 μM bases) dosed with
metal complex (MC) 4 (a), 5 (b) or 6 (c) and visible light (14 J/cm2).
Gel mobility shift assays employed 1% agarose gels (0.75 μg/mL
ethidium bromide) electrophoresed in 1 TAE at 8 V/cm for 30 min.
Lane 1, DNA only (-hv); lane 2, DNA only (+hv); lane 3, 5 µM MC
(+hν); lane 4, 20 µM MC (+hv); lane 5, 40 µM MC (+hv); lane 6, 60
µM MC (+hv); lane 7, 80 µM MC (+hv); lane 8, 100 µM MC (+hv);
lane 9, 100 µM MC (-hv). Forms I, II, and IV DNA refer to supercoiled
plasmid, nicked circular plasmid, and aggregated plasmid,
respectively.
that it caused the DNA bands on the gel to disappear at higher
concentrations with no influence on the dark sample. Quenching of
EtBr luminescence, competition for EtBr binding sites, or distortion
of the helix to prevent EtBr binding are normally implicated in the
lack of DNA staining by treated samples, especially at higher
concentrations of the exogenous agent. However, the dark sample
treated with 5 at high concentration did show luminescence from
intercalated EtBr. Therefore, light activation of 5 must influence its
binding mode and/or interaction with EtBr in a substantial way.
It is interesting to note that the Ir(III) complex yielding the
largest PI of the series with no dark toxicity toward cells (i.e.
complex 6) was the only one that showed a clear difference in PS-
induced aggregation of DNA between dark and light-treated
samples. While DNA may not be the intracellular target, these
results do suggest that there is a noticeable difference in the way
complex 6 interacts with a biological target such as DNA when
Experimental section
The details of experiments are described in ESI.†
10 | Dalton Trans., 2016, 00, 1-12
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Burrows, S. R. Forrest and M. E. Thompson, J. Am. Chem.
View Article Online
Soc., 2001, 123, 4304-4312; (c) C. AdaDchOiI,: M10..1A03.9B/Ca6ldDoT,0M24.16EE.
Thompson and S. R. Forrest, J. Appl. Phys., 2001, 90, 5048-
5051.
Conclusions
Six Ir(III) complexes with cyclometalating and diimine ligands
bearing different degrees of -conjugation were synthesized, and
their photophysical and photobiological properties were
systematically investigated. The natures of the optical transitions
were confirmed by TDDFT calculations. Extending the -
conjugation on the diimine ligand mainly influenced ground-state
absorption, while the nature of the emitting triplet excited state was
not affected. In contrast, extending the -conjugation on the
cyclometalating ligand affects both ground-state absorption and the
nature of the emitting triplet excited states. However, the lowest-
energy triplet excited state for complexes 4-6 did not change. This
4
5
(a) R. D. Costa, E. Orti, H. J. Bolink, F. Monti, G. Accorsi and
N. Armaroli, Angew. Chem. Int. Ed., 2012, 51, 8178-8211; (b)
C.-H. Yang, J. Beltran, V. Lemaur, J. Cornil, D. Hartmann, W.
Sarfert, R. Froehlich, C. Bizzarri and L. De Cola, Inorg. Chem.,
2010, 49, 9891-9901.
(a) C. Li, J. Lin, Y. Guo and S. Zhang, Chem. Commun., 2011,
47, 4442-4444; (b) J. Jiang, C. Zhang, W. Lin, Y. Liu, S. Liu,
Y. Xu, Q. Zhao and W. Huang, Macromol. Rapid Commun.,
2015, 36, 640-646.
3
dppn ligand-localized ,* state gave rise to the long-lived, strong
6
7
(a) Q. Zhao, F. Li and C. Huang, Chem. Soc. Rev., 2010, 39,
3007-3030; (b) N. Zhao, Y.-H. Wu, L.-X. Shi, Q.-P. Lin and Z.-
N. Chen, Dalton Trans., 2010, 39, 8288-8295.
excited-state absorption in the visible to the near-IR region.
Complexes 1-4 exhibit strong RSA at 532 nm for ns laser pulses
which is comparable to the benchmark Ir(III) complex reverse
saturable absorbers at 532 nm, while complex 6 shows the potential
as broadband reverse saturable absorber at 500-850 nm. Presumbly
due to the long-lived 3,* state and the weak but broad ground-state
absorption in the near-IR region, complexes 4-6 elicited
photodynamic effects toward cancer cells with low-energy visible
and red light. The phototherapeutic margin for complex 6 is the
largest for an Ir(III) complex PSs reported to date upon broad visible
or red light activation, and its interaction with plasmid DNA
suggests that a photocytotoxicity mechanism other than singlet
oxygen sensitization may be operative. The Ir(III) complexes also
show potential as theranostic agents due to their strong intracellular
luminescence.
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Acknowledgements
W. Sun acknowledges the financial support from the Army Research
Laboratory (W911NF-10-2-0055) for the synthesis and
photophysical studies of the complexes reported in this paper. The
computational part of the work was supported by NSF (DMR-
1411086 and CNS-1229316) to W. Sun and S. Kilina. S.A.M and
H.Y. acknowledge financial support from the Natural Sciences and
Engineering Council of Canada (NSERC), the Canadian Institutes of
Health Research (CIHR), the Canadian Foundation for Innovation
(CFI), the Nova Scotia Research and Innovation Trust (NSRIT), and
Acadia University.
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