Neutral iridium(iii) complexes bearing BODIPY-substituted N-heterocyclic carbene (NHC) ligands: Synthesis, photophysics,: In vitro theranostic photodynamic therapy, and antimicrobial activity

Article abstract of DOI:10.1039/c9pp00142e

The synthesis, photophysics, and photobiological activities of a series of novel neutral heteroleptic cyclometalated iridium(iii) complexes incorporating boron dipyrromethene (BODIPY) substituted N-heterocyclic carbene (NHC) ligands (Ir1-Ir5) are reported. The effect of the substitution position of BODIPY on the NHC ligands, either on C4 of the phenyl ring (Ir1-Ir3) or C5 of the benzimidazole unit (Ir4 and Ir5), and its linker type (single or triple bond) on the photophysical properties was studied. Ir1-Ir5 exhibited BODIPY-localized intense ¹IL (intraligand transition)/¹MLCT (metal-to-ligand charge transfer) absorption at 530-543 nm and ^1,3IL/^1,3CT (charge transfer) emission at 582-610 nm. The nanosecond transient absorption results revealed that the lowest triplet excited states of these complexes were the BODIPY-localized ³π,π? states. Complexes Ir4 and Ir5 exhibited blue-shifted ¹IL absorption and ^1,3IL/^1,3CT emission bands compared to the corresponding absorption and emission bands in complexes Ir1 and Ir3. However, replacing the methyl substituents on N3 of benzimidazole in complexes Ir1 and Ir4 with oligoether substituents in Ir3 and Ir5, respectively, did not impact the energies of the low-energy absorption and emission bands in the corresponding complexes. Water-soluble complexes Ir3 and Ir5 have been explored as photosensitizers for in vitro photodynamic therapy (PDT) effects toward human SKMEL28 melanoma cells. Ir3 showed no dark cytotoxicity (EC₅₀ > 300 μM) but good photocytotoxic activity (9.66 ± 0.28 μM), whereas Ir5 exhibited a higher dark cytotoxicity (20.2 ± 1.26 μM) and excellent photocytotoxicity (0.15 ± 0.01 μM). The phototherapeutic indices with visible light (400-700 nm) activation were >31 for Ir3 and 135 for Ir5. Ir3 and Ir5 displayed ¹O₂ quantum yields of 38% and 22% in CH₃CN, respectively, upon 450 nm excitation. Ir5 was more effective at generating reactive oxygen species (ROS) in vitro. Ir5 was also active against Staphylococcus aureus upon visible light activation, with a phototherapeutic index of >15 and EC₅₀ value of 6.67 μM. These photobiological activities demonstrated that these neutral Ir(iii) complexes are promising in vitro PDT reagents, and substitution at C5 on the benzimidazole group of the NHC ligand was superior to C4 substitution on the phenyl ring.

Full text of DOI:10.1039/c9pp00142e

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Photobiol. Sci., 2019, DOI: 10.1039/C9PP00142E.
Volume 16
Number 12
December 2017
Pages 1737-1846
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Page 1 of 16
Photo Pc hl ee ams ei c ad lo &n Poh to at od bj ui os tl o mg iac ra gl iSn c si ences
View Article Online
DOI: 10.1039/C9PP00142E
ARTICLE
Neutral iridium(III) complexes bearing BODIPY−substituted
N−heterocyclic carbene (NHC) ligands: synthesis, photophysics, in
vitro theranostic photodynamic therapy, and antimicrobial
activity
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
a
b
a
c
c
a
Bingqing Liu, Susan Monro, Mohammed A. Jabed, Colin G. Cameron, Katsuya L. Colón, Wan Xu,
a
bc
a
Svetlana Kilina, Sherri A. McFarland,* and Wenfang Sun*
The synthesis, photophysics, and photobiological activities of a series of novel neutral heteroleptic cyclometalated
iridium(III) complexes incorporating boron dipyrromethene (BODIPY) substituted N-heterocyclic carbene (NHC) ligands (Ir1
–
–
Ir5) are reported. The effect of the substitution position of BODIPY on the NHC ligands, either on C4 of the phenyl ring (Ir1
Ir3) or C5 of the benzimidazole unit (Ir4 and Ir5), and its linker type (single or triple bond) on the photophysical properties
1
1
was studied. Ir1 − Ir5 exhibited BODIPY−localized intense IL (intraligand transition) / MLCT (metal-to-ligand charge transfer)
1
3
absorption at 530-543 nm and IL/ CT (charge transfer) emission at 582-610 nm. The nanosecond transient absorption
3
results revealed that the lowest triplet excited states of these complexes were the BODIPY−localized ,* states. Complexes
1
1
3
Ir4 and Ir5 exhibited blue-shifted IL absorption and IL/ CT emission bands compared to the corresponding absorption and
emission bands in complexes Ir1 and Ir3. However, replacing the methyl substituents on N3 of benzimidazole in complexes
Ir1 and Ir4 with oligoether substituents in Ir3 and Ir5, respectively, did not impact the energies of the low-energy absorption
and emission bands in the corresponding complexes. Water-soluble complexes Ir3 and Ir5 have been explored as
photosensitizers for in vitro photodynamic therapy (PDT) effects toward human SKMEL28 melanoma cells. Ir3 showed no
dark cytotoxicity (EC50 >300 M) but good photocytotoxic activity (9.66 ± 0.28 M), whereas Ir5 exhibited a higher dark
cytotoxicity (20.2 ± 1.26 M) and excellent photocytotoxicity (0.15 ± 0.01 M). The phototherapeutic indices with visible
1
light (400-700 nm) activation were >31 for Ir3 and 135 for Ir5. Ir3 and Ir5 displayed O
2
quantum yields of 38% and 22% in
3
CH CN, respectively, upon 450 nm excitation. Ir5 was more effective at generating reactive oxygen species (ROS) in vitro. Ir5
was also active against Staphylococcus aureus upon visible light activation, with a phototherapeutic index of >15 and EC50
value of 6.67 M. These photobiological activities demonstrated that these neutral Ir(III) complexes are promising in vitro
PDT reagents, and substitution at C5 on the benzimidazole group of the NHC ligand was superior to C4 substitution on the
phenyl ring.
1
. Introduction
Photodynamic therapy (PDT) is an emerging cancer treatment spatiotemporally.3,6,7 During PDT, reactive oxygen species (ROS)
modality that combines a photosensitizer (PS), oxygen, and light are formed through energy (type II) or electron transfer (type I)
to destroy tumors and tumor vasculature and induce an reactions that take place between ground state molecular
1
-5
immune response. PDT is inherently selective because toxicity oxygen and the excited triplet state of the PS. The most
is confined to regions where the PS, oxygen, and light overlap important mediator of the PDT effect is thought to be singlet
1
oxygen ( O
2
) produced through the type II PDT mechanism, but
-


2
superoxide (O ) or hydroxyl radical ( OH), generated by the
a.Department of Chemistry and Biochemistry, North Dakota State University, Fargo,
type I PDT mechanism may also play a role.
Certain transition metal complexes are among the
numerous PSs that have been explored due to their (1)
ND 58108−6050, USA. E-mail: Wenfang.Sun@ndsu.edu
Department of Chemistry, Acadia University, 6 University Avenue, Wolfville, NS
b.
B4P 2R6, Canada.
c.
Department of Chemistry and Biochemistry, University of North Carolina at
Greensboro, Greensboro, NC 27402−6170, USA. E-mail: samcfarl@uncg.edu
interesting
photophysical
properties,
(2)
reduced
†
Electronic Supplementary Information (ESI) available: Details for the
photobiological activity studies; natural transition orbitals (NTOs) representing
transitions contributing to the absorption bands of Ir1 Ir5 in toluene; solvent-
dependent UV-vis absorption and emission spectra at r.t. for Ir1 Ir5; emission
characteristics in different solvents at r.t. for Ir1 Ir5; comparison of the emission
spectra in air-saturated and deaerated (degassed with N for 40 min) toluene
solution of Ir2 Ir5; and time-resolved nanosecond transient absorption spectra of
Ir1 Ir5 in toluene. See DOI: 10.1039/x0xx00000x
photobleaching, and (3) improved kinetic stabilities (compared
8
to organic PSs). Among the metal complexes, Ru(II) systems
-
-
are probably the most widely studied for PDT.
9,10
Our Ru(II)
-
10,11
complex TLD1433,
which incorporates a

-expansive ligand
2
–
derived from imidazo[4,5-f][1,10]phenanthroline appended to
an -terthienyl unit, just successfully completed a Phase 1b
-

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ARTICLE
Photochem. Photobiol. Sci.
10
clinical trial for treating bladder cancer with PDT ISC. Such a strategy has been applied in TLD1433 and other
View Article Online
D
1,33-3 9O I: 10.1039/C9PP00142E
1
(
ClinicalTrials.gov Identifier: NCT03053635). This example Ru(II) complexes for PDT applications,
but is relatively rare
40-44
demonstrates the utility of lowest-energy ,* triplet states, in for Ir(III) systems.
this case centered on the oligothienyl unit, with extremely long
lifetimes for highly efficient photosensitization.
Borondipyrromethene (BODIPY) and its derivatives, a class
of strong light−harvesting fluorophores with facile wavelength
Some transition-metal complexes possess the distinct tunability, have been extensively explored as potential PSs for
45-48
advantage of having singlet and triplet excited states that can PDT applications in recently years.
be tuned independently for optimization of their respective co-workers have demonstrated
characteristics when appropriate -conjugated ligands are encapsulating BODIPY or aza-BODIPY derivatives bearing
For example, Dong and
that nanoparticles

chosen. We have reported that it is possible to shift the singlet diethylaminophenyl or dimethylaminophenyl substituents
charge-transfer absorption to longer wavelengths (for exhibited very strong pH-triggered photodynamic and
activation by red wavelengths of light) by extending the
conjugation of the organic ligands, while allowing the lowest nude mice.
triplet excited state (T ) to be localized on the -expansive moiety to be attached to a monodentate or polydentate ligand
* nature of the lowest triplet excited to tune the absorption wavelength of the transition-metal PSs

-
photothermal therapy for Hela cells and Hela tumor bearing
4
7,48
Moreover, BODIPY appears as a promising
1

1
2
organic ligands. The
,
3
9-41,49
state results in very long triplet excited state lifetimes, which to the desired spectral region.
However, BODIPY-Ir(III)
have the potential to facilitate ROS production even at low dyads for PDT applications are not well-studied.40,41,49 One of
oxygen tension. Unlike organic PSs, metal complexes possess the few examples used BODIPY tethered cationic
high quantum yields for triplet state formation due to the heavy cyclometalated Ir(III) dyads as PSs for PDT and bioimaging
atom effect (metal-induced rapid ISC), and their triplet excited applications, and their in vitro PDT effects were marginal due to
states are typically long-lived. All of these properties are high dark toxicity (IC50 = 8.16 - 16.70
desirable features for next-generation PSs. lines). Phototherapeutic index (PI) values (1.3-3.8) were very
Ir(III) complexes are also of interest because some have small.40,41
exhibited high efficiency for ROS production via electron or Recently, N−heterocyclic carbene (NHC) based iridium(III)
M toward 1121 or LLC cell
1
4
50-54
energy transfer. Many Ir(III) complexes possess high quantum complexes have attracted great interest.
Unlike the
yields for triplet excited state formation and long-lived triplet numerous well−studied monocationic cyclometalated Ir(III)
+
excited states for efficient ROS generation even under complexes [Ir(C^N)
2
(N^N)] (where N^N refers to the diimine
13
hypoxia. It has been reported that cationic Ir(III) complexes ligand and C^N refers to the cyclometalating ligand),
1
4-18
19,20
can target mitochondria,
lysosomes,
the endoplasmic monoanionic
NHC
ligands,
such
as
1-phenyl-3-
2
1,22
or nuclei23,24 in a variety of cancer cell lines. A alkylbenzimidazole, as the N^N ligand gives rise to neutral
reticulum,
mitochondria-targeted Ir(III) complex PS was reported to show complexes.
54,55
To date, NHC-Ir(III) complexes have largely been
improved PDT effects under hypoxia. These complexes can studied for their catalytic and optoelectronic applications.
also display bright intracellular luminesce, imparting Biological activities were only reported on monocationic Ir(III)
14
50
51-54
theranostic capacity.1
3-20,23-25
Therefore, Ir(III) complexes have complexes with bis(alkylated-NHC) ligands,
15,50
which
emerged as a new platform for theranostic PDT applications. possessed high dark toxicity and could only be activated by UV
However, most of the currently studied Ir(III) complexes absorb light (365 nm). To date, the biological activities of the neutral
light maximally in the UV to blue spectral regions, wavelengths Ir(C^N)
of lesser interest for PDT because of their shallow tissue
2
(NHC) complexes remain unexplored.
Herein, a series of neutral iridium(III) complexes bearing
26
penetration due to tissue scattering and absorption.
BODIPY−NHC ligands (Chart 1) were designed and synthesized
Although it is possible to red-shift the charge transfer to explore their photophysical properties and in vitro PDT
ground-state absorption bands of the cyclometalating effects. These complexes all contain benzo[h]quinoline (bhq) as
+
monocationic Ir(III) complexes [Ir(C^N)
2
(N^N)] (where N^N the cyclometalating ligands, but the NHC ligand varies from
refers to the diimine ligand and C^N refers to the 1−(4−BODIPY−ethynylphenyl)-
cyclometalating ligand) to longer wavelength by introducing 3−methyl−1H−benzo[d]imidazole−3−iumiodide
(L1),
2
7
electron-withdrawing substituents on the diimine ligands or 1−(4−BODIPY−phenyl)−3−methyl−1H−benzo[d]imidazole−3−iu
through benzannulation at the diimine ligands, the molar miodide L2),
extinction coefficients of the charge transfer absorption bands 1−(4−BODIPY−ethynylphenyl)−3−(2−(2−(2−methoxyethoxy)eth
are quite low and the lowest triplet excited state (T ) lifetimes oxy)ethyl−1H−benzo[d]imidazol−3−iumiodide L3),
In contrast, it has been 5−(4−BODIPY−ethynylphenyl)−3−methyl−1H−benzo[d]imidazol
(
1
(
28-32
become much shorter (tens of ns).
shown that bichromophoric transition-metal complexes with

-
−
3−iumiodide
(
L4),
to
3
conjugated organic chromophores result in long-lived
,* T
1
5−(4−BODIPY−ethynylphenyl)−3−(2−(2−(2−
states localized at the organic chromophore with simultaneous methoxyethoxy)ethoxy)ethyl−1H−benzo[d]imidazol−3−iumiodi
red-shifting of the ground-state absorption to the longer de (L5). BODIPY was incorporated into the NHC ligand scaffold
wavelengths. Metal coordination improves
-conjugation at different sites through a single bond (Ir2) or a triple bond (Ir1
across the organic ligands through planarization, facilitating the and Ir3−Ir5) in order to red-shift the absorption of the
interactions between ligands. It also increases the quantum complexes to longer wavelengths. An oligoether chain,
yield of triplet excited-state formation via heavy atom induced 2−(2−(2−methoxyethoxy)ethoxy)ethyl, was appended to Ir3
2
| Photochem. Photobiol. Sci., 2019, 00, 1-14
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Page 3 of 16
Photochemical & Photobiological Sciences
Photochem. Photobiol. Sci.
ARTICLE
and Ir5 to increase the hydrophilicity of the neutral complexes. 2.1. Materials and synthesis
View Article Online
DOI: 10.1039/C9PP00142E
The photophysical properties of these five complexes were
All reagents and solvents were purchased from Alfa Aesar and
systematically investigated by various spectroscopic methods
and simulated by density functional theory (DFT) calculations.
The in vitro theranostic PDT effects of the two water-soluble
complexes Ir3 and Ir5 were investigated. The impact of the
BODIPY attachment site on the photophysics and PDT effects of
these dyads was explored.
2 3
VWR International and used as received. Al O gels (activated,
neutral) and silica gels (230−400 mesh) for column
chromatography were purchased from Sorbent Technology.
Benzo[h]quinoline (C^N ligand) was obtained from Alfa−Aesar
and its Ir(III) µ−chloro−bridged dimers [Ir(C^N) Cl] was
2 2
synthesized according to the literature procedure.56
Compounds 1-(4-bromophenyl)-1H-benzimidazole (compound
5
7
1
in Scheme 1), 4-bromo-2-nitro-N-phenylaminobenzene
58
1
(compound
6
in Scheme 1),
benzenediamine (compound
−iodo−BODIPY were synthesized following the reported
4-bromo-N -phenyl-1,2-
in Scheme 1),59 and
7
60
2
procedures. The synthetic route for the synthesis of ligands L1-
L5 and complexes Ir1-Ir5 are provided in Scheme 1. 1H NMR,
high−resolution mass spectrometry (HRMS), and elemental
analyses were used to characterize these complexes. 1H NMR
spectra were obtained on a Varian Oxford−400 or Bruker−400
Chart 1. Structures of BODIPY-NHC-Ir(III) Complexes Ir1–Ir5
.
3
spectrometer in CDCl using tetramethylsilane (TMS) as the
internal standard. Bruker BioTOF III mass spectrometer was
used for ESI−HRMS analyses. NuMega Resonance Laboratories,
Inc. in San Diego, California conducted elemental analyses.
2
. Experiments section
Scheme 1. Synthetic Route for Ligands L1
Cs CO
TBAF, THF, 0°C, 1 hr.; (iv) 2−Iodo−BODIPY, CuI, Pd(PPh
78 °C, 1 hr., then r.t. overnight, followed by HCl, 100 °C, 1 hr.; (vi) 2−iodo−BODIPY, K
−
L5 and Complexes Ir1
−
(PPh
Ir5. Reagents and conditions: (i) 1−bromo−4−iodobenzene, CuI,
2
3
, 1,10-phenanthroline, DMF, 110°C, 40 hrs.; (ii) CuI, PdCl
2
3
)
2
, ethynyltrimethylsilane, TEA/dioxane, 100 °C, 1 hr.; (iii)
n
)
3 4
, TEA, THF, 50 °C, 16 hrs.; (v) triisopropyl borate, BuLi, anhydrous THF,
−
2 3
CO , Pd(PPh
3
)
4
, toluene, THF, water, 70 °C,
o
overnight; (vii) NaOAc, aniline, 160 C, 7 hrs.; (viii) Na
2
S, S, water, reflux, 6 hrs.; (ix) formic acid, 100 °C, overnight; (x) MeI, THF, 100
o
o
C, 24 hrs.; (xi) 1−iodo−3,6,9−trioxadecane, toluene, 100 C, 96 hrs.; (xii) [Ir(benzo[h]quinoline)
2
(μ−Cl)]
2 2
, Ag O, 1,2−dichloroethane,
reflux, 24 hrs.
Compound 2. A solution of
1 (544 mg, 2 mmol), CuI (19 mg, 0.1
2 3 2
mmol), PdCl (PPh ) (70 mg, 0.1 mmol) in trimethylamine
2
.2. Synthesis of precursor compounds 2−5 and 8−10
This journal is © The Royal Society of Chemistry 20xx
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ARTICLE
Photochem. Photobiol. Sci.
o
(
TEA)/dioxane (2:1, v/v) was degassed with N
temperature for 20 minutes. Then, ethynyltrimethylsilane (0.86 the volatiles under reduced pressure, watDeOr (I:1100.m10L3)9/wCa9sPPa0d0d14e2dE,
mL, 6 mmol) was added and the resulting reaction mixture was and the mixture was extracted with CH Cl (3 10 mL). The
heated at 100 °C for 1 hour under N . After cooling to room combined organic layers were washed with brine (100 mL), and
temperature, the mixture was concentrated and purified by dried over anhydrous MgSO . The solvent was then removed,
2
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at room mixture was heated to 70 C for overnight. After evaporation of
2
2

2
4
column chromatography over silica gel using hexane:ethyl and the crude product was purified by column chromatography
acetate = 1:2 (v/v) as the eluent to give product as pale yellow on silica gel using hexane/ethyl acetate (v/v = 1:1) as the eluent
1
solid. This pale yellow solid (500 mg, 1.8 mmol) was dissolved to obtain orange solid as the target compound (79 mg, 78%). H
3
in THF (10 ml), and THF solution of tetrabutylammonium NMR (400 MHz, CDCl ) δ 8.13 (d, J = 7.5 Hz, 1H), 7.92−7.84 (m,
fluoride (TBAF) (10 mL, 1 M in THF) was added at 0 °C. The 1H), 7.62−7.45 (m, 7H), 7.40−7.29 (m, 5H), 6.04 (s, 1H), 2.61 (s,
reaction mixture was stirred at room temperature for 1 hour. 3H), 2.59 (s, 3H), 1.41 (s, 3H), 1.36 (s, 3H).
Then the solution was diluted with ethyl acetate (EA) and
washed with water and brine consequently. The organic layer mmol) and formic acid (20 mL) was stirred at 100 °C for
was dried over Na SO and concentrated. The residue was overnight. The reaction mixture was cooled to room
Compound 8. The mixture of compound 7 (880 mg, 3.36
2
4
purified by column chromatography over silica gel using temperature and concentrated under reduced pressure to
hexane:ethyl acetate= 1:2 (v/v) as the eluent to give compound afford a crude solid. The crude solid was partitioned between
1
2
as pale yellow solid (360 mg, 81%). H NMR (400 MHz, CDCl
3
)
ethyl acetate (250 mL) and NH
3
/H
2
O (25 mL). The ethyl acetate
SO , and then concentrated
H), 7.57 (dd, J = 6.6, 2.7 Hz, 1H), 7.53 (d, J = 8.4 Hz, 2H), under reduced pressure. The residue was purified by column
.43−7.34 (m, 2H), 3.22 (s, 1H). chromatography (silica gel, eluted with CH Cl ) to get the target
Compound 3. The solution of 2-iodo−BODIPY (200 mg, 0.44 compound as off−white solid (860 mg, 98%). H NMR (400 MHz,
δ 8.14 (s, 1H), 7.91 (dd, J = 6.4, 2.7 Hz, 1H), 7.73 (d, J = 8.4 Hz, layer was combined, dried with Na
2
4
2
7
2
2
1
mmol), compound
mmol), and Pd(PPh
mixed solvent (THF/TEA = 4/1) was heated to 50 C and stirred
3
2 (97 mg, 0.44 mmol), CuI (17 mg, 0.09 CDCl ) δ 8.10 (s, 1H), 8.02 (d, J = 1.5 Hz, 1H), 7.59 (dd, J = 8.2, 7.4
3
)
4
(57 mg, 0.049 mmol) in 10 mL degassed Hz, 2H), 7.52−7.47 (m, 3H), 7.46−7.36 (m, 2H).
o
Compound 9. Following the similar synthetic procedure for
for 16 hours in dark. After cooling to room temperature, the compound
solvent was removed under reduced pressure and the residue compound
2
8
, compound
as the starting material. H NMR (400 MHz, CDCl
9
was obtained with 87% yield using
1
3
)
2 2
was extracted with CH Cl and washed with brine. After δ 8.13 (s, 1H), 8.06−7.96 (m, 1H), 7.59 (dd, J = 10.0, 5.5 Hz, 2H),
removal of the solvent from the organic layer, the crude product 7.49 (dd, J = 11.7, 5.7 Hz, 4H), 7.42 (dt, J = 13.5, 5.1 Hz, 1H), 3.07
was purified by flash column chromatography with (s, 1H).
CH
2
Cl
2
/acetone (v/v = 50/1) being used as the eluent to obtain
Compound 10. Following the similar synthetic procedure
1
a dark red solid (200 mg, 83%). H NMR (400 MHz, with CDCl
δ 8.14 (s, 1H), 7.94−7.86 (m, 1H), 7.67 (d, J = 8.4 Hz, 2H), using compound
3
)
for compound
3
, compound 10 was obtained with 41% yield
1
9
as the starting material. H NMR (500 MHz,
7
2
.62−7.44 (m, 6H), 7.43−7.30 (m, 4H), 6.08 (s, 1H), 2.75 (s, 3H), CDCl
.62 (s, 3H), 1.55 (s, 3H), 1.44 (s, 3H). 7.54−7.45 (m, 7H), 7.42 (d, J = 8.4 Hz, 1H), 7.33−7.28 (m, 2H),
Compound 4. Under -78 C, BuLi (0.5 mL, 2.5 M in hexane) 6.03 (s, 1H), 2.74 (s, 3H), 2.59 (s, 3H), 1.54 (s, 3H), 1.41 (s, 3H).
3
) δ 8.14 (s, 1H), 7.97 (s, 1H), 7.59 (t, J = 7.7 Hz, 2H),
o
n
was added dropwise to the solution of compound
1 (151 mg,
i
0
.56 mmol) and B(O Pr)
3
(157 mg, 0.83 mmol) in THF (10 mL). 2.3. General synthetic procedure for ligands L1−L5.
o
The mixture was stirred at -78 C for 1 h, and then allowed to
warm up to room temperature, and continued stirring for
overnight. The reaction was quenched by addition of
In a round−bottom flask equipped with a gastight Teflon cap,
the precursor NHCs compound , or 10 (1 mmol) was
dissolved in 10 mL of THF (for synthesizing L1 L2, or L4) or in 1
mL of toluene (for synthesizing L3 and L5). Then 1.5 mmol CH
3, 5
,
4
concentrated aqueous NH Cl (10 mL), and the volatiles were
3
I
evaporated in vacuum. Then, HCl (10 mL, 37% aq.) was added
to the solution and the resultant mixture was heated to reflux
for 1 h. After cooling to room temperature, the mixture was
carefully adjusted to pH=7 with NaOH. The white suspension
was extracted with CH Cl . After removal of the solvent, the
2 2
residue was dried under vacuum. The crude product was
(for synthesizing L1, L2, or L4) or 1−iodo−3,6,9−trioxadecane
(
for synthesizing L3 and L5) was added. The mixture was heated
o
to 100 C for 24 h. After the reaction, the solution was allowed
to cool to room temperature. The formed precipitate was
collected by filtration and washed with THF, dichloroethane,
and Et
2
O (10 mL  2) to obtain the corresponding target
purified by column chromatography on silica gel and eluted with
acetone to obtain white solid as the product (47 mg, 36%).
compound.
1
H
L1. A dark red powder was obtained as the product (yield:
NMR (400 MHz, DMSO) δ 8.60 (s, 1H), 8.24 (s, 1H), 8.04 (d, J =
.0 Hz, 2H), 7.79 (d, J = 7.4 Hz, 1H), 7.67 (d, J = 7.9 Hz, 2H), 7.34
1
7
3%). H NMR (400 MHz, DMSO) δ 10.16 (s, 1H), 8.18 (d, J = 8.4
8
Hz, 1H), 8.05−7.71 (m, 8H), 7.65 (d, J = 3.7 Hz, 2H), 7.46−7.40
(dt, J = 13.6, 6.8 Hz, 2H).
(
(
m, 2H), 6.39 (s, 1H), 4.20 (s, 3H), 2.66 (s, 3H), 2.56 (s, 3H), 1.53
s, 3H), 1.43 (s, 3H).
Compound 5. A solution of compound
mmol), 2-iodo-BODIPY (56 mg, 0.12 mmol), and K
4
(44 mg, 0.18
CO (166 mg,
.2 mmol) in toluene (8 mL), THF (8 mL), and water (2 mL) was
2
3
L2. A red powder was obtained as the product (yield: 67%).
H NMR (400 MHz, DMSO) δ 10.15 (s, 1H), 8.16 (d, J = 8.2 Hz,
1
1
degassed with N
2
at room temperature for 20 minutes. After
1
H), 7.94−7.69 (m, 6H), 7.63 (dd, J = 12.4, 7.5 Hz, 4H), 7.46 (d, J
that, Pd(PPh ) (3 mg, 0.0025 mmol) was added, and the
3 4
4
| Photochem. Photobiol. Sci., 2019, 00, 1-14
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 16
Photochemical & Photobiological Sciences
Photochem. Photobiol. Sci.
ARTICLE
=
1
7.6 Hz, 2H), 6.29 (s, 1H), 4.17 (s, 3H), 2.52 (s, 3H), 2.50 (s, 3H),
.38 (s, 3H), 1.36 (s, 3H).
Ir3. A dark purple powder was obtained asVi
(yield: 56%). H NMR (400 MHz, CDCl ) δ 9.36 (d, J = 5.4 Hz, 1H),
3
t
e
h
w
e
Art
p
ic
r
le
o
O
duct
n
li
ne
1
DOI: 10.1039/C9PP00142E
L3. A dark red powder was obtained as the product (yield: 8.39 (d, J = 5.9 Hz, 1H), 8.27 (dd, J = 12.3, 7.0 Hz, 2H), 8.16 (d, J
1
5
3%). H NMR (500 MHz, CDCl
3
) δ 10.72 (s, 1H), 7.97 (d, J = 7.6 = 8.5 Hz, 1H), 8.03 (dd, J = 16.6, 8.0 Hz, 2H), 7.83 (d, J = 8.3 Hz,
Hz, 1H), 7.88 (d, J = 8.7 Hz, 2H), 7.74−7.66 (m, 4H), 7.53 (dd, J = 1H), 7.74 (t, J = 8.2 Hz, 2H), 7.68 (d, J = 8.8 Hz, 1H), 7.56−7.47
5
2
=
3
1
.0, 1.9 Hz, 2H), 7.33−7.27 (m, 4H), 6.07 (s, 1H), 5.09−5.03 (m, (m, 3H), 7.39−7.26 (m, 4H), 7.25−7.00 (m, 6H), 6.87 (d, J = 1.8
H), 4.22−4.14 (m, 2H), 3.74 (dd, J = 5.5, 3.4 Hz, 2H), 3.60 (dd, J Hz, 1H), 6.81 (t, J = 7.6 Hz, 1H), 6.68 (d, J = 7.0 Hz, 1H), 6.28 (d, J
5.4, 3.5 Hz, 2H), 3.54 (dd, J = 5.6, 3.7 Hz, 2H), 3.41 (dd, J = 5.6, = 7.0 Hz, 1H), 5.98 (d, J = 7.1 Hz, 1H), 4.37−4.17 (m, 2H), 3.75
.7 Hz, 2H), 3.28 (s, 3H), 2.73 (s, 3H), 2.60 (s, 3H), 1.53 (s, 3H), (dd, J = 10.5, 5.7 Hz, 2H), 3.39−3.32 (m, 2H), 3.29 (s, 3H),
.42 (s, 3H).
3.02−2.83 (m, 2H), 2.75−2.59 (m, 2H), 2.59 (s, 3H), 2.52 (s, 3H),
L4. A dark red powder was obtained as the product (yield: 2.40 (dd, J = 4.1, 2.8 Hz, 2H), 1.36 (s, 3H), 1.32 (s, 3H). ESI-HRMS
1
+
5
7
6
1
8%). H NMR (400 MHz, DMSO) δ 10.17 (s, 1H), 8.36 (s, 1H), (m/z): calcd. for [C67
.82−7.73 (m, 7H), 7.62 (d, J = 3.6 Hz, 3H), 7.53−7.34 (m, 2H), 1235.4164. Anal. Calcd for C67
.37 (s, 1H), 4.17 (s, 3H), 2.66 (s, 3H), 2.54 (s, 3H), 1.52 (s, 3H), C, 60.57; H, 4.58; N, 6.02. Found: C, 60.74; H, 4.55; N, 5.75.
H
56BF
2
IrN
6
O
3
+H] , 1235.4183; found,
H56BF
2
IrN ·1.6CH Cl ·0.3C H :
6
O
3
2
2
6 14
.41 (s, 3H).
Ir4. A dark purple powder was obtained as the product
1
L5. A dark red powder was obtained as the product (yield: (yield: 57%). H NMR (400 MHz, CDCl
3
) δ 8.27 (d, J = 4.6 Hz, 1H),
1
6
7
7
2
3
5%). H NMR (400 MHz, CDCl ) δ 10.61 (s, 1H), 8.19 (s, 1H), 8.20 (d, J = 5.5 Hz, 1H), 8.06 (d, J = 8.7 Hz, 1H), 8.02−7.93 (m,
.97−7.88 (m, 3H), 7.79−7.68 (m, 4H), 7.64−7.54 (m, 4H), 2H), 7.79−7.68 (m, 3H), 7.54−7.42 (m, 5H), 7.38 (t, J = 8.4 Hz,
.35−7.30 (m, 1H), 6.10 (s, 1H), 5.08−4.98 (m, 2H), 4.24−4.14 (m, 1H), 7.30 (dd, J = 11.9, 7.4 Hz, 2H), 7.22 (dd, J = 6.5, 3.1 Hz, 3H),
H), 3.81−3.72 (m, 2H), 3.67−3.61 (m, 2H), 3.54 (dd, J = 5.4, 3.6 7.11−6.95 (m, 5H), 6.75 (d, J = 5.7 Hz, 1H), 6.68 (dd, J = 11.5, 7.1
Hz, 2H), 3.45 (dd, J = 5.7, 3.7 Hz, 2H), 3.30 (s, 3H), 2.75 (s, 3H), Hz, 2H), 6.28 (d, J = 7.1 Hz, 1H), 5.98 (s, 1H), 3.19 (s, 3H), 2.64 (s,
2
.63 (s, 3H), 1.56 (s, 3H), 1.45 (s, 3H).
3H), 2.52 (s, 3H), 1.43 (s, 3H), 1.34 (s, 3H). ESI-HRMS (m/z):
+
calcd. for [C61
Anal. Calcd for C61
N, 5.00. Found: C, 55.46; H, 5.14; N, 5.26.
H
44BF
2 6
IrN +H] , 1103.3396; found, 1103.3385.
2
.4. General synthetic procedure for iridium complexes Ir1-Ir5.
The mixture of the NHC ligand (L1 L5, 0.12 mmol),
Ir(benzo[h]quinoline) (μ-Cl)] (76 mg, 0.06 mmol), Ag O (56
H44BF
2
6 2 2 6
IrN ·4.8CH Cl ·2C H14: C, 55.55; H, 4.89;
-
Ir5. A dark purple powder was obtained as the product
[
2
2
2
1
(
yield: 65%). H NMR (400 MHz, CDCl
3
) δ 8.34 (d, J = 5.5 Hz, 1H),
mg, 0.24 mmol), and 1,2-dichloroethane (15 mL) was heated to
reflux in the dark for 24 h. After the mixture was cooled to room
temperature, the solvent was removed under reduced pressure
to afford the crude product. Purification of the crude product
was carried out on silica gel column chromatography eluted
8
1
.25 (d, J = 5.4 Hz, 1H), 8.11 (d, J = 8.5 Hz, 1H), 8.07−8.03 (m,
H), 8.02−7.98 (m, 1H), 7.83 (dd, J = 14.7, 8.1 Hz, 1H), 7.75 (dd,
J = 8.9, 2.0 Hz, 2H), 7.67 (d, J = 1.9 Hz, 1H), 7.59−7.46 (m, 5H),
7
1
.46−7.27 (m, 5H), 7.17−7.00 (m, 5H), 6.80 (dd, J = 5.1, 3.6 Hz,
H), 6.74 (t, J = 7.6 Hz, 1H), 6.67 (dd, J = 6.2, 2.5 Hz, 1H), 6.29
with CH
2 2
Cl . The obtained product was further recrystallized in
(dd, J = 6.3, 3.3 Hz, 1H), 6.04 (s, 1H), 4.31−4.24 (m, 2H),
CH Cl / hexane to give the desired pure complex.
2
2
3
.77−3.71 (m, 2H), 3.42−3.37 (m, 2H), 3.33−3.29 (m, 2H), 3.27
Ir1. A dark purple powder was obtained as the product
(
(
[
s, 3H), 3.25−3.22 (m, 2H), 2.96−2.85 (m, 2H), 2.70 (s, 3H), 2.58
1
(yield: 32%). H NMR (400 MHz, CDCl
3
) δ 8.39 (d, J = 5.2 Hz, 1H),
s, 3H), 1.50 (s, 3H), 1.40 (s, 3H). ESI-HRMS (m/z): calcd. for
8
7
9
.27 (d, J = 5.2 Hz, 1H), 8.17 (d, J = 8.2 Hz, 1H), 8.03 (dd, J = 12.1,
.6 Hz, 2H), 7.85−7.66 (m, 3H), 7.57−7.46 (m, 4H), 7.39−7.21 (m,
H), 7.18−6.99 (m, 4H), 6.87 (s, 1H), 6.74 (d, J = 6.9 Hz, 1H), 6.33
+
67
C H
56BF
N
2 6
O
3
Ir+H] , 1235.4183; found, 1235.4169. Anal. Calcd
for C67
H56BF
2
IrN
O
6 3
.0.3H
2
O: C, 64.92; H, 4.60; N, 6.78. Found: C,
6
4.97; H, 4.77; N, 6.49.
(d, J = 7.0 Hz, 1H), 5.98 (s, 1H), 3.26 (s, 3H), 2.55 (s, 3H), 2.53 (s,
3
H), 1.36 (s, 3H), 1.33 (s, 3H). ESI-HRMS (m/z): calcd. for
+
[
C
61
H
44BF
2
IrN
IrN
.09; N, 4.85. Found: C, 55.37; H, 5.42; N, 5.15.
Ir2. A red powder was obtained as the product (yield: 30%).
1H NMR (400 MHz, CDCl
) δ 8.40 (d, J = 5.2 Hz, 1H), 8.30 (d, J =
.5 Hz, 1H), 8.18 (d, J = 8.1 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.97
d, J = 7.8 Hz, 1H), 7.83 (d, J = 7.9 Hz, 1H), 7.78 (d, J = 8.7 Hz, 1H),
6
+H] , 1103.3396; found, 1103.3384. Anal. Calcd 2.5. Photophysical studies.
for C61 44BF
5
H
2
6 2 2 6 6
·5CH Cl ·2.4C H14 (C H14: hexane): C, 55.71; H,
The spectroscopic grade solvents used for photophysical studies
were purchased from VWR International and used as received
without further purification. The ultraviolet−visible (UV−vis)
absorption spectra of complexes Ir1-Ir5 were measured on a
Varian Cary 50 spectrophotometer. Steady−state emission
spectra of complexes Ir1-Ir5 were recorded using a HORIBA
FluoroMax 4 fluorometer/phosphorometer. The relative
actinometry method was used for measuring the emission
3
5
(
7
7
.69 (d, J = 8.7 Hz, 1H), 7.60−7.40 (m, 4H), 7.35 (t, J = 8.1 Hz, 2H),
.22 (d, J = 6.6 Hz, 6H), 7.10 (ddd, J = 22.5, 14.7, 6.6 Hz, 4H), 6.89
(d, J = 6.9 Hz, 1H), 6.79 (d, J = 8.3 Hz, 1H), 6.43 (s, 1H), 6.26 (d, J
quantum yields of Ir1-Ir5. [Ru(bpy)
em = 0.097)61 was used as the reference. The
max = 436 nm,
3 2
]Cl in degassed acetonitrile
=
1
7.3 Hz, 1H), 5.91 (s, 1H), 3.28 (s, 3H), 2.52 (s, 3H), 2.15 (s, 3H),
(λ

.33 (s, 3H), 1.26 (s, 3H). ESI-HRMS (m/z): calcd. for
nanosecond transient absorption (TA) measurements, i.e. the
TA spectra, triplet lifetimes, and quantum yields, were carried
out on a laser flash photolysis spectrometer (Edinburgh LP920).
The excitation source was the third−harmonic output (355 nm)
of a Quantel Brilliant Nd:YAG laser (pulse duration, 4.1 ns;
+
[
C
59
H
44BF
2
IrN
IrN
Found: C, 64.62; H, 4.92; N, 7.19.
6
+H] , 1079.3396; found, 1079.3389. Anal. Calcd
for C59 44BF
H
2
6 2 2 6
·0.4CH Cl ·0.6C H14: C, 65.02; H, 4.61; N, 7.22.
This journal is © The Royal Society of Chemistry 20xx
Photochem. Photobiol. Sci., 2019, 00, 1-14 | 5
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Photo Pc hl ee ams ei c ad lo &n Poh to at od bj ui os tl o mg iac ra gl iSn c si ences
Page 6 of 16
ARTICLE
Photochem. Photobiol. Sci.
repetition rate was 1 Hz). Before measurement, each sample in ESI Fig. S1. The absorption band maxima and molar extinction
View Article Online
DOI: 10.1039/C9PP00142E
solution was purged with nitrogen for 40 min. The singlet coefficients are summarized in Table 1. Ir1-Ir5 exhibited strong
depletion method was followed to calculate the triplet absorption bands with vibronic structure in the region of
6
2
T
excited−state molar extinction coefficients (ε ), and the 280−460 nm, and broad, intense absorption bands centered
6
3
relative actinometry method was used to estimate the triplet between 500-600 nm. The energies and spectral features of
excited−state quantum yields using SiNc in benzene as the these low-energy absorption bands resemble those of the
−
1
−1
64
75
reference (ε590 = 70,000 M cm ,

T
= 0.20).
1,3,5,7-tetramethyl-8-phenyl-BODIPY.
However, the band
maxima of Ir1
compared to the
-
Ir5 are approximately 30-40 nm red-shifted
1
2.6. Computational methodology.
,* absorption band of the 1,3,5,7-
tetramethyl-8-phenyl-BODIPY, and these bands are broader
than that of 1,3,5,7-tetramethyl-8-phenyl-BODIPY. These
characteristics imply that the NHC ligand interacts with the
BODIPY motif and there could be some charge transfer
character in this band (see discussion vide infra).
Inserting a CC bond between the BODIPY motif and the
phenyl ring in complex Ir1 led to a red-shift of approximately 13-
nm compared to that of complex Ir2. This difference was
The ground state geometries were optimized using the Density
6
5
66
Functional Theory (DFT) employing PBE1 functional and the
67
mixed basis set, where LANL2DZ was used for Ir(III) to
incorporate the effective core potential for a heavy element and
6
8
6
-31G*
basis set was used for all remaining atoms. The
solvent effects were modeled by the Conductor-like Polarizable
69
Continuum Model (CPCM) reaction field method for toluene.
Linear response time dependent DFT (TDDFT)70 was
employed to calculate excited state properties of the complexes
by using the same functional and basis set that were used for
the ground state calculations. It was previously reported for
other Ir(III) complexes that both the ground and excited state
calculations using this methodology show good agreement with
attributed to the extended
-conjugation within the NHC-
BODIPY ligand of Ir1. Varying the point of attachment of BODIPY
on the NHC ligand also showed a noticeable effect on the energy
of the low-energy absorption band. Complex Ir4, with BODIPY
attached at C5 of the benzimidazole ring blue shifted the low-
energy absorption band by approximately 8 nm compared to
the corresponding band in Ir1, where BODIPY is appended at C4
of the phenyl ring. Replacement the methyl substituents on C3
of benzimidazole in complexes Ir1 and Ir4 by oligoether
substituents as in Ir3 and Ir5 did not impact the energy of the
low-energy absorption band but slightly attenuated the molar
extinction coefficients for this transition.
7
1
the experimental results, which defined our choice of the
methodology. To meet the same energy range of the
experimental absorption spectra, the lowest 80 states were
calculated by TDDFT. The thermal linewidths of spectra were
obtained using Gaussian function with the broadening
parameter of 0.08 eV, which well fits to the line shape of
experimental absorption spectra.
To better understand the nature of the different
absorption bands, time-dependent density functional theory
To calculate the emission energies, we have optimized the
triplet excited state using the analytical gradient method based
on TDDFT, within PBE1 functional and the mixed LANL2DZ/6-
(TDDFT) calculations were carried out for complexes Ir1−Ir5 in
toluene. As shown in ESI Fig. S1, the calculated spectra matched
well with the experimental spectra. The natural transition
orbitals (NTOs) corresponding to the major transitions
contributing to the major absorption bands of Ir1−Ir5 are
presented in Table 2, and in ESI Tables S1 and S2.
3
1G* basis set. To get a better understanding of the nature of
72
the transitions, natural transition orbitals (NTOs) were
calculated, which is the compact representation of the excited
electron-hole pair obtained from the transition density matrices
calculated by TDDFT. NTOs were visualized by VMD software
using 0.02 iso resolution. All quantum chemical calculations
7
3
®
74
5
were performed using the Gaussian09 software package.
1
8
6
4
x10
x10
x10
x10
Ir1
Ir2
Ir3
Ir4
Ir5
4
4
4
4
2
.7. Photobiological activity studies.
The details of the cellular assays, cell culture, cytotoxicity and
photocytotoxicity cell assays, confocal microscopy, bacterial
survival assays, and in vitro ROS generation measurements are
provided in the Supporting Information.

3
. Results and discussion
2x10
3
.1. Electronic absorption.
The UV−vis absorption spectra of Ir1
toluene, CH CN, THF, and CH Cl at room temperature. The
0
-
Ir5 were measured in
300
400
500
600
700
3
2
2
Wavelength / nm
spectra in toluene (the preferred solvent due to the high
solubility of the neutral complexes in it) are displayed in Fig. 1,
and the normalized spectra in the other solvents are provided
Fig. 1. UV−vis absorption spectra of Ir1−Ir5 in toluene.
6
| Photochem. Photobiol. Sci., 2019, 00, 1-14
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 7 of 16
Photochemical & Photobiological Sciences
Photochem. Photobiol. Sci.
ARTICLE
Table 1. Photophysical parameters for complexes Ir1−Ir5.
View Article Online
DO
d
I: 10.1039/C9PP00142E

abs/nm (log ε


em/nm (τem
/

s);

T1−Tn/nm (τTA
/


s; log εT1−Tn
/
∆
 (ex/nm)
−
1
−1
a
b
−1
−1
c
/L·mol ·cm )
em
L·mol ·cm );
T
e
n.d.f
n.d.f
Ir1
294 (4.86), 335 (4.68), 352
610 (4.96); 0.041 457 (42.1; - ), 679 (48.0; 4.13); 0.51
(
(
4.56), 398 (4.40), 430
4.33), 543 (4.90)
e
Ir2
Ir3
286 (4.78), 347 (4.46), 398
4.29), 530 (4.87)
290 (4.86), 335 (4.69), 352
583 (3.12); 0.063 458 (24.0; - ), 681 (28.7; 3.83); 0.86
(
e
610 (5.26); 0.047 455 (26.1; - ), 690 (32.1; 4.11); 0.51
0.38 (450);
0.37 (534)
(
(
4.57), 397 (4.42), 430
4.33), 543 (4.88)
e
n.d.f
Ir4
322 (4.74), 350 (4.64), 396
4.40), 535 (4.88)
309 (4.87), 350 (4.73), 395
4.50), 535 (4.86)
Absorption band maxima (
em), lifetimes (τem), and quantum yields (
degassed acetonitrile solution of [Ru(bpy) ]Cl
587 (4.77); 0.034 449 (28.0; - ), 699 (30.9; 4.09); 0.39
(
e
Ir5
587 (4.89); 0.010 452 (52.9; - ), 690 (55.2; 4.06); 0.40
0.11 (352); 0.22
(450); 0.08 (534)
(
a
b

abs) and molar extinction coefficients (log ε) in toluene at room temperature. Emission band maxima
−
5
(
em) in toluene at room temperature, c = 1×10 mol/L. The reference used was a
6
1
3
2
(

em = 0.097,

ex = 436 nm) The emission lifetimes reported are the longer-lived
3
3
1
ILCT/ MLCT phosphorescence lifetimes. The short-lived IL fluorescence lifetimes were unable to be reliably measured due to the
c
resolution of our instrument. Nanosecond TA band maxima (

T1−Tn), triplet excited−state lifetimes (τTA), triplet extinction
4
coefficients (log εT1−Tn), and quantum yields (

T
) measured in toluene at room temperature. SiNc in benzene (ε590 = 7×10 L
−
1
−1
= 0.20)64 was used as the reference for calculating
d
mol cm ,

T

T
. Singlet oxygen quantum yields (

∆
) were measured in
e
acetonitrile and are corrected to within ±5%. Not determined due to strong ground-state absorption, which does not satisfy the
f
condition to apply singlet depletion method for estimation of εT1−Tn. Not determined.
As the NTOs in Table 2 indicated, the electrons of the S
1
lifetimes of several nanoseconds and a longer lifetimes of
states were almost exclusively localized on the BODIPY moiety, several microseconds. The emission was only partially
while the holes were delocalized on the entire quenched by air. These characteristics imply that the emission
1
BODIPY−substituted NHC ligands and on the metal d orbitals. of these complexes could be mixed with IL fluorescence and
1
1
3
Therefore, the S
(
(
1
states in Ir1
intraligand charge transfer,
metal-to-ligand charge
−
Ir5 have the mixed π,π* / ILCT charge

transfer,
transfer
phosphorescence
( CT).
This
1
(NHC)*(BODIPY)) / MLCT fluorescence/phosphorescence mixing has been reported for a
4
1
d(Ir)*(BODIPY)) cationic dinuclear Ir(III) complex linked by BODIPY.
configurations, which contributed to the intense low-energy
absorption bands. For the absorption band(s) in the 370-450 nm Table 2. NTOs for lowest-energy transitions of complexes Ir1-
region, the NTOs in ESI Table S1 suggest major ligand-to-ligand Ir5 in toluene.
1
1
charge transfer ( LLCT,

(bhq)*(BODIPY))
/
MLCT
S
S1
n
Hole
Electron
1
Ir1
Ir2
Ir3
Ir4
Ir5
transitions, admixing with some BODIPY-NHC localized IL
intraligand transition, i.e. π,π* / ILCT) characters. In contrast,
according to the NTOs shown in Table S2, the high energy,
strong absorption bands at 280-370 can be predominantly
assigned to the spin-allowed bhq or BODIPY-NHC ligand-
1
1
533 nm
f = 0.633
(
S
1
1
1
1
480 nm
f = 0.507
centered π,π* transitions, mixed with LLCT, MLCT, and minor
1
ILCT configurations.
S
1
3
.2. Photoluminescence.
The steady−state emission spectra for complexes Ir1
measured in degassed toluene, CH CN, THF, and
dichloromethane at room temperature. The normalized
emission spectra in toluene are presented in Fig. 2, and the
emission maxima and lifetimes are compiled in Table 1. The
normalized emission spectra and the emission data in other
solvents are provided in ESI Fig. S3 and Table S3. As shown in
Fig. 2, all of the complexes exhibited broad and structureless
luminescence in toluene ranging from 582 to 610 nm, which
531 nm
f = 0.647
-
Ir5 were
3
S
1
5
15 nm
f = 0.655
S
1
5
13 nm
f = 0.641
1
were mirror images to their corresponding IL absorption bands.
The emission signals showed bi-exponential decays, with a short
This journal is © The Royal Society of Chemistry 20xx
Photochem. Photobiol. Sci., 2019, 00, 1-14 | 7
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Photochem. Photobiol. Sci.
BODIPY on the NHC ligand also affected the emission energy as
View Article Online
DOI: 10.1039/C9PP00142E
it did for the UV-vis absorption, namely, the emission maxima
of Ir4 and Ir5 displayed a blue-shift of 23 nm with respect to
those of Ir1 and Ir3
.
To assign the nature of the emitting states for these
complexes, TDDFT calculations were performed based on the
optimized singlet triplet excited state geometries. The singlet
and triplet excited-state NTOs obtained for Ir1
in Table 3 for S and T states, and in ESI Table S4 for T
Because neither the calculated T energies nor the energy trend
-
Ir5 are compiled
1
2
1
states.
1
matched the experimental results, and the emission lifetimes
were much shorter than the lifetimes obtained from the ns
transient absorption measurement (which will be discussed in
the next section), we believe that the phosphorescence
components of the observed emission from these complexes
are from the T
the fluorescent S
configurations, while the
2
states. Based on the NTOs provided in Table 3,
Fig. 2. Normalized experimental emission spectra of Ir1−Ir5 in
1
1
1
1
states have mixed ILCT/
states are predominantly
ILCT/ MLCT configurations mixed with minor 3
* character.
states and the T states are in energetic proximity,
phosphorescence. The inset shows the therefore, they both contribute to the observed emission.
comparison of emission intensity in air-saturated and deaerated However, these two states are not in thermal equilibrium
,*/ MLCT
deaerated toluene at room temperature. The excitation
wavelength was 543 nm for Ir1 and Ir3, 530 nm for Ir2, and 535
nm for Ir4 and Ir5. The open-headed lines represent the
T
2
3
3
,

calculated S
the calculated T
1
fluorescence and the solid-headed lines represent The S
1
2
2
toluene solutions for Ir1
.
because the emission decay was bi-exponential, with a short
component of less than 10 ns and a longer lifetime of several
microseconds.
Similar to the trend observed from the UV-vis absorption
experiments, the emission maxima of Ir1 and Ir3 were identical
to those of Ir4 and Ir5, respectively. This indicates that the
oligoether chain did not impact the energies of the emitting
states in these complexes. In contrast, the emission of Ir2 with
BODIPY singly-bonded to NHC showed a pronounced blue-shift
3
.3. Transient absorption (TA).
To further understand the triplet excited state characteristics of
Ir1-Ir5, especially the non-emitting T states in these complexes,
1
nanosecond TA were investigated in deaerated toluene at room
temperature. The TA spectra of Ir1-Ir5 at zero-time delay upon 355-
nm excitation are presented in Fig. 3. The TA parameters, such as
(27 nm) compared to that of Ir1, with the CC linker, due to the
shorter π-conjugation and the reduced co-planarity between
BODIPY and NHC ligand in Ir2. The attachment position of
Table 3. NTOs corresponding to the optimized S
1
and T
2
states of complexes Ir1
−Ir5 in toluene.
S
1
T
2
Energy
Hole
Electron
Energy
Hole
Electron
Ir1
Ir2
Ir3
Ir4
647 nm
623 nm
598 nm
644 nm
609 nm
538 nm
621 nm
586 nm
Ir5
605 nm
584 nm
8
| Photochem. Photobiol. Sci., 2019, 00, 1-14
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Page 9 of 16
Photo Pc hl ee ams ei c ad lo &n Poh to at od bj ui os tl o mg iac ra gl iSn c si ences
ARTICLE
Photochem. Photobiol. Sci.
for Ir3 was near 37% and
76
in aqueous solution. The value of

∆
View Article Online
0
.05
.00
largely independent of excitation wavelen
DO
g
t
I
h,
:
10
w
.1
h
0
e
39re/C
as
9P
t
P0
hat r
0
14fo2E
Ir5 ranged from 8-22% as a function of excitation wavelength.
Excitation at 352, 450, and 534 nm yielded values for ∆ of 11,
0
2
2, and 8%, respectively.
Ir3 was the more efficient O
-0.05
-0.10
-0.15
-0.20
-0.25
1
2
generator at all wavelengths
investigated, which could be attributed, in part, to the fact that
its quantum yield for triplet state formation is larger (51 versus
4
chromophore, either on the meso-phenyl versus the
benzimidazole portion of the NHC ligand, impacts the excited
state dynamics and photophysical properties of the complexes.
These structural differences may, therefore, be expected to also
alter their photobiological properties.
Ir1
Ir2
Ir3
Ir4
Ir5
0%). Clearly, the substitution position of the BODIPY
400
500
600
700
800
Wavelength / nm
3
.5. Cytotoxicity and photocytotoxicity toward cancer cells.
Based on their abilities to generate 1O
under cell-free
Fig. 3. Nanosecond transient difference absorption spectra of
complexes Ir1 Ir5 in toluene at room temperature immediately
after 355 nm laser pulse excitation. A355 = 0.4 in a 1-cm cuvette.
−
2
conditions, albeit with modest efficiency, it was anticipated that
both Ir3 and Ir5 would give rise to in vitro photobiological
effects. The human melanoma cell line SKMEL28 was used to
the TA band maxima, triplet excited−state lifetimes and
quantum yields, and triplet extinction coefficients, are compiled
in Table 1. Fitting of the TA signals revealed a long-lived species
probe the cytotoxicities and photocytotoxicities of Ir3 and Ir5
.
Briefly, cells were dosed 1 nM-300 µM of Ir3 or Ir5 (dissolved in
water supplemented with 10% DMSO (v/v) and serially diluted
with phosphate buffered saline (PBS)) and incubated for 16 h
before receiving a dark treatment or a light treatment. The
illumination condition was 100 J cm of either broadband
visible or monochromatic red (625 nm) light. After a dark or light
treatment, the cells were incubated for 48 h, treated with the
(ca. 28-55 s). In addition, the TA spectral features of Ir1-Ir5
were similar, with ground-state bleaching arising from the
1
BODIPY-NHC localized IL absorption. These spectra are also
75
similar to that of the iodo-BODIPY reported in the literature.
-
2
Therefore, the triplet excited states that produced the observed
TA should have the same nature for all of the complexes, likely
3
from the BODIPY localized
,* state. The lifetimes of Ir1-Ir5
77
resazurin cell viability indicator, and incubated an additional
–4 h. The relative cell viabilities for the dark and light
obtained from the decay of the TA signals are distinctively
different from their emission lifetimes, indicating that the
excited states observed by TA in these complexes are different
2
treatments of cells dosed with complex were quantified by the
metabolic reduction of resazurin to its fluorescent product
resorufin (Fig. 4, Table 4).
Ir3 was nontoxic toward SKMEL28 cells (EC50 >300 µM),
while Ir5 was substantially more cytotoxic (EC50 = 20 µM). With
broadband visible light activation, both complexes were
photocytotoxic. The photocytotoxicy of Ir5 was submicromolar,
with an EC50 value of 150 nM and PI of 135, while the visible-
light EC50 value for Ir3 was approximately 10 µM and its PI was
from their emitting excited states. The long TA lifetimes were
3
assigned to excited states with

,

* characters. The NTOs for
states for these
* configuration and localized exclusively
1 1
T states in ESI Table S4 confirmed that the T
3
complexes are of

,
on the BODIPY motif.
3.4. Singlet oxygen generation.
Ir3 and Ir5 were designed with oligoether substituents as R3 31. The photocytotoxicities of both complexes with red light
Chart 1) to increase the hydrophilicities of these neutral Ir(III) were substantially attenuated (5-fold for Ir3 and over 85-fold
(
NHC complexes for improved aqueous solubility (which is a for Ir5), giving rise to much smaller PI values (6 for Ir3 and 2 for
desirable property for in vitro biological testing). Our detailed Ir5).
photophysical and computational analyses of Ir3 and Ir5
(
compared to their corresponding methyl-substituted Ir2 and Table 4. Comparison of EC50 values (M) for SKMEL28 cancer
Ir4 relatives) indicated that the addition of the oligoether cells dosed with complexes Ir3 and Ir5
.
Dark
> 300
Visa
PIb
Redc
53.7 ± 2.1 >6
13.0 ± 0.1
PIb
groups did not alter the general excited state dynamics
observed for these systems. Therefore, the more water-soluble
Ir3 and Ir5 were further investigated for their photobiological
Ir3
Ir5
9.66 ± 0.28
>31
135
20.2 ± 1.3 0.15 ± 0.01
2
a
−2
activities.
Vis−PDT: 16 h drug-to-light interval followed by 100 J cm
broadband visible light (400-700 nm) irradiation,
phototherapeutic index (ratio of dark EC50 to visible-light EC50),
Red-PDT: 16 h drug-to-light interval followed by 100 J cm light
irradiation with 625 nm LEDs.
1
b
PI =
The abilities of Ir3 and Ir5 to generate singlet oxygen ( O
2
)
1
were assessed as quantum yields for O
CH CN relative to [Ru(bpy) ](PF as the standard (
The direct method was employed, whereby sensitized O
2
production (
∆
 ) in
= 56%).
c
−2
3
3
)
6 2

∆
1
2
was
CN
detected by its phosphorescence centered at 1268 nm. CH
3
1
was used as the solvent since O
2
phosphorescence is quenched
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Page 10 of 16
ARTICLE
Photochem. Photobiol. Sci.
by Ir5 in the absence of a light trigger, whereby Ir5ViewwaAsrtiaclte Olenalinset
1
5

more cytotoxic (in the dark) than Ir3.DOI: 10.1039/C9PP00142E
Light-treated complexes Ir3 and Ir5 increased the detected
fluorescence from the DCF product in a dose-dependent
manner over the dark controls and also in comparison to the
positive control THBP (Fig. 5). The highest concentration of
light-treated Ir5 (126 µM) showed a slight decrease in the DCF
fluorescence relative to the second highest concentration (39
µM). This attenuation is typical when the cells begin to lose
viability, which is why it is important to choose a sub-lethal dose
for ROS quantification. Ir5 produced significantly more DCF
fluorescence than Ir3 at the highest concentrations tested.
It is possible that the much larger PI measured for Ir5
Fig. 4. In vitro dose-response curves for complexes Ir3 (left) and
Ir5 (right) in SKMEL28 cells treated in the dark (black) and with
visible (blue) or red (red) light activation.
Despite having much higher cytotoxicity in the dark, Ir5 was
the better in vitro photosensitizing agent according to both the
magnitude of its photocytotoxicity and its phototherapeutic
(
compared to Ir3) may stem from its more efficient light-
margin. This was initially surprising since the value of

∆
for Ir3
1
mediated ROS production. ROS other than O
2
could be involved
was significantly larger than that for Ir5. This discrepancy could
based on the lower value of
∆
 measured for Ir5 under the cell-
1
be due to differences in
2
O production under cell-free
free condition. However, the level of DCF fluorescence is only
raised at relatively high PS concentrations, which seems to
conditions versus the in vitro experiment, or that reactive
1
oxygen species (ROS) other than O
2
may be involved in the
indicate the absence of
a good correlation between
phototoxic mechanism, or due to different cell uptake.
photocytotoxicity and widespread redox imbalance. It seems
more likely that the large difference in phototoxic effects for Ir3
and Ir5 stems from factors that were not directly probed as part
of this study, including differences in the biological target(s) for
each PS and possibly contact-mediated interactions with such
To probe for the involvement of ROS, more broadly
defined, as a possible source of the photocytotoxic activity for
Ir3 and Ir5, SKMEL28 cells were preincubated with the cell-
permeable and highly sensitive 2',7'-dichlorodihydrofluorescein
diacetate (DCFDA) fluorogenic dye before performing
80
targets.
(photo)cytotoxicity assays (Fig. 5). The compounds were tested
Confocal microscopy was used to observe the qualitative
in the range of 4 pM to 126 µM, and a sub-lethal light dose of
effects of Ir3 and Ir5 on SKMEL28 cells with a dark or visible light
-2
5
0 J cm broadband visible light was used for the light condition
-2
treatment of 50 J cm (Fig. 6). A concentration of 50 µM was
with a reduced incubation time of 30 min between compound
addition and light treatment. DCFDA is deacetylated by cellular
esterases to a nonfluorescent compound, which is subsequently
oxidized by ROS to the highly fluorescent DCF product that can
chosen because a marked difference in the dark cytotoxicity of
Ir3 and Ir5 at this concentration was anticipated (since 50 µM is
greater than the dark EC50 value of Ir3, but not of Ir5). The light
-2
treatment was chosen to be 50 J cm (half the dose of the cell
78
be detected by its characteristic emission. ROS that can be
cytotoxicity assays) and imaging was done at 15 min post-
treatment to ensure that cells were imaged under sub-lethal
conditions, where a comparison of cellular morphologies and
uptake might be made.
•
–
detected by this method include superoxide anion (O
2
),
•
hydrogen peroxide (H
2
O
2
), hydroxyl radical (HO ), and singlet
), which react directly or indirectly with the
1
oxygen ( O
2
79
deacetylated probe.
3
The differences in the MLCT emission quantum yields for
Dark treatments were included to quantify baseline ROS
levels under the assay conditions for comparison, and tert-butyl
hydrogen peroxide (TBHP) was used as a positive control (Fig.
Ir3 and Ir5 were apparent in the dark confocal images, where
cellular uptake by Ir3 was detectable as a very weak signal but
no signal was detected for Ir5. Qualitatively, there appeared to
be more cellular debris from dead and dying cells treated with
Ir5 in the dark (Fig. 6a, bottom row) but emission was not
detected. Both compounds caused a general change in the
morphology of a large fraction of the cells treated in the dark,
from the typical elongated and spindle shape to spherical.
5
c). The signal produced by TBHP was relatively weak, but
sufficient, owing to the short incubation that was necessary to
ensure a sub-lethal treatment with photoactivated Ir3 and Ir5
.
ROS generation with Ir3 in the dark was minimal at all
concentrations tested, while cells treated with Ir5 in the dark
showed elevated ROS levels at concentrations greater than 1
µM. This observation may account for the cytotoxicity induced
(a)
12000
0000
Dark
Light
(b)
12000
10000
8000
6000
4000
2000
0
(c)
12000
10000
8000
6000
4000
2000
0
1
8000
6000
4000
2000
0
-5.4 -4.4 -3.4 -2.4 -1.4 -0.4 0.6 1.6 2.1
-5.4 -4.4 -3.4 -2.4 -1.4 -0.4 0.6 1.6 2.1
-4.3 -3.3 -2.3 -1.3 -0.3 0.7 1.4 2.1 2.4
Log (concentration / µM)
Log (concentration / µM)
Log (concentration / µM)
1
0 | Photochem. Photobiol. Sci., 2019, 00, 1-14
This journal is © The Royal Society of Chemistry 20xx
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Page 11 of 16
Photochemical & Photobiological Sciences
Photochem. Photobiol. Sci.
ARTICLE
Fig. 5. Reactive oxygen species assay results for SKMEL28 cells treated with Ir3 (a), Ir5 (b), or the positive control tert-butyl
View Article Online
-
1
hydrogen peroxide TBHP (c) using DCFDA as a ROS probe. Cells were treated in the dark (black bars) or withDO50I: 1
J
0
c
.1
m
039/
v
C
is
9
i
P
b
P
l
0
e
0
l
1
ig42
ht
E
(blue bars). ROS production was measured at 120 min post-treatment.
Dark
Light
DIC
emission
overlay
DIC
emission
overlay
(a)
(b)
Fig. 6. Laser scanning confocal microscopy images of SKMEL28 cells dosed with 50 M Ir3 (top row) or Ir5 (bottom row) in the
−
2
dark (a) or with 50 J cm visible light (b).
With light activation, intracellular emission from Ir3 was showing photobiological activity against SKMEL28 melanoma
much more pronounced (Fig. 6b, top row). If the signal intensity cells, Ir3 was inactive against S. aureus both in the dark and with
is proportional to concentration, then photoactivated uptake of a light treatment. On the other hand, Ir5 was nontoxic to S.
Ir3 results in accumulation in the cytoplasm and mitochondria aureus in the dark but phototoxic with a visible light EC50 of
but not in the nuclei. Interestingly, Ir5 produced luminescence approximately 7 µM (PI > 15). Relatively few examples of metal
in the cellular debris from dead and dying cells that were complexes as PSs for antimicrobial PDT (aPDT) have been
38,81-84
treated with visible light and very faint luminescence from the reported,
making Ir5 notable in that respect.
intact cells. Images of cells treated with Ir3 and light did not The observation that Ir3 was less photobiologically active
exhibit the dark clumps of cellular debris that were observed in (inactive) than Ir5 against S. aureus agreed with the trend in
the images of Ir5, possibly reflecting the differences in cancer cells, further highlighting the discrepancy between the
1
photocytotoxicities and/or aggregation properties (in the
presence of cellular remains) of these two complexes.
2
O quantum yields and phototoxicities for these two
complexes. Why Ir5 is less effective than Ir3 is not known at this
Photoactivated uptake also led to a larger concentration of time, but it appears that factors other than O
1
2
are also
Ir5 in cells (assuming that signal intensity is proportional to important for the phototoxic mechanism against S. aureus. The
concentration), making its detection possible. Given that substitution position of the BODIPY unit on the NHC ligand
luminescence from Ir3 is almost five-fold more efficient than clearly plays a major role in determining the biological and
that from Ir5 (and this difference could be magnified in cells), photobiological activities against the cell lines investigated in
the confocal imaging experiment cannot definitively establish this study. It is plausible that their structural differences affect
that cellular uptake of Ir3 higher than Ir5. In addition, the Stokes membrane permeabilization and contact-dependent chemical
shift associated with Ir3 is larger than that of Ir5, where inner- reactions such as lipid photooxidation as demonstrated by
8
0
filter effects could contribute to the lower signal intensity Baptista and coworkers. Confirming this would be a critical
associated with Ir5. It would be expected that the higher dark next step for rational design of subsequent-generation PSs of
toxicity of Ir5 would be correlated with better cellular uptake, this class.
but additional intracellular compound quantification methods
were not carried out as part of this study.
Table 5. Comparison of EC50 values (
M) for Staphylococcus
aureus dosed with Ir3 or Ir5
.
3
.6. Cytotoxicity and photocytotoxicity toward bacteria.
Dark
Visa
>100
6.67 ± 0.07
PIb
-
>15
The antimicrobial photobiological activities of Ir3 and Ir5 were
also assessed using Staphylococcus aureus (S. aureus) growing
as planktonic cultures (Table 5, Fig. 7). The complexes were
tested at concentrations between 0.4 and 100 µM, and the light
Ir3
Ir5
>100
>100
a35 J cm
−2
broadband visible light irradiation; bPI =
phototherapeutic index (ratio of dark EC50 to visible-light EC50).
-2
treatments were approximately 35 J cm of visible light. Despite
This journal is © The Royal Society of Chemistry 20xx
Photochem. Photobiol. Sci., 2019, 00, 1-14 | 11
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Photo Pc hl ee ams ei c ad lo &n Poh to at od bj ui os tl o mg iac ra gl iSn c si ences
Page 12 of 16
ARTICLE
Photochem. Photobiol. Sci.
1
20
00
120
View Article Online
DOI: 10.1039/C9PP00142E
(a)
(b)
1
100
80
60
40
20
0
80
60
40
20
0
.
4
.1
0.2
0.5
0.8
1.1
1.4
1.7
2.0
-
0
-0
Log (concentration / µM)
Log (concentration / µM)
Fig. 7. Bacterial cell survival dose-response bar graphs for complexes Ir3 (a) and Ir5 (b) in S. aureus, treated in the dark (black) or
-
1
with 35 J cm visible (blue) light. In panel b, the bacterial survival rates were zero when Ir5 concentrations reached

12.5
M,
thus no blue bars were shown for those concentrations.
1
substitution at C5 of benzimidazole leads to lower O
2
quantum
yields but more effective phototoxic effects. Obviously, the
impact of BODIPY substitution position at the NHC ligand is
more pronounced on the photobiological activities than on the
photophysical properties. Future studies are aimed at
quantifying the interaction of the Ir3 and Ir5 with cells to better
understand their stark differences in photocytotoxicity, which is
a critical next-step toward establishing structure-activity
relationships and expanding this new class of neutral Ir(III) NHC
complexes.
4
. Conclusions
New neutral Ir(III) complexes bearing BODIPY-substituted NHC
ligands are reported. Their photophysical characteristics were
systematically investigated via spectroscopic methods and DFT
calculations. All complexes exhibited BODIPY-localized intense
1
1
1
3
IL/ MLCT absorption at 530-543 nm and IL/ CT emission at
5
82-610 nm. However, the lowest triplet excited state of these
3
complexes are the BODIPY−localized
,* states. It was
demonstrated that the position of the BODIPY pendant on the
1
1
1
3
NHC ligand impacted both the IL/ MLCT absorption and IL/ CT
Conflicts of interest
emission bands. Complexes Ir1 and Ir3, with the BODIPY-
ethynyl motif attached at C4 of the phenyl ring of the NHC There are no conflicts to declare.
1
1
ligand, caused a red-shift of the IL/ MLCT absorption and
1
3
IL/ CT emission bands compared to those in Ir4 and Ir5 that
Acknowledgements
have BODIPY-ethynyl at C5 of the benzimidazole unit of the NHC
ligand. Meanwhile, both the lowest singlet excited state and
the emitting states of Ir1 were lowered compared to those in
Ir2, due to the extended π−conjugation induced by the ethynyl
linker in Ir1. In contrast, replacing the methyl substituents on
C3 of benzimidazole in complexes Ir1 and Ir4 by oligoether
substituents in Ir3 and Ir5, respectively, did not impact the
energies of the lowest singlet and emitting excited states in the
corresponding complexes.
W. Sun and S. Kilina acknowledges the financial support from
the National Science Foundation (DMR-1411086) for materials
synthesis, characterization and computational simulation of the
optical spectra. For computational resources and administrative
support, authors thank the Center for Computationally Assisted
Science and Technology (CCAST) at North Dakota State
University. The photobiological research reported in this
publication was supported by the National Cancer Institute of
the National Institutes of Health under Award Number
R01CA222227. The content is solely the responsibility of the
authors and does not necessarily represent the official views of
the National Institutes of Health. S. A. M. also acknowledges
support from the University of North Carolina at Greensboro,
the Natural Sciences and Engineering Council of Canada, the
Canadian Foundation for Innovation, the Nova Scotia Research
and Innovation Trust, and Acadia University. We also thank
Huimin Yin at Acadia University for her help in the
photobiological studies.
Ir3 and Ir5 were photobiologically active toward SKMEL28
melanoma cells with visible light activation, with Ir5 possessing
a
much larger PI and higher photocytotoxicity. The
photobiological trends in cancer cells did not correlate with cell-
1
free O
2
quantum yields. The DCFDA assay for intracellular ROS
detection argued against a correlation between phototoxicity
and widespread redox imbalance, but did show that Ir5 was
more effective at ROS production at the high PS concentrations.
Ir5 was also photobiologically active toward S. aureus, while Ir3
1
was not. While ROS other than O
2
may play a role in the
phototoxic mechanism toward both cancer cells and bacterial
cells, we hypothesize that factors such as cell membrane
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Photochem. Photobiol. Sci.
Table of Contents Synopsis
View Article Online
DOI: 10.1039/C9PP00142E
Neutral Ir(III) complexes bearing BODIPY−substituted N−heterocyclic carbene ligands exhibited moderate to strong
photodynamic therapeutic effects toward SKMEL28 cells and antimicrobial activity upon broadband visible light
activation.
4
8
6
4
2
x10
x10
x10
x10
4
4
4

Ir3
Ir5
0
300
400
500
600
700
Wavelength / nm
1
6 | Photochem. Photobiol. Sci., 2019, 00, 1-14
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