A novel photosensitizer based on a ruthenium(ii) phenanthroline bis(perylenediimide) dyad: synthesis, generation of singlet oxygen and in vitro photodynamic therapy

Article abstract of DOI:10.1039/C8NJ02944J

In this study, a novel photosensitizer having two perylenediimide units and a phenanthroline ruthenium(ii) coordination moiety (Ru-BP) has been developed for photodynamic therapy (PDT) of cancer cells. This new compound was prepared via reactions of two n

Full text of DOI:10.1039/C8NJ02944J

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ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
rsc.li/njc

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A novel photosensitizer based on a ruthenium(II) phenanthroline
bis(perylenediimide) dyad: synthesis, generation of singlet oxygen and in
vitro photodynamic therapy
Nuray Esra Aksakala, Hasan Hüseyin Kazanb, Esra Tanrıverdi Eçika, Fatma Yuksela*
a
Department of Chemistry, Faculty of Science, Gebze Technical University, Gebze, Kocaeli,
Turkey
b Department of Biological Sciences, Middle East Technical University, Ankara, Turkey
* Author for correspondence:
Prof. Dr. Fatma Yuksel, Department of Chemistry, Gebze Technical University, P.O.Box: 141,
Gebze 41400, Kocaeli, Turkey
Tel:
00 90 262 6053016
00 90 262 6053005
Fax:
e-mail : fatma@gtu.edu.tr
KEYWORDS: Ruthenium(II) complex, Perylene diimide, Photodynamic Therapy, Singlet
Oxygen, Leukemia cells

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ABSTRACT
In this study, a novel photosensitizer having two perylenediimide and phenanthroline
ruthenium (II) coordination moieties (Ru-BP) has been developed for photodynamic therapy
(PDT) of cancer cells. This new compound was prepared via reaction of two newly designed
molecules, namely 5,6,12,13-tetrakis(4-(tert-butyl)phenoxy)-2-(2,6-diisopropylphenyl)-9-(4-
hydroxyphenyl)anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetraone
(P6)
and bis(2,2’-bipyridyl)-(4,7-dichlorophenanthroline)ruthenium(II) complex (7). The singlet
oxygen productions of P6 and Ru-BP were investigated by chemical method using trap
molecule as 1,3-diphenylisobenzofurane. Additionally, photodynamic therapy efficacy of the
novel Ru-BP complex and P6 were evaluated in vitro. Ru-BP significantly decreased viabilities
of human chronic myeloid leukemia cells under red light but not in dark, pointing the
complex, itself, was not cytotoxic and singlet oxygen formation was required for the
initiation of cell death mechanisms. Thus, Ru-BP can be effectively used as a photosensitizer
in photodynamic therapy, which makes the novel Ru-BP a promising singlet oxygen
generator for further biological applications.

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Introduction
Photodynamic therapy (PDT) has emerged as an innovative and non-invasive therapeutic
modality for several types of cancers.1-4 The primary components of PDT are a
photosensitizer (PS), molecular oxygen and a light source. Following light excitation of the
PS, an excited triplet state is formed which acts as an energy donor to molecular oxygen
(3O2) to product reactive singlet oxygen (1O2).5,6 The sensitized 1O2 gives irreversible damage
to tumour cells in the irradiated area. Photosensitizers are generally prepared from highly
conjugated organic dyes and their transition metal complex, which can be monitored by
various optical imaging techniques.2,7-10 Compared to organic chromophores, transition
metal complexes constitute another big family of medicinal agents.11,12 Additionally, merging
of organic chromophores with charge transfer complexes often leads to attractive
properties such as unusual singlet and triplet state photophysics and related excited state
dynamics.13 Among these compounds, Ru (II)-based complexes are drawing great attention
in the development of new photosensitizers due to their excellent biocompatibility and
tuneable photophysical/chemical and excited-state properties.7,12-16 Most of the Ru(II) PSs
are based on polypyridyl ligands.12,17,18 These complexes have long emission lifetimes from
the triplet metal-to-ligand charge transfer state (MLCT) quenched by triplet (ground state)
dioxygen and exhibit intriguing PDT effects via the type II process.15,17,18 As in the most of the
complex preparation, Ru(II)-based complexes also require a light-harvesting chromophore
such as BODIPY (boron dipyrromethene), perylene or other cyclometaleted ligands.7 The
perylene unit, one of the most desirable molecules having large π-conjugation system, is a
candidate to improve the optical properties of the complexes, to provide a red-shift of MLCT
absorption band and an increased molar extinction coefficient of the metal complexes.15,19,20
Furthermore, perylenes have high photochemical stability and fluorescence quantum yields

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in common aliphatic and aromatic solvents, resistivity towards oxidative degradation and
decompositions.19-22 Perylene chromophores are mainly used in the diimide form which
receive widespread attention because of the molecular structure, rigid polycyclic aromatic
core substituted from imide (peri) positions.21 The disadvantage of perylene unit is its low
solubility and aggregation that caused from its planar skeleton. In order to overcome the
problem, perylene has been substituted from the bay positions in most of the studies.22
Although many ruthenium(II) polypyridyl complexes and perylene dyes have been
independently investigated as photosensitizers in the literature,8,20 combination of these
two units is very limited.15 The success of the combination has encouraged us to focus on
designing a novel complex bearing two perylenediimide (PDI) and a Ru(II) polypyridyl units at
the same time in order to enhance photooxygenation ability. In the present study, a novel
bis(perylenediimide) ruthenium(II) complex (Ru-BP) was prepared via reactions of two newly
designed molecules, 5,6,12,13-tetrakis(4-(tert-butyl)phenoxy)-2-(2,6-diisopropylphenyl)-9-
(4-hydroxyphenyl)
anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetraone
(P6) and 4,7-dichlorophenanthroline ruthenium(II) complex (7) (Figure 1). Generally,
ruthenium complexes coordinated by phenanthroline derivatives are substituted from 5- or
5,6- positions with several chromophores and a few perylene substituted derivatives of
those are also connected from 5-position of phenanthroline moiety.23 The present study can
be accepted as the first example of modification of two perylenediimide unit attached from
4,7-positions of phenanthroline metal complex.15,19,20 The photophysical and singlet oxygen
generation properties of the novel Ru-BP was investigated via absorption and fluorescence
spectroscopies. In addition, the photodynamic therapy efficacy of Ru-BP complex was
assessed in human chronic myeloid leukemia cells in vitro. Overall, this study presents novel
and fully characterized Ru-BP complex with fundamental photodynamic therapy efficiency.

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O
O
N
O
N
O
O
O
O
O
O
N
O
O
O
O
O
N
O
O
O
O
N
N
Ru
N
N
N
N
-
2PF6
Ru-BP
Fig. 1 Structure of new bis(perylenediimide) ruthenium(II) complex (Ru-BP).
Results and discussion
Syntheses and characterizations
The synthesis of Ru-BP is depicted in Scheme 1. First, the synthesis of new ruthenium
complex (7) was carried out via reaction between 4,7-dichloro-1,10-phenanthroline (5)24 and
ruthenium(II) bis(bipyridinyl) complex (6)25 in ethanol/H2O reaction medium. Without
advanced purification steps, the crude material was used for further reaction. Next,
synthesis of perylene diimide P6 was conducted in five steps (Scheme 1). After bromination
of P1 to obtain P2,26 preparation of diimide P327 was employed using 2,6-diisopropylaniline
as aromatic amine. Then, 4-tert-butylphenol was used to substitute bay positions of P3 in
order to prepare P4.28 After synthesizing monoimide monoanhydride P529 in KOH basic
medium, desired perylenediimide P6 was prepared using 4-aminophenol as a reagent. Single
molecular ion peak in the mass spectrum supports the purity of the compound. (Fig. S3). In
the FT-IR spectrum of P6 the disappearance of anhydride band at 1770 cm-1 confirms the
assigned structure. Also, C=O bands of imide group was observed at 1672 and 1705 cm-1 (Fig.

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1
S4). Additionally, the H-NMR and 13C-NMR spectra are consistent with the molecular
structure of P6 (Fig. S5,S6).

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O
O
O
O
O
O
O
O
Cl
Cl
Cl
N
N
Ru
N
O
O
N
Ru
N
NH
NH
O
O
NH
NH
N
N
NH2
NH2
Cl
Cl
N
N
b
c
N
N
d
2
N
F
P
2
a
Cl
1
5
4
O
O
6
O
3
7
OH
O
O
O
O
O
O
O
O
O
O
O
N
O
O
O
O
O
N
O
O
O
O
N
O
O
e
f
g
h
i
Br
Br
Br
Br
OR
OR
RO
RO
Br
Br
Br
Br
RO
RO
OR
OR
RO
RO
OR
OR
O
O
O
O
N
O
N
O
N
N
P1
P2
P5
P3
P4
P6
j
O
O
N
N
O
O
O
O
O
O
O
O
O
O
O
O
N
N
O
O
O
O
N
N
N Ru
N
N
-
N
2PF6
Ru-BP
Scheme 1 Synthesis of Ru-BP. Reagents and conditions: (a) Meldrum’s acid (2.3 equiv.)
,CH(OMe)3 , reflux, 3h (b) diphenyl ether, reflux, 30 min (c) POCl3, reflux, 2h 23 (d) 6,24 EtOH/
H2O, reflux, 16 h; (e) Br2, H2SO4, rt→120 ̊
C, 96 h25 (f) 2,6-diisopropylaniline (2 equiv.),
propionic acid, reflux, 24 h26 (g) 4-tert butylphenol (8 equiv.), K2CO3, NMP, reflux, 24 h27 (h)
KOH, t-BuOH, reflux, 30 min28 (i) 4-amino phenol (1 equiv.) , propionic acid, reflux, 24 h (j), rt,
72h.

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Nucleophilic substitution reaction of P6 and 7 leads to Ru-BP as a dark purple solid (Scheme
1). The mass spectrum of Ru-BP confirms the molecular structure. The molecular ion peak is
observed at as [M-2PF6]+. Also, the peak observed at m/z 3204 belongs to [M-PF6]+ (Fig. S7).
In the FT-IR spectrum of Ru-BP the expected imide C=O bands appear at 1671 and 1705 cm-1
similar as P6 and the absence of hydroxyl peak at around 3400 cm-1 supports the structure.
The peak around 830 cm-1 belongs to the PF6 counter ion30 of the structure (Fig. S8). The
–
1H-NMR (Fig. S9) and 13C-NMR spectra (Fig. S10) of Ru-BP are consistent with the structure
of Ru-BP. Integration ratios and chemical shift values were assigned in Fig. S9 and Fig. S10.
Aromatic protons of the structure appear between 8.7-6.8 ppm. Distinctly, compared to the
1H-NMR of P6, specific phenanthroline protons of the complex Ru-BP arise at 8.6 ppm.
Tertiary protons of diisopropyl amino group were observed at 2.7 ppm. Methyl protons of
phenoxy cores give a singlet at 1.26 ppm while diisopropyl groups’ appear at 1.11 ppm as
doublet.
Also, Ru-BP is highly soluble in common organic solvents such as dichloromethane,
chloroform, tetrahydrofuran (THF) and dimethylsuphoxide (DMSO) and it is stable below 200
°C according to the thermogravimetric analysis (TGA) (Fig S11).
Photophysical properties of Ru-BP in comparison with P6
Spectral characterization of Ru-BP was carried out recording absorption, excitation and
emission spectra in dimethylsulphoxide (DMSO) at room temperature. To study the effect of
the Ru(II) phenanthroline chromophore, same spectroscopic studies were also carried for
newly synthesized P6. The UV-vis absorption spectra of P6 and Ru-BP are shown in Figure 2.
Absorption maxima together with molar extinction coefficients are depicted in Table 1. The
spectra of P6 and Ru-BP show a similar profile bearing four maxima observed approximately

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at 260, 285, 450 and 580 nm. The overlap of characteristic π- π* transition of PDI and MLCT
transitions appeared between 440 and 590 nm.31 The increase in the absorption intensity at
260-290 nm region indicates the existence of Ru(II) bipyridyl moiety in the main structure.
Additionally, molar extinction coefficient of Ru-BP is higher than the P6’s as indicated in
Table 1. The ground state absorption spectra of P6 and Ru-BP were also recorded at
different concentrations (Fig. S12 and S13). The emission properties of P6 and Ru-BP were
studied in DMSO at room temperature (Table 1). The patterns of emission behavior of P6
and Ru-BP were similar, and their emission maxima were 614 and 615 nm. For both
compounds, the excitation spectra were similar to the corresponding absorption spectra and
the both excitation and absorption spectra were mirror image of their own fluorescence
emission spectra. The Stoke’s shift was found to be 34 nm for P6 and 33 nm for Ru-BP. The
fluoroscence lifetimes (τF) of P6 and Ru-BP was measured using the time correlated single
photon counting (TCSPC) technique in DMSO. Obtained lifetime spectra are shown in Fig
S16. The lifetimes were found to be 2.09 ns for P6 and 1.04, 5.65 for Ru-BP. The absorption
and emission profiles of Ru-BP is suitable for in vivo photooxidation and deeper penetration
in tissue.1,3

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2
1
0
Ru-BP
P6
300
400
500
600
700
Wavelength (nm)
Fig. 2 UV-vis absorption spectra of P6 and Ru-BP in DMSO.
Table 1 Photophysical and photochemical properties of P6 and Ru-BP a.
Compound
P6
λab, nm (Log εb)
λem, nm
614
τF (ns)c
dφΔ
267 (4.74), 283 (4.77), 449 (4.33), 580 (4.78)
264 (11.85), 290 (11.80), 451 (4.62), 582 (4.94)
2.09
0.01
(CHISQ=0.9899979)
1.04 (23.6 %) 5.65 (76.4 %)
(CHISQ = 0.9366626)
615
0.30
Ru-BP
a
b
c
dimethylsulphoxide Molar extinction coefficients Lifetime dSinglet Oxygen Quantum
Yield

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A
Absorption
Excitation
Emission
200
100
0
0.2
0.0
400
500
600
700
800
900
Wavelength (nm)
0.2
Absorption
Emission
Excitation
B
100
0.1
0.0
400
0
500
600
700
800
Wavelength (nm)
Fig. 3 UV-vis, fluorescence emission and excitation spectra of P6 (0.5 µM) (A) and Ru-BP (2.0
µM) (B)

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Photosensitizing property of Ru-BP
The mechanism of type II PDT is based on energy transfer from triplet state of the
photosensitizers to molecular oxygen for generating singlet oxygen (1O2) which can instan-
1
taneously damage biomolecules to initiate cell death.6 Investigating the generation of O2 is
highly significant to pre-study PDT application at the molecular level. In this work, singlet
oxygen generation capacity of novel bis(perylenediimide) ruthenium (II) complex (Ru-BP)
was performed using diphenylisobenzofuran (DBPF), selective singlet oxygen trap molecule,
in DMSO. A solution of the Ru-BP (2.0 µM) and DPBF (35.0 µM) was kept for 20 min in the
dark to eliminate any inputs to the absorbance signal of DPBF from obscure dark reactions.
Absorption band of DPBF at 414 nm did not show any change proved the lack of dark
reactions. The mixture of the Ru-BP and DPBF was irradiated with red light (λ= 632 nm, 2.5
mW /cm2) for a time interval 0 to 24.0 min. Photo-induced production of singlet oxygen from
Ru-BP was monitored gradual decrease in the absorption intensity of DPBF (Fig. 4). The data
for Ru-BP was plotted as the change in absorption of the trap molecule at 414 nm versus
irradiation time (Fig.4). Also, singlet oxygen measurements of the P6 (2.0 µM) and
methylene blue (2.0 µM) as the reference molecule were performed under the same
experimental conditions (Fig. S14, S15). Singlet oxygen quantum yields (ΦΔ) of free ligand P6
and Ru-BP were calculated according to the literature.32 The singlet oxygen quantum yield of
Ru-BP(ΦΔ=0.30) is thirty times higher than the free ligand P6 (ΦΔ=0.01) (Fig. S15). The
attractive increase of the ΦΔ would be explained by heavy atom effect that has been widely
known as the most popular way to facilitate the intersystem crossing (ISC) of the
chromophore.7 In the present work, singlet oxygen generation showed good efficiency when
compared with reported ruthenium(II) or ruthenium perylene photosensitisers.14,15 The

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photostability upon repetitive excitation and is desirable in PDT application.1,7 The solution
of Ru-BP without DPBF were triggered with light for 20 min and no change was observed in
the absorbance intensity of compound in the red region of the spectrum (Fig. S17). It is
obvious that this complex was not degraded under the condition of singlet oxygen
measurements.
1.2
0 s
1.2
dark
5 s
y = -0.0195x + 1.0825
1
0.8
0.6
0.4
0.2
0
0.8
0.4
10 s
15 s
20 s
25 s
30 s
0
10
20
30
Time (s)
350
400
450
500
550
600
650
700
Wavelength (nm)
Fig. 4 Decrease in absorbance spectrum of DPBF (35.0 µM) in the presence of Ru-BP (2.0
µM) in DMSO and absorbance decrease of DPBF at 414 nm with time in DMSO in the
presence of Ru-BP.
In vitro photodynamic therapy
In the present study, we also aimed to study the photodynamic therapy efficacy of Ru-BP
after chemical characterization. Ruthenium complexes and their in vitro and/or in vivo
efficacy have been widely used in photodynamic therapy studies for effective treatment of
cancer.16,17,33 To check the efficacy of Ru-BP in terms of photodynamic therapy, we used

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human chronic myeloid leukemia cell line, K562. We treated cells with increasing
concentrations of Ru-BP and incubated the cells either under red light (λ= 632 nm, 2.5 mW
/cm2) or in dark for 5 h. Next, we determined the cell viabilities by MTT assay. According to
results (Fig. 5), Ru-BP complex significantly decreased cell viabilities if only the cells were
incubated under red light but not in dark. The ability of Ru-BP in the production of singlet
oxygen by red light source was demonstrated in Fig. 4 Singlet oxygen, whether intracellular
or extracellular, is able to trigger apoptosis, a type of well-regulated cell death mechanism.34
Fig. 5 proves that Ru-BP, itself, was not enough to trigger cell death in dark but a red light
source was needed to produce singlet oxygen and cause death of cancer cells. However, this
data is not enough to determine the cell death type, whether apoptotic or not, which
requires further molecular studies. Also, P6, itself, at maximum concentration of Ru-BP
tested was not successful to induce cell death in dark or under red light, confirming the
chemical characterization studies of P6, which was shown to produce limited singlet oxygen
compared to Ru-BP, on cells. The inhibitory concentration 50 (IC50) value for Ru-BP was
calculated as below 5.0 µM while could not be calculated when they were treated in dark,
pointing the success of light-excited Ru-BP in the initiating cell death.

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Cytotoxicity assay
150
100
50
Dark
****
Red Light
*
**
****
ns
****
****
0
Ru-BP ( M)
Fig. 5 In vitro photodynamic therapy efficacy. *p<0.05; **p<0.01; ****p<0.0001.
To further show the ability of Ru-BP in the induction of cell death in the presence of red
light, we used Trypan Blue staining. It is a dye widely used in cell culture and cannot
penetrate into living cells while it internalizes into and stains the nonviable cells blue.35 In
this study, we treated cells with Trypan Blue in absence or presence of Ru-BP and under light
or in dark. According to the results, the blue colour that is a sign of nonviable cells was only
available for Ru-BP-treated cells under red irradiation. Ruthenium complexes have been
widely used in photodynamic therapy studies. However, some problems, such as, high IC50
value,36 increased toxicity of photosensitizer without irradiation,37 long incubation time of
photosensitizer38 and irradiation under lower wavelengths preventing deep tissue
penetration39 have been faced during these studies. The present study is critical in terms of
overcoming listed drawbacks of ruthenium-based photodynamic therapy studies owing to
low cytotoxicity of Ru-BP in dark (Fig. 5), relatively low IC50 value (<5 µM), short incubation

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time to induce cell death (5 h) and irradiation with red light which increases the tissue
penetration depth. Still, further in vivo studies are needed to conclude the effectiveness of
Ru-BP in clinics.
Fig. 6 Trypan Blue staining of Ru-BP-treated or –untreated cells in dark or under red light.
Red arrows point the blue-coloured cells. Scale: 200 µm.
Experimental Section
General methods
Perylene-3,4,9,12-tetracarboylic
dianydride,
1,10-phenanthroline,
tetrabutylammonium hexafluorophosphate and RuCl3.H2O was obtained from Sigma-Aldrich.
All reagents and solvents were of reagent-grade quality, obtained from commercial

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suppliers. Infrared spectra were recorded between 4000 and 650 cm-1 by using a Perkin
Elmer Spectrum100 FT-IR spectrometer with an attenuated total reflection (ATR) accessory
featuring a zinc selenide (ZnSe) crystal. The mass spectra were recorded on a MALDI (Matrix
Assisted Laser Desorption Ionization) BRUKER Microflex LT using 1,8,9-anthrasenetriol
(dithranol) as a matrix. The NMR spectra were recorded on a Varian INOVA 500 MHz
spectrometer. Electronic absorption spectra were recorded with a Shimadzu 2101 UV
spectrophotometer and the fluorescence excitation and emission spectra were recorded on
a Varian Eclipse spectrofluorometer using 1 cm pathlength cuvettes at room temperature.
Fluorescence lifetimes were measured by a time correlated single photon counting (TCSPC)
method using FLUOROLOG-3 spectrofluorometer (Horiba JobinYvon, Edison, NJ) equipped
with a NanoLED and a standard air cooled R928 PMT detector. Photo-irradiations were done
using a red LED (λ= 632 nm, 2.5 mW /cm2) and samples were irradiated with the light source
from a 10.0 cm distance.
Singlet Oxygen Measurements
Singlet oxygen quantum yields (ΦΔ) were calculated according to the literature.32 The
relative quantum yields were calculated with reference to Methylene Blue (MB) in DMSO as
0.52.40 Air saturated DMSO was obtained by bubbling air for 30 minutes. The absorbance of
DPBF was adjusted around 1.0-1.1 in air saturated DMSO. Then, the sample was added to
cuvette and sample’s absorbance was adjusted around 0.1-0.2. After, taking some
measurements in dark, we exposed the cuvette to red LED at the peak absorption
wavelength for 5 s. Absorbance was measured for several times after each irradiation. Then,
slope of absorbance maxima of DPBF at 414 nm versus time graph for each sample was
calculated. Singlet oxygen quantum yield was calculated according to the equation:

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ΦΔ (samp) = ΦΔ (ref) × [m (samp)/m(ref)]×[F(ref)/F(samp)]
“m” is the slope of difference in change in absorbance of DPBF (414 nm) with the irradiation
time, “F” is the absorption correction factor, which is given by F = 1 – 10-OD (OD at the
irradiation wavelength).
Synthesis of 7. Compound 5 and 6 were prepared according to the reported procedures.24,25
A mixture of 5 (52 mg, 0.21 mmol) and 6 (0.1 g, 0.21 mmol) was refluxed in Ethanol/H2O (15
mL/7 mL) for 16 h.(Scheme 1) After cooling, NH4PF6 (0.6 g, 3.68 mmol) was added to the
mixture. Resulting orange solid was washed with water and diethyl ether. ATR-IR νmax (cm-1):
3325, 3123, 1606, 1560, 1469, 1446, 1412, 1395, 1336, 1316, 1245, 1219, 1085, 821, 760,
730 cm-1. MS: MALDI-TOF (dithranol): m/z: 662 [M-2PF6]+ , 629 [M-2PF6-Cl]+ (Fig. S1, S2).
Synthesis of P6. From P1 to P5 literature procedure26-29 was employed. A mixture of 4-
aminophenol (0.01 g, 0.1 mmol) and (55.0 mg, 0.05 mmol) of P5 was refluxed under Ar
atmosphere in 5 mL propionic acid. After 24 h, the mixture was extracted with H2O/CH2Cl2.
Preparative thin layer chromatography on silica gel (CH2Cl2) yielded 25 mg (42% yield) of P6.
1H-NMR (CDCl3) δ 8.28 (s, 2H), 8.23 (s, 2H), 7.41(t, J = 8.0 Hz, 2H), 7.25-7.21 (m, 10H), 7.08 (d,
J = 8.5 Hz, 2H), 6.88 (t, J = 10 Hz, 5H), 6.83 (d, J = 8.5 Hz, 4H), 2.70 (septet, J = 7.0 Hz, 2H),
1.26 (d, J = 4.0 Hz, 36 H), 1.11 (d, J = 7.0 Hz, 12H). 13C-NMR (CDCl3): 164.051, 163.578,
156.241, 156.211, 155.921, 153.076, 152.958, 147.638, 147.558, 145.832, 133.392, 130.9,
129.846, 129.609, 127.969, 126.894, 124.093, 122.933, 122.75, 121.077, 120.878, 120.496,
120.362, 120.031, 119.545, 119.411, 116.415, 34.58, 31.638, 29.922, 29.29, 24.229. ATR-IR
νmax (cm-1): 3406, 3063, 3039, 2962, 2930, 2869, 1705, 1672, 1585, 1504, 1464, 1447, 1407,
1363, 1341, 1315, 1283, 1220, 1173, 882, 832 cm-1. UV (DMSO): λmax = 267, 283, 449, 580
nm. MS: MALDI-TOF (dithranol): m/z: 1235 [M+], 1257 [M+ 2H] (Fig. S3-S6).

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Synthesis of Ru-BP. A mixture of 7 (0.02 g, 0.03 mmol), P6 (75.0 mg, 0.06 mmol) and K2CO3
(8.2 mg, 0.06mmol) was stirred at room temperature under Ar in DMF (Scheme 1). After 72
h, the mixture was extracted with H2O/CH2Cl2. Chromatography on silica gel (CH2Cl2 →
CH2Cl2 /EtOH (50/1) yielded 60 mg (60% yield) of Ru-BP. 1H-NMR (CDCl3) δ 8.61 (s, 2H), 8.30-
8.27 (m, 8H), 8.24 (s, 4H), 7.93-7.91 (m, 4H), 7.86-7.83 (m, 4H), 7.59 (d, J = 6.0 Hz, 2H), 7.47-
7.38 (m, 14 H), 7.25-7.20 (m, 21H), 6.99 (d, J = 6.0 Hz, 2H), 6.86-6.82 (m, 15 H), 2.7 (septet, J
= 6.5 Hz, 4H), 1.26 (s, 72 H), 1.11 (d, J = 6.5 Hz, 24H). 13C-NMR (CDCl3) 181.369, 180.874,
163.607, 163.557, 163.491, 162.62, 157.399, 156.399, 156.336, 156.122, 153.066, 152.878,
152.672, 152.572, 148.184, 147.585, 145.817, 137.826, 137.564, 137.477, 133.918, 133.413,
133.375, 131.507, 130.883, 129.7, 129.615, 129.194, 128.773, 128.083, 127.104, 126.88,
126.704, 126.649, 124.094, 123.87, 123.682, 123.59, 123.422, 123.004, 122.376, 122.268,
122.164, 122.094, 121.375, 120.725, 120.633, 120.545, 120.336, 120.3, 120.05, 119.628,
119.489, 119.458, 119.327, 119.276, 116.872, 110.364, 34.715, 34.578, 34.436, 33.395,
32.142, 31.835, 31.779, 31.652, 31.5, 29.917, 29.665, 29.578, 29.496, 29.461, 29.386,
29.298, 24.972, 24.239, 22.91, 14.338. ATR-IR νmax (cm-1): 3067, 2962, 2906, 2869, 1705,
1671, 1585, 1501, 1466, 1405, 1363, 1339, 1281, 1206, 1173, 1110, 1014, 834, 761, 729 cm-
1. UV (DMSO): λmax = 264, 290, 448, 551, 582 nm. MS: MALDI-TOF (dithranol): m/z: 3060 [M-
2PF6], 3204 [M-PF6 ] (Fig. S7-S10).
Cell lines and cell culture
In this study, human chronic myeloid leukemia, K562 cell line was used for photodynamic
therapy studies. Cells were maintained in RPMI 1640 medium (Biochrom AG, Germany) in
the presence of 10% FBS (v/v; Biochrom AG, Germany) and 1% gentamycin (v/v; Biological
Industries, Israel) at 37⁰C and 5% CO2.

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Cell viability assay
Cell viabilities were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium
bromide (MTT) assay. 30x103 cells were seeded into two different 96-well plates and
incubated for 24h. Next, cells were treated DMSO to check the solvent effect and increasing
concentration of Ru-BP (0.3125-20 µM). One of the plates was incubated in dark while the
other was incubated under red LED for 5 hours. Then, 10 µl of MTT solution (5mg/ml) was
added onto cells and incubated for 4h. Finally, cells were disrupted with SDS-HCl solution (1g
SDS in 0.01M HCI in 10ml final volume) overnight and the microplates were read by
microplate spectrophotometer (Multiskan GO, Thermo Fisher Scientific, USA) at a
wavelength of 570 nm. Optical densities were converted to % viability by using untreated
cells as 100% and all the treatments were correlated to untreated group. Data were
analyzed by GraphPad Prism 7.0 software (GraphPadInc, USA). The results were considered
significant at the level of 0.05.
Trypan Blue staining
To further demonstrate the cell death, 480x103 cells were treated into two different 6-well
plates. One of the well of each plates was treated with 20.0 µM of Ru-BP and one of them
was treated with DMSO as control. One of the plates was incubated in dark while the other
was treated red light for 5 h. Next, cells were incubated with Trypan Blue (10%) for 5 min
and washed by PBS twice. Next, cells were imaged under a light microscope.
Statistical analyses
All biological experiments were conducted triplicate as three independent experiments. Data
were analysed by Two-way ANOVA and was evaluated as significant when p<0.05.

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CONCLUSIONS
In this work, we demonstrated design, synthesis and photooxygenation of a novel ruthenium
phenanthroline bis(perylenediimide) complex (Ru-BP). The newly synthesized Ru-BP and its
1
starting compound, P6 were fully characterized by FT-IR, MALDI-TOF, H and 13C-NMR, UV-
vis spectroscopy analyses. The novel Ru-BP possesses two PDI core in a ruthenium(II)
complex that is the first example of bis(perylenediimide) substituted phenanthroline metal
complex. The photophysical and photochemical properties of the novel Ru-BP was
investigated via absorption and fluorescence spectroscopies in comparison with P6. Ru-BP
exhibited favourable photophysical properties in the red region with good molar extinction
coefficient. It is obvious from the singlet oxygen quantum yields that ruthenium complex
form of P6 leads to a respectable photosensitizer ability. Moreover, novel Ru-BP complex
displayed significant photodynamic therapy efficacy in vitro. Ru-BP efficiently decreased the
survival of human chronic myeloid cells, K562 under red light but not in dark. This result also
confirms the production of singlet oxygen by Ru-BP in vitro by which cell death mechanisms
were activated. The novel Ru-BP complex is also a strong candidate for in vivo photodynamic
therapy studies due to its low toxicity in dark, low IC50 value, short incubation time and
possible longer tissue penetration depth. Overall, the results described herein clearly
indicate that the novel complex Ru-BP is a potential singlet oxygen generator for PDT
processes. Thus, our in vivo animal model studies are under progress.
ACKNOWLEDGEMENTS
This work was financially supported by the Gebze Technical University Research Project
Foundation, BAP-2017 -A102-15.
REFERENCES

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Page 22 of 24
View Article Online
DOI: 10.1039/C8NJ02944J
1 R. Bonnett, Chemical Aspects of Photodynamic therapy, Gordon & Breach, London, UK,
2000.
2 J. P. Celli, B. Q. Spring, I. Rizvi, C. L. Evans, K. S. Samkoe, S. Verma, B. W. Pogue, T. Hasan,
Chem. Rev., 2010, 110, 2795–2838.
3 M. Ethirajan, Y. Chen, P. Joshi, R. K Pandey, Chem. Soc. Rev., 2011, 40, 340–362.
4 S. H. L. Higgins, K. J. Brewer, Angew. Chem., Int. Ed., 2012, 51, 11420–11422.
5 S. G. Awuahaband, Y. You, RSC Adv., 2012, 2, 11169-11183.
6 J. F. Lovell, T. W. B. Liu, J. Chen, G. Zheng, Chem. Rev., 2010, 110, 2839-57.
7 J. Zhao, W. Wu, J. Sun, S. Guo, Chem. Soc. Rev., 2013, 42, 5323-5351.
8 F. Yukruk, A. L. Dogan, H. Canpinar, D. Guc, E. U. Akkaya, Org. Lett., 2005, 7, 2885-2887.
9 A. Wang, W. Jin, E. Chen, J. Zhou, L. Zhou, S. Wei, Dalton Trans., 2016, 45, 3853-3862.
10. A. Kamkaew, S. H. Lim, H. B. Lee, L. V. Kiew, L. Y. Chungc, K. Burgess, Chem. Soc. Rev.,
2013, 42, 77-88.
11. C. G. Hartinger, P. J. Dyson, Chem. Soc. Rev., 2009, 38, 391–401.
12 Q. X. Zhou, W. H. Lei, J. R. Chen, C. Li, Y. J. Hou, X. S. Wang, B. W. Zhang, Chem. Eur. J.,
2010, 16, 3157 – 3165.
13 F. N Castellano, Acc. Chem. Res., 2015, 48, 828-839.
14 Z. Lv., H. Wei, Q. Li, . Su, S. Liu, K. Y. Zang, W. Lv, Q. Zao, X. Li, W. Huang, Chem. Sci., 2018,
9, 502–512.
15 P. J. S. Maiaa, I. Aguiarb, M. S. Vellosoc, D. Zhangd, E. R. Santosb, J. R. Oliveirac, J. C.
Junqueirac, M. Selked, R. M. Carlosb, J. Photochem. Photobiol., A, 2018, 353, 536–545.
16. P. Kaspler, S. Lazic, S. Forward, Y. Arenas, A. Mandela, L. Lilge, Photochem. Photobiol. Sci.,
2016, 15, 481–495.
17 H. Huang, B. Yu, P. Zhang, J. Huang, Y. Chen, G. Gasser, L. Ji, H. Chao, Angew. Chem., Int.
Ed., 2015, 54, 14049.
ꢀ
18 J. Liu, Y. Chen, G. Li, P. Zhang, C. Jin, L. Zeng, L. Ji, H. Chao, Biomaterials, 2015, 56, 140.
19 K. Kodama, A. Kobayashi, T. Hirose, Tetrahedron Lett., 2013, 54, 5514–5517.
ꢀ

Page 23 of 24
New Journal of Chemistry
View Article Online
DOI: 10.1039/C8NJ02944J
20 E. R. Santos, J. Pina, T. Venâncio, C. Serpa, J. M. G. Martinho, R. M. Carlos, J. Phys. Chem.
C, 2016, 120, 22831−22843.
21 F. Würthner, C. R. Saha-Möller, B. Fimmel, S. Ogi, P. Leowanawat, D. Schmidt, Chem.
Rev., 2016, 116, 962−1052.
22. F. Würthner, C. Thalacker, A. Sautter, Adv. Mater., 1999, 11, 754-758.
23 R. K. Dubey, M. Niemi, K. Kaunisto, K. Stranius, A. Efimov, N. V. Tkachenko, H.
Lemmetyinen, Inorg. Chem., 2013, 52, 9761−9773.
24 F. Molock, D. W. Boykin, J. Heterocycl. Chem., 1983, 20, 681-686.
25 Y. Nakabayashi, Y. Watanabe, T. Nakao, O. Yamauchi, Inorg. Chim. Acta, 2004, 357, 2553.
26 W. Qiu., S. Chen, X. Sun, Y. Liu, D. Zhu, Org. Lett., 2006, 8, 867-870.
27 Z. B. Hill, D. B. Rodovsky, J. M. Legera, G. P. Bartholomew, Chem. Comm., 2008, 6594-96.
28 H. Quante, P. Schlichting, U. Rohr, Y. Geerts, K. Müllen, Macromol. Chem. Phys., 1996,
197, 4029-44.
29 L. D. Wescott, D. L. Mattern, J. Org. Chem., 2003, 68, 10058-66.
30 J. G. Wang, Y. L. Shang, Mater. Res. Innovations, 2013, 17, 58-61.
31 F. Würthner, Chem. Commun., 2004, 1564–1579.
32 N. Adarsh, M. Shanmugasundaram, R. R. Avirah, D. Ramaiah, Chem. Eur. J., 2012, 18,
12655-62.
33 L. Zheng, S. Kuang, G. Li, C. Jin, L. Ji, H. Chao, Chem. Commun., 2017, 53, 1977-1980.
34 M. Reithmuller, N. Burger, G. Bauer, Redox Biology, 2015, 6, 157-168.
35 N. Barekzi, M. Laroussi, G. Konesky, S. Roman, Plasma Process Polym., 2016, 13, 1189-
1194.
36 W. Li, Q. Xie, L. Lai, Z. Mo, X. Peng, E. Leng, D. Zhang, H. Sun, Y. Li, W. Mei, S. Gao,
Photodiagn. Photodyn., 2017, 18, 83-94.
37 K. Qiu, J. Wang, C. Song, L. Wang, H. Zhu, H. Huang, J. Huang, H. Wang, L. Ji, H. Chao, ACS
Appl. Mater Interfaces, 2017, 9, 18482-18492.
38 H. Huang, P. Zhang, B. Yu, C. Jin, L. Ji, H. Chao, Dalton Trans., 2015, 44, 17335-17345.
39 H. Yu, J. Liu, Z. Hao, J. He, M. Sun, S. Hu, L. Y, H. Chao, Dyes Pigments, 2017, 136, 416-426.
40 N. Adarsh, R. R. Avirah, D. Ramaiah, Org. Lett. 2010, 12, 5720-5723.

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