A novel water soluble axially substituted silicon(IV) phthalocyanine bearing quaternized 4-(4-pyridinyl)phenol groups: Synthesis, characterization, photophysicochemical properties and BSA/DNA binding behavior

Article abstract of DOI:10.1016/j.poly.2020.114937

The novel axially bis[4-(4-pyridinyl)phenol] substituted silicon(IV) phthalocyanine (3) and its quaternized derivative (3Q) were designed and synthesized. These phthalocyanines were fully characterized by elemental analysis and different spectroscopic met

Full text of DOI:10.1016/j.poly.2020.114937

Journal Pre-proofs
A novel water soluble axially substituted silicon(IV) phthalocyanine bearing
quaternized 4-(4-pyridinyl)phenol groups: Synthesis, characterization, photo‐
physicochemical properties and BSA/DNA binding behavior
Shaya Y. Al-Raqa, Khaoula Khezami, Esra Nur Kaya, Mahmut Durmuş
PII:
S0277-5387(20)30594-5
DOI:
Reference:
https://doi.org/10.1016/j.poly.2020.114937
POLY 114937
To appear in:
Polyhedron
Received Date:
Revised Date:
Accepted Date:
2 September 2020
10 November 2020
16 November 2020
Please cite this article as: S.Y. Al-Raqa, K. Khezami, E.N. Kaya, M. Durmuş, A novel water soluble axially
substituted silicon(IV) phthalocyanine bearing quaternized 4-(4-pyridinyl)phenol groups: Synthesis,
characterization, photophysicochemical properties and BSA/DNA binding behavior, Polyhedron (2020), doi:
https://doi.org/10.1016/j.poly.2020.114937
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A novel water soluble axially substituted silicon(IV) phthalocyanine
bearing quaternized 4-(4-pyridinyl)phenol groups: Synthesis,
characterization, photophysicochemical properties and BSA/DNA
binding behavior
Shaya Y.Al-Raqaa, Khaoula Khezamib,c, Esra Nur Kayab, Mahmut Durmuşb*
aTaibah University, Department of Chemistry, Al-Madinah Al Munawrah, P.O Box 344, Saudi
Arabia
bGebze Technical University, Department of Chemistry, Gebze, 41400, Kocaeli, Turkey.
cUniversity of Carthage, Faculty of Science of Bizert, Tunisia.
-------------------------------------------------------------------------------------------------------
*Correspondence to: Mahmut Durmuş, Tel: 00902626053019, Fax: 00906053001
e.mail: durmus@gtu.edu.tr
1

Abstract
The novel axially bis[4-(4-pyridinyl)phenol] substituted silicon(IV) phthalocyanine (3) and its
quaternized derivative (3Q) were designed and synthesized. These phthalocyanines were fully
characterized by elemental analysis and different spectroscopic methods, such as FT-IR, UV–Vis,
MALDI-TOF and 1H NMR. The photophysical and photochemical properties, such as electronic
absorption, fluorescence emission, fluorescence lifetimes, singlet oxygen generation and
photostability, of these phthalocyanines were investigated. These properties were examined in
DMSO for the non-ionic phthalocyanine (3) and in both DMSO and aqueous solution for the ionic
phthalocyanine (3Q). The addition of bis[4-(4-pyridinyl)phenol] groups as axial ligands showed
an improvement of the photophysical and photochemical properties. In addition, a spectroscopic
investigation of the binding behavior of the quaternized silicon(IV) phthalocyanine complex to
bovine serum albumin (BSA) and DNA was also studied in this work.
Keywords: Silicon(IV) phthalocyanine, fluorescence, singlet oxygen, photosensitizer, DNA/BSA
binding.
2

1. Introduction
Phthalocyanines have been found to be a successful form of second generation photosensitizer in
recent decades due to their low dark toxicity, strong absorption in the phototherapeutic window
and simple chemical modification [1-5]. Most studies have focused on their activities against
cancer and several photosensitizers based on phthalocyanine have been authorized for clinical
testing to date [1].
Owing to their interesting photophysicochemical properties, phthalocyanine complexes have been
intensively studied with exciting results for their potential application in photodynamic therapy
(PDT) [6-8]. The most important disadvantage of phthalocyanines in these applications is their
insolubility in specific solvents and the presence of aggregation due to plain ring interactions.
Peripheral, non-peripheral or axially substituted phthalocyanines may be prepared to resolve these
limitations [9-15]. Most studies have shown that amino functionalized photosensitizers typically
exhibit some biological activity. Amino groups often include tertiary amines or quaternary
ammonium salts, such as piperidinyl, pyridyl, morpholinyl, piperazinyl, dimethylaminoethyl and
their respective methylated classes [16-26]. Water-soluble and cationic phthalocyanine dyes, in
particular, are extremely powerful substances for use in healthcare and medical fields. Because of
their ability to bind to DNA through intercalation or large and minor groove interactions etc. and
their photo-activation properties, phthalocyanine derivatives seem to be promising anticancer
agents [27-31].
The aim of this study is to prepare a fully water soluble phthalocyanine and to investigate its
photophysical and photochemical properties for photodynamic therapy. For this purpose, the
axially bis[4-(4-pyridinyl)phenol] substituted silicon(IV) phthalocyanine (3) was designed and
3

synthesized for the first time. This compound was converted to its quaternized derivative (3Q) to
obtain a water soluble phthalocyanine photosensitizer (Scheme 1). The photophysical properties of
the singlet state and photochemical properties of both new synthesized SiPcs (3 and 3Q) were
determined. The bovine serum albumin (BSA) and DNA binding behaviors of the studied water
soluble silicon(IV) phthalocyanine (3Q) were also investigated in water.
2. Experimental
2.1. Equipment
FT-IR spectra were recorded on a Perkin Elmer Spectrum 100 spectrometer. Electronic absorption
spectra in the UV-visible region were recorded with a Shimadzu 2101 UV spectrophotometer.
Positive ion and linear mode MALDI-MS of the novel phthalocyanines were obtained in 2,5-
dihydroxybenzoicacid (DHB) as the MALDI matrix, using a nitrogen laser accumulating 50 laser
shots, with a Bruker Microflex LT MALDI-TOF mass spectrometer. 1H NMR spectra were
recorded on a Varian 500 MHz spectrometer in DMSO-d6 and CDCl3 solution. Fluorescence
excitation and emission spectra were recorded on a Varian Eclipse spectrofluorometer using a 1
cm path length cuvette at room temperature. Fluorescence lifetimes were measured by a time
correlated single photon counting (TCSPC) method using a FLUOROLOG-3 spectrofluorometer
(Horiba JobinYvon, Edison, NJ) equipped with a NanoLED and a standard air cooled R928 PMT
detector. Elemental analyses were performed with a Thermo Finnigan Flash 1112 Instrument
(Waltham, MA, USA). Photo-irradiations were done using a General Electric quartz line lamp
(300W). A 600 nm glass cut off filter (Schott) and a water filter were used to filter off ultraviolet
and infrared radiations respectively. An interference filter (Intor, 670 nm with a band width of 40
nm) was additionally placed in the light path before the sample. Light intensities were measured
with a POWER MAX5100 (Molelectron detector incorporated) power meter.
4

2.2. Photophysical parameters
2.2.1 Fluorescence quantum yields and lifetimes
Fluorescence quantum yields (ΦF) were determined in DMSO for the non-ionic phthalocyanine
and in both DMSO and water+Triton-X solutions for the quaternized cationic phthalocyanine by a
comparative method using equation 1 [32, 33]:
F . AStd . n 2
(1)
Φ
F
 Φ (Std)
F
F
Std . A . nS2td
where F and FStd are the areas under the fluorescence emission curves of the samples (3 and 3Q)
and the standard, respectively. A and AStd are the respective absorbances of the samples and
n 2
2
standard at the excitation wavelengths, respectively.
and
are the refractive indices of the
n Std
solvents used for the samples and standard, respectively. The unsubstituted ZnPc (ΦF = 0.20) [34]
was employed as the standard in DMSO. The absorbance of the solutions at the excitation
wavelength ranged between 0.04 and 0.05.
Fluorescence lifetime measurements were performed with the FLUOROLOG-3
spectrofluorometer instrument using the TCSPC technique. A NanoLED was used as the light
source and a standard air cooled R928 PMT as the detector. The instrumental response function
was measured with the use of a Ludox® (Sigma-Aldrich) colloidal solution at the excitation
wavelengths of the samples. Signals were acquired using a Datastation FluoroHub-B single photon
counting controller and data analysis was performed using the commercially available DAS 6
decay analysis software package from Horiba JobinYvon. The goodness of fit was assessed by
5

minimizing the reduced chi squared function, each trace contained 10000 points and the reported
lifetime values were the result of at least three independent measurements.
2.3. Photochemical parameters
2.3.1. Singlet oxygen quantum yields
Singlet oxygen quantum yield () determinations were carried out using the experimental set-
up described in the literature [35,36] in DMSO for the axially di-substituted silicon(IV)
phthalocyanine (3) and in both DMSO and water + TX-100 for the quaternized cationic
phthalocyanine (3Q). Typically, a 3 mL portion of the respective unsubstituted or peripheral
tetra- (3 and 3Q) substituted silicon(IV) phthalocyanine solutions (C= 1x10-5 M) containing
the singlet oxygen quencher was irradiated in the Q band region with the photo irradiation set-
up described in the references [35, 36]. The  values were determined in air using the relative
method with ZnPc (in DMSO) or ZnPcSmix (in aqueous media) as standards. 1,3-
Diphenylisobenzofuran (DPBF) or 9,10-anthracenediyl-bis(methylene)dimalonic acid
(ADMA) were used as chemical quenchers for singlet oxygen in DMSO and aqueous media,
respectively. Equation 2 was employed for the determination of the  values:
Std
abs
R . I
Std
Δ
Φ
Δ
 Φ
Std
R
. Iabs
(2)
ΦSΔtd
ΦSΔtd
where
is the singlet oxygen quantum yield for the standards ZnPc (
= 0.67 in DMSO)
ΦSΔtd
[37] and ZnPcSmix
(
= 0.45 in aqueous media) [36], R and RStd are the DPBF (or ADMA)
photobleaching rates in the presence of the respective samples (3 and 3Q) and standards,
6

Std
abs
I
respectively. Iabs and
are the rates of light absorption by the samples (3 and 3Q) and
standards, respectively. To avoid chain reactions induced by the quenchers (DPBF or ADMA)
in the presence of singlet oxygen, the concentration of the quenchers (DPBF or ADMA) was
lowered to ~3 x 10-5 M [38]. Solutions of the sensitizer (C = 1 x 10-5 M) containing the quencher
(DPBF or ADMA) were prepared in the dark and irradiated in the Q band region. DPBF
degradation at 417 nm and ADMA degradation at 380 nm were monitored. A light intensity of
6.21 x 1015 photons s-1 cm-2 was used for the  determinations.
2.3.2. Photodegradation quantum yields
Photodegradation quantum yield (Φd) determinations were carried out using the experimental set-
up described in the literature [35,36]. Photodegradation quantum yields were determined using
equation 3 in both DMSO and water+Triton-X solutions.
(C0
 C
t
). V . N
A
Φd 
(3)
Iabs.S. t
where C0 and Ct are the sample concentrations before and after irradiation, respectively. V is the
reaction volume, NA is Avogadro’s constant, S is the irradiated cell area and t is the irradiation
time, Iabs is the overlap integral of the radiation source light intensity and the absorption of the
samples. A light intensity of 2.17 x 1016 photons s-1 cm-2 was employed for the Φd determinations.
2.4. Binding of quaternized silicon(IV) phthalocyanine (3Q) to BSA protein
The binding of the water soluble quaternized silicon(IV) phthalocyanine derivative (3Q) to BSA
protein was studied by spectrofluorometry at room temperature in aqueous solutions. An
aqueous solution of BSA (fixed concentration) was titrated with varying concentrations of
quaternized silicon(IV) phthalocyanine solutions. BSA was excited at 280 nm and the
7

fluorescence emission spectra were recorded between 290 and 450 nm. The steady diminution
of the fluorescence emission of BSA with the increase in quaternized phthalocyanine (3Q)
concentrations was recorded and used in the determination of the binding constant and
stoichiometry of the binding step values on BSA, according to equation 4 [39].


(F -F)
0
-
(4)
log
= logK + nlog Pc






b
(F-F )
where F0 and F are the fluorescence emission intensities of BSA in the absence and presence of the
quaternized silicon(IV) phthalocyanine (3Q), respectively. F is the fluorescence emission
intensity of BSA saturated with quaternized silicon(IV) phthalocyanine (3Q). Kb is the binding
constant. n is the stoichiometry of the binding step on a BSA molecule. [Pc] is the concentration


(F0 -F )
(F -F )
log




of the quaternized phthalocyanine. Plots of
against log [Pc] would provide values for
n (from the slope) and Kb (from the intercept).
The fluorescence intensity for BSA decreased on addition of the quaternized silicon(IV)
phthalocyanine (3Q) solutions and these reductions were related to the quaternized phthalocyanine
concentrations by the Stern-Volmer relationship (Equation 5):
FBSA
BSA
SV
0
= 1 + K [Pc]
(5)
FBSA
BSA
SV
k
and
is given by Equation 6:
BSA
(6)
K
 k q τF(BSA)
SV
FBSA
and FBSA are the fluorescence intensities of BSA in the absence and presence of the
0
where
BSA
SV
K
quaternized phthalocyanine (3Q) respectively.
is the Stern-Volmer quenching constant, kq
8

is the bimolecular quenching constant and F(BSA) is the fluorescence lifetime of BSA (F(BSA)
=
F0BSA / FBSA
BSA
SV
K
10 ns) [40-42]. The
values were obtained from plots of
versus [Pc] and the kq
values can be determined from equation 6.
2.5. Binding of quaternized silicon(IV) phthalocyanine derivative (3Q) to DNA
DNA binding experiments were conducted by titrating 1.00 x 10-5 M Pc solutions (2 mL) in a
quartz cuvette with a DNA stock solution (0.01 mL, 1.74 x 10-4 M) and by recording the changes
in intensity of the emission spectrum at 699 nm. All solutions were prepared in water buffer (pH
7.4).
2.6. Synthesis
2.6.1. 4-(4-Pyridinyl)phenol (2)
56 mg (0.454 mmol) of 4-pyridine boronic acid, 100 mg (0.454 mmol) of 4-iodophenol, 29.8
mg (0.025 mmol) of Pd (PPh3)4, and 633 mg (1.94 mmol) of Cs2CO3 were dissolved in a
1,4-dioxane/water (5:1) solution and heated to reflux temperature until complete conversion
of the starting material to the product. This blend was extracted with ethyl acetate/water after
cooling to room temperature. Anhydrous MgSO4 was used for drying the organic phase and
then filtered off. The crude product was purified by column chromatography on silica gel using
a CH2Cl2/ethanol (10:3) solvent system as the eluent. Yield: 35 mg (45%), m.p.: 256 °C. FT-
IR(ATR), vmax/cm-1: 3358 (-OH), 3026 (Aromatic-CH), 1582 (C=N), 1532 (C=C), 1170 (C-N-C).
1H NMR (DMSO-d6, , ppm): 9.87 (s, 1H, OH), 8.58 (brd, 2H, Ar-H), 7.68 (d, 4H, Ar-H, J = 8.09
Hz), 6.90 (d, 2H, Ar-H, J = 8.62 Hz). Elemental analysis C11H9NO: C, 77.18; H, 5.30; N, 8.18;
found: C, 77.07; H, 5.24; N, 8.11%. MS: (MALDI-TOF), m/z calcd.: 171.20 for C11H9NO; found:
171.46 [M]+, 154.36 [M-OH]+.
9

2.6.2. Axially bis[4-(4-pyridinyl)phenoxy)] silicon(IV) phthalocyanine (3)
Dichloro(phthalocyaninato)silicon (100 mg, 0.163 mmol) [43] and 4-(pyridine-4-yl phenol (2)
(70 mg, 0.408 mmol) were dissolved in dry toluene (15 mL) and excess sodium hydride (12
mg, 0.539 mmol) was added to the mixture, which was then heated at 110 ℃ under an argon
atmosphere. After 24 h, the solvent was evaporated. The crude product was purified by column
chromatography on silica gel using a dichloromethane (DCM): ethanol (100:1) solvent system
as the eluent. The pure product was collected as a blue solid. Yield: 66 mg (46%), m.p.: > 300
°C. FT-IR(ATR), vmax/cm-1: 3063-3023 (Ar-CH), 1600 (C=N), 1515 (C=C), 1166 (C-N-C), 1080
(
Si-O-C). UV-vis (DMSO), λmax, nm (log ε): 685 (5.22), 614 (4.49), 355 (4.79). 1H NMR (CDCl3,
500 MHz, δ, ppm): 9.66 (m, 8H, Ar-H-Pc), 8.41 (m, 8H, Ar-H-Pc), 8.25 (d, 4H, Ar-H, J = 8.25
Hz), 6.71 (d, 4H, Ar-H, J = 4.65 Hz), 5.94 (d, 4H, Ar-H, J = 8.40 Hz), 2.58 (d, 4H, Ar-H, J = 8.22
Hz). Elemental analysis for C54H32N10O2Si: C, 73.62; H, 3.66; N, 15.90; found: C, 73.64; H, 3.69;
N, 15.88%. (MALDI-TOF-MS) (m/z) calcd.: 880.98 for C54H32N10O2Si; found: [M - C11H9NO
(44-(4-pyridinyl)phenol)]+: 709.39 .
2.6.3. Axially bis[(N-methyl-4-(4-pyridinyl)phenoxy] silicon(IV) phthalocyaninato iodide (3Q)
Compound 3 (50 mg, 0.056 mmol) was dissolved in CHCl3 (3 mL) and methyl iodide (1 mL)
was added to this solution. The reaction mixture was stirred at room temperature for 3 days.
After this time, the product was precipitated from acetone. The product was washed with
ethyl acetate, CHCl3, n-hexane, CH2Cl2 and diethyl ether in an argon atmospheric. The
effective product was well dried. Yield: 22 mg (55%), m.p.: 300 °C; FT-IR(ATR), vmax/cm-1 :
3040-3033 (Ar-CH), 2811 (Al-CH), 1588 (C=N), 1488 (C=C), 1169 (C-N-C), 1082 (Si-O-C); UV-
vis (DMSO) λmax, nm (log ε): 686 (5.18), 614 (4.44), 353 (4.92); 1H NMR (DMSO-d6, 500 MHz,
δ, ppm): 9.72 (brd, 8H, Ar-H-Pc), 8.58 (brd, 8H, Ar-H-Pc), 8.50-8.47 (brd, 4H, Ar-H), 7.71-7.54
10

(brd, 4H, Ar-H), 6.59-6.42 (brd, 4H, Ar-H), 4.03 (s, 6H, CH3), 2.57 (brd, 4H, Ar-H). Elemental
analysis C56H38N10O2SiI2: C, 57.74; H, 3.29; N, 12.02; found: C, 57.64; H, 3.23; N,
11.98%. (MALDI-TOF-MS) (m/z) calcd. for C56H38N10O2SiI2: 1164.86; found: 583.36 [M+2/2],
572.51 [M-C24H22N2-2I], 556.36 [M-C24H22N2O-2I].
3. Results and discussion
3.1. Synthesis and characterization
The synthetic approach for the new silicon(IV) phthalocyanine compounds 3 and 3Q is
described in Scheme 1. Compound 2 was obtained via the Suzuki-Miyaura cross-coupling
reaction between 4-pyridine boronic acid and 4-iodophenol. The new silicon(IV)
phthalocyanine 3 was synthesized from SiPcCl2 by the reaction of 2 in the presence of NaH at
reflux temperature in toluene. For quaternization of silicon(IV) phthalocyanine 3, an excess
amount of methyl iodide was used as the quaternization agent and the reaction was achieved
in chloroform at room temperature. This structures of the novel silicon(IV) phthalocyanines
were characterized by different spectroscopic techniques, namely FT-IR, UV–Vis, 1H NMR,
MALDI-TOF and elemental analysis.
The disappearance of the -OH vibrational band for the pyridyl group in the FT-IR spectrum of
compound 2 proved the formation of phthalocyanine 3. Concerning the FT-IR spectra of
compounds 3 and 3Q, specific vibrational bands for Si-O-C stretching [44] were detected at
1080 and 1082 cm-1, respectively for these phthalocyanines. Vibrations belonging to the
aromatic groups were observed between 3063 and 3023 cm-1 for 3 and between 3040 and 3033
cm-1 for 3Q. Also, a new vibration (-CH3) caused by the quaternization was observed at 2811
cm-1 and verified the formation of the quaternized phthalocyanine 3Q.
11

1
In the H NMR spectrum of phthalocyanine 3, the disappearance of the -OH signal for the
starting pyridinyl compound (2) and the presence of signals belonging to the aromatic protons
on the phthalocyanine macrocycle, observed at δ 9.66 and 8.41 ppm, are evidence for the
formation of the described compound, according to the literature [45-47]. Signals belonging to
the aromatic protons at δ 2.58 ppm were observed at a high field due to the magnetic anisotropy of
the delocalized 18π electrons on the phthalocyanine ring, as described for SiPcs [48]. The 1H NMR
spectrum of 3Q shows similar signals to the starting SiPc 3, except for the -CH3 protons which
appear as a singlet peak at δ 4.03 ppm (Fig. 1), identifying the formation of the quaternized
product. These results confirmed the structures of compounds 3 and 3Q.
The MALDI-TOF spectra of 3 and 3Q indicated that the molecular weight values of these
phthalocyanines are in accordance with the suggested molecules. The main peak of compound
3 (Fig. 2) was observed at 709.39 as [M - C11H9NO (4-(4-pyridinyl)phenol)]+. Besides, the
molecular ion peaks for compound 3Q were identified at 583.36 as [M+2/2], 572.51 as [M-
C24H22N2-2I] and 556.36 as [M-C24H22N2O-2I] (Fig. 3).
3.2. Photophysical and photochemical studies
3.2.1. Ground state electronic absorption spectra
Phthalocyanines typically show distinctive bands in the UV and visible region of their electronic
spectra. Two fundamental bands for π → π * transitions are detected: one is named as the Q band
and the other is named as the Soret or B band. While the Q bands are detected at around 650-750
nm, the B bands are detected at around 300-350 nm in the electronic spectra [49,50].
Compound 3 and its quaternized derivative 3Q showed peaks at 685 and 686 nm for the Q band,
respectively, in DMSO. The B bands were observed at 355 and 353 nm, respectively in DMSO.
12

3.2.2. Aggregation studies
Aggregation is generally represented as a coplanar connection of rings that develop from monomer
to dimer, and to complexes of a higher order. The extent of aggregation depends on the
concentration of the solution, nature of the solvent, nature of the substituents, complexed metal
ions and temperature [51,52].
In this study, the aggregation behaviors of the phthalocyanines (3 and 3Q) were investigated in
various solvents, namely DMSO, DMF, THF, toluene, chloroform and dichloromethane (Fig. 4A)
for the non-ionic phthalocyanine and in DMSO, water and water-TX 100 for the ionic
phthalocyanine complex (Fig. 4B). The studied non-ionic phthalocyanine 3 did not show any
aggregation in the different solvents. The spectra of 3Q in aqueous solutions display approximately
a 44 nm red-shifted band that can be related, because of exciton theory, to the formation of J-type
aggregates and points to the formation of self-assembled units with a slipped cofacial design. In
contrast, the incorporation of 3Q in water with quantities of triton X-100 induces a sharpening of
the absorption band at 686 nm, which clearly indicates the suppression of aggregation after the
interaction of the host-guest by the addition of triton X-100 [53]. The composed aggregates were
destroyed by the addition of some drops of triton X-100, thus the elevation of this phthalocyanine's
Q band intensity was increased after this addition (Fig. 4B).
Another proof for the formation of J-type aggregation for the studied phthalocyanine 3Q is the
gradual addition of methanol to a solution of 3Q in water, which caused an increase in the
absorption of the monomer band at 686 nm and a decrease in that of the J-band at 730 nm, followed
by its disappearance (Supporting Information, Fig. S4). These spectral changes suggest clearly that
the addition of a coordinating solvent (such as methanol) caused disaggregation of the J-aggregated
species as a result of the competitive coordination of the solvent molecules.
13

Also, an acidic solution (such as HCl solution) was added to a solution of phthalocyanine 3Q in
DMSO to determine the change of symmetry (from D4h to D2h) of the phthalocyanine ring due to
protonation of the inner nitrogen atoms in the phthalocyanine ring by the addition of acid, because
protonation can also cause a red-shift of the Q band in the UV-Vis spectrum of this phthalocyanine.
However, no new red-shifted band was observed by the addition of the acidic solution, which
indicates the formation of the red-shifted band for phthalocyanine 3Q is not related to any change
of symmetry due to the protonation of the phthalocyanine ring and thus indicates that the red-
shifted peak is formed due to the J-aggregation of the molecules of phthalocyanine 3Q (Supporting
Information, Fig. S5).
The UV-vis spectra of these Pcs were also measured at different concentrations in DMSO. The
Beer–Lambert law was obeyed for both of the axially di-substituted silicon(IV) phthalocyanines
3 and 3Q at concentrations ranging from 1 × 10−5 to 1 × 10−6 M in DMSO (Fig. 5). The
spectroscopic parameters are summarized in Table 1.
3.2.3. Fluorescence spectra
The fluorescence activity was examined in DMSO for 3 and in both DMSO and aqueous
solutions for 3Q. The axially di-substituted silicon(IV) phthalocyanine 3 did not show any
fluorescence emission in DMSO. This case is expected to be due to the formation of an
intramolecular photoinduced electron transfer (PET) system between the bis[4-(4-
pyridinyl)phenol] groups and the phthalocyanine center, as this compound then showed a
fluorescence emission in DMSO after quaternization of the nitrogen atoms of the pyridine
groups due to inhibition of the intramolecular PET. Fluorescence emission maxima were
observed at 684 nm in DMSO and at 693 nm in water +Triton X-100 for compound 3Q. The
excitation spectra are similar to the absorption spectra and both are mirror images of the
14

emission spectra for the phthalocyanine complex 3Q, indicating that the molecule did not show
any degradation during excitation. The fluorescence emission, absorption and excitation spectra
of 3Q are supplied in Fig. 6 and the obtained data are given in Table 1.
3.2.4. Fluorescence quantum yields and lifetimes
For the monitoring of photosensitizer molecules in a body, the photosensitizer should display some
fluorescence behavior. Hence the determination of the photosensitizers' fluorescence properties,
such as fluorescence quantum yield (ΦF) and fluorescence lifetime (τF) values are crucial for PDT
research. The fluorescence quantum yield (ΦF) of the ionic phthalocyanine 3Q was investigated in
DMSO and water +Triton X-100 and the obtained data are summarized in (Table 2).
The ΦF value of the non-ionic quaternized phthalocyanine 3 was not calculated due to the non-
fluorescence behavior in DMSO. The ΦF values were found to be 0.57 and 0.29 for
compound 3Q in DMSO and water +Triton X-100 solution, respectively. The quaternized axially
di-substituted silicon(IV) phthalocyanine 3Q exhibit a higher fluorescence quantum yield than both
the unsubstituted ZnPc (ΦF = 0.20) and the silicon(IV) Pc (ΦF = 0.44) in DMSO.
The fluorescence lifetimes (τF) of 3Q were established by applying a time correlated single photon
counting (TCSPC) technique in both DMSO and water-TX 100 solutions, and the data results are
given in Table 2. An associated spectrum for fluorescence lifetime measurements of 3Q in DMSO
is given in Fig. 7. The fluorescence lifetime values of 3Q were determined to be 4.61 and 4.46 ns
in DMSO and water +Triton X-100, respectively. Although these values are higher than the
unsubstituted ZnPc (τF: 3.99 ns), they are less than the unsubstituted SiPc derivative (τF: 5.37) in
DMSO.
3.2.5. Singlet oxygen generation
15

The PDT mechanism depends on the conversion of energy between the triplet state of a
photosensitizer and singlet oxygen (1O2) produced by ground state molecular oxygen. The singlet
oxygen generation measurements were carried out in DMSO for the phthalocyanine 3 and in both
DMSO and water+Triton X-100 solution for its quaternized derivative 3Q by measuring the
photoinduced decomposition of 1,3 diphenylisobenzofuran (DPBF) for both 3 and 3Q, and 9,10-
anthracenediyl-bis(methylene)dimalonic acid (ADMA) for 3Q as the scavenger. The reduction of
the DPBF absorbance at 417 nm for phthalocyanine 3 and the ADMA absorbance at 378 nm for its
quaternized derivative 3Q were monitored (Fig. 8). The ΦΔ values were obtained as 0.10 for
compound 3 and 0.22 for compound 3Q in DMSO, as shown in Table 2. Any shifts in the Q band
intensities of compounds 3 and 3Q during the ΦΔ analysis were not noticed, which proved the
studied phthalocyanines were not degraded during the singlet oxygen measurements by the light
irradiation. The non-ionic phthalocyanine indicated a low ΦΔ value in DMSO (ΦΔ = 0.10) (Fig.
S1). It is assumed that the photoinduced electron transfer (PET) process between the Pc macrocycle
and the pyridinyl groups quenches the excited singlet oxygen state of non-ionic phthalocyanines,
this leads to a reduced production of singlet oxygen. After quaternization, the investigated
phthalocyanine exhibited higher singlet oxygen generation (almost 0.22) due to the presence of a
lone pair electrons for the nitrogen atoms that inhibit the intramolecular PET process effectively,
leading to higher singlet oxygen generation. The ΦΔ value of 0.68 was obtained in aqueous solution.
Comparing the singlet oxygen quantum yields between 3 and the unsubstituted SiPcCl2 (ΦΔ = 0.15
[44]), it was found that the yield for 3 was lower than that for the unsubstituted SiPcCl2, but it
increased to 0.22 after quaternization.
3.2.6. Photodegradation studies
16

The photostability determination of the photosensitizers under light irradiation plays an efficient
role for PDT. The degradation of molecules through light irradiation is commonly referred to by
the photodegradation quantum yield (Φd) and relies on the structure, light intensity, type of solvent
and concentration [54].
In the present work, the studied ionic and non-ionic phthalocyanines 3 and 3Q showed
photodegradation without phototransformation during light irradiation (Figs. 9 and S2). The Φd
values were found to be 2.15x 10-5 and 1.85 x10-5 in DMSO for 3 and 3Q, respectively and these
results are similar to other phthalocyanine derivatives [55]. Compared to the unsubstituted zinc(II)
phthalocyanine (Φd = 2.61 × 10−5 [56]), the values for both 3 and 3Q in DMSO were found to be
lower.
The quaternized ionic SiPc showed less stability against to light irradiation in water Triton X-100
solution than in DMSO because the Φd value of this compound was found to be higher in the water
Triton X-100 solution than in DMSO.
3.2.7. Binding properties of quaternized silicon(IV) phthalocyanine to BSA protein
The dominant protein in blood is bovine serum albumin, which exhibits crucial roles in the delivery
of drugs. Analysis of the BSA binding properties of photosensitizers is one of the defining ways of
drug delivery to specific tissues by the blood stream [57]. Therefore, the binding properties of the
newly synthesized quaternized silicon(IV) phthalocyanine 3Q derivative to BSA protein were
studied by spectrofluorometry at room temperature in an aqueous solution. The water solution of a
fixed concentration of BSA (3.00 x 10-5 M) was titrated with varying concentrations of the 3Q
solution. BSA was excited at 280 nm and the fluorescence emission spectra were reported between
290 and 500 nm for quaternized phthalocyanine-BSA solution. The fluorescence emission peak of
BSA at 348 nm decreased on increasing the phthalocyanine concentrations due to the interaction
17

of the phthalocyanine molecules with the tryptophan residues on the BSA protein (Figure 10). BSA
and 3Q showed a fluorescence quenching response; hence the Stern–Volmer quenching constant
(KSV) could be established. The plot slope, shown in Fig. 10-inset, represents KSV and this value
was found as 0.97 x 105 M-1 (Table 3) for the interaction of 3Q with BSA. The bimolecular
quenching constant (Kq) was obtained for 3Q by equation 6 (in the supplementary information)
using an approximate fluorescence lifetime of BSA. The Kq value of 3Q was found to be 0.97 x
1013 M−1 s−1, (Table 3), which exceed the proposed value for diffusion-controlled (dynamic)
quenching (1010 M-1 s-1 according to the Einstein–Smoluchowski approximation), indicating that
the quenching mechanism in this case is static at room temperature [58]. In addition, fluorescence
lifetimes were calculated as 4.46 and 4.43 ns for 3Q and 3Q+BSA, respectively.
Static quenching does not reduce the lifetime so even the fluorescent molecules are detected and
the uncomplex fluorophores reveal the unquenched lifetime [59].
In the supporting material, the binding constant (Kb) and number of binding sites (n) on BSA were
determined using equation 4. The plot slope, shown in Fig. 10-B, gave the value of n and the plot
intercept gave the value of Kb. For studied phthalocyanine 3Q, these values were calculated as 0.92
and 3.09 x 10-6 M-1, respectively (Table 3). The Kb and n values were found to be similar to the Pc-
BSA interactions in aqueous solutions for other phthalocyanine derivatives [60-62]. In this study,
the acquired n value was equivalent to 1, indicating that a 1:1 complex was formed
stoichiometrically between the 3Q and BSA.
3.2.8. Binding of water soluble quaternized silicon(IV) phthalocyanine complex (3Q) to DNA
DNA is an important biomolecular target for the treatment of genetic diseases, especially cancer.
In the UV–Vis region, the spectral change by the interaction of compounds with DNA indicates
the binding mode. The binding of molecules to DNA generally are associated with changes in
18

absorbance and/or red or blue shifts in wavelength [63]. In general, the intercalative mode of
interaction of compounds with CT-DNA results in the formation of an isosbestic point because of
strong stacking of aromatic rings with the bases of DNA [64]. DNA binding experiments were
performed by titrating 1.00 x 10-5 M of 3Q in a water solution (2 mL) with 1.74 x 10-4 M of a CT-
DNA (ε: 6200 M−1 cm−1) stock solution in water (0.010 mL) and the changes in the UV–vis
absorption spectra of 3Q were recorded. Although the Q band of 3Q at 692 nm decreased in
intensity, the other Q band at 719 nm increased on addition of CT-DNA in an aqueous solution
(Fig. 11). The appearance of an isosbestic point at 703 nm supports the intercalation mode of
interaction between 3Q and CT-DNA.
A significant aspect of fluorescence is that there is a direct connection between the spectroscopic
observations and the molecular features of the sample. Therefore, an appropriate way to display
the 3Q binding with CT-DNA is to see adjustments to the emission spectra. Fig. 12 shows the
fluorescence quenching of 3Q on the addition of CT-DNA in a water solution at room temperature.
The measurements were conducted using a constant amount of 3Q (1 x 10-5 M) whilst increasing
the amount of CT-DNA (0–4.24 µM). On addition of CT-DNA, the fluorescence emission peak of
Q3 at 699 nm decreased gradually, indicating the formation of an interaction between 3Q and CT-
DNA. The binding constant (Kb) and binding stoichiometry (n) values estimated from the binding
plot of the phthalocyanine 3Q with CT-DNA are given in Table 3.
19

4. Conclusions
To summarize, the syntheses of the axially di-substituted silicon(IV) phthalocyanine (3) and its
quaternized derivative (3Q) are reported for the first time. These novel silicon(IV) phthalocyanines
(3 and 3Q) were characterized by FT-IR, UV-Vis, 1H NMR and MALDI-TOF spectroscopic
techniques and by elemental analysis. The aggregation properties of the novel phthalocyanines
were measured in DMSO in the concentration range 1ꢀ×ꢀ10−5 to 1 ×ꢀ10−6 M and the Lambert-Beer
law was obeyed. Furthermore, the aggregation behaviors of 3 and 3Q were measured in different
solvents. The photophysical and photochemical properties were determined in DMSO for the non-
ionic phthalocyanine 3 and in both DMSO and water+TX solution for the ionic counterpart, 3Q.
After quaternization, the novel silicon(IV) phthalocyanine derivative 3Q displayed better
photophysical and photochemical activities due to the inhibition of the impact of an intramolecular
photoinduced electron transmission (PET) between the substituents and the phthalocyanine for
phthalocyanine 3. As a result, these phthalocyanines, and especially the quaternized derivative 3Q,
offer potential as candidate photosensitizers for the treatment of cancer by photodynamic therapy.
Fluorescence and both fluorescence and absorption spectroscopies were also explored with regard
to the bovine serum albumin (BSA) and DNA binding activities of the water-soluble quaternized
silicon(IV) phthalocyanine (3Q), respectively. The gathered results propose that there is an
interaction between BSA or DNA molecules and 3Q.
Acknowledgments
Khaoula Khezami gives thanks to TUBITAK for the fellowship 2216, RESEARCH
FELLOWSHIP PROGRAMME FOR INTERNATIONAL RESEARCHERS.
20

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24

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Table 1. Absorption, excitation and emission spectral data for 3 and 3Q in DMSO and
water+TX.
Compound
Solvent
Q band
(log ε)
Excitation Emission Stokes shift
λmax (nm)
λEx (nm)
-----------
683
λEm (nm)
-----------
684
ΔStokes (nm)
3
DMSO
685
5.22
5.18
5.26
---------
3Q
DMSO
686
2
3Q
Water+TX
DMSO
686
687
693
7
Std SiPca
Std ZnPcb
672, 701
672
4.71, 4.47 672
678
6
DMSO
5.10
672
682
10
aData from Ref [65].
bData from Ref [56].
25

Table 2. Photophysical and photochemical data for 3 and 3Q in both DMSO and water +TX
Compound
3
Solvent
DMSO
ΦF
τF(ns)
ΦΔ
Φd (x 10-5)
-
-
0.1
2.15
3Q
DMSO
0.57
4.61
0.22
1.85
3Q
Water+TX
DMSO
0.29
4.46
0.68
3.08
Std SiPc
0.44 a
5.37 c
0.15 a
-
Std ZnPc
DMSO
0.20b
3.99c
0.67 d 2.61 e
a Data from Ref [44].
b Data from Ref [34].
c Data from Ref [37].
d Data from Ref [66].
e Data from Ref [56].
26

Table 3. Binding and fluorescence quenching data for the interaction of BSA and DNA with 3Q
in aqueous solution.
Compound
Ksv
(x105 M-1)
0.97
Kq
(x1013 M−1 s−1)
0.97
Kb
(x10-6 M-1)
3.09
n
3Q-BSA
3Q-DNA
0.92
1.57
7.32
16.41
1.62
27

Figure Captions
Scheme 1. Synthetic pathway for the axially di-substituted silicon(IV) phthalocyanine 3 and its
quaternized 3Q derivative.
Figure 1. 1H NMR spectrum of 3Q in DMSO-d6.
Figure 2. MALDI-TOF mass spectrum of 3.
Figure 3. MALDI-TOF mass spectrum of 3Q.
Figure 4. Electronic absorption spectrum (A) of 3 and (B) 3Q in different solvents.
Figure 5. UV–Vis electronic absorption spectrum of (A) 3 and (B) 3Q at different
concentrations in DMSO.
Figure 6. Absorption, excitation and emission spectra for 3Q in DMSO.
Figure 7. Time correlated single photon counting (TCSPC) fluorescence decay curve of 3Q in
DMSO.
Figure 8. Electronic absorption changes during the determination of singlet oxygen quantum yield
for: (A) 3Q in DMSO, (B) 3Q in water+TX using DPBF and ADMA as a singlet oxygen quencher,
respectively.
Figure 9. Electronic absorption spectral changes during the investigation of the photodegradation
quantum yield of 3Q (A) in DMSO and (B) water +TX.
Figure 10. (A) Fluorescence emission spectral changes of BSA by the addition of different
concentrations of 3Q in water. Inset: Stern–Volmer plots for quenching of BSA by 3Q in water.
28

[BSA] = 3.00 x 10-5 M and [Pc] = 0, 1.66 x 10-6, 3.33 x10-6, 5.00 x 10-6, 6.66 x 10-6, 8.33 x10-6 M.
(B) determination of the 3Q-BSA binding constant (kb) and number of binding sites (n) on BSA.
Figure 11. Electronic titration spectra of 1.00 x10-5 M 3Q with CT-DNA (1.74 x 10-4 M) at pH 7.4
in water. Inset, the plot of absorbance at 684 nm versus the CT-DNA concentration.
Figure 12. Fluorescence emission spectral changes of A) 3Q (1.00 x 10-5 M) by the addition of
CT-DNA at pH 7.4 in water B) 3Q (1.00 x 10-5 M) (blue) and water (red). Excitation wavelength
= 660 nm.
29