Axially paraben substituted silicon(IV) phthalocyanines towards dental pathogen Streptococcus mutans: Synthesis, photophysical, photochemical and in vitro properties

Article abstract of DOI:10.1016/j.jphotochem.2015.03.010

A series of silicon(IV) phthalocyanines axially substituted with methylparaben, ethylparaben, propylparaben and butylparaben groups were synthesized and studied as photosensitizers for antimicrobial photodynamic therapy. The absorption, fluorescence, phot

Full text of DOI:10.1016/j.jphotochem.2015.03.010

Journal of Photochemistry and Photobiology A: Chemistry 306 (2015) 31–40
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Axially paraben substituted silicon(IV) phthalocyanines towards dental
pathogen Streptococcus mutans: Synthesis, photophysical,
photochemical and in vitro properties
b,**
Gökçe Canan Taşkın a, Mahmut Durmuş a, Fatma Yüksel a, Vanya Mantareva
,
Vesselin Kussovski c, Ivan Angelov b, Devrim Atilla a,
*
a
Gebze Technical University, Department of Chemistry, P.O. Box 141, Gebze, Kocaeli 41400, Turkey
b
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev, Bld. 9, 1113 Sofia, Bulgaria
The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev, Bld. 26, 1113 Sofia, Bulgaria
c
A R T I C L E I N F O
A B S T R A C T
series of silicon(IV) phthalocyanines axially substituted with methylparaben, ethylparaben,
Article history:
A
Received 12 January 2015
Received in revised form 13 March 2015
Accepted 15 March 2015
propylparaben and butylparaben groups were synthesized and studied as photosensitizers for
antimicrobial photodynamic therapy. The absorption, fluorescence, photodegradation and singlet
oxygen generation properties of the synthesized Si(IV) phthalocyanines were studied. In vitro
antibacterial photodynamic therapy was investigated against cariogenic pathogenic bacterium
Streptococcus mutans. Axially propylparaben substituted Si(IV) phthalocyanine showed significant
Available online 17 March 2015
Keywords:
Silicon phthalocyanine
Parabens
Antimicrobial photodynamic therapy
Pathogenic bacteria
Streptococcus mutan
photodynamic efficacy (log 4) at concentration of 10 m ,
M and mild irradiation (60 mW cmꢀ2, 50 J cmꢀ2
LED 637 nm). The low inactivation capacity was observed for the other studied Si(IV) phthalocyanines.
The uptake and localization properties of axially propylparaben substituted Si(IV) phthalocyanine
showed a sufficient level of accumulation in planktonic cultured bacterium and a complete penetration
depth into the biomass of the 48-h bacterial biofilm.
ã 2015 Elsevier B.V. All rights reserved.
1. Introduction
Last decade phthalocyanines (Pcs) have been recognized as the
most widely studied photosensitizers for photodynamic inactiva-
Phthalocyanine (Pc) derivatives have a great attention in
photodynamic therapy (PDT) of cancer disease as photosensitizers
due to their strong absorption in the red visible region, low dark
toxicity and high efficiency of singlet oxygen generation [1,2].
Antimicrobial photodynamic therapy (aPDT) is very actual as a
promising approach for treatment of resistant pathogenic micro-
organisms in the so called post-antibiotic era [3,4]. Thus, aPDT has
more advantages over the conventional chemotherapeutics such as
fast outcome after treatment, safety for human health, lack of the
drug resistance and minimal risk for side effects [5]. APDT involves
application of non-toxic photosensitizer (PS) in the unexcited state,
which after irradiation with red or infrared light (630–850 nm)
leads to generation of singlet oxygen and other reactive oxygen
species (ROSs) which are toxic to the cells [1,5,6].
tion of microorganism [3,7] and several studies showed that
positively charged Pcs are efficient in the photodynamic inactiva-
tion of both Gram-positive and Gram-negative bacteria [8–13]. The
number of studies have been published during 90th years of last
century concerning differently substituted Si(IV) phthalocyanines
for aPDT [14]. Recently Roncucci et al., patented several metal
phthalocyanines including silicon(IV) Pcs as antibacterial compo-
sitions in 2013. The silicon phthalocyanine (Pc4) has been
extensively studied and nowadays Pc4 undergoes phase I for
clinical applications [15–17].
Parabens, the alkyl esters of p-hydroxybenzoic acid, are one of
the most functional preservative agents used in drugs, cosmetics,
foodstuffs as well as in dental materials [18,19]. Nevertheless some
recent findings of the high level of parabens accumulated in the
breast cancer tissues, their negative influence on human health has
not been proven and they have been still in usage as additives in
the cosmetic products [19–21]. Moreover the restricted applica-
tions of parabens in some trademarks assume the development of
the alternative antibacterial agents. An effective solution for
suppression of bacterial growth is aPDT [22].
* Corresponding author. Tel.: +90 262 6053020.
** Corresponding author. Tel.: +359 9606 181.
E-mail addresses: mantareva@orgchm.bas.bg (V. Mantareva), datilla@gtu.edu.tr
(D. Atilla).
http://dx.doi.org/10.1016/j.jphotochem.2015.03.010
1010-6030/ã 2015 Elsevier B.V. All rights reserved.

32
G.C. Taşkın et al. / Journal of Photochemistry and Photobiology A: Chemistry 306 (2015) 31–40
The aPDT is under clinical consideration for treatment of dental
MS(m/z)calcd. forC50H34N8O6Si:870.94, found:870.52[M]+ (Fig. in
Supplementary file).
caries, which is one of the most common infectious diseases
worldwide [6,7,14]. Some results in the literature indicated that
parabens are potential antibacterial agents against immobilized or
planktonicbacteriafoundinoralcavity[18]. Streptococcus mutans (S.
mutans)isaGram-positivebacteriumwhichismostlyfoundbetween
adjacent teeth or in the deep crevices on the biting surface of teeth
[23]. Parabens are known as effective inhibitors of glycolysis in S.
mutans [19,23]. Experimental aPDT with disulfonated aluminium
phthalocyanine has been shown to be effective against biofilms of
cariogenic bacteria and against human supragingival dental plaque
microbes in the planktonic phase [24].
The present study aims the synthesis of four silicon(IV)
phthalocyanines axially substituted with methylparaben, ethyl-
paraben, propylparaben and butylparaben groups. The growth
crystals with two of them namely methylparaben- and butylpar-
aben- substituted Si(IV) Pcs were studied. All different parabens
substituted Si(IV) Pc were evaluated with their photophysical
(absorption and fluorescence) and photochemical (photostability
and singlet oxygen generation) properties. The photodynamic
effectiveness of the obtained Si(IV) Pcs were investigated against
cariogenic bacterium S. mutans as planktonic and biofilm cultured.
2.2.2. Phthalocyanine 3c
This compound was synthesized according to the procedure
described for 3b. Silicon(IV) phthalocyanine dichloride (0.5 g,
0.82 mmol), propyl-4-hydroxybenzoate (1.47 g, 8.17 mmol) and
2 mL dry pyridine in 50 mL dry toluene to give 3c as a blue solid
(98 mg,12.9%)1HNMR(500 MHz,CD2Cl2):
d9.58–9.60(m,8H,Pc-H),
8.34–8.36(m, 8H, Pc-H), 6.19(d, J:7.8 Hz, 4H, p-C6H4), 3.68 (t, 4H,
OCH2),2.38(d,J:7.8 Hz,4H,p-C6H4),1.28–1.35 (m,4H,-CH2-),0.63(t,
6H, -CH3) (Fig. S6 in Supplementary file). Micro TOF-ESI/MS (m/z)
calcd. for C52H38N8O6Si: 898.99, found: 921.26 [M + Na]+ (Fig. S5 in
Supplementary file).
2.2.3. Phthalocyanine 3d
This compound was synthesized according to the procedure
described for 3b. Silicon(IV) phthalocyanine dichloride (0.5 g,
0.82 mmol), butyl-4-hydroxybenzoate (1.58 g, 8.17 mmol) and
2 mL dry pyridine in 50 mL dry toluene to give 3d as a blue solid.
(105 mg,13.5%).1H NMR(500 MHz, CD2Cl2):
d 9.58–9.60 (m, 8H, Pc-
H), 8.33–8.35 (m, 8H, Pc-H), 6.18 (d, J:7.8 Hz, 4H, p-C6H4), 3.72 (t, 4H,
OCH2), 2.38 (d, J:7.8 Hz, 4H, p-C6H4),1.25–1.30 (m, 4H, -CH2-),1.01–
1.09 (m, 4H, -CH2-), 0.67 (t, 6H, -CH3) (Fig. S8 in Supplementary file).
Micro TOF-ESI/MS (m/z) calcd. for C54H42N8O6Si: 927.05, found:
949.29[M + Na]+ (Fig. S7 in Supplementary file).
2. Experimental
2.1. Chemicals
2.3. Equipment
Methanol, tetralin (98%), toluene, tributhylamine and pyridine
were obtained from Merck. Tetrahydrofuran (THF) and dimethyl
sulfoxide (DMSO) for UV spectroscopy were purchased from Sigma–
Aldrich. Toluene and methanol were distilled from sodium benzo-
phenoneketylandsodium,respectively.Tributhylaminewasdistilled
fromCaH2andpyridinewasdistilledfromNaOH.Allsolidsweredried
under vacuum in such glassware overnight. Thin layer chromatogra-
phy (TLC) was carry out using Merck precoated silica gel 60 F254 TLC
plates. The photobiological studies were carried out with the stock
solutions of the Si(IV) Pcs which were prepared in concentrations
ꢁ2 mMindimethylformamide(DMF)andstoredinthedarkandcool
place. The dilutions were performed in sterile 0.01 M phosphate-
NMR spectra were recorded on Varian 500 MHz spectrometer
using the deuterated CD2Cl2. Electrospray ionization (ESI) mass
spectrometry was carried out on Bruker Micro TOF-ESI/MS
spectrometer. Absorption spectra were recorded on Shimadzu
2101 UV–vis spectrophotometer. The fluorescence studies were
performed with Varian Eclipse and Fluorolog-3 fluorimeters. The
bacterial biofilm was examined with confocal laser scanning
microscope (CLSM) of Leica Microsystems (Model: Leica TCS SPE)
with Leica LAS AF software. General Electric quartz line lamp
(300 W) was used for photoirradiations studies. 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 (Into,
670 nm for 3a–d 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 incor-
porated) power meter.
bufferedsaline(PBS)to 5and10 mMfinalconcentrationsoftheSi(IV)
Pcs prior photobiological experiments. The assessment of the drug
concentrationswascarriedoutonthebasisoftherecordedabsorption
spectra.
2.2. Synthesis
2.4. Photophysical and photochemical parameters
The starting compounds such as 1,3-diiminoisoindoline (1) [25–
27] and silicon(IV) phthalocyanine dichloride (Si(IV) PcCl2) (2) [26]
were prepared according to the procedure given in the literature.
Synthesis of axially methylparaben substituted silicon(IV) phthalo-
cyanine (3a) was published in a Chinese scientific journal [28]. 1H
NMR and mass spectra of 3a were given in Supplementary file as
Figs. S5 and S1 respectively.
2.4.1. Fluorescence quantum yields
Fluorescence quantum yields (FF) were determined in DMF by
the comparative method using Eq. (1) [29,30],
FStdAStdn2
FStdAn2Std
FF
¼
FFðStdÞ ¼
(1)
where F and FStd are the areas under the fluorescence emission
curves of the samples (3a–d) and the standard, respectively. A and
2.2.1. Phthalocyanine 3b
Silicon(IV)phthalocyaninedichloride(0.5g, 0.82 mmol), ethyl-4-
hydroxybenzoate (1.36 g, 8.18 mmol) and 2 mL dry pyridine in 50 mL
drytoluenewasrefluxedfortwodays.Afterevaporationofthesolvent
in vacuo the residue was dissolved in CH2Cl2 and it was purified by
preparative thin layer chromatography on silica gel plates using
CH2Cl2/EtOH(50:1)aseluenttogivedesiredproduct.Thebluecolored
product was crystalized from chloroform (102 mg, 14.3%). 1H NMR
A
Std are the respective absorbances of the samples and standard at
the excitation wavelengths and n and nstd are the refractive indices
of solvents used for the sample and standard, respectively.
Unsubstituted ZnPc (FF = 0.20) [31] was employed as the standard
in DMSO.
2.4.2. Singlet oxygen quantum yields
(500 MHz,CD2Cl2):d9.58–9.60(m,8H,Pc-H),8.34–8.36(m,8H,Pc-H),
Singlet-oxygen quantum yield
(FD) determinations were
6.18(d,J:7.8 Hz,4H,p-C6H4),3.76(q,4H,OCH2),2.38(d,J:7.8 Hz,4H,p-
carried out using the experimental setup described in the
C6H4),0.92(t,6H,-CH3)(Fig.S4inSupplementaryfile).MicroTOF-ESI/

G.C. Taşkın et al. / Journal of Photochemistry and Photobiology A: Chemistry 306 (2015) 31–40
33
literature [30,32]. Typically, a 2 mL portion of the respective axially
paraben substituted silicon(IV) phthalocyanine (3a–d) solutions
(C = 1.00 ꢂ10ꢀ5 M) that contained the singlet oxygen scavenger
was irradiated in the Q-band region with the photoirradiation
setup described in the literature [30,32]. The FD values were
determined in air using the relative method with 1,3-diphenyli-
sobenzofuran (DPBF) as a singlet oxygen chemical scavenger in
DMF using Eq. (2).
conditions (5% CO2) at 37 ꢃC for 48 h on solid medium Trypticase1
Soy agar, supplemented with 0.3% yeast extract.
2.6.2. Biofilm assay
Biofilm assay was performed on coverslips, which were placed
in commercial presterilized polystyrene flat bottomed 12-well cell
culture test plates (Switzerland). A standard bacterial suspension
(1 mL, 107 CFU mLꢀ1) prepared after serial dilutions was applied
onto the surface of the discs placed in each well of the plate
followed by incubation for 1.5 h at 37 ꢃC to promote cellular
adherence to surface of the discs. The blank control wells with the
discs at the same conditions but without bacterial cells were
inoculated. After the initial adhesion phase, the cell suspensions
were aspirated and the discs were gently washed with PBS to
remove loosely adherent cells. In the biofilm phase formation of S.
mutans an addition of 4 mL Tryptic soy broth (Difco Laboratories,
MD, USA) was placed in each well. The plates were covered and
incubated for 48 h at 37 ꢃC to form the microbial biofilm for in vitro
experiments.
Std
RI
FSDtd
(2)
abs
FD
¼
RStdIabs
FSDtd is the singlet oxygen quantum yield for the standard
unsubstituted ZnPc (FDStd = 0.56 in DMF) [33]. R and RStd are the
DPBF photobleaching rates in the presence of the respective axially
paraben substituted silicon(IV) phthalocyanine (3a–d) and stan-
dard, respectively; Iabs and ISabtds are the rates of light absorption by
the samples (3a–d) and standard, respectively. The concentrations
of DPBF in the solutions were calculated using the determined
value of loge= 4.36 at 417 nm in DMF. The light intensity used for
FD determinations was found to be 6.58 ꢂ 1015 photons sꢀ1 cmꢀ2
.
2.6.3. In vitro photodynamic study
Bacterial suspensions (1 mL) were incubated with the axially
parabens substituted SiPcs (3a–3d) for 15 min, by using the freshly
prepared stock solutions in DMF (0.5% DMF from the total volume)
2.4.3. Photodegradation quantum yields
Photodegradation quantum yield (Fd) determinations were
carried out using the experimental set-up described in literature
[30,32]. Photodegradation quantum yields were determined using
Eq. (3),
with two different concentrations after dilution (5.0
mM and
10 M SiPcs). The incubation was carried out on magnetic stirrer in
m
the dark at room temperature. The bacterial suspensions were with
cell densities between 106 and 107 CFU mLꢀ1 in PBS. After
ðC0 ꢀ CtÞVNA
incubation time was passed an aliquot (200 mL) of the suspension
Fd
¼
(3)
IabsSt
was placed in a standard palette where the irradiation was
performed. The samples were irradiated with a LED 637 nm with
fluence rate 60 mW cmꢀ2 and total light dose 50 J cmꢀ2. The power
density was controlled with photometer before and after irradia-
tion. The biofilm of bacteria cells developed for 48-h incubation at
37 ꢃC by using coverslips were washed with PBS and were
where C0 and Ct are the samples (3a–d) concentrations before and
after irradiation respectively, V is the reaction volume, NA is the
Avogadro’s constant, S is the irradiated cell area, t is the irradiation
time and Iabs is the overlap integral of the radiation source light
intensity and the absorption of the samples (3a–d). A light
intensity of 2.19 ꢂ1016 photons sꢀ1 cmꢀ2 was employed for Fd
determinations.
incubated with 5 mM and 10 mM SiPcs for 1.5 h in dark place.
The samples were washed again after incubation and were
positioned for irradiation with 637 nm. The samples of four control
groups were collected: (1) with SiPc, but no light (dark toxicity), (2)
without SiPc, but illuminated, (3) only bacterial suspension (no
SiPc, no light) and (4) bacteria incubated with DMF (5% from the
total volume). After irradiation, the aliquot of cells (0.1 mL) was
taken off and serially diluted (10-fold) with PBS. The same action
was repeated for the control samples (1–4). Aliquots (0.1 mL) were
spread over Trypticase1 Soy agar. The number of colonies (CFU) on
each plate was counted following 48 h incubation at 25 ꢃC.
2.5. X-ray crystallography
Intensity data were recorded on a Bruker APEX II QUAZAR
diffractometer using monochromatized Mo
Ka X-radiation
(l
= 0.71073 Å). Absorption correction was performed by multi-
scan method implemented in SADABS [34] and space groups were
determined using XPREP implemented in APEX2 [35]. Structures
were determined using the direct methods procedure in SHELXS-
97 and refined by full-matrix least squares on F2 using SHELXL-97
[36]. All non-hydrogen atoms were refined with anisotropic
displacement factors. The C–H hydrogen atoms were placed in
calculated positions and the displacement parameters of the H
atoms were fixed at Uiso(H) = 1.2Ueq of their parent atoms. The final
geometrical calculations were carried out with PLATON [37], and
MERCURY [38] programs and the molecular drawings were done
with DIAMOND [39] program. Structure determinations have been
deposited with the Cambridge Crystallographic Data Centre with
references CCDC 976697 and 976698 for compounds 3a and 3d,
respectively.
2.6.4. Uptake study
The uptake study was carried out with four axially paraben
substituted Si(IV) Pc (3a–3d) by using a previously described
chemical extraction procedure [9]. The quantification of the
number of the sensitizer molecules accumulated into S. mutans
cells was carried out by the means of fluorescence measurements
of the collected samples. The estimation of the uptake for 5 mM 3a–
3d into bacterial cells after 15 min incubation in the dark was
carried out for the cellular suspensions with different densities
(105–108 CFU mLꢀ1). The collected samples for fluorescence
measurements were as followed: (1) the supernatant of phosphate
buffered saline (PBS) after incubation, (2) the PBS after first and (3)
after second cell wash and (4) THF: 2% SDS (9:1) solution after
cellular extraction. The collected samples (1–4) were diluted with
THF and measured at excitation wavelength 637 nm. All measure-
ments were carried out by using previously described set-up for
fluorescence measurements [40]. The results were presented as
number of the photosensitizer molecules per one cell by
processing the obtained values of fluorescence intensities and
2.6. Antimicrobial photodynamic therapy
2.6.1. Bacterial strain
The Gram-positive cariogenic bacterium S. mutans strain 20523
from the collection DSMZ – Germany was used for antimicrobial
PDT studies. The bacteria were incubated in microaerophilic

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G.C. Taşkın et al. / Journal of Photochemistry and Photobiology A: Chemistry 306 (2015) 31–40
referring to the calibration curves taken for SiPc (3a–3d) in the
solvent mixtures used for fluorescent measurements.
means was compared by two-tailed unpaired Student’s test. The
values of P < 0.05 were considered as significant.
3. Results and discussions
2.6.5. Confocal laser scanning microscopy
The biofilms of S. mutans developed for 48 h incubation at 37 ꢃC
on coverslips were used for study of penetration depth and
localization of SiPcs. The biofilm images were processed via the
Leica LAS AF software provided with the CLSM as previously
described in Ref. [41]. The oil immersion of 63ꢂ objective
(NA = 1.23) was used. The bacterial biofilms were characterized
with a BacLight Live-Dead bacterial viability kit, L13152 (Molecular
Probes, Europe BV). The kit contains two fluorescence stains:
SYTO9 and propidium iodide. The preparation and application of
the dyes were according to the product information list. The viable
cells were detected by fluorescence from the SYTO9 stain (exc.
488 nm, em: 525 nm). The dead cells were detected by fluores-
cence from the propidium iodide (exc: 520 nm, em: 620 nm). The
3.1. Synthesis and characterization
The synthesis of the target compounds involves the nucleo-
philic displacement of two chlorides substituents from SiPc(Cl)2 by
reaction of paraben derivatives in dried toluene and pyridine at
reflux temperature. Synthetic pathway for the synthesis of axially
di-parabens substituted Si(IV) phthalocyanines (3a–3d) was
presented in Scheme 1. The proposed structures of newly
synthesized Si(IV) phthalocyanine derivatives (3b–3d) were
confirmed by 1H NMR spectra. 1H NMR spectra of the axially-
paraben substituted Si(IV) Pcs showed intense multiple signals at
around d9.5 and 8.3 ppm belonging to the aromatic Pc ring protons.
The close proximity of the ligands to the Pc ring delocalization
induced large up-field shifted resonances for the ligand protons.
Thus four aromatic hydrogen atoms belonging to ligands gave a
images of a 0.150 mm slices were taken by scanning the biofilms of
bacterial specie (S. mutans). The thickness of biofilm was evaluated
by the native fluorescence of bacterial cells (exc. 488 nm, em. 520–
580 nm). The biofilm was incubated for 1.5 h with SiPc then after
washing in PBS it was covered with a coverslip. The fluorescence of
Si(IV) Pcs inside the biofilm was imaged by excitation with 633 nm
laser and emission between 660 and 740 nm to observe the
phthalocyanine penetration depth into biomass. The whole biofilm
doublet at around
d 2.4 ppm for 3b–3d. The novel Si(IV)
phthalocyanine derivatives were also characterized by ESI/MS
mass spectroscopy. Molecular ion peaks were compatible with
calculated values for 3b–3d. Mass (Figs. S3, S5 and S7) and 1H NMR
(Figs. S4, S6 and S8) spectra of compounds 3b–3d were given in the
supplementary file. IR spectra of 3b–3d were also confirmed the
proposed structures.
was scanned on slices of 0.150 mm following the red fluorescent
signal as well as the auto fluorescence. The co- localization of dye
into the biomass was evaluated.
3.2. X-ray crystallography of the axially paraben substituted Si(IV)
phthalocyanines
2.6.6. Statistics
The uptake and in vitro photoinactivation experiments were
carried out in triplicate and the data were presented as a
mean ꢄ standard deviation (SD). The difference between two
The crystal structures of 3a and 3d were determined by X-ray
crystallography. The data collection and refinement details were
given in Table 1. 3a had triclinic system, P-1 space group while 3d
Scheme 1. Synthetic pathway for the preparation of silicon(IV) phthalocyanines axially substituted with different paraben groups.

G.C. Taşkın et al. / Journal of Photochemistry and Photobiology A: Chemistry 306 (2015) 31–40
35
Table 1
while the N1—Si—N1# and the N2—Si—N2# bond angles were
linear with 180,00 (7)ꢃ. The O—Si—O bond angles were also linear
[18.000(6)ꢃ], and the angle between the main plane of Pc ring and
benzyl ring of axially substituted paraben derivative was 49,29ꢃ in
3a and 45,85ꢃ in 3d (Fig. depicted S9 in the supporting file). The
Si-N [av. 1910(ꢄ0.002) Å for 3a and 1908 (ꢄ0.016) Å for 3d] and the
Si—O [1.7315(10) Å for 3a and 1,7168 (19) Å for 3d] bond lengths
were compatible with those previously observed for the similar
SiPc derivatives which were axially substituted with several alkoxy
groups [42–44] (The selected bond and conformational parameters
were given in Table S1 in the supporting file).
X-ray crystallographic data and refinement parameters for the compounds 3a and
3d.
Compound
3a
3d
Empirical formula
Formula weight
Temperature (K)
Crystal system
Space group
a (Å)
C48H30N8O6Si
842.89
120(2)
Triclinic
P-1
7.6630(13)
10.8722(19)
12.013(2)
104.948(9)
94.127(10)
98.972(10)
948.5(3)
1
1.476
0.130
436
28.37
16928
4700
0.0386
4700/0/287
0.0416
C54H42N8O6Si
927.05
120(2)
Orthorhombic
Pbca
21.979(9)
8.501(4)
b (Å)
c (Å)
23.726(10)
a
b
g
(ꢃ)
(ꢃ)
(ꢃ)
The crystal packing investigations of 3a and 3d showed that
there was no classic hydrogen bonds in both unit cells, but there
Volume (Å3)
4433.(3)
4
1.389
0.118
1936
25.02
29828
3903
0.1045
3903/0/314
0.0523
0.1276
1.066
were several C—Hꢅ ꢅ ꢅ
p and p–p intermolecular interactions which
Z
were stabilizing the packing of both unit cells, the selected
intermolecular interactions (<4.0 Å) were listed in Table S2. In a
unit cell of 3a, there were additionally two different kinds of
intermolecular C—Hꢅ ꢅ ꢅO interactions between the ester oxygen
atoms of paraben groups and phthalocyanine rings less than 3.5 Å
and the phthalocyanine rings were packed vertically (Fig. S10). In
the case of 3d, these interactions were not observed probably due
to longer alkyl chain (n-butyl), instead the intermolecular C—Hꢅ ꢅ ꢅN
interaction was observed. Therefore, the phthalocyanine rings
were packed in the zigzag chain-like arrangement along a axis
(Fig. S11).
Density (calc, Mg/m3)
Absorption coeff. (mmꢀ1
F(000)
)
umax (ꢃ)
Reflections collected
Independent reflections
Rint (merging R value)
Data/Restrains/Parameters
R (F2>2 F2)
s
wR (all data)
0.1095
1.028
Goodness-of-fit on F2
had orthorombic system, Pbca space group. Although 3a was
synthesized previously [28], its crystal structure was given in this
study.
In structures, the phthalocyanine molecule sit on an inversion
centre (Fig. 1) and Si atom which laid on a centre of symmetry was
six-coordinate with four inner nitrogen atoms on Pc core and two
oxygen atoms on the paraben’s units in an octahedral environ-
ment. The Pc rings of both structures were essentially planar and
the SiN4 coordination was perfectly square planar, the N1—Si—
N2 bond angles were nearly perpendicular [av. 90,00 (ꢄ0.55)ꢃ]
3.3. UV–vis absorption and fluorescence properties
Four studied axially paraben substituted Si(IV) phthalocyanines
(3a–3d) were soluble in most of organic solvents such as toluene,
tetrahydrofuran, dichloromethane, chloroform and N,N-dimethyl-
formamide and partly in DMSO. Electronic absorption spectra of
the 3a–3d were measured in the above solvents and the Q-bands
were observed with maxima at around 680 nm (Table 2). The B
bands were also observed at around 355 nm. The sharp Q-bands
observed in all studied solvents were an evidence of the formation
Fig. 1. Crystal structures of compounds 3a and 3d with the atom-numbering scheme. Displacement ellipsoids were drawn at the 50% probability level. The hydrogen atoms
have been omitted for clarity (Symmetry code{#} for 3a:-x + 1, -y + 1, -z; for 3d: -x, -y + 1 -z + 1).

36
G.C. Taşkın et al. / Journal of Photochemistry and Photobiology A: Chemistry 306 (2015) 31–40
Table 2
Absorption, excitation and emission spectral data for the axially parabens substituted silicon (IV) phthalocyanine in DMF.
Compound
Q band lmax, (nm)
log
e
Excitation lEx, (nm)
Emission lEm, (nm)
Stokes shift DStokes, (nm)
3a
3b
3c
683
682
683
682
670
5.10
5.16
5.29
5.35
5.37
684
685
684
683
670
690
690
690
690
676
6
5
6
7
6
3d
ZnPca
a
Data from Ref. [33].
of non-aggregated species. The further photophysical and photo-
chemical studies were performed in DMF. Fig. 2 showed the
electronic absorption spectra of 3a–3d in DMF at the concentration
of 10ꢀ5 M. The absorption intensities and extinction coefficients of
the studied phthalocyanines grow bigger with the growth of the
hydrocarbon length on the paraben groups (Fig. 2 and Table 2). The
hydrocarbon length did not show any effect on the maxima of
absorption wavelength of these compounds.
All studied phthalocyanines showed similar fluorescence
emission spectra in DMF. Fig. 3 showed the absorption, fluores-
cence emission and excitation spectra of compound 3d as an
example in DMF. Fluorescence emission and excitation maxima of
3a–3d in DMF were listed in Table 2. All studied Si(IV)
phthalocyanines showed the same fluorescence emission maxima
(690 nm) in DMF. The observed Stokes shifts for 3a–3d were
similar with unsubstituted zinc phthalocyanine used as a standard
compound (Table 2).
The excitation spectra of axially paraben substituted silicon(IV)
phthalocyanines (3a–3d) were related to absorption spectra and
both were mirror images of the fluorescence spectra in DMF
(Fig. 3). The proximity of the wavelength in absorption and
excitation spectra of each component indicated that the nuclear
configurations of the ground and excited states are similar and not
affected by the excitation wavelength for all studied compounds.
The phthalocyanines possess an optimal fluorescence behavior
which is suitable for imaging of the studied object. The
that the ground-state dimers were the physical quenchers for the
monomeric molecules in the excited state [45]. The obtained two
life-times with a difference of two orders of magnitude measured
in DMF suggested the formation of physical aggregates (Fig. 4A).
The contribution of the shorter life-time component evaluated
from the fluorescence decay curves was about 96 ꢄ1% and the
measured life-times were between 5.6 and 7.1 ns. The mono-
exponential curves with life-times between 5.8 and 6.2 ns were
obtained for studied phthalocyanines 3a–3d in THF (Fig. 4B). The
life-time values were similar without dependence on the
hydrocarbon chain of the substituted parabens. A mono-exponen-
tial curve was also observed for the axially hydroxyl substituted
SiPc (tF = 5.4 ns in THF) used as a reference compound. The
obtained values decreased with 0.2–0.3 ns for excitation with a
picosecond diode at 654 nm. The values of the excited state life-
times of 3a–3d in DMF as compared to tF in THF showed an
essential increasing for SiPc 3a. The value of tF was 1.5 ns higher in
DMF than that in THF (5.6 ns). The obtained values of life-time of
fluorescence were of the same order of magnitude for the other
three axially paraben substituted Si(IV) phthalocyanines (3b–3d).
The obtained values were not dependent on the excitation neither
the observation wavelengths.
3.4. Singlet oxygen study
The formed singlet oxygen in the presence of axially paraben
substituted silicon(IV) phthalocyanines was determined by fol-
lowing the chemical method based on the chemical quenching of
1,3-diphenylisobenzofuran (DPBF). The intensity of DPBF absor-
bance at 417 nm decreased with light irradiation (Fig. 5). The
intensities of Q bands for 3a–3d did not show any change
indicating that the photodegradation did not occur by the light
exposure during the singlet oxygen studies. The studied 3a–3d
showed similar FD values (around 0.50) which were slightly lower
than unsubstituted Zn(II) phthalocyanine (Table 3). The FD values
of the studied paraben substituted Si(IV) Pcs were found with
higher FD value than for axial hydroxyl substituted Si(IV) Pc in
DMSO (FD = 0.28) [46]. The increasing of the hydrocarbon chain
from 3a to 3d did not show any quenching effect on singlet oxygen
generation.
fluorescence properties such as fluorescence quantum yield (FF
)
and fluorescence lifetime are also important for PDT
(tF)
applications. The fluorescence quantum yields (FF) of 3a–3d
were determined in DMF and they showed similar FF values
(Table 3) which were higher than that of unsubstituted Zn(II)
phthalocyanine. The increase in the hydrocarbon chain length did
not show any significant effect on the FF values.
The fluorescence decay curves of 3a–3d were recorded for
excitation wavelength of 405 nm and resulted in two exponential
curves of life-times (tF) in DMF (Fig. 4A). This observation could be
explained with the ability of 3a–3d to form non-fluorescent
species at concentrations over 10ꢀ5 M in DMF. It was well observed
Table 3
Photophysical and photochemical data for the axially parabens substituted silicon
(IV) phthalocyanines in DMF.
Compound
FF
Fd (x10ꢀ5
)
FD
3a
3b
3c
0.30
0.27
0.27
0.27
0.17
6.29
0.18
0.15
0.13
2.3
0.47
0.47
0.46
0.48
0.56
3d
ZnPca
a
Fig. 2. Absorption spectra of 3a–3d in DMF at the concentration of 10ꢀ5 M.
Data from Ref. [33].

G.C. Taşkın et al. / Journal of Photochemistry and Photobiology A: Chemistry 306 (2015) 31–40
37
Fig. 3. Absorption, excitation and emission spectra of 3d in DMF (lexc: 650 nm).
3.5. Photostability study
The photodegradation of 3a–3d under light irradiation was
defined by the photodegradation quantum yield Fd). The
(
decreasing of the absorption bands for the DMF solutions of the
studied phthalocyanines were determined by UV–vis spectrosco-
py. The observed spectral changes were shown as an example for
phthalocyanine 3c in DMF (Fig. 6). The obtained Fd values were
presented in Table 3. Generally, Fd values of the studied silicon(IV)
Pc compounds were lower than unsubstituted zinc(II) phthalocy-
anine except for 3a. The Fd value for 3a was higher than for the
other studied silicon(IV) phthalocyanines as well as unsubstituted
zinc(II) phthalocyanine.
3.6. In vitro studies
The photodynamic inactivation studies with axially methyl-
paraben, ethylparaben, propylparaben and butylparaben substi-
tuted Si(IV) Pcs towards S. mutans in suspension (106 CFU mLꢀ1
)
were presented in Fig. 7. Si(IV) Pcs showed the lack of dark toxicity
against planktonic cultured S. mutans at concentrations of 5
and 10 M. The dark toxicities due to the length chains of different
m
M
m
alkyl- parabens were not observed. Among four studied photo-
sensitizers 3a–3d, the photoinactivation was significant only after
treatment with 3c. The other three Si(IV) Pcs (3a,3b and 3d)
Fig. 5. Absorption changes due to photobleaching of DPBF obtained by irradiation
of 3d at a concentration of 10ꢀ5 M for determination of the produced singled oxygen
(Inset: Plot of DPBF absorbance versus time).
Fig. 4. Time-resolved fluorescence of the compounds 3a–3d in DMF (A) and THF
(B). lexc: 405 nm.

38
G.C. Taşkın et al. / Journal of Photochemistry and Photobiology A: Chemistry 306 (2015) 31–40
Fig. 6. Absorption changes during the photodegradation studies of the compound 3c in DMF. (Inset: Plot of the Q band decrease of absorbance versus time). 300 W General
electric quartz line lamp with power density of 18 mW/cm2 was used as a light source.
showed lower photodynamic efficacy (<log 3) at the applied
concentrations (5 M and 10 M).
in the spectral range 650–800 nm at excitation wavelength of
therapeutic light 637 nm by using set-up for fluorescence
measurements, which was described in our previous papers
[40,41]. It was found an inverse behavior of the decreasing
accumulation of 3a–3dthe SiPcs with increase of the cell densities
of suspensions (105–108 CFU mLꢀ1). The calculated amount of 3a–
3d into bacterial cells was estimated between 20% and 30% from
the total amount of the incubated photosensitizer. The incubation
was performed for 15 min than the cells were washed to remove
the non-bounded compound in order to estimate the real level of
saturation.
A high number of the bound 3c molecules per one bacterial cell
at lower cell density were in the agreement with our previous
findings and the literature [9,50,51]. The uptake study with
propylparaben Si(IV) Pc suggested that the accumulation of a
photosensitizer into cells at low cell density will have a substantial
effect on the photodynamic response. The phenomenon of an
inverse dependence of the number of dye molecule on the cell
density was firstly reported by Demidova and Hamblin for
photodynamic sensitizer and Escherichia coli bacterial cells [50].
The chemical structure of the photosensitizer, as the experimental
conditions (incubation time, temperature and concentration) is a
key factor for the uptake and further photodynamic effect [33]. The
m
m
The comparison of the axial propylparaben substituted Si(IV) Pc
3c with the recently studied tetra-methylpyridyloxy substituted Si
(IV)-phthalocyanine (SiPcMe) [47] suggested the higher photo-
inactivation efficiency for the newly studied Si(IV) Pc. It was found
that the bacterial cells were photoinactivated approximately 1 log
at the same experimental conditions. Nevertheless the lower
uptake of 3c as compared to cationic SiPcMe, compound 3c was
with high photocytotoxicity towards S. mutans planktonic cells.
The in vitro experiments on 48-h biofilms showed low ability for
photoinactivation even with the active Si(IV) Pc 3c at the used PDT
protocol, namely concentrations up to 10 mM, prolong incubation
time (1.5 h) and gentle irradiation from a LED 637 nm. In our
previous works with SiPcMe the effect was also negligible towards
most of the treated bacterial strains at different photodynamic
therapeutic conditions [47–49].
3.7. Uptake study
The uptake study was performed with photodynamically
effective propylparaben SiPc 3c as well as for 3a, 3b and 3dwith
S. mutans as planktonic cultured (Fig. 8). The spectra were recorded
Fig. 7. Survival of S. mutans cells (106 cells mLꢀ1) incubated with 5
3a–3d for 15 min and irradiated with LED 637 nm with a light dose 50 J cmꢀ2
m
M and 10
m
.
M
Fig. 8. Uptake of 10
suspension with different cell densities.
mM Si(IV) Pc 3a–3d after 15 min incubation of S. mutans cell

G.C. Taşkın et al. / Journal of Photochemistry and Photobiology A: Chemistry 306 (2015) 31–40
39
Fig. 9. Confocal laser scanning fluorescence images of S. mutans biofilm taken at (A) lexc: 488 nm, lem: 520–580 nm (green autofluorescence) and (B) lexc: 633 nm and lem
:
660–740 nm (red fluorescence of 3c) and (C) overlap of both images. Magnification x63.
optimal incubation time was observed to be between 5 and 30 min
for an optimal level of uptake of a non-charged phthalocyanine
[51]. It is well studied that the binding process occurs as a result of
compound hydrophobicity or by an electrostatic mechanism of
interactions [50,51]. The studied Si(IV) Pcs are non-charged
photosensitizer with hydrophobic nature, which appears favorable
property for penetration into lipophilic cell membrane.
generation able to inactivate bacterial cells. The different alkyl
groups to Si(IV) were not contributed to the inactivation capacity of
the studied axial paraben substituted SiPcs towards bacterium
strain S. mutans.
Acknowledgements
This work was supported by the collaboration project between
Scientific and Technological Research Council of Turkey (TUBITAK)
Project No: 212M053 and Bulgarian Academy of Sciences and
partly by the National Science Fund, Bulgaria (B02/9/2014).
3.8. Localization of 3c in bacterial biofilm
The bacterial biofilm was developed on coverslips and was
characterized by the native fluorescence of the cell chromophores
(lexc: 488 nm; lem: 500–580 nm) by a laser scanning of the biofilm
Appendix A. Supplementary data
(Fig. 9A). The visualization of red fluorescence of phthalocyanine
dye (lexc: 633 nm, lem: 660–740 nm) was obtained for the slices of
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
jphotochem.2015.03.010
biofilm with thickness of 0.150
pictured for determination of the biofilm thicknesses (4–5
m
m (Fig. 9B). In addition z-axis was
m)
m
and the penetration depth of compound 3c into the whole biomass.
The obtained bright fluorescence signal and co-localization image
shown in Fig. 9C suggested that Si(IV) Pc 3c was localized inside
the bacterial cells, but not in the matrix (Fig. 9A–C, dark area). The
assessments of the penetration depth were performed by the
fluorescence mode as well as the transmission mode which
suggested that 3c was distributed into the whole biomass. In case
of bacterial biofilm formed for 48-h, it needed longer incubation
time (1.5 h) than the time (5–30 min) necessary for dye uptake in
cell suspension [4,9,41]. Under the used treatment conditions, SiPc
3c was rapidly bound to S. mutans cells with full penetration into
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