Tetracationic fluorinated zinc(ii)phthalocyanine: Synthesis, characterization and DNA-binding properties

Article abstract of DOI:10.1016/j.dyepig.2012.06.018

Selective replacement of the p-fluorine atom of 4-(2′,3′, 4′,5′,6′-pentafluorobenzyloxy) phthalonitrile with N,N-dimethylaminoetanethiol gave 4-[2′,3′,5′,6′- tetrafluoro-4′-(2-dimethylaminoetanethio)benzyloxy]phthalonitrile. A peripherally tetra-substituted zinc(II) phthalocyanine was achieved by tetramerization of the new perfluorobenzyloxyphthalonitrile in the presence of zinc acetate. The zinc derivative was quaternized with methyl iodide to afford a water-soluble product. The new compounds were characterized by using elemental analyses, ¹H NMR, ¹⁹F NMR, UV-vis and FT-IR spectroscopy and mass spectrometry. The interaction between the quaternized zinc phthalocyanine and calf thymus DNA was investigated in tris buffer (pH 7.4) by absorption and fluorescence spectroscopy. Upon addition of the quaternized zinc phthalocyanine, a change in the thermal denaturation profile of DNA was observed.

Full text of DOI:10.1016/j.dyepig.2012.06.018

Dyes and Pigments 96 (2013) 52e58
Contents lists available at SciVerse ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Tetracationic fluorinated zinc(ii)phthalocyanine: Synthesis, characterization
and DNA-binding properties
,
Mukaddes Özçes¸ meci a, Özgür Bülent Ecevit b, Serdar Sürgün a, Esin Hamuryudan a
*
a Department of Chemistry, Istanbul Technical University, Maslak, 34469 Istanbul, Turkey
b Department of Science, Borough of Manhattan Community College, City University of New York, 10007 NY, USA
a r t i c l e i n f o
a b s t r a c t
Selective replacement of the p-fluorine atom of 4-(20,30,40,50,60-pentafluorobenzyloxy) phthalonitrile with
N,N-dimethylaminoetanethiol gave 4-[20,30,50,60-tetrafluoro-40-(2-dimethylaminoetanethio)benzyloxy]
phthalonitrile. A peripherally tetra-substituted zinc(II) phthalocyanine was achieved by tetramerization
of the new perfluorobenzyloxyphthalonitrile in the presence of zinc acetate. The zinc derivative was
quaternized with methyl iodide to afford a water-soluble product. The new compounds were charac-
terized by using elemental analyses, 1H NMR, 19F NMR, UVevis and FT-IR spectroscopy and mass spec-
trometry. The interaction between the quaternized zinc phthalocyanine and calf thymus DNA was
investigated in tris buffer (pH 7.4) by absorption and fluorescence spectroscopy. Upon addition of the
quaternized zinc phthalocyanine, a change in the thermal denaturation profile of DNA was observed.
Ó 2012 Elsevier Ltd. All rights reserved.
Article history:
Received 5 April 2012
Received in revised form
22 June 2012
Accepted 25 June 2012
Available online 14 July 2012
Keywords:
Fluorinated phthalocyanines
Quaternization
Calf thymus DNA
Nucleophilic substitution
Solubility
Aggregation
1. Introduction
interaction and reaction of metal complexes with DNA continues to
be an important area of research. DNA is highly negatively charged
Phthalocyanines represent an important class of functional dyes
[1]. In addition to their use as advanced materials in various fields,
these macrocyclic compounds have also found application in
medicine. Owing to their strong and long-wavelength absorptions,
high efficiency at generating reactive oxygen species, and ease of
chemical modification, phthalocyanines have emerged as a prom-
ising class of second-generation photosensitisers for photodynamic
therapy (PDT) of cancer [2]. Fluorinated MPcs are currently
receiving a great deal of attention [3]. The presence of fluorine in
the structure of a photosensitizer may enrich it with required
pharmacokinetic features. Photostability, high level of singlet
oxygen production, lipophilicity and selective accumulation in
tumor cells have made the fluorinated porphyrinoids potential
entities for photodynamic therapy [4].
and it interacts strongly with oppositely charged species. Cationic
porphyrins, phthalocyanines, and their analogs have long been of
interest for their interactions with DNA because of their role in the
human body, ability to accumulate in many kinds of cancer cells, as
well as their magnetic and optical properties. Studies on the
interaction of cationic phthalocyanines and their analogs with DNA
have received interest in recent years [4e12]. Water soluble
cationic phthalocyanines and metallophthalocyanines and their
binding to DNA have become important subjects of interest in the
search of new DNA-targeting drugs [13,14].
Phthalocyanines are hardly soluble in common solvents,
because of their large structures. This causes difficulties in their
separation or identification and limits their potential applications
in medicine [4]. Significant effort and resources have been utilized
to introduce various fluorine containing groups to the periphery of
these macrocycles in order to overcome these limitations [15e25].
Moreover, an enhanced solubility of fluorinated phthalocyanines
was found to simplify preparation of various drug formulations,
including emulsions and nanoparticles. The presence of both
superhydrophobic fluorine and hydrophilic NeH groups in the
phthalocyanine structure appears to enrich it simultaneously with
Cancer is one of the leading causes of death in the world.
Although there are exciting developments in the personalized
cancer therapy, such drugs currently represent only a small
percentage of the total anticancer agents. Since the molecular
recognition of DNA is of fundamental importance to life, the
water solubility [26,27]. Aggregation is also
phenomenon in phthalocyanine chemistry. However, bulky
a well-known
* Corresponding author. Tel.: þ90 212 285 6826; fax: þ90 212 285 6386.
E-mail address: esin@itu.edu.tr (E. Hamuryudan).
0143-7208/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dyepig.2012.06.018

M. Özçes¸meci et al. / Dyes and Pigments 96 (2013) 52e58
53
fluorinated substituents on peripheral sites can reduce interactions
between phthalocyanine molecules, lowering their aggregation and
self-oxidation tendencies [4].
(C^N), 1250 (CeOeC); 1H NMR (CDCl3),
(d
, ppm): 7.74 (d,
J ¼ 8.64 Hz, H, AreH), 7.34 (s, H, AreH), 7.28 (d, J ¼ 8.70 Hz, H,
AreH), 5.21 (s, 2H, OCH2), 3.01 (t, 2H, SCH2), 2.54 (t, 2H, NCH2), 2.23
During the last few years we have focused on the preparation of
fluorine containing groups introduced to the periphery of phtha-
locyanines. The introduction of fluoro-substituents on the phtha-
locyanine improves its solubility in common solvents and enhances
electrochemical, spectroelectrochemical and optical properties
[15e20]. We report herein the synthesis, characterization and DNA-
binding properties of new organo-soluble and water soluble
zinc phthalocyanines bearing functionalized polyfluorinated
substituents.
(s, 6H, NCH3); 19F NMR (acetone-d6), (
d
, ppm): ꢂ144.1 (d-o-fluo-
rine), ꢂ164.4 (q-m-fluorine); MS (ESIþ): m/z 378.6 [M-2CH3]þ.
2.3.2. Synthesis of 2,9(10), 16(17), 23(24)-tetrakis-[20,30,50,60-
tetrafluoro-40-(2-dimethylaminoetanethio) benzyloxy]
phthalocyaninato zinc(II) (2)
A mixture of 1 (0.150 g, 0.366 mmol), anhydrous Zn(CH3COO)2
(0.017 g, 0.0915 mmol), and anhydrous DMF (2 mL) were mixed in
a glass tube which was sealed under nitrogen. After the reaction
mixture was heated and stirred at 140 ꢀC for 2 days, the mixture
was cooled to room temperature. Then it was poured into ice water
(200 mL). The creamy precipitate was washed with water and dried
in vacuo. The green product was isolated by column chromatog-
raphy with silica gel using THF/hexane (5:2) as eluent. Yield:
0.070 g (45%), m.p: >200 ꢀC. Anal. calcd. for C76H60F16N12O4S4Zn: C,
2. Experimental
2.1. Materials
Calf-thymus DNA and all other analytical grade chemicals were
purchased from Merck Chemicals and SigmaeAldrich Chemicals.
All solvents were dried and purified as described by Perrin et al.
[28]. All solutions for DNA-binding studies were prepared using
purified water by the Millipore Milli-Q Water system. 4-
Nitrophthalonitrile and 4-(20,30,40,50,60-pentafluorobenzyloxy)
phthalonitrile were prepared according to the reported procedures
[15,29]. Silica gel (Kieselgel 60, 200e400 mesh) was used in the
separation and purification of compounds by column chromatog-
raphy. The homogeneity of the products was tested in each step
by TLC.
53.60; H, 3.55; N, 9.87%. Found: C, 53.71; H, 3.63; N, 9.92. IR nmax
,
,
cmꢂ1: 2952e252 (alkyl CH), 1217 (CeOeC); 1H NMR (CDCl3), (
d
ppm): 7.74 (d, J ¼ 8.32 Hz, 4H, AreH), 7.53 (s, 4H, AreH), 7.39 (d,
J ¼ 8.30 Hz, 4H, AreH), 5.17 (s, 8H, OCH2), 3.30 (t, 8H, SCH2), 2.91 (t,
8H, NCH2), 2.80 (s, 24H, NCH3). 19F NMR (CDCl3), (
d
, ppm): ꢂ143.61
(m, 8F, o-fluorine), ꢂ164.70 (m, 8F, m-fluorine). UVevis (THF): lmax
/
nm (log ): 350 (4.28), 676 (4.57). MS (MALDI-TOF MS) m/z: 1654.47
3
[M-3CH3]þ.
2.3.3. Synthesis of 2,9(10), 16(17), 23(24)-tetrakis-[20,30,50,60-
tetrafluoro-40-(2-dimethylaminoetanethio)benzyloxy]
phthalocyaninato zinc(II)tetraiodide (3)
2.2. Equipment
Compound 2 (0.030 g, 0.018 mmol) was dissolved in chloroform
(30 mL) and methyl iodide (0.010 mg, 0.072 mmol) was added to this
solution. The reaction mixture was stirred first at 50 ꢀC for 3 h and
then at room temperature for 20 h. The resulting suspension was
filtered off, washed with CHCl3 and dried. Yield: 0.020 g (48%), m.p:
>200 ꢀC. Anal. calcd. for C80H72F16N12O4S4I4Zn: C, 42.31; H, 3.20; N,
7.40%. Found: C, 42.43; H, 3.53; N, 7.58. IR nmax, cmꢂ1: 2956e2776
1H NMR spectra were recorded on a Bruker 250 MHz spec-
trometer using TMS as internal reference. 19F NMR spectrum was
recorded on a Varian Unity Inova 500 MHz NMR. IR spectra were
recorded on a PerkineElmer Spectrum One FT-IR (ATR sampling
accessory) spectrophotometer. Electronic spectra were recorded on
a Scinco LabProPlus UV/vis spectrophotometer. Absorbance and
fluorescence spectra for DNA-binding experiments were recorded
in a quartz cuvette using Hitachi U-2910 and FluoroMax-4 (Horiba
Jobin Yvon), respectively. Mass spectra were performed on Varian
711 and Bruker microflex LT MALDI-TOF MS mass spectrometers.
The isotopic patterns for all assigned signals are in agreement with
the calculated natural abundance. Data have been given for the
most abundant isotope only.
(alkyl CH), 1220 (CeOeC); 19F NMR (d-DMSO), (
d
, ppm): ꢂ145.27
(m, 8F, o-fluorine), ꢂ161.75 (m, 8F, m-fluorine). 1H NMR (d-DMSO),
(d, ppm): 9.32e9.04 (m, 8H, AreH), 7.86 (s, 4H, AreH), 5.83 (s, 8H,
OCH2), 3.55 (t, 8H, SCH2), 3.31 (s, 36H, NCH3), 2.97 (s, 8H, NCH2).
UVevis (DMSO): lmax/nm (log ): 360 (4.25), 683 (4.55). MS (MALDI-
3
TOF MS) m/z: 2181.66 [M-6CH3]þ, 1420.23 [M-6CH3I]þ.
2.4. DNA-binding studies
2.3. Synthesis
2.4.1. Absorbance and fluorescence studies
2.3.1. Synthesis of 4-[20,30,50,60-tetrafluoro-40-(2-
All titrations for DNA-binding experiments were conducted at
pH 7.4 in a 10 mM Tris buffer containing 50 mM NaCl. The final
concentration of ct-DNA was 1e20 mM. The concentration of ct-
DNA was determined by UV absorbance at 260 nm using the
molar absorptivity constant of 13,200 Mꢂ1 cmꢂ1. In order to
determine dilution effects, a control experiment was performed in
which the DNA solution was replaced by buffer solution. Absorp-
tion spectra were recorded in the region of 350e825 nm, and
fluorescence spectra were recorded in the region of 660e760 nm
after excitation at 655 nm. Titrations of the Pc with ct-DNA were
dimethylaminoetanethio)benzyloxy] phthalonitrile (1)
4-(20,30,40,50,60-pentafluorobenzyloxy)phthalonitrile
(1
g,
3.085 mmol) was dissolved in dry DMF (20 mL) under nitrogen
atmosphere and N,N-dimethylaminoethanethiol (0.320 g,
3.085 mmol) was added to the solution. After stirring 10 min, finely
ground anhydrous K2CO3 (0.639 g, 4.628 mmol) was added por-
tionwise within 2 h with efficient stirring. The reaction mixture was
stirred under nitrogen at 50 ꢀC for 48 h. Then the solution was
poured into ice-water (200 mL). The precipitate was collected by
filtration, washed first with water until the filtrate was neutral and
then extracted with chloroform (3 ꢁ 200 mL); the chloroform
extracts were combined, dried over MgSO4, and the solvent was
removed at reduced pressure. The desired product was isolated by
column chromatography with silica gel using methanol/chloroform
(1:100) as eluent. Yield: 0.189 g (15%), mp: 53 ꢀC. Anal. calcd. for
C19H15F4N3OS: C, 55.74; H, 3.69; N, 10.26%. Found: C, 55.59; H, 3.42;
N, 10.43. IR nmax, cmꢂ1: 3088 (AreCH), 2918e2776 (alkyl CH), 2231
performed by adding small aliquot (10 mL) of a concentrated DNA
solution to the Pc solution at constant concentration. All solutions
were allowed to equilibrate for 10 min before measurements were
recorded.
2.4.2. Thermal-denaturation studies
Ct-DNA and Pc in buffer solution were heated in the tempera-
ture range of 30 ꢀCe85 ꢀC at
a
rate of 0.2ꢀ/min. Melting

54
M. Özçes¸meci et al. / Dyes and Pigments 96 (2013) 52e58
temperatures were monitored at 260 nm wavelength using Hitachi
U-2910 spectrophotometer.
phthalocyanine (2) with methyl iodide in CHCl3 (Scheme 2)
[31e36]. With regard to the solubility of phthalocyanines, the green
product 2 is soluble in most of the polar organic solvents such as
CHCl3, CH2Cl2, THF, acetone; whilst the cationic derivative 3 is fully
soluble in water and DMSO as expected.
3. Results and discussion
Elemental analysis results and the spectral data (FT-IR, 1H NMR,
19F NMR, UVevis and MS) for the newly synthesized compounds
were consistent with the assigned formulations.
3.1. Synthesis and characterization
The synthetic procedure for the new compounds is outlined in
Scheme 1. The phthalonitrile derivative 1 was obtained by base-
catalyzed aromatic nucleophilic displacement of fluoride ion from
4-(20,30,40,50,60-pentafluorobenzyloxy)phthalonitrile with the eSH
function of N,N-dimethylaminoethanethiol using K2CO3 as the base
in dry DMF at 50 ꢀC under N2 for 48 h (Scheme 1). The substitution
regioselectivity was always quite high only at the para-fluorine of
pentafluorophenyl group with Oꢂ, Sꢂ or Nꢂ nucleophiles [30].
Cyclotetramerization of the dinitrile 1 to the tetra-substituted
zinc phthalocyanine (2) was accomplished in the presence of
anhydrous Zn(CH3COO)2 in DMF at 140 ꢀC for 2 days in a sealed
tube. Compound 2 was isolated by column chromatography on
silica gel using THF/hexane (5:2) mixtures as an eluent. As a natural
consequence of the presence of single substituents on each benzo
group, the phthalocyanine 2 is a mixture of four structural isomers
(D2h:Cs:C2v:C4h). The presence of isomers might be verified by the
slight broadening encountered in the UVevis absorption bands and
broadening of the signals in the 1H NMR spectrum. No attempt was
made to separate the isomers of 2. Tetra-cationic zinc phthalocya-
nine (3) was obtained from the reaction of corresponding
In the FT-IR spectrum of compound 1, stretching vibrations of
C^N groups at 2231 cmꢂ1, aliphatic CH groups at 2918e2776 cmꢂ1
,
aromatic CH groups at 3088 cmꢂ1, and ether (CeOeC) units at
1250 cmꢂ1 appeared at expected frequencies. In the 1H NMR spec-
trum of compound 1 in CDCl3, the aromatic protons are present as
a doublet, singlet, doublet at
CH2 protons of pentafluorobenzyl group appear at
a singlet andthealiphatic protonsof the side chainsof 1 exhibited the
eSCH2 protons as a triplet at : 3.01 ppm. The eNCH2 protons reso-
nate at : 2.54 ppm and eNCH3 protons at : 2.23 ppm as a singlet.
The 19F NMR spectrum of 1 in acetone-d6 is revealed only two
fluorine peaks at
d: 7.74, 7.34, 7.28 ppm, respectively. The
d: 5.21 ppm as
d
d
d
d
: ꢂ144.1 and ꢂ164.4 ppm, thus supporting the
idea that para-fluorine atom was replaced with 2-
dimethylaminoetanethio group. Phthalocyanines 2 and 3 also
have very similar 19F NMR spectra for the fluorinated peripheral
substituents. In the 19F NMR spectrum of compound 2 in CDCl3, the
ortho and meta fluorine atoms are present as a multiplet at
and 164.70 ppm, respectively. In d-DMSO compound 3 gives the
signals as a multiplet at : 145.27 and 161.75 ppm, respectively.
d: 143.61
d
Scheme 1. Synthetic route of the phthalonitrile derivative (1) and ZnPc (2); (i) N,N-dimethylaminoethanethiol, DMF and K2CO3; (ii) Zn(CH3COO)2 and DMF.

M. Özçes¸meci et al. / Dyes and Pigments 96 (2013) 52e58
55
Scheme 2. Synthetic route of the quaternized ZnPc (3).
The cyclotetramerization of compound 1 can be seen clearly by
the absence of the C^N peaks at 2231 cmꢂ1, on the FT-IR spectrum
on formation of compound 2. The FT-IR spectrum of 2 showed
similar characteristics. Also, no major change was found in the FT-IR
spectrum of 3 after quarternization.
The Beer-lambert law was obeyed for compound 2 in the concen-
trations ranging from 2.0 ꢁ 10ꢂ6 to 2.0 ꢁ 10ꢂ5 mol dmꢂ3 at
maximum absorption (
l
¼ 676 nm). The Beer-Lambert relationship
is shown as an inset for 2 in Fig. 1. As the concentration is increased,
the absorption maxima of the Q band also increased and the blue
shift of Q band absorptions is not observed.
The 1H NMR spectrum of tetra substituted zinc(II) phthalocya-
nine derivative (2) was almost identical with that of the starting
compound 1 except for some signal broadening and some small
shifts in the positions of some signals. It is probable that the
phthalocyanine derivative obtained as a mixture of positional
isomers which are expected to show chemical shifts that differ
slightly from each other.
3.2. Aggregation behavior of quaternized zinc phthalocyanine (3) in
aqueous solutions
The UVevis spectrum of the quaternized zinc phthalocyanine
(3) in DMSO is very similar to corresponding non-quaternized
derivative 2 in THF. However, the electronic spectrum of 3 in
water showed some differences from that in DMSO as a result of
The mass spectrum of 1 was obtained by MS (ESI) technique. In
the mass spectrum of 1, fragment ion corresponding to the loss of
[M-2CH3]þ at m/z: 378.6 was easily identified. Also, MALDI-TOF MS
technique was used to identify compound 2 and 3. In the mass
spectrum of 2, the fragment ion peak corresponding to the loss of
[M-3CH3]þ was observed at m/z: 1654.47. In the case of 3, the
fragment ion peaks corresponding to the loss of [M-6CH3]þ and [M-
6CH3I]þ were observed at m/z: 2181.66, 1420,23 respectively.
Electronic spectra are especially fruitful to establish the struc-
ture of the phthalocyanines. The phthalocyanines exhibit typical
electronic spectra with two strong absorption regions, one in the
UV region at about 300e400 nm (B-band) and the other one is in
the visible region at 600e700 nm (Q-band), both correlate to pep*
transitions. The characteristic Q-band transition of 2 was observed
as a single band at 676 nm in THF. In the spectrum of the quater-
nized zinc phthalocyanine (3) in DMSO the Q-band was observed at
683 nm. B-bands of these phthalocyanines (2e3) appeared at
around 350e360 nm. Generally in Pcs increasing the concentration
leads to aggregation, which is easily observed by the values of the Q
band absorptions. In this study, complex 2 did not show aggrega-
tion in THF at different concentrations, because of the bulky nature
of 20,30,50,60-tetrafluoro-40-(2-dimethylaminoetanethio)benzyloxy
groups on the peripheral sites of macrocycle ring [20]. Fig. 1 shows
the changes in the visible spectrum of 2 in THF with concentration.
Fig. 1. UVevis spectra of 2 in THF at different concentrations: 2 ꢁ 10ꢂ6 (A), 4 ꢁ 10ꢂ6
(B), 6 ꢁ 10ꢂ6 (C), 8 ꢁ 10ꢂ6 (D), 1 ꢁ 10ꢂ5 (E), 2 ꢁ 10ꢂ5 (F) mol dmꢂ3
.

56
M. Özçes¸meci et al. / Dyes and Pigments 96 (2013) 52e58
differing solvent. First, the B-bands are slightly shifted to shorter
wavelength. Second, the intensity of the Q-bands is much lower.
Third, for 3, the Q-bands absorption is present as a shoulder, while
the absorption of the aggregated species around 645 nm is the main
peak (Fig. 2) [37,38].
Aggregation behavior of Pc is depicted as a coplanar association
of rings progressing from monomer to dimer and higher order
complexes and it is dependent on concentration, nature of solvent
and substituents, metal ions and temperature [19,39,40].
Aggregation is not desired in MPc complexes since aggregates
are generally photoinactive. This problem is particularly serious in
polar media such as water, which tends to self-associate and repel
the hydrophobic
p systems to form aggregates. Therefore, hydro-
philic and nonaggregated phthalocyanines have special importance
and have received much current attention. One of the strategies to
reduce the aggregation of phthalocyanines in aqueous media
involves the use of surfactants or other substances which can create
a microheterogeneous environment such as a micelle or liposome.
It has been found that phthalocyanines exist mainly as monomeric
species in these environments, giving a relatively high photo-
activity [41]. Addition of a drop of Triton X-100 to an aqueous
solution of 3 resulted in the decrease in the high energy band due to
the aggregates and the increase in the low energy band due to the
monomer (683 nm for 3) (Fig. 3).
Fig. 3. UVevis spectra of 3 in water and water containing Triton X-100.
is considered to be indicative of an intercalation as a result of a
pep
stacking between DNA bases and Pc. On the other hand, small
changes in the absorption spectra usually indicate external stacking
or electrostatic binding. In the absorption titration spectrum of 3, as
shown in Fig. 4, a small blue shift and hypochromicity were
observed in the Q-band upon addition of various amounts of ct-
DNA to 3. Addition of ct-DNA to 3 caused also a small bath-
ochromic shift in the wavelength maximum of the B-Band, and
a decrease in the intensity of the spectrum. The wavelength shifts in
the Q and B-Bands were not large enough to be considered indic-
ative of an intercalation. The insignificant blue and red shifts in the
Q- and B-Bands, respectively, were attributed to an electrostatic
binding between the cationic Pc and the negatively charged DNA
phosphate backbone.
3.3. DNA-binding studies
3.3.1. Absorbance studies
Absorption titration experiments were performed to investigate
the binding of Pc to ct-DNA. It is very important to understand the
binding modes of Pc to DNA. There are three possible known
binding modes of phthalocyanines to DNA: intercalation, external
binding and external binding with stacking. Intercalation is the
process where the phthalocyanine molecules insert themselves in
between base pairs of DNA. In general intercalation involves
a relatively hydrophobic, polar, planar region of the DNA binder, but
electrostatic, dipoleedipole, and H-binding interactions between
the intercalator and DNA are also important. Electrostatic and H-
bonding interactions are the main binding forces in external
binding. Some of the factors which inhibit intercalation and favor
external binding are steric hindrance and lack of planarity [42].
The binding mode of Pc to DNA is one of the crucial factors
affecting the degree of the wavelength shift and also whether
hypochromism or hyperchromism occurs. A considerable red shift
3.3.2. Fluorescence studies
In order to calculate the binding constants of Pc to DNA,
generally, either fluorescence or absorbance titration is utilized.
2.0
1.5
1.0
0.5
0.0
300
400
500
600
700
800
Wavelength(nm)
Fig. 4. Absorbance titration spectra of 3 (5
mM) upon addition of increasing amounts of
Fig. 2. UVevis absorption spectra of 3 in water and DMSO.
ct-DNA (0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, and 33
mM) (from top to bottom).

M. Özçes¸meci et al. / Dyes and Pigments 96 (2013) 52e58
57
3.3.3. Thermal-denaturation studies
DNA melting profile is very informative about helix stability.
Thermal denaturation studies on DNA in the presence of a ligand
can also be employed to differentiate between electrostatic and
intercalative binding modes. The difference between the melting
temperatures of DNA in a buffer solution and DNA with a ligand is
usually large if the binding of the ligand to DNA occurs through
intercalation. On the other hand, if the binding mode of the ligand
to DNA is non-intercalative, the difference in the melting temper-
atures is generally smaller. In our experiments, DNA melting profile
is found to be sensitive to the addition of 3, as shown in Fig. 6.
Addition of 5
mM of 3e35 mM of ct-DNA increased the melting
temperature of ct-DNA by about 8 ꢀC. This small change in the
melting temperature is also indicative of a non-intercalative
binding between ct-DNA and 3.
4. Conclusion
In this work, we have synthesized and characterized a novel Zn
phthalocyanine substituted with four 20,30,50,60-tetrafluoro-40-(2-
dimethylaminoetanethio)benzyloxy substituents on peripheral
positions and produced tetra-cationic water soluble Zn phthalo-
cyanine. Using absorption and fluorescence spectroscopic tech-
niques, DNA binding properties of tetra-cationic ZnPc were also
investigated and the change in the melting point of DNA was
determined with thermal denaturation profiles. Titrametric inves-
tigation indicate that electrostatic binding between cationic
phthalocyanine and anionic DNA phosphate groups is occurring to
Fig. 5. Scatchard plots obtained from the competitive fluorescence titration data for
binding of 3 to DNA. With 2e11 M ethidium bromide, -: 0 M, C: 0.1 M, :: 0.2 M.
m
m
m
m
Absorbance titration data is very useful for determining the
absorption coefficient of bound Pc, if the binding mode between
the Pc and DNA is intercalative. However, in our experiments, only
a small change was observed in the Q and B-Bands of the absorp-
tion spectrum upon binding to DNA which suggests a non-
intercalating binding mode. Therefore, a fluorescence titration
method was used to determine the apparent binding constant
(Kapp). In this method, ethidium bromide, an intercalating agent, is
used as a spectroscopic probe and a competitive fluorescence
titration is carried out to monitor the decrease in the fluorescence
intensity of ethidium bromide bound to DNA in the presence of 3.
The intercalating ability of 3 can be calculated by using the amount
of decrease in fluorescence intensity of ethidium bromide bound to
DNA. Fluorescence titration data obtained by this method were
used for the Scatchard plot analysis shown in Fig. 5. The apparent
binding constant of 3 to DNA calculated from the Scatchard plot
a large extend and pep stacking between DNA and phthalocyanine
moieties takes place afterward.
In summary, the findings of this study will help us to enhance
our understanding of the binding modes of the phthalocyanines, in
particular polyfluorinated substituted metallo-phthalocyanines, to
DNA. In addition, these data will form the basis of our research
efforts on the use of these novel phthalocyanines as promising
candidates in photodynamic therapy.
Acknowledgments
(6.20 ꢁ 105
M
ꢂ1) is found to be smaller than 106, which is an
average value for the apparent binding constant of small inter-
calating compounds. This result suggests that the mode of binding
for 3 to ct-DNA is most likely to be non-intercalative.
This work was supported by the Research Fund of the Istanbul
Technical University.
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