Water soluble {2-[3-(diethylamino)phenoxy]ethoxy} substitutedzinc(II) phthalocyanine photosensitizers

Dilek Çakır a, Cem Göl b, Volkan Çakır a, Mahmut Durmuş b,Zekeriya Bıyıklıoğlu a,n, Halit Kantekin a

a Department of Chemistry, Faculty of Sciences, Karadeniz Technical University, 61080 Trabzon, Turkeyb Gebze Institute of Technology, Department of Chemistry, PO Box 141, Gebze, 41400, Kocaeli, Turkey

a r t i c l e i n f o

Article history:Received 1 September 2014Received in revised form11 October 2014Accepted 20 October 2014Available online 29 October 2014

Keywords:PhthalocyanineWater solublePhotodynamic therapyPhotosensitizerBSA bindingDNA interaction

a b s t r a c t

The new peripherally and non-peripherally tetra-{2-[3-(diethylamino)phenoxy] ethoxy} substituted zincphthalocyanines (2a and 3a) were synthesized by cyclotetramerization of phthalonitrile derivatives(2 and 3). 2-[3-(diethylamino)phenoxy] ethoxy group was chosen as substituent because the quaterni-zation of the diethylamino functionality on the structure of this group produced water soluble zincphthalocyanines (2b and 3b). The water solubility is very important for many different applications suchas photosensitizers in the photodynamic therapy of cancer because the water soluble photosensitizerscan be injected directly to the body and they can transport to cancer cells through blood stream. The newcompounds were characterized by using elemental analysis, UV–vis, IR, 1H NMR, 13C NMR and massspectroscopies. The photophysical and photochemical properties of these novel photosensitizercompounds were examined in DMSO (both non-ionic and ionic complexes) and in PBS (for ioniccomplexes) solutions. The investigation of these properties is very important for the usage of thecompounds as photosensitizers for PDT because determination of these properties is the first stage ofpotential of the compounds as photosensitizers. The bovine serum albumin (BSA) and DNA bindingbehaviour of the studied water soluble zinc (II) phthalocyanines were also investigated in PBS solutionsfor the determination of biological activity of these compounds.

& 2014 Elsevier B.V. All rights reserved.

1. Introduction

Phthalocyanines (Pcs) are the synthetic analogues of naturallyoccuring porphyrin family. Phthalocyanines are known as multi waydye staffs and are being applied in variety of industrial and medicalfields such as nonlinear optical devices [1], optical data storage[2,3], solar cell [4,5], electrochromic displays [6,7], chemical sensors[8,9], gas sensors [10,11], liquid crystals [12,13], semiconductors[14], photocatalysts [15]. One of the new and important practicalapplications of metallophthalocyanines containing diamagneticmetals such as Zn2þ , Al3þ , Ga3þ is their use as photosensitizersfor photodynamic therapy (PDT) of cancer due to their long tripletlifetimes and high efficiency of photogeneration of cytotoxic singletoxygen [16–20].

Unsubstituted phthalocyanines are practically insoluble incommon organic solvents and water, which restrict the investiga-tion and application of phthalocyanines. Therefore, the solubility

of phthalocyanines has been improved by introducing crownethers, alkyl, alkoxy, phenoxy, alkylthio, tertiary butyl, macrocyclicgroups and amino substituents to their peripheral positions[21–29].

Phthalocyanine compounds can be quaternized through aminegroups on substituents to reduce aggregation and increase theirsolubility and biological efficacy [30–32]. Water solubility playsvery important role for a potential photosensitizer [30] in PDTapplications. The advantages of MPcs bearing cationic substituentsover those with neutral and anionic substituents are numerous[33] such as improving water solubility, prevent aggregation [34],efficiency as PDT agents [35], enhancing cell uptake [36] andselectively localized in the cell mitochondria and causing toapoptosis [37].

The target of our ongoing research is to synthesis of water-soluble zinc phthalocyanine photosensitizers as potential PDTagents. For this reason, in this study, we have investigatedphotophysical (fluorescence quantum yields and lifetimes) andphotochemical (singlet oxygen and photodegradation quantumyields) properties of newly synthesized peripherally and non-peripherally tetra-substituted non-ionic (2a and 3a) and cationic

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Journal of Luminescence

http://dx.doi.org/10.1016/j.jlumin.2014.10.0440022-2313/& 2014 Elsevier B.V. All rights reserved.

n Corresponding author. Tel.: þ90 462 377 36 64; fax: þ90 462 325 31 96.E-mail address: zekeriya_61@yahoo.com (Z. Bıyıklıoğlu).

Journal of Luminescence 159 (2015) 79–87

(2b and 3b) zinc phthalocyanines for the first time. The bovineserum albumin (BSA) and DNA binding behaviour of the studiedwater soluble zinc (II) phthalocyanines were investigated in PBSsolutions.

2. Experimental

The used materials, equipments, the photophysical and photo-chemical parameters were supplied as supplementary information.

2.1. Synthesis

2.1.1. 3-{2-[3-(Diethylamino)phenoxy]ethoxy}phthalonitrile (3)3-Nitrophthalonitrile (1.32 g, 7.65�10�3 mol) was dissolved

in anhydrous DMF (0.023 L) under nitrogen atmosphere and2-[3-(diethylamino)phenoxy]ethanol (1) (1.6 g, 7.65 �10�3 mol)was added to this solution. After stirring for 10 min, finely groundanhydrous K2CO3 (4.2 g, 30.6 �10�3 mol) was added in portion-wise over 2 h with stirring. The reaction mixture was stirred at50 1C for four days under nitrogen atmosphere. Then 0.2 L waterwas added and the aqueous phase extracted with chloroform(3�0.07 L). The combined extracts were dried over anhydrousMgSO4 and then filtered. Solvent was evaporated and the productwas crystallized from ethanol. Yield: 0.9 g (35%), mp: 103–104 1C.IR (KBr pellet), ν/cm�1: 3087 (Aromatic-H), 2971-2868 (AliphaticC-H), 2230 (C�N), 1611, 1583, 1501, 1463, 1376, 1357, 1279, 1215,1142, 1071, 1025, 997, 829, 794, 753, 687. 1H NMR (CDCl3), (δ:ppm):7.66 (t, 1H, Ar-H), 7.39 (d, 2 H, Ar-H), 7.12 (t, 1 H, Ar-H), 6.35 (d, 1 H,Ar-H), 6.20 (m, 2 H, Ar-H), 4.50 (m, 2 H, –CH2-O), 4.39 (m, 2 H,–CH2-O), 3.34 (m, 4H, –CH2-N), 1.15 (t, 6H, –CH3). 13C NMR (CDCl3),(δ:ppm): 161.38, 159.73, 149.42, 134.81, 130.30, 125.74, 120.62,117.59, 115.64, 113.28, 109.46, 105.89, 100.63, 99.07, 68.70, 66.05,44.64, 12.85. MS (ESI), (m/z): Calc.: 335; Found: 336 [MþH]þ .Elemental analysis, Calcd. for C20H21N3O2: Found: C 70.71, H 5.88,N 12.30%, requires C 71.62, H 6.31, N 12.53%.

2.1.2. 2(3),9(10),16(17),23(24)-Tetrakis-{2-[3-(diethylamino)phenoxy]ethoxy}phthalocyaninato zinc (II) (2a)

4-{2-[3-(Diethylamino)phenoxy]ethoxy}phthalonitrile (2) (0.3 g,0.89�10�3 mol), anhydrous Zn(CH3COO)2 (0.081 g, 0.44�10�3

mol) and five drops of 1.8-diazabicyclo[5.4.0]undec-7-ene (DBU) in0.003 L of dry n-pentanol was stirred and heated at 160 1C in a glasssealed tube for 12 h under N2 atmosphere. After cooling to roomtemperature the green crude product was precipitated with ethanoland then dried in vacuo. Furthermore, purification was achievedusing column chromatography with basic alumina as column mate-rial and CHCl3:CH3OH (100:6) as solvent system. Yield: 0.173 g (55%).IR (KBr pellet), ν/cm�1: 3065 (Aromatic-H), 2965-2868 (AliphaticC-H), 1608, 1571, 1489, 1449, 1393, 1372, 1338, 1276, 1213, 1140, 1093,1069, 956, 822, 745, 686. 1H NMR (CDCl3), (δ:ppm): 7.53 (m, 4 H, Ar-H), 7.25-7.17 (m, 8 H, Ar-H), 6.79 (m, 4 H, Ar-H), 6.36 (m, 12 H, Ar-H),4.27-4.10 (m, 16 H, CH2-O), 3.33 (m, 16 H, –CH2-N), 1.15(t, 24 H, CH3). 13C NMR (CDCl3), (δ:ppm): 160.23, 159.17, 149.36,138.64, 131.19, 130.24, 129.07, 124.65, 122.63, 117.37, 105.48, 103.52,100.95, 99.21, 66.44, 44.62, 12.94. UV–vis (DMSO), λmax(logε)nm: 356(4.94), 615 (4.57), 682 (5.23). MS (ESI), (m/z): Calc.: 1407; Found:1408 [MþH]þ , Elemental analysis, Calcd for C80H84N12O8Zn: Found:C 68.40, H 5.80, N 12.40%, requires C 68.29, H 6.02, N 11.95%.

2.1.3. 1(4),8(11),15(18),22(25)-Tetrakis-{2-[3-(diethylamino)phenoxy]ethoxy}phthalocyaninato zinc (II) (3a)

The synthetic method resembles that of compound 2a. Yield:0.126 g (48%). IR (KBr pellet), ν/cm�1: 3071 (Aromatic-H), 2964-2862 (Aliphatic C-H), 1609, 1572, 1488, 1448, 1373, 1334, 1270,1214, 1171, 1141, 1078, 1023, 984, 886, 800, 742, 686. 1H NMR

(CDCl3), (δ:ppm): 8.85 (m, 8 H, Ar-H), 7.37 (m, 4 H, Ar-H), 6.56-6.18(m, 16 H, Ar-H), 4.88-4.69 (m, 16 H, CH2-O), 3.44 (m, 16 H, –CH2-N),1.10 (m, 24 H, CH3). 13C NMR (CDCl3), (δ:ppm): 160.32, 155.82,152.99, 150.01, 149.48, 141.10, 130.36, 117.40, 108.55, 106.90, 105.64,105.41, 100.85, 99.50, 67.09, 44.61, 12.88. UV–vis (DMSO), λmax

(logε)nm: 313 (4.75), 374 (4.65), 632 (4.62), 702 (5.36). MS (ESI),(m/z): Calc.: 1407; Found: 1408 [MþH]þ . Elemental analysis,Calcd for C80H84N12O8Zn: Found: C 68.80, H 5.70, N 11.70%,requires C 68.29, H 6.02, N 11.95%.

2.1.4. 2(3),9(10),16(17),23(24)-Tetrakis-{2-[3-(N-diethylmethylamino)phenoxy] ethoxy}phthalocyaninato zinc (II)iodide (2b)

Zinc(II) phthalocyanine 2a (0.040 g, 0.028 �10�3 mol) wasdissolved in 0.005 L of chloroform and 0.0023 L methyl iodidewas added to this solution. The reaction mixture was stirred atroom temperature for seven days. The green precipitate was filteredoff, washed with chloroform, acetone and diethyl ether. Finally,water-soluble quaternized zinc phthalocyanine were dried in vacuo.Yield: 0.045 g (80%). IR (KBr pellet), ν/cm�1: 3022 (Aromatic-H),2975-2876 (Aliphatic C-H), 1603, 1486, 1448, 1393, 1331, 1290, 1227,1056, 957, 868, 823, 773, 744, 688, 657. UV–vis (DMSO), λmax(logε)nm: 360 (4.77), 614 (4.29), 683 (4.97). MALDI-TOF-MSm/z: Calc.: 1974; Found: 368 [M-4Iþ2]þ4. Elemental analysis, Calcdfor C84H96N12O8ZnI4: Found: C 51.46, H 4.28, N 9.28%, requires C51.09, H 4.90, N 8.51%.

2.1.5. 1(4),8(11),15(18),22(25)-Tetrakis-{2-[3-(N-diethylmethylamino)phenoxy] ethoxy}phthalocyaninato zinc (II)iodide (3b)

The synthetic method resembles that of compound 2b. Yield:0.039 g (70%). IR (KBr pellet), ν/cm�1: 3016 (Aromatic-H), 2925-2851 (Aliphatic C-H), 1602, 1586, 1485, 1448, 1388, 1329, 1248,1168, 1080, 982, 872, 801, 744, 688. UV–vis (DMSO), λmax(logε)nm:324 (4.68), 383 (4.62), 629 (4.57), 699 (5.33). MALDI-TOF-MS m/z:Calc.: 1974; Found: 367 [M-4Iþ1]þ4. Elemental analysis, calcdfor C84H96N12O8ZnI4: Found: C 51.50, H 4.20, N 9.32%, requiresC 51.09, H 4.90, N 8.51%.

3. Results and discussion

3.1. Synthesis and characterization

Peripherally and non-peripherally tetra-substituted zinc(II)phthalocyanines (2a and 3a) and their water soluble cationicderivatives (2b and 3b) were synthesized according to the routeshown in Scheme 1 and Scheme 2, respectively. The phthalonitrilederivatives 2 and 3 were obtained by the aromatic nucleophilicsubstitution reaction of 2-[3-(diethylamino)phenoxy]ethanol(1) with 4-nitrophthalonitrile and 3-nitrophthalonitrile, respec-tively. The cyclotetramerization of the phthalonitrile derivatives2 and 3 in the presence of a few drops DBU as a strong base andanhydrous Zn(CH3COO)2 at reflux temperature under a nitrogenatmosphere in n-pentanol afforded the zinc phthalocyanines (2aand 3a). Water soluble ionic zinc(II) phthalocyanines (2b and 3b)were synthesized from the reaction of corresponding zinc(II)phthalocyanines (2a and 3a) with methyl iodide (as quaternizationagent) in chloroform. After reaction with methyl iodide, thequaternized zinc phthalocyanines (2b and 3b) showed excellentsolubility in water.

In the IR spectra of phthalonitrile derivatives 2 and 3, thecharacteristic C�N stretching vibrations were observed at 2229and 2230 cm�1, respectively. After cyclotetramerization reaction,these sharp peaks disappeared in the IR spectra of compounds 2aand 3a. On the other hand, the IR spectra of zinc(II)

D. Çakır et al. / Journal of Luminescence 159 (2015) 79–8780

phthalocyanines 2a and 3a are very similar. No major changes inthe IR spectra were observed after quaternization.

In the 1H NMR spectrum of compound 3, the aromatic protonswere indicated as two triplets at 7.66 and 7.12 ppm, two doubletsat 7.39 and 6.35 ppm, one multiplet at 6.20 ppm. Aliphatic –CH2-O,–CH2-N and –CH3 protons were observed as multiplets at4.50-4.39, 3.34 ppm and a triplet at 1.15 ppm, respectively.

The 1H NMR spectra of peripherally and non-peripherally tetra-substituted zinc (II) phthalocyanines 2a and 3a showed aromaticprotons at between 7.53-6.36 ppm for complex 2a and 8.85-6.18 ppmfor complex 3a. Aliphatic protons of zinc phthalocyanine complexeswere also observed at between 4.27-1.15 ppm for complex 2a and4.88-1.10 ppm for complex 3a. Examining the 1H NMR spectra of zinc

phthalocyanine complexes 2a and 3a in CDCl3 exhibited the expectedchemical shifts.

In the mass spectrum of phthalonitrile derivative 3, the presenceof molecular ion peaks at m/z¼336 [MþH]þ , confirmed theproposed structures. The molecular ion peaks of the phthalocyaninecomplexes 2a and 3a were observed at m/z¼1408 [MþH]þ . In themass spectra of water soluble zinc phthalocyanine complexes 2band 3b, the molecular ion peaks were observed at m/z¼368[M-4Iþ2]þ4 and 367 [M-4Iþ1]þ4 respectively, confirmed the pro-posed structures.

The UV–vis spectra of the phthalocyanine complexes exhibitcharacteristic Q and B bands. Two principle π-πn transitions areobserved for phthalocyanines: a Q-band which is a π-πn transition

NN

N

N

N

N

N

N

OOH

N

OO

N

CN

CN OO

N

CN

CN

NN

N

N

N

N

N

N

OR

ORRO

RO

OR

OR

RO

RO

N

O

CN

CN

O2NCN

CN

NO2

Scheme 1. The synthesis of the zinc(II) phthalocyanines. (i) K2CO3, N2, DMF. (ii) Zn(CH3COO)2, n-pentanol, DBU, 160 1C .

D. Çakır et al. / Journal of Luminescence 159 (2015) 79–87 81

from the highest occupied molecular orbital (HOMO) to the lowestunoccupied molecular orbital (LUMO) of the complexes. The Q bandabsorptions due to the π-πn transitions for were observed at682 nm for complex 2a, 702 nm for complex 3a, 683 nm forcomplex 2b and 699 nm for complex 3b DMSO (Table 1). Synthe-sized zinc phthalocyanines showed red-shifted absorption peaksafter substitution of 2-[3-(diethylamino)phenoxy]ethanol groups onthe phthalocyanine framework. The Q bands of the non-

peripherally substituted phthalocyanines were red-shifted whencompared to the corresponding peripherally substituted counter-parts in DMSO (Fig. 1). The red-shifts were 20 nm between 2a and3a and 16 nm between 2b and 3b. The observed red spectral shiftsare typical of Pcs with substituents at the non-peripheral positionsand have been explained in the literature [38,39] The B bands werebroad due to the superimposition of the B1 and B2 bands in the360 nm region.

3.2. Aggregation studies

The aggregation behaviour of the newly synthesized phthalo-cyanines in different solutions and different concentrations in

NN

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NOR

ORRO

RO

Zn

NN

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NOR'

OR'R'O

R'O

Zn

+4

4I

R=

N

OR'=

N

O

NN

N

N

N

N

N

N

Zn

OR

OR

RO

RO

NN

N

N

N

N

N

N

Zn

OR'

OR'

R'O

R'O +4

4I

CH3-ICHCl3, rt

CHCl3, rtCH3-I

Scheme 2. The synthesis of the water soluble zinc(II) phthalocyanines.

Table 1Absorption, excitation and emission spectral data for zinc phthalocyanine com-plexes (2a, 3a, 2b and 3b) in DMSO, PBS and PBS with Triton X-100 solutions.

Compound Solvent Q bandλmax,(nm)

log ε ExcitationλEx, (nm)

EmissionλEm, (nm)

StokesshiftΔStokes,(nm)

2a DMSO 682 5.24 687 695 132b DMSO 683 4.98 686 693 10

PBS 641, 683 4.43,4.28

— — —

PBSþTX 683 4.76 689 696 133a DMSO 702 5.37 706 713 113b DMSO 699 5.33 700 706 7

PBS 651, 693 4.76,4.70

— — —

PBSþTX 699 5.20 701 716 17Std-ZnPca DMSO 672 5.14 672 682 10

a Data from reference [48].

Fig. 1. Absorption spectra of the studied zinc phthalocyanines (2a-b and 3a-b) inDMSO at the concentration of 1�10�5 M.

D. Çakır et al. / Journal of Luminescence 159 (2015) 79–8782

DMSO were examined using UV–vis spectroscopy in this study.The UV–vis spectra of the synthesized phthalocyanines (2a, 2b, 3aand 3b) were measured in most common organic solvents (DMSO,DMF, THF, toluene, chloroform and dichloromethane) for determi-nation of aggregation properties of these compounds in order toselect suitable solvent for photophysical and photochemical prop-erties. The studied non-ionic phthalocyanines (2a and 3a) did notform aggregates in DMSO, DMF, THF, toluene, chloroform anddichloromethane solutions. The ionic complexes (2b and 3b) didnot also form aggregates in DMSO, DMF and methanol. However,they showed aggregation in aqueous solutions (Fig. 2). DMSO wasselected among the studied organic solvents for further studiesabout photophysical and photochemical properties of newlysynthesized phthalocyanines. Moreover, the non-aggregated solu-tions were observed in this solvent for all studied phthalocyaninesand the small amount of this solvent can be used for biologicalapplications without any toxic effect.

The aggregation behaviour of the zinc phthalocyanines (2a, 2b,3a and 3b) were also studied at different concentration in DMSO. Itis concluded that Beer-Lambert Law could be applied for thesynthesized phthalocyanine compounds.

Cofacial aggregation (H-aggregation) was determined in PBSby the UV–vis absorption spectra of quaternized phthalocyanines(2b and 3b) as evidenced by the presence of two non-vibrationalpeaks in the Q band region (Fig. 2). The lower energy (red-shifted)bands at 683 nm for 2b and 693 nm for 3b are due to the monomericspecies, while the higher energy (blue-shifted) bands at 641 nm for

2b and 651 nm for 3b are due to the aggregated species.Triton X-100 (%1) which is a surfactant was added to the PBS

solutions of the quaternized zinc phthalocyanines to prove aggre-gation. Addition of triton X-100 (%1) to a PBS solution of quater-nized zinc phthalocyanines (2b and 3b) resulted considerableincrease in intensity of the low energy side of the Q band(Fig. 3), suggesting that the molecules were aggregated and theaddition of triton X-100 broke up the aggregates between the Pcmolecules.

3.3. Fluorescence spectra

The fluorescence emission, absorption and excitation spectra ofnewly synthesized zinc (II) phthalocyanine complexes were stu-died in DMSO and the spectra were given in Fig. 4 for compound3b as an example. All substituted zinc (II) phthalocyanine com-plexes showed similar fluorescence behaviour in DMSO. Fluores-cence emission and excitation maxima were listed in Table 1. Theobserved Stokes' shifts were within the region observed for typicalzinc (II) phthalocyanine complexes. The excitation spectra weresimilar to absorption spectra and both were mirror images of thefluorescent spectra for all studied zinc (II) phthalocyanine com-plexes suggesting that the molecules did not show any degrada-tion during excitation in DMSO. The fluorescence behaviours of

0

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0.6

300 400 500 600 700 800

Wavelength (nm)

Abs

orba

nce

2b3b

Fig. 2. Absorption spectra of the quaternized ionic phthalocyanine compounds 2band 3b in PBS solution at the concentration of 1�10�5 M.

Fig. 3. Absorption spectra of the substituted zinc phthalocyanine a) 2b, b) 3b in PBS and PBSþTX solutions at the concentration of 1�10�5 M.

0

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400

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550 600 650 700 750 800

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a.u

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1.8

Abs

orba

nceExcitation

Emission

Absorption

Fig. 4. Absorption, excitation and emission spectra for compound 3b in DMSO.Excitation wavelength¼660 nm.

D. Çakır et al. / Journal of Luminescence 159 (2015) 79–87 83

quaternized zinc (II) phthalocyanines were also examined in PBSbut, they did not show any fluorescence in PBS solution. It could bedue to the formation of aggregates among the zinc phthalocyaninemolecules in PBS solution. It is known that the aggregates are notfluorescent.

3.4. Fluorescence quantum yields and lifetimes

The ΦF values of the studied zinc (II) phthalocyanine com-pounds (2a-b and 3a-b) are typical for MPc compounds. Thesevalues ranged from 0.05 to 0.23 in DMSO. Instead of compound 2b,the ΦF values of all substituted zinc(II) phthalocyanines (2a, 3a and3b) were lower than the unsubstituted zinc (II) Pc (Std-ZnPc)in DMSO as seen in Table 2. The quaternization of the zinc(II)phthalocyanines increased the ΦF values when compared thestudied zinc (II) Pc compounds. It could be suggested that thelone pair electrons of the nitrogen atoms on the substituents innon-quaternized complexes (2a and 3a) are engaged when theyquaternized. In addition, peripherally substituted zinc(II) phthalo-cyanine compounds (2a-b) showed higher ΦF values than non-peripherally substituted counterparts (3a-b) suggesting not asmuch of quenching of the excited singlet state by peripheralsubstitution compared to the non-peripheral substitution. Theseresults suggest that the substitution of the phthalocyanine frame-work and quaternization of the substituents affect on the ΦF valuesof these compounds.

Fluorescence lifetime (τF) refers to the average time a moleculestays in its excited state before emission, and its value is directlyrelated to that of ΦF. The longer lifetime is caused by the higherquantum yield of fluorescence. The τF values are higher forperipherally tetra-substituted zinc complexes (2a-b) when com-pared to non-peripherally tetra-substituted complexes (3a-b) inDMSO. The quaternization of the zinc phthalocyanine complexescaused increasing of the τF values, Table 2. Moreover, the τF valuesof substituted zinc (II) Pc compounds are typical for zinc (II) Pccomplexes [40].

The natural radiative lifetime (τ0) and the rate constants forfluorescence (κF) values of substituted zinc (II) phthalocyaninecompounds (2a-b and 3a-b) are also given in Table 2. The τ0 valuesof the substituted zinc (II) phthalocyanine compounds are higherthan unsubstituted zinc (II) Pc compound (Std-ZnPc) except forcompound 3a which is slightly lower than Std-ZnPc in DMSO. Inaddition, the τ0 values of quaternized ionic zinc(II) phthalocyaninederivatives (2b and 3b) are marginally higher than non-ioniccounterparts (2a and 3a). On the contrary τ0 values of studiedcompounds, the rate constants for fluorescence (kF) of studied zinc(II) phthalocyanine compounds are lower than unsubstituted zinc(II) phthalocyanine (Std-ZnPc) except for compound 3a which isslightly higher in DMSO. The kF value of compound 2b is thelowest among the studied zinc(II) phthalocyanine complexes.

3.5. Singlet oxygen generation properties

Energy transfer between the triplet state of photosensitizers andground state molecular oxygen leads to the production of singletoxygen. There is a necessity of high efficiency of transfer of energybetween excited triplet state of photosensitizers and ground state ofoxygen to generate large amounts of singlet oxygen, essential for PDT.The ΦΔ values were determined using a chemical method (using theabsorption bands of DPBF in DMSO and ADMA in PBS as quenchers).The disappearance of DPBF or ADMA was monitored using UV–visspectrophotometer (Fig. 5a using DPBF in DMSO for complex 2a andFig. 5b using ADMA in PBS for complex 3b). Many factors areresponsible for the magnitude of the determined quantum yield ofsinglet oxygen including; ability of substituents and solvents toquench the singlet oxygen, triplet excited state energy, the tripletexcited state lifetime and the efficiency of the energy transferbetween the triplet excited state and the ground state of oxygen.There was no decrease in the Q band of the ZnPc derivatives duringΦΔ determinations (Fig. 5) which confirms that all these complexesdid not degrade used light irradiation (30 V) for singlet oxygenstudies. The ΦΔ values of the substituted and unsubstituted zinc (II)phthalocyanine compounds in DMSO are given in Table 2. The ΦΔ

values of tetra-substituted zinc (II) Pc compounds (2a-b and 3a-b) arelower than the ΦΔ value of unsubstituted zinc (II) Pc compoundexcept for compound 3bwhich has the highestΦΔ value among themin DMSO. Especially, the ΦΔ values of quaternized zinc (II) phthalo-cyanine (2b and 3b) derivatives are relatively higher than their non-ionic counterparts (2a and 3a) in DMSO.

Table 2 shows that lower ΦΔ values are observed in PBS solutionscompared to in DMSO. The low ΦΔ values in PBS compared to DMSO

Table 2Photophysical and photochemical parameters of zinc phthalocyanine complexes(2a, 3a, 2b and 3b) in DMSO and PBS and PBS solutions.

Compound Solvent ΦF τF (ns) akF (s�1)(x108)

τ0(ns)

Φd

(x 10�4)ΦΔ

2a DMSO 0.13 0.91 1.43 6.98 1.08 0.542b DMSO 0.23 2.97 0.78 12.91 1.23 0.57

PBS — — — — 5.66 0.113a DMSO 0.05 0.31 1.49 6.70 2 0.553b DMSO 0.13 0.96 1.36 7.36 4.82 0.85

PBS — — — — 4 0.17Std-ZnPc DMSO 0.20b 1.22c 1.47c 6.80c 0.26c 0.67c

a kF is the rate constant for fluorescence. Values calculated using kF¼ΦF/τF.b Data from reference [41]c Data from reference [48]

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Abs

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0 s

180 s

360 s

540 s

720 s

900 s

Fig. 5. UV–vis absorption changes for the studied phthalocyanines during thedetermination of singlet oxygen quantum yield. a) compound 2a in DMSO and b)compound 3b in PBS at a concentration of 1�10�5 M. (Inset: Plots of DPBF orADMA absorbance versus time).

D. Çakır et al. / Journal of Luminescence 159 (2015) 79–8784

was explained by the fact that singlet oxygen absorbs at 1270 nm,and water, which absorbs around this wavelength has a great effecton singlet oxygen lifetime, while DMSO which exhibits little absorp-tion in this region [41] resulting in large ΦΔ values in DMSO.

3.6. Photodegradation studies

Degradation of the molecules under irradiation can be used tostudy their stability and this is especially important for thosemolecules intended for use in photocatalysis. The collapse of theabsorption spectra without any distortion of the shape confirmsphotodegradation not associated with phototransformation intodifferent forms of MPc absorbing in the visible region. The spectralchanges observed for all the complexes (2a-b and 3a-b) confirmedphotodegradation occurred without phototransformation (Fig. 6 asan example for complex 2a in DMSO).

All the studied zinc Pc complexes showed about the samestability with Φd of the order of 10�4. The Φd values, found in thisstudy, are similar with zinc Pc complexes having different sub-stituents on the phthalocyanine ring in literature [16]. Stable zincphthalocyanine complexes show Φd values as low as 10�6 andthese values are the order of 10�3 for unstable molecules havebeen reported [16]. All studied zinc phthalocyanine complexes(2a-b and 3a-b) showed higher Φd values when compared to theunsubstituted ZnPc (Std-ZnPc) in DMSO suggesting that thesubstitution of the 2-[3-(diethylamino)phenoxy]ethanol groupson the phthalocyanine framework decreasing the stability of thezinc Pc complexes. The ionic zinc Pc complexes (2b and 3b)showed higher Φd values than non-ionic complexes (2a and 3a)suggesting that quaternization of these groups resulted in thedecrease in the stability of the ionic zinc Pc complexes.

3.7. Binding of quaternized zinc (II) phthalocyanine derivatives toBSA protein

Fig. 7 shows the changes in the fluorescence emission spectra ofBSA at the presence of different concentrations of 2b as an examplein PBS. The quaternized ionic zinc Pc complexes are mixtures ofaggregated and unaggregated species. The total concentrations ofthe complexes are mixture of the monomer and aggregated species.The BSA fluorescence at 348 nm is mainly attributable to trypto-phan residues in the macromolecule. BSA and the respectivequaternized ionic zinc phthalocyanine complexes exhibit recipro-cated fluorescence quenching on one another; hence it was possibleto determine Stern-Volmer quenching constants (KSV). The slope ofthe plots shown at Fig. 8 gave KSV values which are listed in Table 3.These values suggest that BSA fluorescence quenching is moreeffective for quaternized peripherally substituted Pc complex (2b)than quaternized non-peripherally substituted Pc complex (3b) in

PBS. Using the approximate fluorescence lifetime of BSA [42,43], thebimolecular quenching constant (kq) was determined usingequation 7 in supporting information. These values are of the orderof 1013 M�1 s�1, which exceed the proposed value of 1010 M�1 s�1

for diffusion-controlled (dynamic) quenching (according to the

0.0

0.3

0.6

0.9

1.2

1.5

1.8

300 400 500 600 700 800Wavelength (nm)

Abs

orba

nce

0 s

60 s

120 s

180 s

240 s

y = -0.0005x + 1.6406R = 0.9912

0

0.6

1.2

1.8

0 50 100 150 200 250Time (s)

Abs

orba

nce

Fig. 6. UV–vis absorption changes during the photodegradation studies of thecompound 2a in DMSO showing the disappearance of the Q-band at 60 s intervals.(Inset: Plot of absorbance versus time).

290 340 390 440 490

Inte

nsity

(a.u

.)

Wavelength (nm)

[Pc]=0

[Pc]=saturated

Fig. 7. Fluorescence emission spectral changes of BSA (C¼3.00�10�5 M) onaddition of varying concentrations of 2b (0, 1.66�10–6, 3.33�10–6, 5.00�10–6,6.66�10–6, 8.33�10�6 M) in PBS.

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

0.E+00 1.E-06 2.E-06 3.E-06 4.E-06 5.E-06 6.E-06 7.E-06 8.E-06 9.E-06[Pc]

Io/I

2b

3b

Fig. 8. Stern-Volmer plots of substituted zinc phthalocyanines quenching of BSA inPBS. [BSA]¼3.00�10�5 M in water. [Pc]¼0, 1.66�10–6, 3.33�10–6, 5.00�10–6,6.66�10–6, 8.33�10�6 M.

Table 3Binding and fluorescence quenching data for interaction of BSA with quaternizedzinc phthalocyanines complexes (2b and 3b) in PBS.

Compound KBSASV /105 (M�1) kq /1013 (M�1 s�1) Kb /10�6 (M�1) n

2b 13.74 13.74 7.23 1.143b 11.79 11.79 8.20 1.15

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

-5.9 -5.8 -5.7 -5.6 -5.5 -5.4 -5.3 -5.2 -5.1 -5.0log[Pc]

log(

[Fo-

F]/F

-F∞

)

2b3b

Fig. 9. Determination of studied zinc phthalocyanine compounds-BSA bindingconstant (and number of binding sites on BSA). [BSA]¼3.00�10�5 M and [Pc]¼0,1.66�10–6, 3.33�10–6, 5.00�10–6, 6.66�10–6, 8.33�10�6 M in PBS.

D. Çakır et al. / Journal of Luminescence 159 (2015) 79–87 85

Einstein-Smoluchowski approximation) at room temperature [44].This also, is an indication that the mechanism of BSA quenching byquaternized Pc complexes (2b and 3b) is not diffusion-controlled (i.e., not dynamic quenching, but static quenching). The kq value ishigher for quaternized peripherally substituted Pc complex (2b)than quaternized non-peripherally (3b) substituted zinc Pc com-plexes in PBS. The binding constants (Kb) and number of bindingsites (n) on BSA were obtained using equation 5 in supporting

information and the results are shown in Table 3. The slope of theplots shown at Fig. 9 gave n values and the intercepts of these plotsgave Kb values. The values of Kb and n are typical of MPc-BSAinteractions in aqueous solutions [45,46]. The higher Kb value forquaternized non-peripherally substituted Pc complex (3b) impliesthat non-peripherally tetra-substituted zinc Pc complex binds morestrongly to BSA than the other quaternized zinc Pc (2b). Thedecrease in the intrinsic fluorescence intensity of tryptophan withquaternized Pc concentration indicates that these complexes readilybind to BSA, which implies that the Pc molecules reach subdomainswhere tryptophan residues are located in BSA. This also suggeststhat the primary binding sites of these molecules are very close totryptophan residues, since the occurrence of quenching requiresmolecular contact.

3.8. Binding of quaternized zinc (II) phthalocyanine derivatives toDNA

In order to determine the binding parameters of positivelycharged phthaocyanines with DNA, the titration of quaternizedzinc (II) phthalocyanines with DNA solution was performed bymonitoring of changes in optical spectrum. DNA binding experi-ments were performed by titrating 1.00�10�5 M of quaternizedzinc (II) phthalocyanines in PBSþTriton X-100 (1% to preventaggregation) solutions (2 mL) with 1.74�10�4 M DNA stock solu-tion in PBS (0.1 mL) and the changes in the UV–vis absorptionspectra of quaternized zinc (II) phthalocyanines were recorded.Fig. 10 demonstrates the changes in the absorption spectrum of 3bupon titration with DNA as an example. The linear decrease in theabsorbance at 699 nm with increasing concentrations of DNA aswell as sharp saturation indicates an interaction occurringbetween quaternized zinc (II) phthalocyanines and DNA (Fig. 10,inset).

The emission intensities of the quaternized zinc (II) phthalo-cyanines were reduced by the addition of DNA which indicatesthat an interaction occurring between quaternized zinc (II) phtha-locyanines and DNA molecules (Fig. 11). A linear decrease wasobserved in the emission at 717 nm by the increasing concentra-tions of DNA (Fig. 11, inset).

The interaction of studied quaternized zinc phthalocyanines andDNA is expected to be electrostatic in origin. In order to confirmthis, the effect of addition of a strong electrolyte such as NaCl [47]was investigated and monitored by both absorption and fluores-cence spectroscopies. Both absorption and fluorescence spectralintensities were increased by the addition of NaCl (Fig. 12) due todissociation of Pcs-DNA complexes. The changes may be explainedas a competition between positively charged quaternized zinc (II)

0.0

0.4

0.8

1.2

1.6

Wavelength (nm)

Abs

orba

nce

0 μl

100 μl

200 μl

300 μl

400 μl

300 400 500 600 700 800

Fig. 10. The observed spectral changes of 1.00�10�5 M 3b by the addition of DNA(1.74�10�4 M) at pH 7.4 in 1xPBS buffer containing 1% Triton X-100. Inset, the plotof absorbance at 699 nm versus the DNA concentration.

0

200

400

600

Wavelength (nm)

Inte

nsity

(a.u

.)

0 μl

100 μl

200 μl

300 μl

400 μl

685 715 745 775 805 835

DNA

Fig. 11. Emission spectra changes of 3b (1.00�10�5 M) by the addition of DNA atpH 7.4 in 1x PBS buffer containing 1% Triton X-100. Inset, the plot of emission at716 nm versus the ratio of DNA added at each step to the total [3b] concentration.Excitation wavelength: 675 nm.

0.0

0.4

0.7

1.1

1.4

Wavelength (nm)

Abs

orba

nce

0

200

400

600

300 400 500 600 700 800 685 715 745 775 805 835Wavelength (nm)

Inte

nsity

(a.u

.)

Fig. 12. a) Electronic absorption and a) fluorescence emission spectral changes of 3b in PBS solutions by the addition of NaCl.

D. Çakır et al. / Journal of Luminescence 159 (2015) 79–8786

phthalocyanines and NaCl for nucleic acid sites with sodium ionbinding prevailing. The results clearly indicated that the importantrole is electrostatic interactions between DNA and Pc molecules.

4. Conclusion

In the presented work, a range of new zinc phthalocyanines(2a and 3a) bearing four {2-[3-(diethylamino)phenoxy]ethoxy}groups and their water soluble quaternized derivatives (2b and3b) were synthesized. New zinc phthalocyanines were character-ized by standard methods (UV–vis, IR, 1H NMR, 13C NMR, MSspectroscopic data and elemental analysis). However, the synthe-sized non-ionic zinc (II) phthalocyanines exhibited good solubilityin most common organic solvents and the quaternized ionicderivatives also showed excellent solubility in aqueous media.On the other hand, the quaternized ionic derivatives formedaggregated species in aqueous solutions indicated with the broadpeaks at Q band region in the UV–vis spectra of these complexes inspite of possessing of good solubility in aqueous media. Thephotophysical and photochemical properties of the synthesizedzinc (II) phthalocyanines were investigated in DMSO for non-ioniccomplexes and in both DMSO and PBS solutions for quaternizedionic complexes. The bovine serum albumin (BSA) and DNAbinding behaviour of the studied water-soluble quaternized zinc(II) phthalocyanines (2b and 3b) were also investigated by fluor-escence and both fluorescence and absorption spectroscopies,respectively. An interaction occurred between BSA or DNA mole-cules and water soluble zinc (II) phthalocyanines was determined.In conclusion, all of these results demonstrated that newlysynthesized zinc (II) phthalocyanines especially water-solublederivatives can be good candidates for photodynamic therapy ofcancer treatment.

Acknowledgement

This study was supported by The Scientific & TechnologicalResearch Council of Turkey (TÜBİTAK, project no: 111T963).

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.jlumin.2014.10.044.

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