New peripherally and non-peripherally tetra-substituted watersoluble zinc phthalocyanines: Synthesis, photophysics andphotochemistry
Volkan Çakır a, Dilek Çakır a, Mehmet Piskin b, Mahmut Durmus c, Zekeriya Bıyıklıo�glu a, *
a Department of Chemistry, Faculty of Sciences, Karadeniz Technical University, 61080 Trabzon, Turkeyb Çanakkale Onsekiz Mart University, Çanakkale Vocational School of Technical Sciences, Department of Food Technology, 17100 Çanakkale, Turkeyc Gebze Technical University, Department of Chemistry, PO Box 141, Gebze, 41400, Kocaeli, Turkey
a r t i c l e i n f o
Article history:Received 2 January 2015Received in revised form12 February 2015Accepted 13 February 2015Available online 24 February 2015
The synthesis and photochemical properties of peripherally and non-peripherally 1,3-bis[3-(dimethy-lamino)phenoxy]prop-2-oxy tetra-substituted zinc phthalocyanines (2a and 3a) and their quaternizedderivatives (2b and 3b) were reported in this study. Photochemical properties of quaternized ionic zinc(II) phthalocyanines (2b and 3b) were investigated in both dimethyl sulfoxide (DMSO) and aqueoussolutions, while non ionic derivatives were only studied in DMSO. The quaternized compounds exhibitexcellent solubility in water, making them potential photosensitizers for use in photodynamic therapy(PDT) of cancer. This study also showed that the water-soluble quaternized Zn(II) phthalocyaninesstrongly bind to blood plasma proteins such as bovine serum albumin (BSA). On the other hand, theinteractions of the novel water soluble phthalocyaines with DNA were also examined.
Phthalocyanines have been studied extensively over the last fewdecades for their applications in material science [1,2]. In recentyears, the applications of metallophthalocyanine complexes haveextended to optical limiting devices [3e5], molecular electronics[6], liquid crystals [7,8], gas sensors [9], semiconductor materials[10], photovoltaic cells [11,12]. Their strong absorbtion in the regionof biological optical window (600e800 nm), efficiency in gener-ating singlet oxygen, lack of dark toxicity, flexibility in structuralmodifications including the central transition metal and photo-stability make them promising candidates for cancer treatment byphotodynamic therapy (PDT) [13e18]. PDT is based on productionof cytotoxic singlet oxygen after irradiation of photosensitizer andachieved success in clinical practice for treatment of various dis-eases, mainly cancer [19].
Applications of phthalocyanines are restricted owing to theirinsolubility in common organic solvents and water. It has beenfound that suitable functional groups in the peripheral benzene
ax: þ90 462 325 31 96.ıo�glu).
rings of the phthalocyanine structure can improve the solubility inprotic or non-protic solvents [20e23]. Water solubility playsimportant role in PDT applications because the blood itself is ahydrophilic system. The water-soluble drug can be directly injectedinto the patient's blood stream [24e26]. Water-soluble phthalo-cyanines consist of sulfonates [27], carboxylates [28,29] and qua-ternized amino groups [30e33] on the peripheral and non-peripheral positions.
Incorporation of non-transition metals like zinc in the center ofthe phthalocyanine (Pc) ring results in complexes with high tripletstate quantum yields and long triplet lifetimes, which are requiredfor efficient photosensitization [34].
Water soluble phthalocyanines exist as loosely associated ag-gregates that are not chemically bound in aqueous solution andwhich can be dissociated by surfactants or by non-aqueous solvents[35e37]. Aggregation occurs as a result of solvent effects that alterthe chemical properties of the metallophthalocyanine complexesleading to co-planar association of the aromatic rings [38e43].Although the aggregation tendency of Pcs in aqueous medium isproblematic, water solubility is an additional advantage for appli-cation in, for example, PDT, since they can be injected directly intothe bloodstream.
V. Çakır et al. / Journal of Organometallic Chemistry 783 (2015) 120e129 121
In this study, we reported the synthesis of novel tetra-substituted zinc(II) phthalocyanines at the peripheral and non-peripheral position with 1,3-bis[3-(dimethylamino)phenoxy]propan-2-ol groups (2a and 3a) (Scheme 1) and their quaternizedderivatives (2b and 3b) (Scheme 2). The spectroscopic, photo-physical and photochemical properties of 2a and 3a inDMSOand for2b and 3b in bothDMSO and aqueousmedium, (phosphate bufferedsaline solution PBS, pH 7.4) were also investigated to give an indi-cation of the potential of the complexes as photosensitizers for PDTapplications. The association of the MPc complexes with bovineserum albumin (BSA) were investigated since hydrophilic dyes bindpreferentially to serum proteins, such as BSA and serum albumin isone of the key components in the body that influences drug delivery[44]. Among the different Pcs employed for DNA-binding studies,the positively charged Pcs are the most efficient ones in terms ofbinding and cleaving DNA as compared with negatively chargedones. According to this perspective, the goal of this work is to
O2N CN
CN
N
N
2
N
O
O
N
O CN
CN
N
NN
NN
NN
N
Zn
RO OR
ORRO
i
ii
N
O
O
N
R=
2a
Scheme 1. The synthetic pathway for preparation of peripherally and non-peripherally tetrapentanol, DBU, 160 �C.
synthesize quaternized zinc (2b and 3b) phthalocyanines whichhave the potential use for photolysis of DNA in tumor cells. We alsoreport herein the DNA binding properties of novel water solublezinc phthalocyanines bearing 1,3-bis[3-(dimethylamino)phenoxy]propan-2-ol tetra-substituted substituents.
Experimental
The used materials, equipments the photochemical formulasand parameters were supplied as Supplementary Information.
Scheme 2. The synthetic pathway for the preparation of the water soluble zinc(II) phthalocyanine complexes.
V. Çakır et al. / Journal of Organometallic Chemistry 783 (2015) 120e129122
(dimethylamino)phenoxy]propan-2-ol 1 (2 g, 6.06 mmol) wasadded to this mixture. After stirring for 30 min at 60 �C, finelyground anhydrous K2CO3 (3.34 g, 24.2 mmol) was added portionwise within 2 h. The reaction mixture was stirred under N2 atmo-sphere at 60 �C for 4 days. At the end of this time, the reactionmixture was poured into ice-water (170 g) and stirred at roomtemperature for further 2 h to yield a crude product. Aqueous phasewas extracted with chloroform (3 � 40 mL). The combined extractswere dried over anhydrous sodium sulfate and filtered. Finally, pureproduct (2) was obtained by column chromatography which wasplaced aluminum oxide using CHCl3:CH3OH (100:4) solvent systemas eluent. Yield: 0.82 g (30%). IR (KBr pellet), nmax/cm�1: 3084(AreH), 2929e2877 (Aliph. CeH), 2231 (C^N), 1611 (C=N), 1574,1503, 1449, 1354, 1287, 1238, 1153, 1057, 996, 824, 753, 686. 1HNMR. (CDCl3), (d:ppm): 7.57 (s, 1H, AreH), 7.21 (t, 3H, AreH),6.46e6.39 (m, 7H, AreH), 4.44 (m, 1H, -CH-), 4.20 (s, 4H, CH2eO),2.98 (s, 12H, -CH3). 13C NMR. (CDCl3), (d:ppm): 157.77, 150.37,1141.78, 132.99, 128.37, 124.60, 121.35, 117.93, 115.07, 111.69, 110.31,104.53, 100.75, 98.03, 67.31, 65.53, 39.00. MS (ESþ), (m/z): 457[MþH]þ. Elemental analysis calcd (%) for C27H28N4O3: C 71.03, H6.18, N 12.27%; found: C 71.44, H 6.40, N 12.66%.
A mixture of 4-{1,3-bis[3-(dimethylamino)phenoxy]-propan-2-oxy}phthalonitrile 2 (0.35 g, 0.76 mmol), anhydrous Zn(CH3COO)2(70 mg, 0.38 mmol) and catalytic amount of DBU in 3.5 mL ofanhydrous n-pentanol was heated and stirred at 160 �C in a sealed
glass tube for 12 h under N2 atmosphere. After cooling to roomtemperature, the reaction mixture was precipitated by the additionof ethanol. The obtained green product was filtered off, washedwith ethanol and diethyl ether and then dried in vacuo. Purificationof the crude product was accomplished by column chromatographywhich was placed aluminum oxide using CHCl3:CH3OH (100:2)solvent system as eluent. Yield: 125 mg (35%). IR (KBr pellet) nmax/cm�1: 3059 (AreH), 2920e2849 (Aliph. CeH), 1611, 1573, 1491,1450, 1394, 1354, 1231, 1152, 1122, 1090, 1047, 996, 825, 746. 1HNMR. (CDCl3), (d:ppm): 7.78 (m, 4H, AreH), 7.13 (m, 24H, AreH),6.62 (m, 16H, AreH), 4.51 (m, 4H, -CH-), 4.36 (s, 16H, CH2eO), 2.90(s, 48H, -CH3). UVevis (DMSO): lmax, nm (log ε): 683 (5.19), 615(4.40), 357 (4.77). MS (ESþ), (m/z): 1930 [MþK]þ. Elemental anal-ysis calcd (%) for C108H112N16O12Zn: C 68.58, H 5.97, N 11.85%;found: C 68.86, H 5.48, N 12.12%.
A mixture of 3-{1,3-bis[3-(dimethylamino)phenoxy]-propan-2-oxy}phthalonitrile 3 (0.4 g, 0.87 mmol), anhydrous Zn(CH3COO)2(80mg, 0.43mmol) and catalytic amount of DBU in 4mL anhydrousn-pentanol was heated and stirred at 160 �C in a sealed glass tubefor 12 h under N2 atmosphere. After cooling to room temperature,the reaction mixture was precipitated by the addition of ethanol.The obtained green product was filtered off, washed with ethanoland diethyl ether and then dried in vacuo. Purification of the crudeproduct was accomplished by column chromatography which wasplaced aluminum oxide using CHCl3:CH3OH (100:1) solvent system
V. Çakır et al. / Journal of Organometallic Chemistry 783 (2015) 120e129 123
A mixture of zinc phthalocyanine 3a (30 mg, 0.015 mmol),2.7 mL methyl iodide in 2 mL chloroform was stirred at roomtemperature for 4 days. The green precipitate was filtered off,washed with chloroform, acetone, diethyl ether and then the greenproduct was dried in vacuo. Yield: 36mg (80%). IR (KBr pellet) nmax/cm�1: 3017 (AreH), 2922-2851 (Aliph. CeH), 1606, 1491, 1397, 1327,1255, 1229, 1181, 1090, 1044, 965, 947, 778, 747, 687. 1H NMR. (D2O),(d:ppm): 7.46e7.10 (m, 44H, AreH), 4.70 (m, 20H, eCHe, CH2eO),3.51e3.41 (m, 72H, eCH3). UVevis (DMSO): lmax, nm (log ε): 700(5.17), 630 (4.30), 384 (4.36), 323 (4.40). MS (ESþ), (m/z): 1981 [M-8Ie2CH3]þ.
Results and discussion
Synthesis and characterization
The synthetic procedure for the new zinc(II) phthalocyaninecompounds were outlined in Schemes 1 and 2. The substitutedphthalonitrile derivatives 2 [45] and 3 were obtained by the aro-matic nucleophilic substitution reaction between 4-nitrophthalonitrile or 3-nitrophthalonitrile and 1,3-bis[3-(dime-thylamino)phenoxy]propan-2-ol 1, respectively. In this reactionK2CO3 and anhydrous DMF were used as a base and solvent,respectively.
Cyclotetramerization of the phthalonitrile derivatives 2 and 3 tothe peripherally and non-peripherally tetra-substituted zinc (II)phthalocyanines 2a and 3a were accomplished by the refluxing ofphthalonitrile derivatives (2 and 3) in n-pentanol in the present ofanhydrous Zn(CH3COO)2 and 1,8-diazabicyclo[5.4.0]undec-7-ene(DBU) at 160 �C for 12 h in a sealed tube. Zinc phthalocyanines2a and3awere purified by column chromatography on basic alumina using[(CHCl3:CH3OH, 100:2) or (CHCl3:CH3OH, 100:1)] solvent mixturesas eluents, respectively. Tetra-cationic water soluble zinc phthalo-cyanines 2b and 3bwere obtained by the reaction of correspondingphthalocyanines 2a and 3a with methyl iodide in CHCl3 at roomtemperature while the novel non-ionic zinc phthalocyanines 2a and3a are soluble in most of the polar organic solvents such as CHCl3,
CH2Cl2, THF, acetone, DMF, DMSO; the tetra-cationic derivatives 2band 3b are fully soluble in water and DMSO as expected. Thestructures of the target compounds were confirmed using UVevis,IR, 1H NMR (for 2a and 3a), 13C NMR (for 2 and 3), MS spectroscopicdata and elemental analysis. The analyses are consistent with thepredicted structures as shown in the experimental section.
In the IR spectrum of compound 3, stretching vibrations at2231 cm�1 for C^Ngroups, at 3084 cm�1 for aromatic CH groups, at2929-2877 cm�1 for aliphatic CH groups appeared at expected fre-quencies. In the 1H NMR spectrum of compound 3, the aromaticprotons were obtained at 7.57 (s), 7.21 (t), 6.46e6.39 (m) ppm andaliphatic protonswere obtained at 4.44 (m), 4.20 (s), 2.98 (s) ppm. Inthe 13C NMR spectrum of compound 3, the aromatic carbon atomswere observed at between at 157.77e98.03 ppm. The nitrile carbonatoms for compound 3were also observed at 117.93 and 111.69 ppm.In the mass spectum of compound 3, the presence of molecular ionpeak atm/z ¼ 457 [MþH]þ, confirmed the proposed structures.
The cyclotetramerization of the phthalonitrile derivatives 2 and3 to the zinc (II) phthalocyanine derivatives 2a and 3a can be seenclearly by the disappearance of the peaks at 2236 and 2231 cm�1
belonging to the C^N vibrations, respectively. The IR spectra of 2aand 3a showed similar characteristics. The 1H NMR spectra ofperipherally and non-peripherally tetra-substituted zinc (II)phthalocyanines 2a and 3a were almost identical with that of thestarting compounds 2 and 3 except for some signal broadening andsome small shifts in the positions of some signals. The mass spectraof compounds 2a and 3a, which showed peaks at m/z ¼ 1930[MþK]þ and 1891 [M]þ, respectively support the proposed formulafor these compounds.
The quaternized water soluble tetra-peripherally and non-peripherally substituted cationic zinc phthalocyanines 2b and 3bare soluble inwater, DMF and DMSO as expected. These compounds2b and 3b were obtained by the reaction of corresponding zincphthalocyanines 2a and 3a with methyl iodide in chloroform atroom temperature. No major change in the IR spectra was foundafter quaternization. The NMR spectra of water soluble zincphthalocyanines 2b and 3b showed phthalocyanine ring protonsand aliphatic protons as unresolved multiplets due to the aggre-gation in D2O. In the mass spectra of compounds 2b and 3b, thepresence of molecular ion peaks atm/z ¼ 1996 [M-8IeCH3]þ for 2band 1981 [M-8Ie2CH3]þ for 3b confirmed the proposed structuresof the targeted complexes.
Electronic spectra are especially important to establish thestructure of phthalocyanines. The phthalocyanines exhibit typicalelectronic spectra with two strong absorption regions, one in theUV region at about 300e400 nm (B band) and the other one is inthe visible region at 600e700 nm (Q band), both correlate to p/p*transitions [46,47]. The UVevis spectra of the zinc phthalocyanines2a and 3a in DMSO showed characteristic Q band absorptions at683 and 703 nm, respectively. B band absorptions of the zincphthalocyanines 2a and 3a were also observed at 357 and 369 nm,respectively.
The UVevis spectra of the quarternized zinc phthalocyanines 2band 3b showed characteristic absorbtions in the Q band region at684 nm for 2b, 701 nm for 3b in DMSO. B band regionwas observedat 359 and 323 nm in DMSO, respectively. A shoulder was observedat 615 and 630 nm for complexes 2b and 3b, respectively.
The red-shifts were observed for zinc phthalocyanines followingsubstitution. The Q bands of the non-peripherally substitutedphthalocyanines showed extra red-shifted absorptions whencompared to the corresponding peripherally substituted counter-parts in DMSO (Fig. 1). The red-shifts are 20 nm between 2a and 3aand 17 nm between 2b and 3b. The observed red spectral shifts aretypical of Pcs with substituents at the non-peripheral positions andhave been explained in the literature [48,49].
0
0.2
0.4
0.6
0.8
1
300 400 500 600 700 800
Abso
rban
ce
Wavelength (nm)
2b3b
Fig. 2. Electronic absorption spectra of the compounds 2b and 3b in PBS at the con-centration of 10 � 10�6 M.
0
0.2
0.4
0.6
0.8
1
300 400 500 600 700 800
Abso
rban
ce
Wavelength (nm)
in PBS
in PBS+Triton x-100
Fig. 3. Electronic absorption spectra of the compound 2b in PBS and PBSþTXT solu-tions at the concentration of 10 � 10�6 M.
V. Çakır et al. / Journal of Organometallic Chemistry 783 (2015) 120e129124
Aggregation studies
In this study, the aggregation behavior of the newly synthesizedphthalocyanines were examined in different solutions (DMSO,DMF, THF, toluene, chloroform and dichloromethane) and atdifferent concentrations in DMSO using UVevis spectroscopy. TheUVevis spectra of the synthesized phthalocyanines (2a, 2b, 3a and3b) were measured in different solvents for determination of ag-gregation properties of these compounds in different solvents andselection of suitable solvent for photochemical properties. Thestudied non-ionic phthalocyanines (2a and 3a) did not form ag-gregates in DMSO, DMF, THF, toluene, chloroform and dichloro-methane solutions. The ionic complexes (2b and 3b) did not alsoform aggregates in DMSO and DMF and methanol. However, theyshowed aggregation in aqueous solutions. The monomeric solu-tions were obtained for all studied zinc (II) phthalocyanines inDMSO. For this reason, this solvent was preferred as a solvent forfurther photochemical studies. On the other hand, the smallamount of DMSO can be used for biological applications withoutany toxic effect [50,51].
In PBS, the non-vibrational peaks were observed at the Q bandregion (Fig. 2). The lower energy (red-shifted) bands at 680 nm for2b is due to the monomeric species, while the higher energy (blue-shifted) bands at 637 nm for 2b is due to the aggregated species(Fig. 2). The non-peripherally substituted ionic Zn(II) phthalocya-nine compound (3b) showed only one peak at around Q band re-gion confirmed that this complex did not form aggregates betweenthe Pc molecules in PBS (Fig. 2). This different aggregation behav-iour of these two phthalocyanines must be attributed to moresterically hindrance of the substituents on the non-peripheral po-sition than peripheral position of phthalocyanine core.
Addition of triton X-100 (TXT) (0.1 mL) which is a surfactantusing for prevent of aggregation to a PBS solution of quaternizedzinc phthalocyanine (2b) resulted considerable increase in in-tensity of the low energy side of the Q band (Fig. 3), suggesting thatthe molecules were aggregated and that addition of triton X-100broken up the aggregates between the Pc molecules.
The aggregation behavior of the newly synthesized zincphthalocyanines (2a, 2b, 3a and 3b) were also studied at differentconcentrations in DMSO (Fig. S1 in Supplementary information).When the concentration was increased, the intensity of absorptionof the Q band was also increased and there was not observe anynew band formation (normally blue or red shifted bands wereobserved for H-type or J-type aggregation of phthalocyanine
0
0.4
0.8
1.2
1.6
300 400 500 600 700 800
Abso
rban
ce
Wavelength (nm)
2a
2b
3a
3b
Fig. 1. Electronic absorption spectra of the studied phthalocyanine (2aeb) and (3aeb)compounds in DMSO at the concentration of 10 � 10�6 M.
molecules) which is an evidence for the formation of monomericsolutions in the studied concentrations range(12 � 10�6�2 � 10�6 M).
Singlet oxygen generation properties
The amount of the singlet oxygen quantum yield (FD) is anindication of generated singlet oxygen by a photosensitizer mole-cule. The determination of the FD values is immense importantbecause this value says the potential of the compounds as photo-sensitizers in photocatalytic applications such as PDT. In this study,the FD values of studied zinc phthalocyanines were determined bychemical method. 1,3-diphenylisobenzofuran (DPBF) and 9,10-antracenediyl-bis(methylene)dimalonoic acid (ADMA) were usedas singlet oxygen quenchers in DMSO and aqueous solutions (PBS),respectively. The UVevis absorption spectral changes during thesinglet oxygen quantum yield determinations were given in Fig. 4as an example for compound 3b in DMSO. Any importantchanges were not observed at the intensity of the Q band and for-mation of new bands during FD determinations, confirming thatcomplexes were not degraded used light irradiation (30 V) duringsinglet oxygen studies. The FD values of the studied zinc phthalo-cyanines are given in Table 1. While the FD values of studied non-ionic phthalocyanines (2a and 3a) were lower, these values are
Table 1Absorption spectral and photochemical data for the studied phthalocyanine com-pounds (2a, 2b, 3a and 3b) in different solvents.
Compound Solvent Q band lmax, (nm) log ε Fd/10�5 FD
V. Çakır et al. / Journal of Organometallic Chemistry 783 (2015) 120e129 125
higher for ionic counterparts (2b and 3b) when compared tounsubstituted zinc(II) phthalocyanine in DMSO. According to theseresults, the substitution of phthalocyanine core with 1,3-bis[3(dimethylamino) phenoxy]propan-2-ol groups was decreasedthe singlet oxygen generation. In the addition of this, the quater-nization of the nitrogen atoms on the substituents was increasedthe produced singlet oxygen due to engaging of the lone pairelectrons on the nitrogen atoms after quaternization or presence ofsulfate anion as counterion in quaternized phthalocyanine com-pounds. The non-peripherally substituted zinc (II) phthalocyaninecomplexes showed higher FD values than peripherally substitutedcounterparts in both DMSO and PBS solutions. It could be attributedto long wavelength absorption of non-peripherally substitutedphthalocyanines compared to peripherally substituted derivatives.
Table 1 shows that lower FD values were observed in PBS(which was mainly water) solutions as compared to FD values inDMSO for quaternized ionic zinc (II) phthalocyanines. The low FD
in aqueous solutions compared to other solvents such as deuter-ated water and DMSO was explained [52] by the fact that singletoxygen absorbs light at 1270 nm and water also absorbs light ataround this wavelength while DMSO exhibits little absorption inthis region. Another factor for the lower singlet oxygen generationin aqueous solution compared to in DMSO for quaternized ioniczinc (II) phthalocyanines can be explained by aggregation effect.The non-peripherally substituted ionic complex (3b) showedhigher singlet oxygen generation than peripherally derivative (2b)in PBS solution because the non-peripherally complex did notshow aggregation in this solvent. The newly synthesized zinc (II)phthalocyanines in this study, especially quaternized ionic de-rivatives, showed relatively higher FD values in DMSO andaqueous solutions and these values make them as potential sen-sitizers for PDT applications. The zinc(II) phthalocyanines con-taining similar groups on the phthalocyanine framework weresynthesized and their singlet oxygen quantum yield values werealso determined in our previously work [14]. The zinc (II) phtha-locyanines studied in this work contain totally eight nitrogenatoms on the substituted groups while similar analogs given inliterature [14] contain four nitrogen atoms. Both non-ionic zinc (II)phthalocyanines and their quaternized derivatives studied in thisstudy generated more singlet oxygen than similar analogs inDMSO and aqueous solutions.
0
0.2
0.4
0.6
0.8
1
1.2
300 400 500
Abso
rban
ce
Wavelen
0 sec5 sec10 sec15 sec20 sec25 sec30 sec
00.20.40.60.8
11.2
0 5
DPBF
Abs
orba
nce
Fig. 4. Electronic absorption changes for the compound 3b during the determination of sinDPBF absorbance versus time).
Photodegradation studies
The stability determination of the molecules under light irra-diation is especially important for those molecules intended for useas photocatalytic applications. Generally, phthalocyanines exhibitoptimal stability against to the light irradiation. In this study, thephotodegradation studies of the phthalocyanines were measuredusing by UVevis spectroscopy. The spectral changes during thesestudies for all the phthalocyanines (2a, 2b, 3a and 3b) confirmedphotodegradation occurred without phototransformation becauseonly decreasing was observed to the Q band intensities of themolecules. Any changes (generally new band formation at around500 nm) did not observe the shape of the UVevis spectra of thesecompounds (Fig. 5 as an example for phthalocyanine 3b). All thestudied non-ionic zinc (II) phthalocyanines (2a and 3a) and theirquaternized ionic derivatives (2b and 3b) showed about the samestability with Fd of the order of 10�5 in DMSO (Table 1). The Fdvalues have been reported as low as 10�6 for stable zinc phthalo-cyanines and the order of 10�3 for unstable counterparts in theliterature [34]. The studied zinc (II) phthalocyanine complexes (2aand 3a) and their quaternized derivatives (2b and 3b) exhibitmoderate stability in DMSOwhich indicated that these compoundsare suitable for photocatalytic applications such as PDT. All studiedzinc(II) phthalocyanine complexes showed higher Fd values thanunsubstituted zinc(II) phthalocyanine. It means the substitution ofthe phthalocyanine core with 1,3-bis[3-(dimethylamino)phenoxy]propan-2-ol groups decreased the stability of the compounds. The
600 700 800gth (nm)
y = -0.0167x + 1.0702R² = 0.9969
10 15 20 25 30Time (sec)
glet oxygen quantum yield in DMSO at a concentration of 10 � 10�6 M. (Inset: Plot of
Fig. 5. Electronic absorption changes during the photodegradation studies of the compound 3b in DMSO showing the disappearance of the Q-band at 5 s intervals. (Inset: Plot ofabsorbance versus time). 300 W General Electric quartz line lamp was used as a light source. Power density was 18 mW/cm2 and used energy was 100 W.
V. Çakır et al. / Journal of Organometallic Chemistry 783 (2015) 120e129126
studied peripherally substituted phthalocyanines (2a and 2b)showed lower Fd values than non-peripherally derivatives in allstudied solutions. It means the substitution of the groups on theperipheral position resulted more stable zinc phthalocyaninederivatives.
Binding properties of quaternized ionic zinc (II) phthalocyanines toBSA protein
A study of the fluorescence quenching of the BSA protein wascarried out by the addition of increasing concentrations of watersoluble quaternized zinc (II) phthalocyanines (2b and 3b) to a fixedconcentration of BSA (3.00 � 10�5 M), and the concentrations ofzinc (II) phthalocyanines in the resulting mixture were 0,1.66 � 10�6, 3.33 � 10�6, 5.00 � 10�6, 6.66 � 10�6, 8.33 � 10�6 M.The studied ionic zinc phthalocyanines were formed mixtures ofaggregated and unaggregated species in PBS. The total concentra-tions of the complexes were mixture of the monomer and aggre-gated species. The fluorescence emission spectral changes of BSA bythe addition of different concentrations of ionic zinc phthalocya-nines (2b and 3b) in PBS solutions were recorded in Fig. 6 as anexample for 2b. The BSA fluorescence at 348 nm is mainly attrib-utable to tryptophan residues in the macromolecule. The decreasein the intrinsic fluorescence intensity of tryptophan residue of BSAby the addition of quaternized zinc (II) phthalocyanine indicatesthat these complexes readily bind to BSA, which implies that thephthalocyanine molecules reach sub domains where tryptophanresidues are located in BSA. This also suggests that the primarybinding sites of these molecules are very close to tryptophan resi-dues, since the occurrence of quenching requiresmolecular contact.BSA and the respective quaternized ionic zinc phthalocyaninesexhibited reciprocated fluorescence quenching on one another;hence it was possible to determine SterneVolmer quenching con-stants (KSV). The plot of Fo/F versus zinc phthalocyanine concen-tration ([Pc]) gave a linear for the studied zinc phthalocyanines (2band 3b) from SterneVolmer relationship (Inset in Fig. 6 for complex2b) using Equation 3 in Supporting information.
The obtained KSV values of the studied ionic zinc phthalocya-nines (2b and 3b) were listed in Table 2, suggest that BSA fluores-cence quenching is more effective for quaternized peripherally
substituted ZnPc complex (2b) than quaternized non-peripherallysubstituted ZnPc complex (3b) in PBS.
The KSV values of complexes 2b and 3b are in the order of 105
and these values are similar with phthalocyanine derivativesbearing different metals and substituents [29e31].
The binding constants (Kb) and number of binding sites (n) onBSAwere obtained using Equation (3) and the results are shown inTable 2. The slopes of the plots shown at Fig. 7 gave n values and theintercepts of these plots gave Kb values. The values of Kb and n aretypical of MPceBSA interactions in aqueous solutions [53,54]. Sincethe fluorescence lifetime of BSA is on the order of 10�8 s [55], theapparent quenching coefficients (Kq) for ionic zinc (II) phthalocy-anines are on the order of 1013 M�1 s�1, much higher than thenormal value for dynamic quenching (ca. 1010 M�1 s�1) [56]. Thisstrongly suggested that the fluorescence quenching of BSA byquaternized zinc phthalocyanine complexes (2b and 3b) wasmainly through a static quenching mechanism [57,58]. The kqvalues are larger for quaternized peripherally substituted ZnPccomplex (2b) than quaternized non-peripherally substituted ZnPccomplex (3b) in PBS. The higher Kb value for quaternized non-peripherally substituted ZnPc complex (3b) implies that this com-plex binds more strongly to BSA than the quaternized peripherallysubstituted ZnPc complex (2b), probably due to less aggregationtendency of the non-peripheral substituted complex (3b) thanperipherally substituted complex (2b). The number of binding sites(n) values of peripherally and non-peripherally substituted zincphthalocyanine complexes (2b and 3b) are also given in Table 2. Allsubstituted quaternized zinc phthalocyanine complexes form 1:1adducts quaternized BSA.
Binding of water soluble quaternized zinc (II) phthalocyanines toDNA
In order to determine the binding parameters of studied qua-ternized zinc (II) phthalocyanines (2b and 3b) with DNA, thetitration of these phthalocyanines with DNA solution was done bymonitoring of changes in optical spectrum. DNA binding experi-ments were performed by titrating 1.00 � 10�5 M of quaternizedzinc (II) phthalocyanines in PBS þ Triton X-100 (a few drops toprevent aggregation) solutions (2 mL) with 1.74 � 10�4 M DNAstock solution in PBS (0.005 mL) and the changes in the UVevis
290 340 390 440 490
Inte
nsity
(a.u
.)
Wavelength (nm)
[Pc]=0
[Pc]=saturated
y = 161122x + 1R² = 0.9738
0
0.5
1
1.5
2
2.5
3
0 0.000005 0.00001
Io/I
[Pc]
Fig. 6. Fluorescence emission spectral changes of BSA (3.00 � 10�5 M) on the addition of different concentrations of Pc in PBS solutions. [2b] ¼ 0, 1.66 � 10�6, 3.33 � 10�6,5.00 � 10�6, 6.66 � 10�6, 8.33 � 10�6 M. (Inset: SterneVolmer plots for quenching of BSA by complex 2b).
V. Çakır et al. / Journal of Organometallic Chemistry 783 (2015) 120e129 127
absorption spectra of quaternized zinc (II) phthalocyanines wererecorded. Fig. 8 demonstrates the changes in the absorption spec-trum of 2b upon titration with DNA as an example. The lineardecrease in the absorbance at 684 nm with increasing concentra-tions of DNA indicate that an interaction between quaternized zinc(II) phthalocyanines and DNA was occurred (Fig. 8, inset).
Fig. 9 shows that the emission intensity of the quaternized zinc(II) phthalocyanines (2b and 3b) is reduced by the addition of DNAto the phthalocyanine solutions which indicates that an interactionoccurring between quaternized zinc (II) phthalocyanines (2b and3b) and DNA molecules. A linear decrease was observed in theemission at 696 nm by the increasing concentrations of DNA (Fig. 9,inset).
The interaction of studied quaternized zinc(II) phthalocyanineand DNA is expected to be electrostatic in origin. In order to confirmthis, the effect of addition of a strong electrolyte such as NaCl [59]was investigated and monitored by both absorption and fluores-cence spectroscopy. Both absorption and fluorescence spectral in-tensities were increased by the addition of NaCl (Fig. 10) due todissociation of Pcs-DNA complexes. The changes may be explainedas a competition between positively charged quaternized zinc (II)phthalocyanines (2b and 3b) and NaCl for nucleic acid sites withsodium ion binding prevailing. The results clearly point to theimportant role of electrostatic interactions between DNA and Pcmolecules.
Conclusion
In conclusion, this work is described the synthesis, character-ization, aggregation behavior, and photochemical properties of newtetra-peripherally and non-peripherally 1,3-bis[3-(dimethylamino)phenoxy]propan-2-ol substituted zinc phthalocyanines and their
Table 2Binding and fluorescence quenching data for interaction of BSA with 2b and 3bderivatives in PBS.
Compound KBSASV
(�105 dm3 mol�1)kq(�1013 s�1)
Kb(�10 �6 dm3 mol�1) n
2b 1.61 1.61 6.30 1.183b 0.78 0.78 8.31 1.33
water soluble derivatives which are good candidates as photosen-sitizers to for PDT. The effect of substituent position and quater-nization on these properties is also presented. It was shown thatquaternization of nitrogen atom on the substituents enhancedwater solubility of the zinc phthalocyanine compounds. The non-ionic and ionic zinc (II) phthalocyanine complexes (2aeb and3aeb) were found to be monomeric in DMSO. While the periph-erally substituted ionic complexes showed aggregation, the non-peripherally substituted counterpart did not show aggregation inPBS solution. The addition of triton X-100 to PBS solution of thestudied phthalocyanines eliminated the aggregates between the Pcmolecules for complex 2b. The value of FD ranged from 0.43 to 0.75in DMSO and from 0.23 to 0.41 in PBS solution gives an indication ofthe potential of these complexes as photosensitizers for PDT ofcancer. The studied 1,3-bis[3-(dimethylamino)phenoxy] propan-2-ol substituted zinc(II) phthalocyanine complexes (2a and 2b) andtheir quaternized derivatives (2be3b) less stable than unsub-stituted zinc(II) phthalocyanine in DMSO. This study reveals thatthe water-soluble quaternized zinc phthalocyanine complexes (2band 3b) bind strongly to serum albumin according to the fluores-cence quenching of BSA with studied water-soluble phthalocya-nines and it means they can easily transfer in the blood. Afterinjection into the blood stream, these photosensitizers will have to
Fig. 7. Determination zinc phthalocyanine-BSA binding constant (Kb) and number ofbinding sites (n) on BSA. [BSA] ¼ 3.00 � 10�5 M and [Pc] ¼ 0, 1.66 � 10�6, 3.33 � 10�6,5.00 � 10�6, 6.66 � 10�6, 8.33 � 10�6 M in PBS solution.
0.0
0.2
0.4
0.6
0.8
300 400 500 600 700 800
Abs
orba
nce
Wavelength (nm)
0 µl
20 µl
40 µl
60 µl
80 µl
100 µl
120 µl
140 µl
160 µl
180 µl
200 µl
220 µl
240 µl
y = -0.002x + 0.6266R² = 0.9794
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 40 80 120 160 200 240
Abs
orba
nce
DNA (µl)
Fig. 8. Electronic titration spectra of 2b (1.00 � 10�5 M) with DNA (1.74 � 10�4 M) at pH 7.4 in 1xPBS buffer containing 1% Triton X-100. Inset, the plot of absorbance at 684 nmversus the DNA concentration.
0
100
200
300
400
500
600
670 690 710 730 750 770 790 810 830 850
Inte
nsity
(a.u
.)
Wavelength (nm)
0 μl
40 μl
80 μl
120 μl
160 μl
200 μl
240 μl
y = -2.2036x + 543.57R² = 0.9964
0
100
200
300
400
500
600
0 50 100 150 200 250
Inte
nsity
(a.u
.)
DNA (μl)
Fig. 9. Emission spectra changes of 2b (1.00 � 10�5 M) by the addition of DNA at pH 7.4 in 1x PBS buffer containing 1% Triton X-100. Inset, the plot of emission at 696 nm versus theratio of DNA added at each step to the total [2b] concentration.
0.0
0.1
0.2
0.3
0.4
300 400 500 600 700 800
Abso
rban
ce
Wavelength (nm)
Addition of NaCl
(A)
0
100
200
300
400
500
600
670 690 710 730 750 770 790
Inte
nsity
(a.u
.)
Wavelength (nm)
Addition of NaCl
(B)
Fig. 10. A) Electronic absorption and B) fluorescence emission spectral changes of 2b-DNA complex by the addition of NaCl in PBS solutions.
V. Çakır et al. / Journal of Organometallic Chemistry 783 (2015) 120e129128
V. Çakır et al. / Journal of Organometallic Chemistry 783 (2015) 120e129 129
encounter serum albumin, which presents a reason for their BSAbinding study. The newly synthesized zinc(II) phthalocyanineswere also interact with DNA as expected electrostatic interactions.
Acknowledgement
This study was supported by The Scientific & TechnologicalResearch Council of Turkey (TÜB_ITAK, project no: 111T963).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jorganchem.2015.02.021.
References
[1] C.C. Leznoff, A.B.P. Lever (Eds.), Phthalocyanines: Properties and Applications,vols. 1e4, VCH Publishers, New York, 1989, 1993, 1996.
[2] N.B. McKeown, Phthalocyanine Materials: Synthesis, Structure and Function,Cambridge University Press, Cambridge, 1998.
[3] J.W. Perry, K. Mansour, I.Y.S. Lee, X.L. Wu, P.V. Bedworth, C.T. Chen, D. Ng,S.R. Marder, P. Miles, T. Wada, M. Tian, H. Sasabe, Science 273 (1996)1533e1536.
[4] M. Hanack, T. Schneider, M. Barthel, J.S. Shirk, S.R. Flom, R.G.S. Pong, Coord.Chem. Rev. 219 (2001) 235e258.
[5] Y. Chen, M. Hanack, Y. Araki, O. Ito, Chem. Soc. Rev. 34 (2005) 517e529.[6] J. Simon, P. Bassoul, Design of Molecular Materials, Supramolecular Engi-
neering, John Wiley Sons Ltd, West Sussex, 2000.[7] S.R. Flom, in: K. Kadish, K.M. Smith, R. Guilard (Eds.), The Porphyrin Handbook,
vol. 19, 2003, p. 179. Boston.[8] M. Durmus, S. Yesilot, V. Ahsen, New. J. Chem. 30 (2006) 675e678.[9] G. Guillaud, J. Simon, J.P. Germain, Coord. Chem. Rev. 180 (1998) 1433e1484.
[10] M. Bouvet, Boston, in: K. Kadish, K.M. Smith, R. Guilard (Eds.), The PorphyrinHandbook, vol. 19, 2003, p. 37.
[11] M.A. Loi, H. Neugebauer, P. Denk, C.J. Brabec, N.S. Sariciftci, A. Gouloumis,P. V�azquez, T. Torres, J. Mater. Chem. 13 (2003) 700e704.
[12] D.M. Guldi, A. Gouloumis, P. V�azquez, T. Torres, Chem. Commun. (2002)2056e2057.
[13] S. Makhseed, A. Tuhl, J. Samuel, P. Zimcik, N. Al-Awadi, V. Novakova, DyesPigm. 95 (2012) 351e357.
[14] D. Çakır, V. Çakır, Z. Bıyıklıo�glu, M. Durmus, H. Kantekin, J. Organomet. Chem.745e746 (2013) 423e431.
[15] J.P. Longo, S.P. Lozzi, A.R. Simioni, P.C. Morais, A.C. Tedesco, R.B. Azevedo,J. Photochem. Photobiol. B 94 (2009) 143e146.
[16] H. Kolarova, P. Nevrelova, R. Bajgar, D. Jirova, K. Kejlova, M. Strnad, Toxicol.Vitro 21 (2007) 249e253.
[19] P. Agostinis, K. Berg, K.A. Cengel, T.H. Foster, A.W. Girotti, S.O. Gollnick, et al.,CA Cancer J. Clin. 61 (2011) 250e281.
[20] Z. Bıyıklıo�glu, H. Kantekin, J. Organomet. Chem. 693 (2008) 505e509.[21] O. Bekircan, Z. Bıyıklıo�glu, I. Acar, H. Kantekin, H. Bektas, J. Organomet. Chem.
693 (2008) 3425e3429.[22] Z. Bıyıklıo�glu, S.Z. Yıldız, H. Kantekin, J. Organomet. Chem. 695 (2010)
1729e1733.
[23] C. G€ol, M. Durmus, Synth. Met. 162 (2012) 605e613.[24] Z. Bıyıklıo�glu, H. Kantekin, Polyhedron 27 (2008) 1650e1654.[25] F. Dumoulin, M. Durmus, V. Ahsen, T. Nyokong, Coord. Chem. Rev. 254 (2010)
2792e2847.[26] S. Wei, J. Zhou, D. Huang, X. Wang, B. Zhang, J. Shen, Dyes Pigm. 71 (2006)
61e67.[27] A. Ogunsipe, T. Nyokong, J. Photochem. Photobiol. A 173 (2005) 211e220.[28] J. Chen, N. Chen, J. Huang, J. Wang, M. Huang, Inorg. Chem. Commun. 9 (2006)
313e315.[29] M. Çamur, M. Bulut, J. Organomet. Chem. 695 (2010) 45e52.[30] Z. Bıyıklıo�glu, Synth. Met. 161 (2011) 508e515.[31] Z. Bıyıklıo�glu, H. Kantekin, Synth. Met. 161 (2011) 943e948.[32] Z. Bıyıklıo�glu, M. Durmus, H. Kantekin, J. Photochem. Photobiol. A 222 (2011)
87e96.[33] Z. Bıyıklıo�glu, Syn. Met. 162 (2012) 26e34.[34] T. Nyokong, Coord. Chem. Rev. 251 (2007) 1707e1722.[35] A. Harriman, M.C. Richoux, J. Chem. Soc. Faraday Trans. 2 (1980) 1618e1626.[36] M.J. Stillman, T. Nyokong, in: C.C. Leznoff, A.B.P. Lever (Eds.), Phthalocyanines:
Properties and Applications, vol. 1, VCH, New York, 1989, p. 139.[37] L.D. Rollman, R.T. Iwamoto, J. Am. Chem. Soc. 90 (1980) 1455e1463.[38] R.D. George, A.W. Snow, J.S. Shirk, W.R. Barger, J. Porphyr. Phthalocyanines 2
(1998) 1e7.[39] K. Kameyama, A. Satake, Y. Kobuke, Tetrahedron Lett. 45 (2004) 7617e7620.[40] A.C.H. Ng, X. Li, D.K.P. Ng, Macromolecules 32 (1999) 5292e5298.[41] A. Ogunsipe, T. Nyokong, M. Durmus, J. Porphyr. Phthalocyanines 11 (2007)
635e644.[42] M. Idowu, T. Nyokong, J. Lumines. 129 (2009) 356e362.[43] S. Moeno, T. Nyokong, J. Photochem. Photobiol. A Chem. 203 (2009) 204e210.[44] A.A. Bhattacharya, S. Curry, N.P. Franks, J. Biol. Chem. 275 (2000)
38731e38738.[45] V. Çakır, D. Çakır, Z. Bıyıklıo�glu, H. Kantekin, J. Incl. Phenom. Mac. Chem. 78
(2014) 61e70.[46] Y. Arslano�glu, E. Hamuryudan, Dyes Pigm. 75 (2007) 150e155.[47] M. €Ozçesmeci, €O.B. Ecevit, S. Sürgün, E. Hamuryudan, Dyes Pigm. 96 (2013)
10192e10195.[49] M. Konami, M. Hatano, A. Tajiri, Chem. Phys. Lett. 166 (1990) 605e608.[50] H. Li, T.J. Jensen, F.R. Fronczek, M.G.H. Vicente, J. Med. Chem. 51 (2008)
215e224.[52] A. Ogunsipe, J.Y. Chen, T. Nyokong, New. J. Chem. 28 (2004) 822e827.[53] S.M.T. Nunes, F.S. Sguilla, A.C. Tedesco, Braz. J. Med. Biol. Res. 37 (2004)
273e284.[54] R. Nilsson, D.R. Kearns, J. Phys. Chem. 78 (1974) 1681e1683.[55] I.E. Borisevitch, T.T. Tominaga, H. Imasato, M. Tabak, J. Lumin. 69 (1996)
65e76.[56] S.L. Murov, I. Carmichael, G.L. Hug, Handbook of Photochemistry, second ed.,
Marcel Dekker, New York, 1993.[57] M. Dockal, D.C. Carter, F. Ruber, J. Biol. Chem. 274 (1999) 29303e29310.[58] Y. Peng, F. Huang, J. Wen, B. Huang, X. Ma, Q. Wang, J. Coord. Chem. 61 (2008)
1503e1512.[59] W. Duan, Z. Wang, M.J. Cook, J. Porphyr. Phthalocyanines 13 (2009)
1255e1261.[60] _I. Gürol, M. Durmus, V. Ahsen, T. Nyokong, Dalton Trans. 34 (2007)
3782e3791.[61] N. Kuznetsova, N. Gretsova, E. Kalmkova, E. Makarova, S. Dashkevich, Rus. J.
