Schiff base containing pyridine groups functionalized water-soluble phthalocyanine: Synthesis, photo-physicochemical properties, and bovine serum albumin binding behavior

The novel pyridine bearing schiff base substituted metal-free (9), zinc(II) phthalocyanine (10), and its quaternized derivative (11) were designed and synthesized. These phthalocyanines were fully characterized by spectroscopic methods (FT-IR, UV–Vis, MALDI-TOF, and 1H NMR). The photo-physicochemical properties of these phthalocyanines were investigated in both DMSO and DMF for 10 and in both DMSO and aqueous solution for 11. The addition of pyridine bearing Schiff base groups as peripheral ligands showed an improvement in the photophysical and photochemical properties. In addition, a spectroscopic investigation of the binding behavior of the water-soluble zinc (II) phthalocyanine complex to bovine serum albumin (BSA) was also studied in this work.

In the 1 H NMR spectrum of the quaternized complex 11, the -CH 3 protons were observed as a singlet peak at 4.28 ppm ( Figure S10), identifying the formation of the quaternized product. These results confirmed the structure of compound 11. In addition to 1 H NMR spectrum results, the MALDI-TOF MS data for the substituted metal-free (4, 6, and 9), zinc phthalocyanines (5, 7, and 10), and the quaternized derivative 11 are available for the formulations given. The molecular ion peaks of synthesized Pcs showed parent ions at m/z: 1170 Figure S11a-S11e). The molecular ion peak values of the fragmentation products of the obtained complexes are also indicated in the supplementary file.

Photophysical and photochemical studies 3.2.1. Ground state electronic absorption spectra and aggregation studies
The ground state electronic absorption spectra of phthalocyanines and their metal derivatives in the studied solution are one of the principal pieces of evidence for their formation. The newly synthesized phthalocyanines for H 2 Pc (4, 6, and 9) and metallophthalocyanines (10 and 11) are detected by the characteristic Q-and B-bands in their electronic spectra. The electronic spectral properties of phthalocyanines, determined by the 18π system of the innermost 16-membered ring, form the basis of their chemical and electrical properties that metal-free phthalocyanine compounds with D 4h symmetry corresponding to the π→π* transitions show a single absorption, while nonmetallic phthalocyanines with D 2h symmetry show two absorptions with equal intensity in the same range [37]. The UV-vis spectrum of H 2 Pc 4, 6 in DMSO and 9 in DMSO and DMSO (plus Triton X-100) and DCM (as an example for 11) was given in Figure S12, its zinc derivative 8 in DMSO and DMF, quaternized zinc derivative 11 in DMSO and water (Figure S13), were recorded at room temperature. The logarithmic molar absorption coefficient values of the bands are listed in Table 1. In DMSO, the two characteristic absorption bands at Q band region of the metal-free phthalocyanines were observed at 669 and 664 (Qx and Qy) nm for 4, 699 and 666 nm for 6, and 700 and 669 nm for 9, respectively (Figure S13) (Table 1). However, the B-bands for all H 2 Pc compounds were seen between 330 and 355 nm.
DMSO is known as a strong coordination solvent that prevents aggregation. However, aggregation was observed for 4, 6, and 9 in DMSO. Figure S12 showed that the addition of Triton X-100 to the medium did not affect the solubility though it decreased the aggregation tendency of 9 in the solvent. The UV-vis spectra of the metal free phthalocyanine (9) showed two characteristic absorption bands at Q band region around 697 nm and 672 nm in DCM solution. It was observed that the solubility of 9 increased in the absorption spectra in DCM solution, it was not effective on the decrease of aggregation tendency and solubility in DMSO.
The electronic spectra of ZnPc 10 and quaternized ZnPc 11 showed characteristic absorption bands at around 685-707 nm and 320-370 nm for the Q band and the B band regions which are characteristic of metallophthalocyanines in DMSO (Table 1) [38].
The characteristic single absorption band of zinc phthalocyanines for 5, 7, and newly synthesized 8 was observed at 677 nm, 676 nm, and 675 nm in DMF, respectively ( Figure 1, Table 1). The absorption spectra of the quaternized complex 11 showed a co-surface aggregation in water, as evidenced by the presence of two bands in the Q band region ( Figure 2). These bands appeared at 675 nm (weak) due to monomeric species and low energy (redshifted) at 638 nm (Table 1) due to aggregate species. The incorporation of 11 in water with quantities of TX-100 causes a sharpening of the absorption band at 680 nm, which clearly shows the decrease of aggregation after the interaction of the host-guest by the addition of surfactant [39].  The aggregation behavior of Pc is usually represented as a coplanar relationship of oriented rings from monomer state to dimer state and depends on many variables (such as concentration, solvent and nature of substituents, metal ions, and temperature) [40]. For the metal phthalocyanines, aggregation is often undesirable as it decreases photoactivity [41].
The aggregation behavior of the compounds was studied in DMSO and DMF for 5, 7, and 10 and in water with quantities of TX-100 and DMSO for 11. After the addition of some drops of TX-100 to the aqueous solution of compound 11, the Q band at about 640 nm was observed to shift to 680 nm (Figure 2), which are lower energy for compound 11. However, as shown in Figure 2, the addition of TX-100 did not completely inhibit the aggregation of 11 in water. Comparing the UV-vis spectra of zinc phthalocyanine 11 in water and DMSO ( Figure S13), it was observed that it exhibited lower aggregation in DMSO, which has a lower polarity than water, while it exhibited an H-type aggregation in water [42].
The Beer-Lambert law was followed for all of these complexes at concentrations ranging from 2 × 10 -6 to 12 × 10 -6 M. The results showed that ZnPcs (5, 7, and 10) (in DMF and DMSO) (as an example for 10, in DMSO and DMF was given in Figure 3 and Figure S14) and the quaternized derivative of ZnPc 11 in DMSO ( Figure 3) did not display aggregation.

Fluorescence spectra
Fluorescence properties of the synthesized phthalocyanine compounds (5, 7, and 10) were investigated in DMSO and DMF, the quaternized metallophthalocyanines 11 in water containing TX-100, and DMSO. The absorption, fluorescence emission, and excitation spectra of complexes 10 in DMSO and 11 in water containing TX-100 are shown in Figure 4 as an example. Fluorescence emission peaks are also listed in Table 1.  The Stokes shifts of ZnPc complexes were observed in the ∼10-14 nm range. The ZnPcs (5, 7, and 10) indicated similar fluorescence behavior in DMF and DMSO. The excitation and absorption spectra were similar to each other and were both mirror images of the fluorescence spectra for complexes (5, 7, and 10) in DMSO and DMF. Hence, these results suggested that the nuclear configurations of the ground are similar to the excited states and are not affected by the excitation of ZnPcs. In water media, while complex 11 was not fluorescent property due to high aggregation tendency it showed fluorescence property in DMSO. Also, 9 did not give emission in the studied organic solvents due to its aggregation. Since aggregation has no emission behavior, the emission properties of compounds with high aggregation tendency in the solutions are very low or not observed [43].

Fluorescence quantum yields
Fluorescent molecules have recently gained importance in PDT applications, as they provide the opportunity to monitor how they progress in the body and whether they accumulate in cancer cells. For this reason, the fluorescence properties of photosensitizers 5, 7, and 10 were investigated. In addition, due to the low solubility of Compounds 4, 6, and 9 in DMSO and DMF solvents and their high aggregation tendency, the fluorescence properties of these compounds could not be investigated. Table 2 shows the Φ F of ZnPc complexes (5, 7, and 10) in DMSO and DMF and for the quaternized ZnPc 11 in both DMSO and water containing TX-100. The fluorescence quantum yield (Φ F ) value of the newly studied zinc Pc complex 10 was slightly lower than the unsubstituted zinc Pc (Φ F = 0.20) in DMSO and was also lower than its quaternized derivative 11 substituted with quaternized imine conjugated pyridine group in aqueous media [44]. The quaternized complex 11 had a high Φ F value in water (plus 0.1 mL TX-100) compared to DMSO due to the aggregation in the first solvent. The fluorescence quantum yield of the Schiff base substituted complex 10 was not significantly increased compared to compounds 5 and 6 according to the literature [31].

Singlet oxygen quantum yields
Singlet oxygen causing irreversible destruction of cells within the irradiated tumor area is a measure of the effectiveness of the PDT procedure. The amount of singlet oxygen produced is the most important indicator of using it as a photosensitizer. Singlet oxygen quantum yield (Φ Δ ) is a measure of singlet oxygen generation efficiency and the Φ Δ values were obtained using Eq. (5) (given Sup. File). Information about the singlet oxygen measurement conditions was given in the supplementary file. Singlet oxygen quantum yields were studied in organic solvents (DMSO and DMF) for the studied ZnPcs (5, 7, 10) using 1,3-Diphenylisobenzofuran (DPBF) as a quencher and water (plus 0.1 mL TX-100) for the quaternized ZnPc 11 using 9,10-anthracenediyl-bis(methylene)dimalonic acid (ADMA) as a quencher. The disappearance of DPBF or ADMA was monitored using a UV-vis spectrophotometer in Figure 5 (a) using DPBF in DMSO and Figure 5 (b) using ADMA in water plus TX-100 for complex (11). The Φ Δ values of the studied Pcs (5, 7, 10, and 11) and standard ZnPc are listed in Table 2. There were no changes in the intensities of the Q band absorptions of the ZnPc derivatives during the Φ Δ determination process.
The Φ Δ value of ZnPc Schiff base bearing 10 was found to be 0.78 in DMSO. This value of 10 was higher than the ZnPc Schiff base bearing in the literature [30,31] and Std-ZnPc (Φ Δ = 0.67). Comparing DMSO and DMF, the Φ Δ value of ZnPc 10 in DMSO is higher than in DMF. This situation may have resulted from the amine group's attempt to quench the singlet oxygen in DMF. The novel ZnPc (10) bearing the Schiff base produced higher singlet oxygen generation in DMF with a Φ Δ value of 0.64 compared to the Std-ZnPc (Φ Δ = 0.57), ZnPcs 5 (Φ Δ = 0.58) and 6 (Φ Δ = 0.62). According to these results, the presence of Schiff base as a ligand increases the Φ Δ with the increase of intersystem transition [46,47]. The fact that ZnPc 10 has a higher Φ Δ value than its quaternized form 11 in DMSO suggests that the quaternization of ZnPc complexes causes a decrease in Φ Δ values. In addition, Table 2 shows that low Φ Δ values were observed for 11 in water containing TX-100 compared to DMSO. The absorption of both singlet oxygen and water around 1270 nm has a great effect on the lifetime of singlet oxygen. This explains why the Φ Δ value in water is lower than the one in deuterated water and DMSO [46]. Φ Δ of 11 showed higher than singlet quantum yields compared to previously studied quaternized zinc analogs bearing the pyridine group in DMSO [42].

Photodegradation quantum yields
Photodegradation is a photochemical method used to determine the stability of phthalocyanines under the influence of applied light which is critical for molecules designed for use as photosensitizers in PDT. It is expected that the concentration of the drug molecule used in photocatalytic applications such as PDT will not change during the treatment process, that is, it will be stable. The photodegradation quantum yield (Φ d ) values for the complexes listed in Table 2 are of the order of 10 -4 . Stable ZnPc molecules showing Φ d values between 10 -6 and 10 -3 have been reported [48]. The change in the absorbance values observed in the Q-band during the Φ d measurement of 10 and 11 in DMSO is presented in Figure 6. The synthesized compounds were found to be less stable than standard ZnPc in DMSO (0.26 × 10 -4 ) and DMF (0.23 × 10 -4 ) [45]. Compared with the photodegradation quantum yields of compounds 5 and 6, an increase in the photodegradation quantum yields in DMSO and DMF was observed with the incorporation of imine bond-conjugated pyridine groups into the structure.
When the pyridine substituted phthalocyanines were in terms of solvent effect, it was seen that the stability in DMSO was higher than that of DMF. Quaternization of pyridine groups caused a decrease in the stability of the ionic zinc Pc complex 11. Compound 11 is less stable in water containing TX-100 ( Figure S15) than in DMSO.

Binding properties of quaternized zinc(II) phthalocyanine to BSA protein
Bovine serum albumin (BSA), is the predominant protein in the blood, it has a very important role in the delivery of drugs. One of the studies to determine drug delivery to specific tissues through the bloodstream is the analysis of the BSA binding properties of photosensitizers [49,50]. Accordingly, the binding properties of the novel quaternized ZnPc 11 to BSA protein were investigated by spectrofluorometric at room temperature in PBS [51] in an aqueous solution. The PBS of a fixed concentration of BSA (3.00 × 10 -5 M) was titrated with varying concentrations of the 11 solution. BSA was excited at 280 nm and the fluorescence emission spectra were reported between 290 and 500 nm for compound 11-BSA solution. The fluorescence emission peak of BSA at 348 nm decreased by increasing the phthalocyanine concentrations due to the interaction of the phthalocyanine molecules with the tryptophan residues on the BSA protein (Figure 7).
When the fluorescence quenching studies of water-soluble ZnPc and BSA in the literature [51][52][53] were examined, it was understood that compound 11 was more effective in the fluorescence quenching of BSA. The Kq value was obtained for 11 and as shown in Table 3, the fact that the value of kq (0.97 × 10 13 M -1 s -1 ) is higher than the recommended value for dynamic quenching (10 10 M -1 s -1 ) [53] indicates that the quenching mechanism is static. A bimolecular quenching constant (Kq) of 11 was acquired by Equation (6) (in supplementary file) using an approximate fluorescence lifetime of BSA [54]. In fluorescence quenching of 11 by BSA in PBS, Stern-Volmer kinetics consistent with diffusion-controlled bimolecular reactions were investigated. The fluorescence emission changes in BSA upon binding of complex 11 are observed in Figure  7. The slope of the graph demonstrated in Figure 7 gave the Stern-Volmer constant (K SV ) value indicated in Table 3.     Compound K sv (10 5 M -1 ) k q (10 13 M -1 s -1 ) 11 6.08 6.08

Conclusion
In this study, a novel Schiff base substituted phthalocyanine complex (9 and 10) carrying pyridine moieties and its watersoluble quaternized derivative (11) were synthesized and characterized. In the synthesis of targeted Pcs, phthalocyanine compound with aldehyde functional group, which is suitable for Schiff base reaction by various primary amines, was chosen as the starting material. Photophysical and photochemical measurements showed that the Schiff base substituted derivative containing pyridine moieties did not affect the singlet oxygen production value in DMSO, but increased in DMF.
When the effect of quaternization on these properties was examined in DMSO and water containing TX-100 for PDT applications, it was determined that quaternized phthalocyanine (11) had effective photophysical and photochemical properties related to photosensitization, gave more important values in DMSO.
As a result of the study to define the stability of the Pc molecule under light irradiation, it was determined that the newly synthesized phthalocyanine compounds (10 and 11) used in solvent systems (DMSO and DMF for 10, DMSO, and water-containing TX-100 for 11) have suitable photodegradation stability.
The interactions between BSA and the quaternized zinc phthalocyanine (11) were also investigated in this study. The result of the fluorescence quenching studies of BSA presented that the water-soluble quaternized zinc phthalocyanine complex (11) showed strong binding to serum albumin and was easily transferable in blood. Consequently, all these results displayed that the novel ZnPc 10 and notably its water-soluble form 11 can be acceptable candidates for PDT of cancer treatment.
FT-IR spectra (KBr pellets) were measured with a Perkin Elmer Spectrum One Spectrometer. Absorption spectra in the UV-visible region were obtained with a Shimadzu 2001 UV spectrophotometer.
Fluorescence spectra were done using a Varian Eclipse spectrofluorometer using 1 cm pathlength cuvettes at room temperature. 1 H NMR spectra were recorded in D 2 O (water soluble zinc phthalocyanine) and DMSO-d 6 ( metal free and zinc phthalocyanine) solutions on a Varian 500 MHz spectrometer.
Photo-irradiations were done using a General Electric quartz line lamp (300 W). A 600 nm glass cut off filter (Schott) and a water filter were used to filter off ultraviolet and infrared radiations respectively. An interference filter (Intor, 700 nm with a bandwidth of 40 nm) was additionally placed in the light path before the sample. Light intensities were measured with a POWER MAX5100 (Molelectron detector incorporated) power meter. The mass spectra were acquired on a Bruker Daltonics (Bremen, Germany) MicroTOF mass spectrometer equipped with an electrospray ionization (ESI) source. The instrument was operated in positive ion mode using a m/z range of 50-3000. The capillary voltage of the ion source was set at 6000 V and the capillary exit at 190 V. The nebulizer gas flow was 1 bar and drying gas flow 8 mL/min.

Photophysical and photochemical studies 2.1. Fluorescence quantum yields
Fluorescence quantum yields (Φ F ) were determined by the comparative method (Eq. 1) [S1], where F and F Std are the areas under the fluorescence emission curves of the samples and the standard, respectively. A and A Std are the respective absorbances of the samples and standard at the excitation wavelengths, respectively. 2 n and 2 Std n are the refractive indices of solvents used for the sample and standard, respectively. Unsubstituted ZnPc (in DMSO) (Φ F = 0.20) [S2], (in DMF) (Φ F = 0.17) [S3], was employed as the standard. Both the samples and standard were excited at the same wavelength. The absorbance of the solutions at the excitation wavelength ranged between 0.04 and 0.05.

Singlet oxygen quantum yields
Singlet oxygen quantum yield (Φ ∆ ) determinations were carried out using the experimental set-up described in the literature [S5-S8]. Quantum yields of singlet oxygen photogeneration were determined in air (no oxygen bubbled) using the relative method (Eq. 2) with ZnPc as reference. 1,3-Diphenylisobenzofuran (DPBF) for organic solvent and 9,10-antracenediylbis(methylene)dimalonoic acid (ADMA) for aqueous solution were used as chemical quencher for singlet oxygen, using equation 2 (2) where is the singlet oxygen quantum yields for the standard ZnPc ( = 0.67 in DMSO) and =0.56 in DMF) [S8], and ZnTSPc ( Std Ä Ö = 0.30 in aqueous solution in the presence of Triton X) [S9]. R and R Std are the quencher photobleaching rates in the presence of the samples and standard, respectively. I abs and Std abs I are the rates of light absorption by the samples and standard, respectively. Typically, a 3 mL portion of the respective unsubstituted ZnPc, ZnTSPc or synthesized phthalocyanines (5, 7, 10, and 11) solutions containing the singlet oxygen quencher was irradiated in the Q band region with the photo irradiation set-up described in the references [S5,8]. To avoid chain reactions induced by the quenchers (DPBF or ADMA) in the presence of singlet oxygen, the concentration of the quenchers (DPBF or ADMA) was lowered to ~3 × 10 −5 M [S10]. Solutions of the sensitizer (C = 1 × 10 −5 M) containing the quencher (DPBF or ADMA) were prepared in the dark and irradiated in the Q band region. DPBF degradation at 417 nm and ADMA degradation at 380 nm were monitored. The light intensity of 1.74 × 10 15 photons s -1 cm -2 was used for Φ ∆ determinations.

Photodegradation quantum yields
Photodegradation quantum yield (Φ d ) determinations were carried out using the experimental set-up described in the literature [S6-S7]. Photodegradation quantum yields were determined using formula 3, where "C 0 "and "C t " are the sample concentrations before and after irradiation respectively, "V" is the reaction volume, "N A ", the Avogadro's constant, "S", the irradiated cell area and "t", the irradiation time, "I abs " is the overlap integral of the radiation source light intensity and the absorption of the samples. A light intensity of 5.35 × 10 15 photons s -1 cm -2 was employed for Φ d determinations.

Binding properties of quaternized zinc(II) phthalocyanine to BSA protein
The binding of quaternized zinc (II) phthalocyanine complex (11) to BSA was studied by spectrofluorometry at room temperature in PBS solution. The PBS of a fixed concentration of BSA (3.00 × 10 -5 M) was titrated with varying concentrations of the 11 solution. BSA was excited at 280 nm and the fluorescence emission spectra were recorded between 290 and 450 nm. The steady diminution of the fluorescence emission of BSA with the increase in the 11 concentrations was recorded. The fluorescence intensity for BSA decreased by addition of the quaternized zinc (II) phthalocyanine (11)