Effect of Some Substituents Increasing the Solubility of Zn(II) and Al(III) Phthalocyanines on Their Photophysical Properties

Water solubility of phthalocyanines (Pcs) usually increases by the introduction of charged or carboxy substituents in the peripheral positions of the macrocycle. As a result, such structural changes influence their photophysical and photochemical properties as photosensitizers. Phthalocyanines substituted with four or eight terminal carboxyl groups and having in some cases additional eight positive charges (water soluble phthalocyanines) were studied in order to evaluate the spectroscopic and photophysical effects of these side residues on the chromophore properties. The quantum yield of singlet oxygen (1O2) generation, the triplet-triplet absorption, and the transient absorption spectra were measured and linked to the structure of the substituents. It was shown that charged substituents did not change the quantum yields of 1O2 generation but decrease its lifetimes. The introduction of the charged substituents not only increases the water solubility but also significantly changes absorption, fluorescence, and transient absorption spectra of water soluble Pcs.


Introduction
Phthalocyanines are purely synthetic analogues of porphyrins. They have a great potential as photosensitizers in photodynamic therapy (PDT) for the treatment of malignancies and other diseases [1][2][3][4]. As photosensitizers, Pcs have the advantage of high molar absorption [5,6], resistance to chemical and photochemical degradation, and long lifetimes of 1 O 2 with high quantum yields [7]. Moreover, Pcs have absorption and emission in the range of 660-750 nm, making them sensitive to the light, which can penetrate deeply into living tissues [8]. Propensity to aggregation due to molecular stacking resulting in low quantum yields of 1 O 2 [9,10] and limiting solubility in aqueous media is the disadvantage of these dyes.
The chemical structure of Pcs can be modified by the introduction of substituents in the peripheral positions of the tetraaza isoindole macrocycle [11], as well as by the coordination of metal ions with the central nitrogen atoms and the addition of axial ligands in the fifth and sixth coordinative positions of such molecules [12,13]. Primary goal of such modifications of Pcs is to tune the water solubility and aggregation [14,15] without significant changes in the photophysical properties. Introduction of several identical groups, for example, carboxylic groups, does not break significantly the symmetry of the Pc molecules but usually reduces their aggregation. In this work, we report the spectroscopic and photophysical characteristics of several new Pcs derivatives that can potentially serve as photosensitizers for PDT with favorable water solubility. We examined Al(III) tetra-4-carboxymethyl sulfanyl (1), Zn(II) tetra-4-carboxyhexylsulfamidophthalocyanine (2) having terminal carboxyl groups as well as Al(III) and Zn(II) octakis-4,5-[2-(N-carboxymethyl-N,N-dimethylammonium)ethoxycarbonyl] phthalocyanine octachlorides (3 and 4) having eight charged substituents ( Figure 1) in order to evaluate the spectroscopic and photophysical effects of these side residues on the chromophore properties. The quantum yield of 1 O 2 generation, the measurements of the triplet-triplet absorption, and the picosecond transient absorption spectra (ps-TAS), which are crucial in determining the feasibility of using these dyes for PDT, are reported and linked to the structure of the substituents.

Chemicals and Reagents. Dimethylformamide (DMF;
Sigma, Germany), D 2 O, and double-distilled water were used as solvents.
Compounds 1 and 2 are soluble in polar solvents especially DMSO and DMF as well as aqueous acetonitrile and alcohols. Compounds 3 and 4 are soluble in water, concentrated mineral acids, and their aqueous solutions, alcohols, and polar aprotic solvents. UV-vis absorption spectra of their aqueous solutions show an aggregation, to a lesser extent in the case of aluminum complex 3.

Absorption and Steady-State
Fluorescence. The ground state absorption spectra were recorded using a spectrophotometer Shimadzu UV-2501PC at room temperature. The steady-state fluorescence spectra were measured using 1 cm × 1 cm quartz cells with a combination of a cw-Xenon lamp (XBO 150) and a monochromator (Lot-Oriel, bandwidth 10 nm) for excitation and a polychromator equipped with a cooled CCD matrix as a detector system (Lot-Oriel, Instaspec IV) [18].

Time-Resolved Singlet Oxygen Luminescence Measurement.
These measurements were carried out using a nanosecond Nd, YAG laser (BM Industries, Evry Cedex, France) coupled with an optical parametric oscillator (BMI). The 1 O 2 emission was detected using a liquid nitrogen cooled Ge-diode (North Coast, Inc., Santa Rosa, CA). For wavelength selection, a combination of a silicon filter and an interference filter with the center wavelength of 1270 nm was placed in front of the diode [19]. The optical density of the sample was adjusted to 0.2 at the excitation wavelengths in the 600-680 nm region. Tetraphenylporphyrin served as a reference standard for the determination of the singlet oxygen quantum yield (Φ1 O 2 ) with value 0.75 [20].

Laser Flash Photolysis.
In the triplet-triplet absorption measurements, the samples were excited using the same instrumentation as described above. A continuous wave monitoring beam was formed by light from XBO lamp that passed through a monochromator tuned to 488 nm. The beam traversed through the solutions of studied compounds perpendicular to the excitation beam. The intensity of the monitoring beam was measured using an avalanche Siphotodiode equipped with an interference filter for 488 nm. Oxygen was removed by bubbling the samples with nitrogen (purity 99,0%) for ca. 40 min at room temperature [21].

Picosecond Transient Absorption Spectroscopy.
To measure ps-TAS, a white light continuum, generated in a cell with a D 2 O/H 2 O mixture using intense 25 ps pulses from a Nd3, YAG laser (PL 2143A, Ekspla) at 1064 nm, was used for the monitoring beam. Before passing through the sample, the monitoring beam was split to provide the reference spectrum. The transmitted as well as the reference beams were focused into two optical fibers and were recorded simultaneously at different strips on a CCD-matrix (Lot-Oriel, Instaspec IV). Tunable radiation from an OPG/OPA (Ekspla PG 401/SH, tuning range 200-2300 nm) pumped by third harmonic of the same laser was used as an excitation beam. The mechanical delay line allowed the measurement of light-induced changes of the absorption spectrum at different delays up to 15 ns after excitation. The OD of all samples was 1.0 at the maximum of the absorption band of the lowest energy. The analysis of the experimental data was performed using the compensation method [22].

Results
In the present work, the photophysical parameters of Zn(II) and Al(III) Pcs complexes 1-4 containing different side residues were studied. All Pcs have terminal carboxyl groups with spacers of different lengths, and two of them (3 and 4) have eight positive charges in molecule.
The chemical structures of the studied compounds are shown in Figure 1.
The tetracarboxy-phthalocyanines 1 and 2 are not soluble in water. However, the introduction in benzene rings of macrocycle of charged substituents leads to the increase in the solubility of the resulting compounds 3 and 4. To compare photophysical properties of compounds 1 and 2 with watersoluble Pcs 3 and 4 in water-like solutions, Pcs complexes 1 and 2 were dissolved in 0.7% DMF in H 2 O (or in D 2 O) mixture. Such amount of DMF is appropriate to keep cells alive that is important for further testing of compounds as potential photosensitizers in PDT.

Singlet Oxygen Measurements.
Several solvents were used in the measurement of the quantum yield of 1 O 2 generation due to the different solubility of the studied compounds in aqueous media. Pure DMF and 0.7% DMF in D 2 O were used for 1 and 2 samples; D 2 O was used for Pcs 3 and 4. Tetraphenylporphyrin and tetraphenylporphyrin tetrasulfonate were used as references in organic and water solutions, respectively.
The comparison of the quantum yields of 1 O 2 allows finding compounds with similar behaviours (Table 1) On the other hand, 1 and 2 are not soluble in D 2 O. The "0.7% DMF in D 2 O" solvent was used to model the behaviour of the photophysical properties of these compounds in aqueous solutions. The Φ1 O 2 decreased to 0.09 for 2 and increased up to 0.16 for 1 compared with the results for these compounds obtained in DMF. The central metal seems to affect the solubility also. Aqueous-like solvents are more suitable for compounds 1 and 3 with Al(III) due to the presence of axial ligand.
The other characteristic of the photophysical properties is the lifetime of 1 O 2 , whose well-known value in D 2 O is 60 s [23]. But for all investigated compounds, the lifetimes of 1 3 and 4, but the Φ1 O 2 for these compounds was the same as that for 1 and 2 solved in DMF. The Pcs 2 and 4 were chosen for additional experiments due to their better fluorescence (compared to these with PcAl(III)) which was required in some techniques.
First, the energy transfer from the first excited triplet state

Laser Flash
Photolysis. The method of laser flash photolysis was used for indirect determination of 1 O 2 formation via triplet lifetime measurement. This is possible because the triplet lifetimes can be related to the singlet oxygen generation. After activation of the first excited singlet state 1 via light absorption, a part of the molecules undergo transition to the first excited triplet state 1 with a quantum yield Φ [24]. The subsequent deactivation could proceed via phosphorescence ( phos ) or nonradiative ( nonrad ) transition. The fraction of triplet states deactivated via interaction with molecular oxygen is referred to as .
The triplet lifetime in the presence of oxygen O 2 is given by O Removal of dissolved oxygen by bubbling the solution with N 2 leads to an increase of the triplet lifetime. In this case, the lifetime is calculated as In the presence of oxygen, the triplet lifetime of Pc 2 in DMF was measured to be = 0.34 s; the lifetime of Pc 4 in D 2 O was 6.0 s. (Table 2).
After oxygen removal, the triplet lifetimes of the compounds dissolved in the same solvents increased to = 52.6 s for Pc 2 and to 8.9 s for Pc 4 as seen in Table 2.  The value of can be obtained finally from O 2 and N 2 : (see [24]). This parameter characterizes the efficiency of the energy transfer from the first excited triplet state 1 to molecular oxygen. Since in case of Pc 2 in DMF value is nearly unity, this means that energy transfer to the molecular oxygen is predominant and takes place almost lossless. For Pc 4, this value is only 0.3. However, the value is substantially lower for Pc 4; the Φ1 O 2 for 2 in DMF and 4 in D 2 O are the same. Therefore, the charged side moieties in compound 4 have no influence on the Φ1 O 2 , but these moieties have influence on the photophysical properties resulting in the redistribution of the energy transfer. Also such a strong reduction of the triplet lifetime may result from the interaction between Pc core and side moieties. To determine the photophysical behavior of our compounds, more details were investigated by ps-TAS method.

Picosecond Transient Absorption Spectroscopy.
It was shown that the ps-TAS of Pc 2 was different from the spectrum of Pc 4. Spectra of Pcs 2 and 4 contain only one peak at 690 and 629 nm, respectively ( Figure 3). Each peak was characterized by an individual decay component. Compound 2 in DMF has two decay components at 690 nm with lifetimes of 177 and 2183 ps; also, Pc 4 in D 2 O has two decay components at 629 nm with lifetimes of 57 and 286 ps ( Figure 3). The amplitude ratio of the first and the second components (A1/A2) for these compounds was approximately the same: 2.58 and 2.25 for Pcs 2 and 4, respectively (Table 3). It means that the shorter decay component makes primary contribution to the transient absorption spectrum. Presumably, the blue shift of the peak maximum and the significant decrease in the decay components lifetimes could be linked to the decrease of 1 O 2 lifetime for water-soluble Pc 4.

Conclusions
The introduction of the charged substituents not only increases the water solubility but also significantly changes absorption, fluorescence, and transient absorption spectra of water-soluble Pcs. It was shown that charged substituents did not change the quantum yields of 1 O 2 generation but they decreased its lifetimes. It could mean that the increase in the solubility in water by charged side moieties does not influence significantly the Φ1 O 2 , which is important for