Solvent Effect , Photochemical and Photophysical Properties of Phthalocyanines with Different Metallic Nuclei

Photophysical and photochemical properties of lithium phthalocyanine (1), gallium(III) phthalocyanine chloride (2), titanium(IV) phthalocyanine dichloride (3) and iron(II) phthalocyanine (4) were investigated in dimethyl sulfoxide (DMSO), tetrahydrofuran (THF) and DMSO-THF mixtures. The influence of the central metal on these properties was analyzed according to solvent type, axial ligands and their paramagnetic and diamagnetic effect. Fluorescence lifetimes were recorded using a time correlated single photon counting setup (TCSPC) technique. In order to demonstrate the generation of reactive oxygen species under light irradiation, the indirect method (applying 1,3-diphenylisobenzofuran (DPBF) as chemical suppressor) and the direct method (analyzing the phosphorescence decay curves of singlete oxygen at 1270 nm) were employed. Compounds 1, 2 and 3 showed a monomeric behavior in all media while compound 4 presented low aggregation in DMSO, but a very pronounced aggregation behavior in THF. Steady-state fluorescence anisotropy was compared with emission spectra and complex 4 presented values beyond the expected limits.


Phthalocyanines
(Pcs) are aromatic macromolecules applied in numerous areas such as electrochromic displaying systems [1,2], solar cells [3][4][5], optical storage devices [6] and photodynamic therapy (PDT) [7][8][9].Chemical and physical properties of phthalocyanines may be modified by the nature of the substituents on the benzene rings or the nature of the axial ligand in the central metal [10].As it is known, phthalocyanine compounds have high aggregation tendency due to their high π-π stacking interactions [11].This feature hinders not only the solubility of compounds but also their photophysical, photochemical and spectroscopic properties in solution.Generally, aggregation depends on some factors, such as concentration, temperature, nature of the substituents, nature of the solvents and complexed metal ions [12,13].For example, large metals such as Aluminum (Al) and Indium (In) are able to hold axial ligands and diminish intermolecular interactions between macrocycles, therefore decreasing aggregation in solution.
The influence of central metal ions on the photophysical and photochemical parameters of benzothiazole-substituted phthalocyanines was investigated in tetrahydrofuran (THF) solution by Nas and co-workers [14].The authors described a large red shift in the Q absorbance band attributed to highly deformed phthalocyanine skeleton by the large size of central lead ion, which did not fit into the cavity of phthalocyanine molecule.Additionally, the variety of metal ions in the framework of the Pc ring affects the fluorescence quantum yields and lifetime values of compounds.
Nas and co-workers [15] published a study on the photophysical and photochemical properties of oxadiazol tetra-substituted phthalocyanine with different metals.The aggregation behavior and electronic absorption spectra of the compounds were studied in different solvents (DMF, DMSO, chloroform, dichloromethane, THF and toluene).The unmetallated phthalocyanine compound showed no aggregation in chloroform, dichloromethane and THF.However, this compound formed aggregates in other used solvents, especially in DMSO, indicating the influence of the solvent effect.The   values for unmetallated and zinc(II) oxadiazole substituted phthalocyanine were lower than those of unsubstituted zinc(II) phthalocyanine, suggesting more quenching due to the substitution and the interaction with the solvent.
The solubility and aggregation processes of peripherally/non-peripherally zinc and indium phenylphenoxy substitued phthalocyanines were investigated by Ali and co-workers [16].They reported that the indium phthalocyanine derivative presents a red shifted Q-band when compared to the corresponding zinc phthalocyanine, in DMSO, suggesting a non-planar effect of a larger central indium atom.In addition, the indium phthalocyanine presented no fluorescence emission spectra as a mirror image of the excitation spectra, which can be attributed to the large indium atom in the cavity phthalocyanine macrocycle.The zinc phthalocyanine derivative showed strong fluorescence, while the indium macrocycles showed very low fluorescence emission due to the heavy metal atom effect.Göksel [17] described the study of a new phthalocyanine biotin derivative to understand the influence of the biomolecule and heavy central metal atoms on the photophysical and photochemical properties.The UV-vis spectra of the compounds were obtained in different solvents and the results demonstrated that the aggregation behavior of the macrocycles was dependent on the concentration, nature of solvent, nature of substituents, complexed metal ions, and temperature.The fluorescence lifetime values of the substituted phthalocyanines were lower than the unsubstituted phthalocyanines in DMSO, suggesting more quenching due to the presence of heavy central metal atom.
In the present study, the aggregation behavior as well as photophysical and photochemical properties of four commercial metal phthalocyanines were investigated.The studied compounds (Figure 1) were di-lithium phthalocyanine (1), gallium(III) phthalocyanine chloride (2), titanium(IV) phthalocyanine dichloride (3) and iron(II) phthalocyanine (4).These metals were chosen because of their different features.Lithium does not show any axial ligands and presents two metal ions, because of by the fact of the Li + be an alkaline metal, it is monovalent, and each metal atom interact with one of the central nitrogen atoms of macrocycle.Gallium and titanium show one and two axial ligands, respectively.Iron is a unique metal that presents paramagnetic behavior.

Equipment
UV-visible region absorption spectra were obtained with an Agilent Cary 60 UV-Vis Spectrophotometer.Fluorescence excitation and emission spectra were recorded on an Agilent Cary Eclipse Fluorescence Spectrophotometer using 1 cm path length quartz cuvette at room temperature.Fluorescence lifetimes (  ), Steady-State Fluorescence Anisotropy and Phosphorescence decay curves at 1270 nm were recorded on a PicoQuant FluoTime 300 Fluorescence Lifetime Spectrometer.Fluorescence lifetimes (  ) were recorded using a time correlated single photon counting setup (TCSPC).The decays were analyzed by means of PicoQuant FluoFit Global Fluorescence Decay Analysis Software.Phosphorescence decay curves at 1270 nm were recorded in the equipment equipped with a nearinfrared photomultiplier tube (NIR PMT) Hamamatsu, model H10330B.Photo-irradiations were done using a picosecond pulsed diode laser (LDH-P-635 driven by PDL 800-B, 635 nm, 80 MHz repetition rate, 72 ps pulse width, PicoQuant GmbH).

Sample preparation
All solutions were prepared by saturation and filtration.Compounds concentration were adjusted and all measurements were carried out at similar absorbance rate (~0.10-0.20).These conditions were chosen in order to avoid aggregated species.All study was carried out at room temperature.

2.4
Photophysical and photochemical parameters

Fluorescence lifetimes and steady-state fluorescence anisotropy
Fluorescence lifetimes (  ) were directly recorded using a time correlated single photon counting setup (TCSPC).
Steady-state fluorescence anisotropy spectra were obtained by changing the detection polarization on a fluorescence path parallel or perpendicular to the polarization of the excitation light (635 nm).Anisotropy [18] values were calculated according to the following Equation (1): where  ǁ and  Ʇ are the observed fluorescence intensities parallel (emission polarizer parallel to the polarized excitation) and perpendicular (emission polarizer perpendicular to the polarized excitation) to the electric vector of the excitation light, respectively.In the spectrometer, G is the G-factor.

Singlet oxygen quantum yields
Singlet oxygen quantum yield (  ) determinations were carried out using the direct method, and experiments were performed at room temperature in the same conditions.Sample absorbance was adjusted to 0.05 at 635 nm (the laser excitation wavelength used), and   values were determined in the air in THF using Equation [19] (2), where  ∆  is the singlet oxygen quantum yield for the standard unsubstituted ZnPc ( ∆  = 0.53 in THF) [20];  and   are the integral of phosphorescence emission curve of singlet oxygen in 1270 nm of the studied compounds (1-4) and standard compounds, respectively.
Furthermore, the chemical method using DPBF as a quencher was also employed.This method is largely used in the literature [13,[21][22][23][24][25].However, the chemical method was only employed in order to demonstrate the generation of reactive oxygen species under light irradiation.Therefore,   values were calculated solely for the direct method.

UV-vis absorption spectra
The phthalocyanines studied have two typical and significant absorption bands in the UV-vis region.One of them is known as "B" or "Soret" band, at 275-389 nm, due to the transitions from the deeper  levels to the LUMO [26,27].The other characteristic band is known as "Q" band, at 600-720 nm, as a result of the transitions from the first excited state -HOMO to  * -LUMO [28].The electronic absorption spectrum of the studied compounds is shown in Figure 2. The narrow and sharp Q bands evidenced the monomeric behavior [29,30] for compounds 1-3, while FePc (4) presents an aggregation behavior.
The Q band of the lithium, gallium and titanium phthalocyanine complexes (1-3) presents a red-shifted in DMSO according to the central metal [25,31].This suggests the existence of non-planar effect with the size increment of the central atom, which was also observed in THF (Figure 3).Complex 1 revealed the presence of an extra band at 688 nm in THF.This band can be explained by the demetalation tendency of dilithium phthalocyanines [32].The Q bands did not change extensively when the studied solvents were changed or proportionated, except for compound 3 (Figure 4 (A)).For this compound, the intensity in the B absorption band changes as the THF partition was increased, which may be because of the influence of the titanium metal, since the two axial ligands in the overlapping of the two sub bands (B1 and B2) [27].This effect was not observed in the other compounds.Figure 4 (B) shows compound 2 as an example.
Compounds 1-3 presented no aggregation behavior in the studied solvents.Table 1 presents the maximum intensity wavelength in absorption and emission spectral, in all solvent conditions.On the other hand, compound 4 shows intense aggregation behavior when THF proportion was increased (Figure 5).This may be attributed to the lower coordinating ability of THF than DMSO with the central metal.The paramagnetic central ion also influences the absorption spectra and photophysical parameters [13,25].Similar intense single broaded Q bands of Iron (II) phthalocyanines are observed in other solvents as previously described [33,34].

Fluorescence spectra
Fluorescence maximum wavelength, in all solvent conditions studied, is listed in Table 1.The observed Stokes shifts were between 4-7 nm for complex 1, 4-6 nm for complex 2 and 2-10 nm for complex 3. Furthermore, the fluorescence spectra of the previously mentioned compounds were mirror images of their respective absorption spectra [35].
Absorption and emission spectra of the studied compounds are shown in Figure 6, in the same solvent conditions.Among compounds 1-3, complex 3 presented the most significant Stokes shift.This may be due to the titanium metal ion and its two axial ligands out of the plane [33].Complex 4 presented a different behavior compared to the other compounds, with larger Stokes shifts in DMSO, DMSO 80% /THF 20% and DMSO 40% /THF 60% (Figure 6).

Lifetimes and anisotropy measurements
Fluorescence Lifetime determines the time available for the fluorophore to interact with its environment.The mean of lifetime is defined as the average time the molecule remains in the excited state until it returns to the ground state [36].
Values of   are presented in Table 1.When comparing the studied compounds (1-4) in all solvent conditions, compound 1 has the largest   value.In larger proportions of DMSO, the   order is 1 > 2 > 3 > 4.However, when THF proportion becomes higher, this order changes to 1 > 3 > 2 > 4.These results show the influence of solvent on the metal phthalocyanines   .Another solvent influence evidence is the change in lifetime values when the compounds were analyzed individually.The lifetime in compounds 1 and 3 increases when the THF amount increases.This fact suggests that the compounds are better solvated and stabilized in this media [37].The opposite is observed for compound 2. The decrease of   in THF may be attributed to the small amounts of aggregates in the solution in addition to the solvent effect.The lifetime values for compound 4 are shorter than the other studied complexes.This behavior is already expected, once this complex showed aggregation behavior [13] in studied conditions and has a paramagnetic metallic nucleus.However, complex 4 exhibited higher   values in prevalence to THF media.When DMSO was absent, complex 4 presented higher   values than complex 2. Steady-state fluorescence anisotropy was compared with emission spectra of the studied compounds (Figure 7).In complex 1, positive regions appear interspersed with negative regions in almost all solvents, with the exception of DMSO 20% /THF 80% and THF, which did not show anisotropy in emission region.For the media that presented anisotropy, r values ranged from -0.1 to 0.1.Complex 2 presented the same behavior, although just THF did not present anisotropy.Complex 3 showed anisotropy values in all studied media, but with r values ranging from -0.4 to 0.4, reaching the limiting anisotropy values [38].Complex 4 was studied in DMSO, DMSO 60% /THF 40% and DMSO 40% /THF 60% because of the previously mentioned aggregation behavior.However, r values extrapolated the limits of the anisotropy value

Singlet oxygen generation
Singlet oxygen (1O2) is the main species responsible for killing diseased cells in the treatment of tumor cancers by Photodynamic Therapy [39].In the literature [23], it is reported that paramagnetic metals, such as copper and cobalt, have short-lived triplet states and consequently low singlet oxygen generation.However, phthalocyanines with diamagnetic metals, such as zinc (Zn 2+ ), aluminum (Al 3+ ), gallium (Ga 3+ ) and silicon (Si 4+ ) [9,24] have long triplet lifetimes and consequently better singlet oxygen generation.The paramagnetic feature not only influences photochemical parameters, but also photophysical properties [25].Studies of triplet lifetimes were not carried out, although it is known that lithium, gallium and titanium have generally good triplet lifetimes through previous studies [40][41][42].
In order to demonstrate the generation of reactive oxygen species under light irradiation [43][44], we employed the chemical method using DPBF as a quencher.In this method, UV-vis spectra were obtained to monitor the disappearance of DPBF absorbance at 417 nm.The results for complex 2 is shown in DMSO (A) and THF (B) in Figure 8.  Rate constants were calculated in relation of DPBF consumption.The constants are in accordance with Figure 9, where k = 0.037 s -1 for compound 1, k = 0.043 s -1 for 2, k = 0.049 s -1 for 3 in DMSO and k = 0.025 s -1 for 1, k = 0.076 s -1 for 2 and k = 0.044 s -1 for 3 in THF.As expected, the rate constant for compound 4 was almost null (approximately 2.0 x 10 -4 s -1 ).Also, the singlet oxygen quantum yield (  ) was determined using the direct method, which analyzes phosphorescence decay curves at 1270 nm using NIR PMT accessory.The   values cannot be obtained in DMSO [45] and the values were obtained only in pure THF.Decay curves are represented in Figure 10.The   value of compound 1 (LiPc) was determined as   = 0.44, compound 2 (GaClPc) as   = 0.63, and compound 3 (TiCl 2 Pc) as   = 0.57.These values correspond with phosphorescence decay curves.Compound 4 (FePc), as expected, was unable to generate singlet oxygen in quantitative amounts.These values are in agreement with DPBF consumption as observed in Figure 9 (B).Compounds 2 and 3 presented higher values of   than the standard compound (  = 0.53).All studied central metal ions are able to generate appreciable singlet oxygen amounts, with exception of compound 4, due to the paramagnetic behavior.

CONCLUSION
The aggregation behavior of the studied compounds was investigated in DMSO, THF and DMSO-THF mixtures.Compounds 1, 2 and 3 showed a monomeric behavior in all media.This evidence is supported by single and narrow Q band.Compound 4 presented low aggregation in DMSO, but a very pronounced change in this behavior was observed in THF.The photophysical results showed the influence of central metal ion on fluorescence lifetime values.Complex 4 presented the lowest values due to the presence of aggregate species in the studied solvents, besides the paramagnetic feature of complex metal ion.
When DMSO was the main solvent, the   order is 1 > 2 > 3 > 4.However, when THF proportion becomes higher, this order changes to 1 > 3 > 2 > 4. The influence of solvent in photophysical media was observed.The lifetime in compounds 1 and 3 increases significantly when the THF amount increases and this fact suggests better solvated and stabilized in this media of these compounds.Steady-state fluorescence anisotropy was compared with emission spectra and complex 4 presented values beyond the expected limits.Complex 1-3 presented high quantum generation yield of singlet oxygen, which demonstrated that these metals are recommended to photodynamic therapy.

Figure 1 .
Figure 1.The chemical structure of the studied compounds 1-4.

Figure 8 .
Figure 8. UV-vis spectra of solution containing complex 2 in DMSO (A) and THF (B) and the changes in DPBF, under constant irradiation by red LED light obtained after each six-second irradiation period.(Inset: Plot of DPBF absorbance versus time).
b Show no mirror images.