br Results and discussion br It is
3. Results and discussion
It is well-known that the formation of phthalocyanine H-aggregates results in a blue-shift of the low-energy Q-band, reduces phthalocya-nines' fluorescence quantum yield and diminishes PDT activity [43–47]. Thus, it was important to evaluate aggregation behavior of the Pc1 and Pc2 in water as physiological solvent. The UV–vis and MCD spectra of tetracationic and octacationic zinc phthalocyanines Pc1 and Pc2 in water are shown in Fig. 1.
In both cases, the Q-band can be identified as a strong MCD A-term centered at 695 nm (Pc1) or 679 nm (Pc2). It is clear from the UV–vis and MCD spectra that in water the degree of aggregation of tetra-cationic Pc1 is significantly higher than that in Pc2. In the case of Pc2, the Q-band shoulder centered at 613 nm can be clearly seen in its UV–vis spectrum and is associated with the positive MCD signal at 617 nm. In contrast, a broad band at ~650 nm was observed in the case of Pc1 in water, and is associated with a very strong positive signal in the MCD spectrum centered at 634 nm. From the MCD spectra, the intensity ratio of signals between the positive portion of the A-term at 667 nm and shoulder at 617 nm is clearly much larger for Pc2 com-pared to the same ratio between positive signals at 686 (A-term) and 634 (shoulder) nm for Pc1. The appearance of the broad, positive MCD signal at 634 nm in the case of Pc1 is indicative of the formation of H-aggregates in solution for this compound [48–52]. The formation of Erdafitinib in Pc1 was further confirmed by its UV–vis spectra taken in methanol and DMSO solutions, where the monomeric form of Pc1 dominates the spectrum (Fig. 2).
The formation of H-aggregates in the case of Pc1 can be easily ex-plained by its lower positive charge compared to Pc2 where its octa-cationic nature precludes π-π interactions between two phthalocyanine cores due to strong electrostatic repulsion. As expected, the introduc-tion of four electron donating groups to α-positions on the core of Pc1 resulted in a significant red-shift of the Q-band compared to Pc2 (705 nm in DMSO for Pc1 monomer vs. 682 nm in DMSO for Pc2 monomer).
3.2. DFT and TDDFT calculations
In order to correlate the red-shifts in Pc1 versus Pc2 with their electronic structures, Density Functional Theory (DFT) calculations
Fig. 2. UV–vis spectra of Pc1 in methanol and DMSO.
Fig. 3. DFT-predicted energy level diagram for Pc1 and Pc2.
were conducted on both compounds. The DFT-predicted energy dia-gram is shown in Fig. 3 and frontier orbitals are pictured in Fig. 4.
In both cases, the DFT-predicted highest occupied molecular orbital (HOMO) orbital resembles the classic Gouterman's “a1u” and the lowest unoccupied molecular orbital (LUMO) and LUMO+1 orbitals resemble Gouterman's “eg” pair of orbitals [53,54]. TDDFT-predicted UV–vis spectra of Pc1 and Pc2 are compared to the experimental data in Fig. 5.
In good agreement with the experimental data, the TDDFT-pre-dicted energy of the Q-band in Pc1 (717 nm) is red-shifted compared to that predicted for Pc2 (661 nm). In addition, a set of intra-ligand charge-transfer transitions from the electron-donating alkoxy groups to LUMO and LUMO+1 have been predicted by TDDFT for Pc1 at 457 nm,
which is in good agreement with experimental data on Pc1 and the similar phthalocyanines with electron-donating groups located at the α-positions of phthalocyanine core [55,56].
3.3. Singlet oxygen generation
In order to estimate the potential photodynamic activity of Pc1 and Pc2, singlet oxygen formation yields for these two photosensitizers in DMSO were determined using 1,3-diphenylisobenzofuran (DPBF) as a singlet oxygen scavenger (Fig. 6) . Upon irradiation of Pc1 and Pc2 in DMSO/DPBF solutions at Q-band wavelengths, rapid degradation of the singlet oxygen scavenger was observed while no decrease of Q-band intensity was detected. The singlet oxygen generation abilities of Pc1 and Pc2 are quite high and virtually equivalent to that observed in unsubstituted ZnPc in DMSO, likely indicative of the minor influence of the peripheral substituents on the formation and lifetime of triplet states in these photosensitizers.
3.4. DNA-binding studies
The interactions between Pc1 and Pc2 and DNA have been in-vestigated for SS-DNNA, DS-DNA, and G4-DNA structures, both by UV–vis and fluorescence spectroscopy (Figs. 7 and 8).
Based on these experiments, the binding features of Pc1 and Pc2 to all three tested DNA structures were similar. In the case of octacationic, less aggregative Pc2, the DNA binding follows the general trend ob-served for the other phthalocyanine photosensitizers used in PDT of cancer [57–63]. For all three DNA structures, an approximate 10% decrease in intensity and 7 nm red-shift of the Q-band were observed in the corresponding Pc2 UV–vis spectra. The observed red-shift does not correspond to the monomerization process as solutions of Pc1 in DMSO and MeOH have a higher-energy Q-band. Such change can only be as-sociated either with DNA-phthalocyanine interaction or J-aggregation. The changes in the Q-band intensity and energy were associated with an initial strong decrease of the fluorescence band observed at 693 nm. During saturation titration experiments an increase in fluorescence in-tensity at 698 nm was consistently observed (Fig. 8). Again, such be-havior is indicative of the strong interaction between Pc2 photo-sensitizer and DNA molecules.