Veronika Novakova

Magnesium Azaphthalocyanines: An Emerging Family of Excellent Red-Emitting Fluorophores

Magnesium(II), zinc(II), and metal-free phthalocyanines (Pcs) and azaphthalocyanines (AzaPcs) containing alkylsulfanyl, aryloxy, and dialkylamino peripheral substituents have been synthesized. The complexation of magnesium(II) by metal-free Pcs and AzaPcs has been studied in detail to determine the optimal reaction conditions necessary to ensure a complete conversion. Photophysical and photochemical measurements in tetrahydrofuran showed that magnesium(II) AzaPcs with aryloxy and alkylsulfanyl substituents have excellent fluorescent properties (Φ(F) up to 0.73) and that the corresponding zinc(II) Pcs are efficient singlet oxygen producers (Φ(Δ) up to 0.68). The presence of dialkylamino substituents causes intramolecular charge transfer within the molecule that competes with fluorescence and singlet oxygen formation. Alkylsulfanyl MgAzaPc and ZnAzaPc were the most photostable compounds among the series of studied derivatives. In addition, high molar absorption coefficients (ε ∼ 300,000 M(-1) cm(-1)), absorption (λ(max) ∼ 650 nm), and emission (λ(em) ∼ 660 nm, high Φ(F)) in the red region suggest that these molecules are potential fluorescent probes that are superior to the commercial red cyanine dye Cy5. MgAzaPc, when incorporated into lipidic bilayers of liposomes, maintains excellent fluorescence properties (Φ(F) = 0.64). Water-soluble MgAzaPc with quaternary ammonium peripheral substituents retained a high fluorescence quantum yield even in water (Φ(F) = 0.25). The described properties show that magnesium(II) AzaPcs are excellent red-emitting fluorophores with potential applications as fluorescent probes in sensing or in vitro imaging applications.

Far-Red-Absorbing Cationic Phthalocyanine Photosensitizers: Synthesis and Evaluation of the Photodynamic Anticancer Activity and the Mode of Cell Death Induction

Novel zinc, magnesium, and metal-free octasubstituted phthalocyanine photosensitizers bearing [(triethylammonio)ethyl]sulfanyl substituents in the peripheral or nonperipheral positions were synthesized and investigated for their photophysical properties (ΦΔ value up to 0.91, λmax up to 750 nm) and photodynamic anticancer activity. The photodynamic treatment of 3T3, HeLa, SK-MEL-28, and HCT 116 cancer cells revealed that the magnesium complexes were not active (IC50 > 100 μM), whereas the IC50 values of the zinc complexes typically reached values in the submicromolar range with low toxicity in the dark (TC50 ≈ 1500 μM). The subcellular changes upon photodynamic treatment of the HeLa cells indicated that the studied photosensitizers induced damage primarily to the lysosomes, which was followed by a relocalization and damage to other organelles. The time-lapse morphological changes along with the flow cytometry and caspase activity measurements indicated a predominant involvement of necrosis-like cell death.

Synthesis, Properties and In Vitro Photodynamic Activity of Water‐soluble Azaphthalocyanines and Azanaphthalocyanines

In this work zinc azaphthalocyanines (AzaPcs) from the group of tetrapyrazinoporphyrazines and zinc azanaphthalocyanines from the group of tetra[6,7]quinoxalinoporphyrazines (TQP) with eight diethylaminoethylsulfanyl substituents were synthesized. Tertiary amines were later quaternized with ethyl iodide to obtain water‐soluble photosensitizers (PSs). Quaternized compounds showed high singlet oxygen quantum yields as determined in DMF by monitoring decomposition of 1,3‐diphenylisobenzofuran. In water medium, quaternized AzaPc derivatives appeared in monomeric form in a wide range of concentrations while quaternized TQP derivatives showed aggregation at higher concentrations (over 1 μm). Photodynamic activity was tested on Hep2 cells using light of λ > 640 nm. Both quaternized dyes showed high photodynamic activity (IC50 = 104 and 220 nm for AzaPc and TQP, respectively). Dark toxicity was not detected even at the highest concentration used in in vitro tests (200 μm) which indicates a promising therapeutic index of these new substances. Tested compounds localized inside the cells mainly within the lysosomes thus suggesting an endocytic mechanism of cellular uptake. No localization within mitochondria was detected. A great advantage of TQP derivatives over other PSs is their very strong absorption at 747 nm that allows activation at wavelengths penetrating deeper into human tissues.

Effective Monofunctional Azaphthalocyanine Photosensitizers for Photodynamic Therapy

In this work we present a rational design of the active part of third generation photosensitizers for photodynamic therapy based on phthalocyanine and an azaphthalocyanine core. The preferred zinc complexes of the AAAB type that contain bulky tert-butylsulfanyl substituents (A) and one carboxy group (B) have been synthesized by statistical condensation and fully characterized. The tetramerization was performed using magnesium(ii) butoxide followed by demetalation and insertion of Zn II . Compound 1 synthesized from 4,5-bis(tert-butylsulfanyl)phthalonitrile (A) and 2,3dicyanoquinoxaline-6-carboxylic acid (B) exerted very promising photophysical properties (Q-band absorption at 726 nm, e = 140000 M −1 cm −1 ), which allowed strong absorption of light at long wavelengths where the penetration of the light through human tissues is deeper. The very high singlet oxygen quantum yield of 1 (� � = 0.80) assures efficient photosensitization. As a result of bulky peripheral substituents, compound 1 shows good solubility in organic solvents with a low degree of aggregation, which makes it potentially viable for non-complicated modification. One carboxy group in the final structure of 1 allows simple binding to possible carriers. This compound is suitable for binding to targeting moieties to form the highly active part of a third-generation photosensitizer.

Role of steric hindrance in the Newman-Kwart rearrangement and in the synthesis and photophysical properties of arylsulfanyl tetrapyrazinoporphyrazines.

Conditions for the Newman-Kwart rearrangement of phenols into thiophenols were investigated in relation to the bulkiness of substituents at the 2 and 6 positions of the starting phenol derivative with an emphasis on eliminating side reactions. Thiophenols with different 2,6-disubstitution patterns (including hydrogen, methyl, isopropyl or tert-butyl groups) were used for the synthesis of 5,6-bis(arylsulfanyl)pyrazine-2,3-dicarbonitriles that underwent cyclotetramerization leading to the corresponding zinc tetrapyrazinoporphyrazines (TPyzPz), aza-analogues of phthalocyanines. Several methods for the cyclotetramerization were attempted to eliminate problematic side reactions. Magnesium butoxide was found as the most suitable cyclotetramerization agent and afforded TPyzPzs in reasonable yields of approximately 30% under mild conditions. The varying arrangements of the peripheral substitutions resulting from the different bulkiness of the substituents were demonstrated by the X-ray structures of the pyrazine-2,3-dicarbonitriles. The prepared zinc arylsulfanyl TPyzPzs showed an absorption maximum at a Q-band over 650 nm, fluorescence quantum yields between 0.078 and 0.20, and singlet oxygen quantum yields ranging 0.58-0.69. TPyzPzs with isopropyl groups were found to be the best derivatives in this series as they combined facile cyclotetramerization, no aggregation, and good photophysical properties, which makes them potentially suitable for photodynamic therapy.

Red-emitting dyes with photophysical and photochemical properties controlled by pH.

New unsymmetrical zinc azaphthalocyanines, bearing one substituted aniline as a peripheral substituent, were prepared by using a statistical condensation approach. Both fluorescence and singlet oxygen quantum yields were extremely low in DMF (Φ(F)<0.01, Φ(Δ)<0.02, respectively), but increased after the addition of sulfuric acid, reaching values comparable to controls without aniline substituents (Φ(F)=0.22-0.29, Φ(Δ)=0.40-0.59, respectively). This behavior was attributed to the deactivation of excited states by intramolecular charge transfer from a donor site (aniline), which was blocked after protonation in acidic media. In the protonated form, all of the compounds efficiently emitted light with λ(em) in the region of 662-675 nm. The investigated compounds were anchored to dioleoylphosphatidylcholine (DOPC) unilamellar vesicles and showed response to buffer pH. They were highly fluorescent at low pH values and almost nonfluorescent in neutral solutions. The pK(a) values were determined in DOPC vesicles and ranged between 2.2 and 4.2.

Azaphthalocyanines: Red Fluorescent Probes for Cations

Fluorescent indicators are important tools for the analysis of biological cations. For example, these probes are useful for the quantification of blood electrolytes, assaying iontransport channels, and monitoring of lithium-drug levels. While the interactions of metal cations with phthalocyanines (Pcs) have previously been studied using absorption, magnetic circular dichroism, and NMR spectroscopy, none of these methods is suitable for in vitro and in vivo studies, owing primarily to a lack of sensitivity, and/or to the incompatibility of the instrumentation with biological systems. In this context, we describe the development of new aza analogues of Pc, azaphthalocyanines (AzaPcs), which upon binding to cations fluoresce in the red region of the visible spectrum. The development of probes that absorb and emit red light at wavelengths greater than 630 nm is highly desirable, especially for in vivo applications, because this increases the depth that the excitation light penetrates, and reduces the effects of light scattering and the autofluorescence of endogenous chromophores that often occurs at shorter wavelengths. Recently, we confirmed that alkylamino-functionalized AzaPcs undergo an ultrafast intramolecular charge transfer (ICT) upon excitation. The ICT relaxation pathway of the excited states results in the quenching of the AzaPc fluorescence (i.e., produces the OFF state). Their fluorescence can be switched ON by protonation of the donor center (i.e., of the amino substituent), if it is sufficiently basic. This property led us to develop a new type of AzaPc sensor based on the coordination of cations to the donor center. In a proof of concept study, an aza[15]crown-5 moiety, which is used as the recognition element, was attached to the periphery of the AzaPc macrocycle. This molecule was further functionalized with bulky tert-butylsulfanyl substituents to eliminate AzaPc aggregation. During the synthesis of the sensors, we observed that their fluorescence quantum yields (FF) are sensitive to substituent groups in the ortho position to the crown ether. Therefore, a series of model compounds with a diethylamino donor center (1a–f, Scheme 1) was syn-

Influence of protonation of peripheral substituents on photophysical and photochemical properties of tetrapyrazinoporphyrazines

Octasubstituted zinc tetrapyrazinoporphyrazines with four N,N-dimethylaminophenyls and four phenyl or pyridin-3-yl substituents were synthesized and fully characterized. Their fluorescence quantum yields in DMF or pyridine were very low, almost undetectable, as a consequence of ultrafast intramolecular charge transfer. Titration of their DMF solutions with sulfuric acid led to increase of the fluorescence quantum yields by two orders of magnitude when the full protonation of peripheral substituents was achieved. Intramolecular charge transfer is no longer a favorable way of excited-state relaxation at full protonation of N,N-dimethylaminophenyl substituents because of loss of donor centers (free electron pair on its nitrogen). Similarly, singlet oxygen quantum yields also increased by two orders of magnitude when sulfuric acid was added to tetrapyrazinoporphyrazine solutions in DMF. Protonation at azomethine nitrogens of tetrapyrazinoporphyrazine macrocycle was observed at higher acid concentrations and it led to considerable decrease of fluorescence quantum yields. Octaphenyl zinc tetrapyrazinoporphyrazine and octa(pyridin-3-yl) zinc tetrapyrazinoporphyrazine were used as controls without intramolecular charge transfer. Their fluorescence and singlet oxygen quantum yields were high in DMF and decreased at higher concentrations of sulfuric acid due to protonation of azomethine nitrogens. The results suggest that the photophysical and photochemical properties of studied compounds may be controlled by changes of pH of medium.

Heavy metal effects on physicochemical properties of non-aggregated azaphthalocyanine derivatives

Two series of peripherally substituted azaphthalocyanines (AzaPcs) containing different transition metals (Al(III), Zn(II), Ga(III), In(III) and Fe(II)) were synthesized and studied for their photophysical properties. As confirmed by UV-vis and 1H NMR analyses, the non-aggregation behavior was effectively induced by the applied bulky peripheral substituents which had no effect on the photophysical properties. Tuning the Q-band position was clearly achievable by using different central heavy metals which have considerable effects on the fluorescence quantum yield and singlet oxygen generation efficiency. This comparative study showed an interesting linear relationship between the former and atomic number of the central metal. The indium containing complexes exhibited the best result due to the heavy metal effect and therefore could be promoted as a potential photosensitizer in photodynamic therapy (PDT) application.

Phenol‐Substituted Tetrapyrazinoporphyrazines: pH‐Dependent Fluorescence in Basic Media

Tetrapyrazinoporphyrazines (TPyzPzs) bearing one, two, four or eight 3,5-di(tert-butyl)-4-hydroxyphenol moieties were synthesized as zinc(II) complexes and metal-free derivatives. The deprotonation of the phenol using tetrabutylammonium hydroxide induced the formation of a strong donor for intramolecular charge transfer that switched OFF the red fluorescence (λF ∼660 nm) of the parent zinc TPyzPzs. The changes were fully reversible for TPyzPzs with one to four phenolic moieties, and an irreversible modification was observed for TPyzPzs substituted with eight phenols. The sensors were anchored to lipophilic particles in water, and a pKa approximately 12.5-12.7 was determined for the phenolic hydroxyl based on fluorescence changes in different buffers. In addition, a novel concept for fluorescence OFF-ON-OFF switching in metal-free TPyzPzs bearing phenolic moieties upon addition of specific amounts of base was demonstrated.