Kamil Kopecky

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.

Synthesis and comparison of photodynamic activity of alkylheteroatom substituted azaphthalocyanines

Abstract Optimal reaction conditions were developed for synthesis of octakis(butylamino), octakis(butylsulfanyl) and octakis(butoxy) azaphthalocyanines (AzaPc’s) with central metal Mg, Zn and metal-free. Their photodynamic activity was measured and compared as a dye-sensitised photooxidation of 1,3-diphenylisobenzofurane (DPBF). Compounds with alkylamino substituent are very poor producers of the singlet oxygen and therefore not suitable as sensitizers for photodynamic therapy (PDT). On the other hand, compounds with alkylsulfanyl and alkoxy substituents possess very good photodynamic activity and are suitable for PDT.

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.

Synthesis and singlet oxygen production of azaphthalocyanines bearing functional derivatives of carboxylic acid

New metal-free, magnesium and zinc azaphthalocyanines (AzaPc) were prepared from 6-(3-tert-butylsulfany1-5,6-dicyanopyrazine-2-ylamino)hexanoic acid and its butylester and N,N-diethylamide. Their singlet oxygen production was measured by a dye-sensitized photooxidation of 1,3-diphenylisobenzofuran (DPBF) and ΦΔ values were calculated. It was found that the presence of carboxylic acid in the structure of AzaPc decreases their singlet oxygen production. Singlet oxygen activity of the newly prepared compounds (containing mixed alkylamino and alkylsulfanyl peripheral substituents) lies between purely alkylsulfanyl and alkylamino derivatives of AzaPc. This fact further supports previously proposed structure-activity relationships, where alkylsulfanyl derivatives belong to the best singlet oxygen producers while the presence of alkylamino substituents in the structure rapidly decreases their photodynamic properties.

Protonation and deprotonation of nitrogens in tetrapyrazino-porphyrazine macrocycles

Acid-base properties of zinc phthalocyanine (Pc) and zinc and metal-free tetrapyrazinoporphyrazine (TPPz) macrocycles with eight peripheral tert-butylsulfanyl or diethylamino substituents are investigated in this work by means of UV-vis spectroscopy. The Pc and TPPz are protonated on the azomethine nitrogens in acidic media. The monoprotonated form was found using trifluoroacetic acid and lower amounts of H2SO4. The higher concentrations of sulfuric acid lead to the appearance of the diprotonated form, and possibly also the triprotonated form in the case of Pc. It was found that the pyrazine nitrogens in the TPPz macrocycle undergo protonation at approx. 20-30% of sulfuric acid in THF solution. TPPz with diethylamino peripheral substituents are protonated preferably on these peripheral tertiary amines. Metal-free TPPz can readily lose the hydrogens on central nitrogens in strong basic media (e.g. tetrabutylammonium hydroxide) to directly form a dianion, with no presence of important or detectable amounts of the monoanion species. A different form of deprotonated macrocycle was observed in pyridine suggesting the formation of only a proton transfer complex, not a true ionic form. The acidity of the central nitrogens is dependent on the strength of the electron-donating effect of peripheral substituents. TPPz with tert-butylsulfanyl substituents is a stronger acid than the one with diethylamino substituents.

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.

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 aggregation on interaction of lipophilic, water-insoluble azaphthalocyanines with DOPC vesicles.

Dioleoylphosphatidylcholine unilamellar vesicles made by extrusion technique (LUVETs) were studied as the delivery system for lipophilic water‐insoluble potential photosensitizers for photodynamic therapy (PDT). Two azaphthalocyanines (AzaPcs) with hydrophobic substituents only and two also possessing two charged amino groups were introduced into the study. All compounds are insoluble in water and form aggregates in PBS with tetrahydrofuran as cosolvent. The size of these aggregates depends on the concentration of AzaPc in solution. AzaPcs with tert‐butyl substituents were found to be incorporated into the lipid bilayer of vesicles in the monomeric form even at high concentrations. The stability of LUVETs with incorporated AzaPc was excellent for at least 4 weeks. Therefore, they are suitable for use as a delivery system for these water‐insoluble photosensitizers. Very low amount of AzaPc with n‐octyl substituents incorporated into LUVETs due to its stronger self‐aggregation. Values of binding constants determined for all AzaPcs showed inverse order than expected from their lipophilicities. However, the binding constants followed the order of the strength of aggregation forces. Aggregation of AzaPcs in water medium plays a very important role in the interaction of AzaPcs with LUVETs.