Inhibition of heat-induced protein aggregation by zirconium phthalocyanines.

Specific proteins found in food sources tend to aggregate into fibrils under heat treatment; studying these aggregation processes and developing tools to control protein heat-induced aggregation is an active area of research. Phthalocyanine complexes are known to exhibit antiprionic and anti-fibrillogenic activity. Thus, the anti-fibrillogenic effect of a series of Zr phthalocyanines with different out-of-plane coordinated ligands, namely positively charged (PcZrLys2 ), negatively charged (PcZrCitr2 ), and group able to form disulfide bridges (PcZrS2 ), on the heat-induced aggregation of such proteins as BLG, insulin, and lysozyme was studied. The inhibition of reaction activity up to about 90% was observed in the presence of these compounds for all proteins. The effective concentration of the inhibitor was calculated for the compound with the highest activity (PcZrS2 ) to be 10.6 ± 3.6 and 7.3 ± 1.2 µM/L, respectively. Fluorescence spectroscopy studies demonstrated similar binding constants of three phthalocyanines binding with BLG globule. This is consistent with the results of molecular dynamics simulation, which imply the interaction of the globule with the tetrapyrrole macrocycle of phthalocyanine, leading to the globule stabilization. At the same time, TEM shows that in the presence of phthalocyanine PcZrS2 , thinner and longer fibrils were formed compared to control in all three proteins (BLG, insulin, and lysozyme). Thus, we can conclude that phthalocyanine PcZrS2 affects the amyloid aggregation's general mechanism, which is typical for proteins of different structures. Therefore, the phthalocyanine PcZrS2 is proposed as an anti-amyloidogenic agent suppressing heat-induced aggregation of proteins of different structures, making it potentially suitable for application in the food industry.

Modification of insulin amyloid aggregation by Zr phthalocyanines functionalized with dehydroacetic acid derivatives.

Amyloid fibrils are widely studied both as target in conformational disorders and as basis for the development of protein-based functional materials. The three Zr phthalocyanines bearing dehydroacetic acid residue (PcZr(L1)2) and its condensed derivatives (PcZr(L2)2 and PcZr(L3)2) as out-of-plane ligands were synthesized and their influence on insulin fibril formation was studied by amyloid-sensitive fluorescent dye based assay, scanning electron microscopy, fluorescent and absorption spectroscopies. The presence of Zr phthalocyanines was shown to modify the fibril formation. The morphology of fibrils formed in the presence of the Zr phthalocyanines differs from that of free insulin and depends on the structure of out-of-plane ligands. It is shown that free insulin mostly forms fibril clusters with the length of about 0.3-2.1 µm. The presence of Zr phthalocyanines leads to the formation of individual 0.4-2.8 µm-long fibrils with a reduced tendency to lateral aggregation and cluster formation (PcZr(L1)2), shorter 0.2-1.5 µm-long fibrils with the tendency to lateral aggregation without clusters (PcZr(L2)2), and fibril-like 0.2-1.0 µm-long structures (PcZr(L3)2). The strongest influence on fibrils morphology made by PcZr(L3)2 could be explained by the additional stacking of phenyl moiety of the ligand with aromatic amino acids in protein. The evidences of binding of studied Zr phthalocyanines to mature fibrils were shown by absorption spectroscopy (for PcZr(L1)2 and PcZr(L2)2) and fluorescent spectroscopy (for PcZr(L3)2). These complexes could be potentially used as external tools allowing the development of functional materials on protein fibrils basis.

Gallato Zirconium (IV) Phtalocyanine Complex Conjugated with SiO2 Nanocarrier as a Photoactive Drug for Photodynamic Therapy of Atheromatic Plaque.

A new conjugate of gallato zirconium (IV) phthalocyanine complexes (PcZrGallate) has been obtained from alkilamino-modified SiO2 nanocarriers (SiO2-(CH2)3-NH2NPs), which may potentially be used in photodynamic therapy of atherosclerosis. Its structure and morphology have been investigated. The photochemical properties of the composite material has been characterized. in saline environments when exposed to different light sources Reactive oxygen species (ROS) generation in DMSO suspension under near IR irradiation was evaluated. The PcZrGallate-SiO2 conjugate has been found to induce a cytotoxic effect on macrophages after IR irradiation, which did not correspond to ROS production. It was found that SiO2 as a carrier helps the photosensitizer to enter into the macrophages, a type of cells that play a key role in the development of atheroma. These properties of the novel conjugate may make it useful in the photodynamic therapy of coronary artery disease.

Synthesis and spectral characterization of the first fluorescein-tagged iron(ii) clathrochelates, their supramolecular interactions with globular proteins, and cellular uptake.

A fluorescein-tagged iron(ii) cage complex was obtained in a moderate total yield using a two-step synthetic procedure starting from its propargylamine-containing clathrochelate precursor. An 11-fold decrease in fluorescence quantum yield is observed in passing from the given fluorescein-based dye to its clathrochelate derivative. An excitation energy transfer from the terminal fluorescent group of the macrobicyclic molecule to its quasiaromatic highly π-conjugated clathrochelate framework can explain this effect. The kinetics of the hydrolysis of the acetyl groups of acetylated fluorescein azide and its clathrochelate derivative in the presence of one equivalent of BSA evidenced no strong supramolecular host-guest interactions between BSA and the tested compounds. Study of a chemical stability of the deacetylated iron(ii) clathrochelate suggested the formation of a supramolecular 1 : 1 BSA-clathrochelate assembly. Moreover, an addition of BSA or HSA to its solution caused the appearance of strong clathrochelate-based ICD outputs. The fluorescence emission anisotropy studies also evidenced the supramolecular binding of the fluorescein-tagged iron(ii) clathrochelate to the BSA macromolecule, leading to a high increase in this type of anisotropy. Subcellular uptake of the fluorescein-tagged molecules was visualized using fluorescence microscopy and showed its distribution to be mainly in the cytosol without entering the nucleus or accumulating in any other organelle. An X-rayed crystal of the above propargylamide macrobicyclic precursor with a reactive terminal C[triple bond, length as m-dash]C bond contains the clathrochelate molecules of two types, A and B. The encapsulated iron(ii) ion in these molecules is situated in the center of its FeN6-coordination polyhedron, the geometry of which is intermediate between a trigonal prism (TP) and a trigonal antiprism (TAP). The Fe-N distances vary from 1.8754(6) to 1.9286(4) Å and the heights h of their distorted TP-TAP polyhedra are very similar (2.30 and 2.31 Å); their values of φ are equal to 25.3 and 26.6°. In this crystal, the molecules of types A and B participate in different types of hydrogen bonding, giving H-bonded clathrochelate tetramers through their carboxylic and amide groups, respectively; these tetramers are connected to H-bonded chains.

Fluorescent ß-ketoenole AmyGreen dye for visualization of amyloid components of bacterial biofilms.

Green-emitting water-soluble amino-ketoenole dye AmyGreen is proposed as an efficient fluorescent stain for visualization of bacterial amyloids in biofilms and the detection of pathological amyloids in vitro. This dye is almost non-fluorescent in solution, displays strong green emission in the presence of amyloid fibril of proteins. AmyGreen is also weakly fluorescent in presence to biomolecules that are components of cells, extracellular matrix or medium: nucleic acids, polysaccharides, lipids, and proteins. Thus, the luminescence turn-on behavior of AmyGreen can be utilized for visualization of amyloid components of bacterial biofilm extracellular matrix. Herein we report the application of AmyGreen for fluorescent staining of a number of amyloid-contained bacteria biofilms produced by Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bordetella avium, and Staphylococcus aureus. The effectiveness of AmyGreen was compared to traditional amyloid sensitive dye Thioflavine T. The main advantage of AmyGreen (concentration 10-5 M) is a higher sensitivity in the visualization of amyloid biofilm components over Thioflavine T (10-4 M) as it was revealed when staining E. coli and K. pneumoniae bacterial biofilms. Besides, AmyGreen displays lower cross-selectivity to nucleic acids as demonstrated both in in-solution experiments and upon staining of eukaryotic human mesenchymal stem cells used as amyloid-free negative control over amyloid-rich bacterial biofilms. The results point to a lower risk of false-positive response upon determination of amyloid components of bacterial biofilm using AmyGreen. Co-staining of biofilm by AmyGreen and cellulose sensitive dye Calcofluor White show difference in their staining patterns and localization, indicating separation of polysaccharide-rich and amyloid-rich regions of investigated biofilms. Thus, we suggest the new AmyGreen stain for visualization and differentiation of amyloid fibrils in bacterial biofilms to be used solely and in combination with other stains for confocal and fluorescence microscopy analysis.

Studies of anti-fibrillogenic activity of phthalocyanines of zirconium containing out-of-plane ligands.

Series of phthalocyanines of zirconium containing lysine, citric, nonanoic acid residues and dibenzolylmethane groups as out-of-plane ligands are firstly studied as inhibitors of fibrillogenesis using cyanine-based fluorescent inhibitory assay. It was shown that studied phthalocyanines at concentration of 20µM inhibited aggregation reaction on 38.5-57.6% and inhibitory activity of phthalocyanines depended on the chemical nature of out-of-plane ligand. For the most active compound PcZrLys(2) (zirconium phthalocyanine containing lysine fragment) the efficient inhibitor concentration was estimated to be 37µM. AFM studies have shown that in the presence of PcZrLys(2) the inhibition of fibrils formation and formation of spherical oligomeric aggregates took place. Due to the ability of phthalocyanines to decrease efficiently protein aggregation into the amyloid fibrils, modification of phthalocyanine molecules via out-of-plane substitutions was proposed as approach for design of anti-fibrillogenic agents with required properties.

Ditopic macropolycyclic complexes: synthesis of hybrid phthalocyaninoclathrochelates.

A remetalation (a capping group exchange) reaction of the boronantimony-capped iron(II) clathrochelates with zirconium and hafnium(IV) phthalocyanines in CH2Cl2/CH3OH medium afforded the hybrid phthalocyaninoclathrochelates in a practically quantitative yield. The complexes obtained have been characterized both on the basis of elemental analysis, PD mass spectrometry, IR, UV-vis, 57Fe Mössbauer, and NMR spectroscopies, and crystallographically. An encapsulated iron(II) ion in an intermediate between a trigonal-prismatic and a trigonal-antiprismatic environment of six nitrogen atoms of the macrobicyclic ligand was found to be in a low-spin state. The cyclic voltammograms show irreversible oxidation and reduction waves assignable to Fe+/Fe2+ couples of macrobicyclic fragments and to phthalocyanine macrocycles.