Crystal structure and Hirshfeld surface analysis of bis-(µ-4-tert-but-oxy-4-oxobut-2-en-2-olato)bis-[(4-tert-but-oxy-4-oxobut-2-en-2-olato)ethano-lzinc(II)].

The mol-ecular and crystal structure of the title binuclear Zn2+ complex, [Zn2(C8H13O3)4(C2H5OH)2], with enolated anionic tert-butyl-aceto-acetate and ethanol was analysed. The coordination polyhedra of the Zn atoms are distorted octa-hedra formed by six oxygen atoms that belong to three ligand mol-ecules and a coordinated ethanol mol-ecule. In the crystal phase, alternating layers can be distinguished parallel to the ac plane. A Hirshfeld surface analysis showed that there are no strong inter-molecular inter-actions in the structure. The most significant contributions to the overall crystal packing are from H⋯H inter-molecular contacts.

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.

Trimethine Cyanine Dyes as NA-Sensitive Probes for Visualization of Cell Compartments in Fluorescence Microscopy.

We propose symmetrical cationic trimethine cyanine dyes with ß-substituents in the polymethine chain based on modified benzothiazole and benzoxazole heterocycles as probes for the detection and visualization of live and fixed cells by fluorescence microscopy. The spectral-luminescent properties of trimethine cyanines have been characterized for free dyes and in the presence of nucleic acids (NA) and globular proteins. The studied cyanines are low to moderate fluorescent when free, but in the presence of NA, they show an increase in emission intensity up to 111 times; the most pronounced emission increase was observed for the dyes T-2 in the presence of dsDNA and T-1 with RNA. Spectral methods showed the binding of all dyes to nucleic acids, and different interaction mechanisms have been proposed. The ability to visualize cell components of the studied dyes has been evaluated using different human cell lines (MCF-7, A2780, HeLa, and Hs27). We have shown that all dyes are cell-permeant staining nucleus components, probably RNA-rich nucleoli with background fluorescence in the cytoplasm, except for the dye T-5. The dye T-5 selectively stains some structures in the cytoplasm of MCF-7 and A2780 cells associated with mitochondria or lysosomes. This effect has also been confirmed for the normal type of cell line-human foreskin fibroblasts (Hs27). The costaining of dye T-5 with MitoTracker CMXRos Red demonstrates specificity to mitochondria at a concentration of 0.1 µM. Colocalization analysis has shown signals overlapping of dye T-5 and MitoTracker CMXRos Red (Pearson's Coefficient value = 0.92 ± 0.04). The photostability study shows benzoxazole dyes to be up to ∼7 times more photostable than benzothiazole ones. Moreover, studied benzoxazoles are less cytotoxic at working concentrations than benzothiazoles (67% of cell viability for T-4, T-5 compared to 12% for T-1, and ∼30% for T-2, T-3 after 24 h). Therefore, the benzoxazole T-4 dye is proposed for nucleic acid detection in vitro and intracellular fluorescence imaging of live and fixed cells. In contrast, the benzoxazole dye T-5 is proposed as a good alternative to commercial dyes for mitochondria staining in the green-yellow region of the spectrum.

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.

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.

Novel Hybrid Compound 4-[(E)-2-phenylethenesulfonamido]-N-hydroxybutanamide with Antimetastatic and Cytotoxic Action: Synthesis and Anticancer Screening.

BACKGROUND: One of the most promising strategies to develop multi-targeted anticancer therapeutics is to introduce to the structure of a potential drug two or more pharmacophores (functional groups or structural fragments), which have antiproliferative, proapoptotic or antimetastatic properties acting via different mechanisms. OBJECTIVE: To design, synthesize and perform screening of a novel hybrid anticancer compound. METHOD: A novel hybrid compound 4-[(E)-2-phenylethenesulfonamido]-N-hydroxybutanamide, combining butanehydroxamate and styrenesulfonamide moieties, was designed, synthesized and investigated as a potent antimetastatic and antiproliferative agent. The structure and purity of the synthesized compound were confirmed by 1H NMR, 13C NMR, LC/MS spectroscopy and elemental analysis. The compound was screened for the anticancer activity in vitro against HeLa and in vivo against Lewis lung carcinoma tumor, using an antitumor metalloenzyme inhibitor GM6001 (Ilomastat, Galardin) and Pifithrin-µ as control anticancer agents. RESULTS: It was found that the application of our compound resulted in a high fraction of apoptotic cells in the cell population, along with disruption in the cell cycle profile manifested as arrest of proliferative phases. Furthermore, changes of the morphological properties (i.e., an enhancement of adhesive properties and reduction of the nuclear-to-cytoplasm ratio) were found. The in vivo screening revealed that the compound significantly inhibited the metastasizing process that was manifested by a reduction in the number and volume of metastases. CONCLUSIONS: The obtained results demonstrate that our compound can serve as a base for further structure optimization in order to design new highly-effective antimetastatic and antitumor agents.

Synthesis and crystal structure of N-(4-chloro-phen-yl)-5,7-dimethyl-1,2,4-triazolo[1,5-a]pyrimidin-2-amine.

The title compound, C13H12ClN5, was synthesized by the cyclization of 1-(4,6-di-methyl-pyrimidin-2-yl)-4-phenyl-thio-semicarbazide in the presence of Ni(NO3)2. The mol-ecular structure of the compound is essentially planar. In the crystal, mol-ecules form dimers via pairs of N-H⋯N hydrogen bonds between the H atom of the exocyclic amino group and the N atom at the 4-position of the triazole ring. The resulting dimers are packed into layers which are connected by π-stacking inter-actions between the aromatic systems of the pyrimidine and benzene nuclei, and between the triazole cores.

Crystal structure of hexa-kis-(µ2-4-tert-but-oxy-4-oxobut-2-en-2-olato)trizinc.

The title complex, systematic name hexa-kis-(µ2-4-tert-but-oxy-4-oxobut-2-en-2-olato)-1:2κ(9) O (2),O (4):O (2);2:3κ(9) O (2),O (4):O (2)-trizinc, [Zn3(C8H13O3)6], syn-the-sized from tert-butyl aceto-acetate and di-ethyl-zinc, consists of trinuclear centrosymmetric mol-ecules of an approximate C 3i symmetry. The three metal cations are arranged in a linear fashion, with the central Zn(II) atom located on a centre of symmetry. All three metal cations exhibit a distorted octa-hedral coordination geometry. The terminal Zn(II) cations are chelated by three tert-butyl aceto-acetate ligands and these units are connected to the central Zn(II) atom by the bridging enolate O atoms.

trans-Dichloridobis(4-methoxy-aniline-κN)palladium(II).

In the title compound, [PdCl(2)(C(7)H(9)NO)(2)], the Pd atom is situated on a crystallographic centre of inversion. The coordination environment of the Pd atom shows a slightly distorted square-planar geometry. The crystal structure exhibits weak inter-molecular Pd⋯Cl inter-actions, with Pd⋯Cl distances of 3.6912 (6) Å. A chain-like arrangement of mol-ecules realized by inter-molecular N-H⋯Cl hydrogen bonds is observed along [010].