Thiacalixarene Carboxylic Acid Derivatives as Inhibitors of Lysozyme Fibrillation.

Amyloid fibroproliferation leads to organ damage and is associated with a number of neurodegenerative diseases affecting populations worldwide. There are several ways to protect against fibril formation, including inhibition. A variety of organic compounds based on molecular recognition of amino acids within the protein have been proposed for the design of such inhibitors. However, the role of macrocyclic compounds, i.e., thiacalix[4]arenes, in inhibiting fibrillation is still almost unknown. In the present work, the use of water-soluble thiacalix[4]arene derivatives for the inhibition of hen egg-white lysozyme (HEWL) amyloid fibrillation is proposed for the first time. The binding of HEWL by the synthesized thiacalix[4]arenes (logKa = 5.05-5.13, 1:1 stoichiometry) leads to the formation of stable supramolecular systems capable of stabilizing the protein structure and protecting against fibrillation by 29-45%. The macrocycle conformation has little effect on protein binding strength, and the native HEWL secondary structure does not change via interaction. The synthesized compounds are non-toxic to the A549 cell line in the range of 0.5-250 µg/mL. The results obtained may be useful for further investigation of the anti-amyloidogenic role of thiacalix[4]arenes, and also open up future prospects for the creation of new ways to prevent neurodegenerative diseases.

Electrochemical DNA Sensor for Valrubicin Detection Based on Poly(Azure C) Films Deposited from Deep Eutectic Solvent.

A novel electrochemical DNA sensor was developed for the detection of the anthracycline drug, valrubicin, on the base of poly(Azure C) electropolymerized from the deep eutectic solvent reline and covered with adsorbed DNA from calf thymus. Biosensor assembling was performed by multiple scanning of the potential in one drop (100 µL) of the dye dissolved in reline and placed on the surface of a screen-printed carbon electrode. Stabilization of the coating was achieved by its polarization in the phosphate buffer. The electrochemical characteristics of the electron transfer were determined and compared with a similar coating obtained from phosphate buffer. The use of deep eutectic solvent made it possible to increase the monomer concentration and avoid using organic solvents on the stage of electrode modification. After the contact of the DNA sensor with valrubicin, two signals related to the intrinsic redox activity of the coating and the drug redox conversion were found on voltammogram. Their synchronous changes with the analyte concentration increased the reliability of the detection. In the square-wave mode, the DNA sensor made it possible to determine from 3 µM to 1 mM (limit of detection, 1 µM) in optimal conditions. The DNA sensor was successfully tested in the voltammetric determination of valrubicin in spiked artificial urine, Ringer-Locke solution mimicking plasma electrolytes and biological samples (urine and saliva) with a recovery of 90-110%. After further testing on clinical samples, it can find application in the pharmacokinetics studies and screening of new drugs' interaction with DNA.

A Hyperbranched Polyol Process for Designing and Manufacturing Nontoxic Cobalt Nanocomposite.

A method for the design and synthesis of a metallopolymer composite (CoNP) based on cobalt nanoparticles using the hyperbranched polyol process was developed. It was shown that hyperbranched polyester polyols in a melted state can be both a reducing agent and a stabilizer of metal nanoparticles at the same time. The mechanism of oxidation of hyperbranched polyol was studied using diffuse reflectance IR spectroscopy. The process of oxidation of OH groups in G4-OH started from 90 °C and finished with the oxidation of aldehyde groups. The composition and properties of nanomaterials were determined with FT-IR and UV-Vis spectroscopy, Nanoparticle Tracking Analysis (NTA), thermogravimetric analysis (TG), powder X-ray diffraction (XRD), NMR relaxation, and in vitro biological tests. The cobalt-containing nanocomposite (CoNP) had a high colloidal stability and contained spheroid polymer aggregates with a diameter of 35-50 nm with immobilized cobalt nanoparticles of 5-7 nm. The values of R2 and R1 according to the NMR relaxation method for CoNPs were 6.77 mM·ms-1 × 10-5 and 4.14 mM·ms-1 × 10-5 for, respectively. The ratio R2/R1 = 0.61 defines the cobalt-containing nanocomposite as a T1 contrast agent. The synthesized CoNPs were nonhemotoxic (HC50 > 8 g/mL) multifunctional reagents and exhibited the properties of synthetic modulators of the enzymatic activity of chymosin aspartic proteinase and exhibited antimycotic activity against Aspergillus fumigatus. The results of the study show the unique prospects of the developed two-component method of the hyperbranched polyol process for the creation of colloidal multifunctional metal-polymer nanocomposites for theranostics.

Biofilm Formation by Mutant Strains of Bacilli under Different Stress Conditions.

Bacillus subtilis is traditionally classified as a PGPR that colonizes plant roots through biofilm formation. The current study focused on investigating the influence of various factors on bacilli biofilm formation. In the course of the study, the levels of biofilm formation by the model strain B. subtilis WT 168 and on its basis created regulatory mutants, as well as strains of bacilli with deleted extracellular proteases under conditions of changes in temperature, pH, salt and oxidative stress and presence of divalent metals ions. B. subtilis 168 forms halotolerant and oxidative stress-resistant biofilms at a temperature range of 22 °C-45 °C and a pH range of 6-8.5. The presence of Ca2+, Mn2+ and Mg2+ upsurges the biofilm development while an inhibition with Zn2+. Biofilm formation level was higher in protease-deficient strains. Relative to the wild-type strain, degU mutants showed a decrease in biofilm formation, abrB mutants formed biofilms more efficiently. spo0A mutants showed a plummeted film formation for the first 36 h, followed by a surge after. The effect of metal ions and NaCl on the mutant biofilms formation is described. Confocal microscopy indicated that B. subtilis mutants and protease-deficient strains differ in matrix structure. The highest content of amyloid-like proteins in mutant biofilms was registered for degU-mutants and protease-deficient strains.

Aggregation, Cytotoxicity and DNA Binding in a Series of Calix[4]arene Amphiphile Containing Aminotriazole Groups.

The present work focuses on the study of the aggregation and complexing properties of calixarenes as potential DNA condensation agents for gene delivery. In the current study, 1,4-triazole derivatives of calix[4]arenes 7 and 8 containing monoammonium fragments were synthesized. The synthesized compound's structure was characterized by using various spectroscopic techniques (FTIR, HRESI MS, ¹H NMR and ¹³C NMR). The interactions between a series of calix[4]arene-containing aminotriazole groups (triazole-containing macrocycles with diethylenetriammonium fragments (3 and 4) and triazole-containing macrocycles with monoammonium fragments (7 and 8)) and calf thymus DNA were carried out via UV absorption, fluorescence spectroscopy, dynamic light scattering and zeta potential measurements. The role of the binding forces of calixarene-DNA complexes was analyzed. Photophysical and morphological studies revealed the interaction of the calixarenes 3, 4 and 8 with ct-DNA, which transformed the fibrous structure of ct-DNA to completely condensed compact structures that are 50 nm in diameter. The cytotoxic properties of calixarenes 3, 4, 7 and 8 against cancerous cells (MCF7, PC-3) as well as a healthy cell line (HSF) were investigated. Compound 4 was found to have the highest toxic effect on MCF7 breast adenocarcinoma (IC50 3.3 µM).

Calix[4]arene Polyamine Triazoles: Synthesis, Aggregation and DNA Binding.

Artificial gene delivery systems are in great demand from both scientific and practical biomedical points of view. In this paper, we present the synthesis of a new click chemistry calix[4]arene precursor with free lower rim and new water-soluble calixarene triazoles with 12 amino-groups on the upper rim (one with free phenol hydroxyl groups and two another containing four butyl or tetradecyl fragments). Aggregation in the series of amino-triazole calixarenes of different lipophilicity (calixarene with free phenol hydroxyl groups or butyl and tetradecyl fragments on the lower rim) was studied using dynamic light scattering and fluorescent pyrene probe. It was found that calix[4]arene with a free lower rim, like alkyl-substituted butyl calix[4]arene, forms stable submicron aggregates 150-200 nm in size, while the more lipophilic tetradecyl -substituted calix[4]arene forms micellar aggregates19 nm in size. Using UV-Vis spectroscopy, fluorimetry and CD, it was shown that amino-triazole calix[4]arenes bind to calf thymus DNA by classical intercalation. According to DLS and TEM data, all studied macrocycles cause significant DNA compaction, forming stable nanoparticles 50-20 nm in size. Among all studied calix[4]arenes the most lipophilic tetradecyl one proved to be the best for both binding and compaction of DNA.

Novel PEPPSI-Type NHC Pd(II) Metallosurfactants on the Base of 1H-Imidazole-4,5-dicarboxylic Acid: Synthesis and Catalysis in Water-Organic Media.

Carrying out organic reactions in water has attracted much attention. Catalytic reactions in water with metallosurfactants, which have both a metallocenter and the surface activity necessary for solubilizing hydrophobic reagents, are of great demand. Herein we proposed new approach to the synthesis of NHC PEPPSI metallosurfactants based on the sequential functionalization of imidazole 4,5-dicarboxylic acid with hydrophilic oligoethylene glycol and lipophilic alkyl fragments. Complexes of different lipophilicity were obtained, and their catalytic activity was studied in model reduction and Suzuki-Miyaura reactions. A comparison was made with the commercial PEPPSI-type catalytic systems designed by Organ. It was found that the reduction reaction in an aqueous solution of the metallosurfactant with the tetradecyl lipophilic fragment was three times more active than the commercially available PEPPSI complexes, which was associated with the formation of stable monodisperse aggregates detected by DLS and TEM.

Synthesis, biological evaluation and structure-activity relationship of 2-(2-hydroxyaryl)alkenylphosphonium salts with potency as anti-MRSA agents.

Here we report the synthesis, in vitro antimicrobial activity, preliminary toxicity and mechanism study of a new series of 2-(2-hydroxyaryl)alkenylphosphonium salts with the variation of phosphonium moiety obtained by a two-step synthetic method from phosphine oxides. The salts showed pronounced activity against Gram-positive bacteria, including MRSA strains, and some fungi. Mechanism of action against S. aureus was studied by CV test, TEM and proteomic assay. No cell wall integrity loss was observed while proteomic assay results suggested interference in different metabolic processes of S. aureus. For this series, lipophilicity was determined as a key factor for the inhibition of Gram-positive bacteria growth and S. aureus killing. Biological properties of methylated derivatives were notably different with manifested action against Gram-negative bacteria.

New Calix[4]arene-Fluoresceine Conjugate by Click Approach-Synthesis and Preparation of Photocatalytically Active Solid Lipid Nanoparticles.

New fluorescent systems for photocatalysis, sensors, labeling, etc., are in great demand. Amphiphilic ones are of special interest since they can form functional colloidal systems that can be used in aqueous solutions. A new macrocycle platform for click chemistry and its adduct with o-propargylfluoresceine was synthesized and characterized using modern physical techniques. Nanosized solid lipid nanoparticles (SLNs) from the calixarene-fluoresceine adduct were synthesized through the solvent injection technique and well-characterized in the solution and in solid state using light-scattering and microscopy methods. The maximum fluorescence intensity of the SLNs was found to be in the pH range from 7 to 10. The Förster resonance energy transfer (FRET) efficiency from SLNs to rhodamine 6g was found to be 97.8%. Finally, pure SLNs and the FRET system SLNs-Rh6G were tested in model photocatalytic ipso oxidative hydroxylation of phenylboronic acid under blue LED light. The SLNs-Rh6G system was found to be the best, giving an almost qualitative phenol yield, which was shown by HPLC-UV analysis.

Oxyethylated Fluoresceine-(thia)calix[4]arene Conjugates: Synthesis and Visible-Light Photoredox Catalysis in Water-Organic Media.

Fluorescent derivatives attract the attention of researchers for their use as sensors, photocatalysts and for the creation of functional materials. In order to create amphiphilic fluorescent derivatives of calixarenes, a fluorescein derivative containing oligoethylene glycol and propargyl groups was obtained. The resulting fluorescein derivative was introduced into three different (thia)calix[4]arene azide derivatives. For all synthesized compounds, the luminescence quantum yields have been established in different solvents. Using UV-visible spectroscopy, dynamic light scattering, as well as transmission and confocal microscopy, aggregation of macrocycles was studied. It was evaluated that calixarene derivatives with alkyl substituents form spherical aggregates, while symmetrical tetrafluorescein-containing thiacalix[4]arene forms extended worm-like aggregates. The macrocycle containing tetradecyl fragments was found to be the most efficient in photoredox ipso-oxidation of phenylboronic acid. In addition, it was shown that in a number of different electron donors (NEt3, DABCO and iPr2EtN), the photoredox ipso-oxidation proceeds best with triethylamine. It has been shown that a low molecular weight surfactant Triton-X100 can also improve the photocatalytic abilities of an oligoethylene glycol fluorescein derivative, thus showing the importance of a combination of micellar and photoredox catalysis.

NHC Polymeric Particles Obtained by Self-Assembly and Click Approach of Calix[4]Arene Amphiphiles as Support for Catalytically Active Pd Nanoclusters.

A new polymeric NHC carrier was synthesized by sequential supramolecular self-assembly and copper-catalyzed azide-alkyne cycloaddition (CuAAC) of amphiphilic imidazolium calix[4]arenes with octyl lipophilic fragments. Obtained polytriazole-imidazolium particles were found as monodisperse submicron particles, with the average diameter of 236 ± 34 nm and average molecular weight of 1380 ± 96 kDa. Successful CuAAC polymerization has been proved using IR spectroscopy and high-resolution ESI mass spectrometry. Polymeric particles, as well as aggregates made from precursor macrocycles, were decorated by Pd clusters (2 nm) for further catalytic investigations. Pd nanoclusters, supported on the polymeric surface, were found highly catalytically active in the model reduction of p-nitrophenol, giving reaction rates an order of magnitude higher compared to literature examples. The reaction was recycled using the same catalyst five times without any loss of activity.

Electrochemical DNA Sensor Based on Poly(Azure A) Obtained from the Buffer Saturated with Chloroform.

Electropolymerized redox polymers offer broad opportunities in detection of biospecific interactions of DNA. In this work, Azure A was electrochemically polymerized by multiple cycling of the potential in phosphate buffer saturated with chloroform and applied for discrimination of the DNA damage. The influence of organic solvent on electrochemical properties of the coating was quantified and conditions for implementation of DNA in the growing polymer film were assessed using cyclic voltammetry, quartz crystal microbalance, and electrochemical impedance spectroscopy. As shown, both chloroform and DNA affected the morphology of the polymer surface and electropolymerization efficiency. The electrochemical DNA sensor developed made it possible to distinguish native and thermally and chemically damaged DNA by changes in the charge transfer resistance and capacitance.

The ABC-Type Efflux Pump MacAB Is Involved in Protection of Serratia marcescens against Aminoglycoside Antibiotics, Polymyxins, and Oxidative Stress.

Serratia marcescens is an emerging pathogen with increasing clinical importance due to its intrinsic resistance to several classes of antibiotics. The chromosomally encoded drug efflux pumps contribute to antibiotic resistance and represent a major challenge for the treatment of bacterial infections. The ABC-type efflux pump MacAB was previously linked to macrolide resistance in Escherichia coli and Salmonella enterica serovar Typhimurium. The role of the MacAB homolog in antibiotic resistance of S. marcescens is currently unknown. We found that an S. marcescens mutant lacking the MacAB pump did not show increased sensitivity to the macrolide antibiotic erythromycin but was significantly more sensitive to aminoglycoside antibiotics and polymyxins. We also showed that, in addition to its role in drug efflux, the MacAB efflux pump is required for swimming motility and biofilm formation. We propose that the motility defect of the ΔmacAB mutant is due, at least in part, to the loss of functional flagella on the bacterial surface. Furthermore, we found that the promoter of the MacAB efflux pump was active during the initial hours of growth in laboratory medium and that its activity was further elevated in the presence of hydrogen peroxide. Finally, we demonstrate a complete loss of ΔmacAB mutant viability in the presence of peroxide, which is fully restored by complementation. Thus, the S. marcescens MacAB efflux pump is essential for survival during oxidative stress and is involved in protection from polymyxins and aminoglycoside antibiotics.IMPORTANCE The opportunistic pathogen Serratia marcescens can cause urinary tract infections, respiratory infections, meningitis, and sepsis in immunocompromised individuals. These infections are challenging to treat due to the intrinsic resistance of S. marcescens to an extensive array of antibiotics. Efflux pumps play a crucial role in protection of bacteria from antimicrobials. The MacAB efflux pump, previously linked to efflux of macrolides in Escherichia coli and protection from oxidative stress in Salmonella enterica serovar Typhimurium, is not characterized in S. marcescens We show the role of the MacAB efflux pump in S. marcescens protection from aminoglycoside antibiotics and polymyxins, modulation of bacterial motility, and biofilm formation, and we illustrate the essential role for this pump in bacterial survival during oxidative stress. Our findings make the MacAB efflux pump an attractive target for inhibition to gain control over S. marcescens infections.

Metallo-Supramolecular Coordination Polymers Based on Amidopyridine Derivatives of Pillar[5]arene and Cu(II) and Pd(II) Cations: Synthesis and Recognition of Nitroaromatic Compounds.

Decasubstituted pillar[5]arenes containing amidopyridine fragments have been synthesized for the first time. As was shown by UV-vis spectroscopy, the pillar[5]arenes with p-amidopyridine fragments form supramolecular associates with Cu(II) and Pd(II) cations in methanol in a 2:1 ratio. Using a sol-gel approach these associates are transformed into metallo-supramolecular coordination polymers (supramolecular gels) which were characterized as amorphous powders by scanning electron microscopy (SEM) and dynamic light scattering (DLS). The powders are able to selectively adsorb up to 46% of nitrophenols from water and were incorporated into an electrochemical sensor to selectively recognize them in aqueous acidic solution.

Facile Fabrication of Natural Polyelectrolyte-Nanoclay Composites: Halloysite Nanotubes, Nucleotides and DNA Study.

Complexation of biopolymers with halloysite nanotubes (HNTs) can greatly affect their applicability as materials building blocks. Here we have performed a systematic investigation of fabrication of halloysite nanotubes complexes with nucleotides and genomic DNA. The binding of DNA and various nucleotide species (polyAU, UMP Na2, ADP Na3, dATP Na, AMP, uridine, ATP Mg) by halloysite nanotubes was tested using UV-spectroscopy. The study revealed that binding of different nucleotides to the nanoclay varied but was low both in the presence and absence of MgCl2, while MgCl2 facilitated significantly the binding of longer molecules such as DNA and polyAU. Modification of the nanotubes with DNA and nucleotide species was further confirmed by measurements of ζ-potentials. DNA-Mg-modified nanotubes were characterized using transmission electron (TEM), atomic force (AFM) and hyperspectral microscopies. Thermogravimetric analysis corroborated the sorption of DNA by the nanotubes, and the presence of DNA on the nanotube surface was indicated by changes in the surface adhesion force measured by AFM. DNA bound by halloysite in the presence of MgCl2 could be partially released after addition of phosphate buffered saline. DNA binding and release from halloysite nanotubes was tested in the range of MgCl2 concentrations (10-100 mM). Even low MgCl2 concentrations significantly increased DNA sorption to halloysite, and the binding was leveled off at about 60 mM. DNA-Mg-modified halloysite nanotubes were used for obtaining a regular pattern on a glass surface by evaporation induced self-assembly process. The obtained spiral-like pattern was highly stable and resisted dissolution after water addition. Our results encompassing modification of non-toxic clay nanotubes with a natural polyanion DNA will find applications for construction of gene delivery vehicles and for halloysite self-assembly on various surfaces (such as skin or hair).

Nanoparticles based on the zwitterionic pillar[5]arene and Ag+: synthesis, self-assembly and cytotoxicity in the human lung cancer cell line A549.

For the first time, stable pillar[5]arene/Ag+ nanoparticles, consisting of water-soluble pillar[5]arene containing γ-sulfobetaine fragments and Ag+ ions without Ag-Ag bonds, were synthesized and characterized. The pillar[5]arene/Ag+ (ratio 1:10) nanoparticles obtained were cubic with a rib length of 100 nm and are less cytotoxic than Ag+ ions. The survival of the A549 model cells in the presence of pillar[5]arene/Ag+ (1:10) nanoparticles at a concentration of 30 and 40 µM was 76% and 55%, while in the absence of pillar[5]arene, the cell survival for free Ag+ ions at the same concentration was 30% and 10%, respectively. The results can be used to create new antibacterial materials and 2D biomedical coatings.

New poly-imidazolium-triazole particles by CuAAC cross-linking of calix[4]arene bis-azide/alkyne amphiphiles - a prospective support for Pd in the Mizoroki-Heck reaction.

A new imidazolium amphiphilic calix[4]arene with terminal acetylene fragments in the polar region was synthesized according to a two step scheme including regioselective chloromethylation of distal di-O-butyl calix[4]arene and subsequent interaction with 1-(hex-5-yn-1-yl)-1H-imidazole. The aggregation properties (CAC, the size and zeta potential of aggregates) of alkynyl calix[4]arene as well as of previously synthesized azidopropyl calix[4]arene and their 1 : 1 mixture were disclosed. Macrocycles with azide and alkyne fragments in the polar region were covalently cross-linked under CuAAC conditions in water. Successful cross-linking of molecules has been proven by IR spectroscopy and MALDI-TOF spectrometry. The obtained polymeric particles were studied both in solution and the solid state and the presence of submicron (∼200 nm) and micron (∼1-5 µm) particles with the prevalence of the latter was found. The average molecular weight of the polymer according to the static light scattering data was found to be 639 ± 44 kDa. The obtained polymeric imidazolium-triazole particles were tested as a support for Pd(OAc)2 in the Mizoroki-Heck reaction carried out in both organic and water media. In both solvents (especially in water) the addition of imidazolium-triazole particles to Pd(OAc)2 increased the conversion of 4-iodanisole. It was found that the ratio between the products (1,1 and 1,2-substituted ethylenes) changes drastically on going from DMF to water from 1 : 5 to 1 : 40 when using supported Pd(OAc)2.

Water-soluble betaines and amines based on thiacalix[4]arene scaffold as new cholinesterase inhibitors.

Novel ammonium and betaine derivatives of p-tert-butylthiacalix[4]arene in cone and 1,3-alternate conformation were synthesized with high yields for the first time. The obtained compounds form in water spherical nanoparticles. It was shown by molecular docking calculations and in vitro experiments that amino and betaine derivatives can inhibit acetylcholinesterase and butyrylcholinesterase on the level of pyridostigmine while the toxicity of the obtained compounds is much lower than that of pyridostigmine.

Nanohydrogel Formation within the Halloysite Lumen for Triggered and Sustained Release.

An easy strategy to obtain nanohydrogels within the halloysite nanotube (HNTs) lumen was investigated. Inorganic reverse micelles based on HNTs and hexadecyltrimethylammonium bromides were dispersed in chloroform, and the hydrophilic cavity was used as a nanoreactor to confine the gel formation based on alginate cross-linked by calcium ions. Spectroscopy and electron microscopy experiments proved the confinement of the polymer into the HNT lumen and the formation of calcium-mediated networks. Biological tests proved the biocompatibility of the hybrid hydrogel. The nanogel in HNTs was suitable for drug loading and sustained release with the opportunity of triggered burst release by chemical stimuli. Here, we propose a new strategy based on inorganic reverse micelles for nanohydrogel formation, which are suitable for industrial and biological applications as well as for selective and triggered adsorption and/or release.

Self-assembly of chiral fluorescent nanoparticles based on water-soluble L-tryptophan derivatives of p-tert-butylthiacalix[4]arene.

New water-soluble tetra-substituted derivatives of p-tert-butylthiacalix[4]arene containing fragments of L-tryptophan in cone and 1,3-alternate conformations were obtained. It was shown that the resulting compounds form stable, positively charged aggregates of 86-134 nm in diameter in water at a concentration of 1 × 10-4 M as confirmed by dynamic light scattering, scanning electron microscopy and transmission electron microscopy. It was established that these aggregates are fluorescently active and chiral. A distinctive feature of the compounds is the pronounced dependence of their spectral (emission and chiroptical) properties on the polarity of the solvent and the length of the linker between the macrocyclic and fluorophore parts of the molecule.