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.

Flow-Through Amperometric Biosensor System Based on Functionalized Aryl Derivative of Phenothiazine and PAMAM-Calix-Dendrimers for the Determination of Uric Acid.

A flow-through biosensor system for the determination of uric acid was developed on the platform of flow-through electrochemical cell manufactured by 3D printing from poly(lactic acid) and equipped with a modified screen-printed graphite electrode (SPE). Uricase was immobilized to the inner surface of a replaceable reactor chamber. Its working volume was reduced to 10 µL against a previously reported similar cell. SPE was modified independently of the enzyme reactor with carbon black, pillar[5]arene, poly(amidoamine) dendrimers based on the p-tert-butylthiacalix[4]arene (PAMAM-calix-dendrimers) platform and electropolymerized 3,7-bis(4-aminophenylamino) phenothiazin-5-ium chloride. Introduction of the PAMAM-calix-dendrimers into the electrode coating led to a fivefold increase in the redox currents of the electroactive polymer. It was found that higher generations of the PAMAM-calix-dendrimers led to a greater increase in the currents measured. Coatings consisted of products of the electropolymerization of the phenothiazine with implemented pillar[5]arene and PAMAM-calix-dendrimers showing high efficiency in the electrochemical reduction of hydrogen peroxide that was formed in the enzymatic oxidation of uric acid. The presence of PAMAM-calix-dendrimer G2 in the coating increased the redox signal related to the uric acid assay by more than 1.5 times. The biosensor system was successfully applied for the enzymatic determination of uric acid in chronoamperometric mode. The following optimal parameters for the chronoamperometric determination of uric acid in flow-through conditions were established: pH 8.0, flow rate 0.2 mL·min-1, 5 U of uricase per reactor. Under these conditions, the biosensor system made it possible to determine from 10 nM to 20 µM of uric acid with the limit of detection (LOD) of 4 nM. Glucose (up to 1 mM), dopamine (up to 0.5 mM), and ascorbic acid (up to 50 µM) did not affect the signal of the biosensor toward uric acid. The biosensor was tested on spiked artificial urine samples, and showed 101% recovery for tenfold diluted samples. The ease of assembly of the flow cell and the low cost of the replacement parts make for a promising future application of the biosensor system in routine clinical analyses.

Romanowsky staining: history, recent advances and future prospects from a chemistry perspective.

Romanowsky staining was an important methodological breakthrough in diagnostic hematology and cytopathology during the late 19th and early 20th centuries; it has facilitated for decades the work of biologists, hematologists and pathologists working with blood cells. Despite more than a century of studying Romanowsky staining, no systematic review has been published that explains the chemical processes that produce the "Romanowsky effect" or "Romanowsky-Giemsa effect" (RGE), i.e., a purple coloration arising from the interaction of an azure dye with eosin and not due merely to their simultaneous presence. Our review is an attempt to build a bridge between chemists and biomedical scientists and to summarize the available data on methylene blue (MB) demethylation as well as the related reduction and decomposition of MB to simpler compounds by both light and enzyme systems and microorganisms. To do this, we analyze modern data on the mechanisms of MB demethylation both in the presence of acids and bases and by disproportionation due to the action of light. We also offer an explanation for why the RGE occurs only when azure B, or to a lesser extent, azure A is present by applying experimental and calculated physicochemical parameters including dye-DNA binding constants and electron density distributions in the molecules of these ligands. Finally, we discuss modern techniques for obtaining new varieties of Romanowsky dyes by modifying previously known ones. We hope that our critical literature study will help scientists understand better the chemical and physicochemical processes and mechanisms of cell staining with such dyes.

Editorial of Special Issue "Functional Nanomaterials Based on Self-Assembly".

In recent years, the design and creation of new functional nanosystems and nanomaterials similar in their properties to biological systems showed remarkable progress as an interdisciplinary field of research combining chemistry, biology, and physics [...].

Towards Antibacterial Agents: Synthesis and Biological Activity of Multivalent Amide Derivatives of Thiacalix[4]arene with Hydroxyl and Amine Groups.

Antimicrobial resistance to modern antibiotics stimulates the search for new ways to synthesize and modify antimicrobial drugs. The development of synthetic approaches that can easily change different fragments of the molecule is a promising solution to this problem. In this work, a synthetic approach was developed to obtain multivalent thiacalix[4]arene derivatives containing different number of amine and hydroxyl groups. A series of macrocyclic compounds in cone, partial cone, and 1,3-alternate stereoisomeric forms containing -NHCH2CH2R (R = NH2, N(CH3)2, and OH) and -N(CH2CH2OH)2 terminal fragments, and their model non-macrocyclic analogues were obtained. The antibacterial activity against Gram-positive (Staphylococcus aureus, Bacillus cereus, and Enterococcus faecalis) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacterial strains and cytotoxicity of the obtained compounds were studied. Structure-activity relationships were established: (1) the macrocyclic compounds had high antibacterial activity, while the monomeric compounds had low activity; (2) the compounds in cone and partial cone conformations had better antibacterial activity compared to the compounds in 1,3-alternate stereoisomeric form; (3) the macrocyclic compounds containing -NHCH2CH2N(CH3)2 terminal fragments had the highest antibacterial activity; (4) introduction of additional terminal hydroxyl groups led to a significant decrease in antibacterial activity; (5) the compounds in partial cone conformation had significant bactericidal activity against all studied cell strains; the best selectivity was observed for the compounds in cone conformation. The mechanism of antibacterial activity of lead compounds with terminal fragments -NHCH2CH2N(CH3)2 was proved using model negatively charged POPG vesicles, i.e., the addition of these compounds led to an increase in the size and zeta potential of the vesicles. The obtained results open up the possibility of using the synthesized macrocyclic compounds as promising antibacterial agents.

Peptidomimetics based on ammonium decasubstituted pillar[5]arenes: Influence of the alpha-amino acid residue nature on cholinesterase inhibition.

Cholinesterase inhibitors are a group of medicines that are widely used for the treatment of cognitive impairments accompanying Alzheimer's disease as well as for the treatment of pathological muscle weaknesses syndromes such as myasthenia gravis. The search for novel non-toxic and effective cholinesterase inhibitors for creating neuroprotective and neurotransmitter agents is an urgent interdisciplinary problem. For the first time, the application of water-soluble pillar[5]arenes containing amino acid residues as effective cholinesterase inhibitors was shown. The influence of the nature of aliphatic and aromatic alpha-amino acid residues (glycine, l-alanine, l-phenylalanine and l-tryptophan) on self-assembly, aggregate's stability, cytotoxicity on A549 and LEK cells and cholinesterase inhibition was studied. It was found that the studied compounds with aliphatic amino acid residues showed a low inhibitory ability against cholinesterases. It was established that the pillar[5]arene containing fragments of l-phenylalanine is the most promising inhibitor of butyrylcholinesterase (IC50 = 0.32 ± 0.01 µM), the pillar[5]arene with l-tryptophan residues is the most promising inhibitor of acetylcholinesterase (IC50 = 0.32 ± 0.01 µM). This study has shown a possible application of peptidomimetics based on pillar[5]arenes to inhibit cholinesterase, as well as control the binding affinity to a particular enzyme in a structure-dependent manner.

Voltammetric Sensor for Doxorubicin Determination Based on Self-Assembled DNA-Polyphenothiazine Composite.

A novel voltammetric sensor based on a self-assembled composite formed by native DNA and electropolymerized N-phenyl-3-(phenylimino)-3H-phenothiazin-7-amine has been developed and applied for sensitive determination of doxorubicin, an anthracycline drug applied for cancer therapy. For this purpose, a monomeric phenothiazine derivative has been deposited on the glassy carbon electrode from the 0.4 M H2SO4-acetone mixture (1:1 v/v) by multiple potential cycling. The DNA aliquot was either on the electrode modified with electropolymerized film or added to the reaction medium prior to electropolymerization. The DNA entrapment and its influence on the redox behavior of the underlying layer were studied by scanning electron microscopy and electrochemical impedance spectroscopy. The DNA-doxorubicin interactions affected the charge distribution in the surface layer and, hence, altered the redox equilibrium of the polyphenothiazine coating. The voltametric signal was successfully applied for the determination of doxorubicin in the concentration range from 10 pM to 0.2 mM (limit of detection 5 pM). The DNA sensor was tested on spiked artificial plasma samples and two commercial medications (recovery of 90-95%). After further testing on real clinical samples, the electrochemical DNA sensor developed can find application in monitoring drug release and screening new antitumor drugs able to intercalate DNA.

Electrochemical DNA-Sensor Based on Macrocyclic Dendrimers with Terminal Amino Groups and Carbon Nanomaterials.

The assembling of thiacalix[4]arene-based dendrimers in cone, partial cone, and 1,3-alternate configuration on the surface of a glassy carbon electrode coated with carbon black or multiwalled carbon nanotubes has been characterized using cyclic voltammetry, electrochemical impedance spectroscopy, and scanning electron microscopy. Native and damaged DNA were electrostatically accumulated on the modifier layer. The influence of the charge of the redox indicator and of the macrocycle/DNA ratio was quantified and the roles of the electrostatic interactions and of the diffusional transfer of the redox indicator to the electrode interface indicator access were established. The developed DNA sensors were tested on discrimination of native, thermally denatured, and chemically damaged DNA and on the determination of doxorubicin as the model intercalator. The limit of detection of doxorubicin established for the biosensor based on multi-walled carbon nanotubes was equal to 1.0 pM with recovery from spiked human serum of 105-120%. After further optimization of the assembling directed towards the stabilization of the signal, the developed DNA sensors can find application in the preliminary screening of antitumor drugs and thermal damage of DNA. They can also be applied for testing potential drug/DNA nanocontainers as future delivery systems.

Impedimetric DNA Sensor Based on Electropolymerized N-Phenylaminophenothiazine and Thiacalix[4]arene Tetraacids for Doxorubicin Determination.

Electrochemical DNA sensors are highly demanded for fast and reliable determination of antitumor drugs and chemotherapy monitoring. In this work, an impedimetric DNA sensor has been developed on the base of a phenylamino derivative of phenothiazine (PhTz). A glassy carbon electrode was covered with electrodeposited product of PhTz oxidation obtained through multiple scans of the potential. The addition of thiacalix[4]arene derivatives bearing four terminal carboxylic groups in the substituents of the lower rim improved the conditions of electropolymerization and affected the performance of the electrochemical sensor depending on the configuration of the macrocyclic core and molar ratio with PhTz molecules in the reaction medium. Following that, the deposition of DNA by physical adsorption was confirmed by atomic force microscopy and electrochemical impedance spectroscopy. The redox properties of the surface layer obtained changed the electron transfer resistance in the presence of doxorubicin due to its intercalating DNA helix and influencing charge distribution on the electrode interface. This made it possible to determine 3 pM-1 nM doxorubicin in 20 min incubation (limit of detection 1.0 pM). The DNA sensor developed was tested on a bovine serum protein solution, Ringer-Locke's solution mimicking plasma electrolytes and commercial medication (doxorubicin-LANS) and showed a satisfactory recovery rate of 90-105%. The sensor could find applications in pharmacy and medical diagnostics for the assessment of drugs able to specifically bind to DNA.

Towards Protection of Nucleic Acids from Herbicide Attack: Self-Assembly of Betaines Based on Pillar[5]arene with Glyphosate and DNA.

Herbicides are one of the main parts of pesticides used today. Due to the high efficiency and widespread use of glyphosate-based herbicides, the search for substances reducing their genotoxicity is an important interdisciplinary task. One possible approach for solving the problem of herbicide toxicity is to use compounds that can protect DNA from damage by glyphosate derivatives. For the first time, a method for developing DNA-protecting measures against glyphosate isopropylamine salt (GIS) damage was presented and realized, based on low-toxicity water-soluble pillar[5]arene derivatives. Two- and three-component systems based on pillar[5]arene derivatives, GIS, and model DNA from salmon sperm, as well as their cytotoxicity, were studied. The synthesized pillar[5]arene derivatives do not interact with GIS, while GIS is able to bind DNA from salmon sperm with lgKa = 4.92. The pillar[5]arene betaine derivative containing fragments of L-phenylalanine and the ester derivative with diglycine fragments bind DNA with lgKa = 5.24 and lgKa = 4.88, respectively. The study of the associates (pillar[5]arene-DNA) with GIS showed that the interaction of GIS with DNA is inhibited only by the betaine pillar[5]arene containing fragments of L-Phe (lgKa = 3.60). This study has shown a possible application of betaine pillar[5]arene derivatives for nucleic acid protection according to its competitive binding with biomacromolecules.

PAMAM-Calix-Dendrimers: Second Generation Synthesis, Fluorescent Properties and Catecholamines Binding.

A convenient method for the synthesis of the second generation of PAMAM dendrimers based on a p-tert-butylthiacalix[4]arene core in cone, partial cone and 1,3-alternate conformations was developed. Unusual fluorescence of the obtained PAMAM-calix-dendrimers has been found and explained. The binding ability of the synthesized dendrimers toward catecholamines (dopamine, L-adrenaline and L-noradrenaline) was shown by UV-Vis, fluorescence, 1D and 2D NMR spectroscopy and the binding constants (logKa 3.85-4.74) calculated. As was shown, the PAMAM-calix-dendrimers bind catecholamines by the internal cavities. All the studied hormones were most efficiently bound by the dendrimers bearing a macrocyclic core in 1,3-alternate conformation. The size of the formed supramolecular systems of dendrimer/catecholamine was established by the DLS method. A decrease in hemolytic activity of the PAMAM-calix-dendrimers with an increase in the generation number of a dendrimer was shown for the dendrimers with a core in 1,3-alternate conformation. The prospects for the use of the synthesized dendrimers with the macrocyclic core as drug delivery agents were discussed.

Macrocyclic Ionic Liquids with Amino Acid Residues: Synthesis and Influence of Thiacalix[4]arene Conformation on Thermal Stability.

Novel thiacalix[4]arene based ammonium ionic liquids (ILs) containing amino acid residues (glycine and L-phenylalanine) in cone, partial cone, and 1,3-alternate conformations were synthesized by alkylation of macrocyclic tertiary amines with N-bromoacetyl-amino acids ethyl ester followed by replacing bromide anions with bis(trifluoromethylsulfonyl)imide ions. The melting temperature of the obtained ILs was found in the range of 50−75 °C. The effect of macrocyclic core conformation on the synthesized ILs' melting points was shown, i.e., the ILs in partial cone conformation have the lowest melting points. Thermal stability of the obtained macrocyclic ILs was determined via thermogravimetry and differential scanning calorimetry. The onset of decomposition of the synthesized compounds was established at 305−327 °C. The compounds with L-phenylalanine residues are less thermally stable by 3−19 °C than the same glycine-containing derivatives.

Fluorescein-Labeled Thiacalix[4]arenes as Potential Theranostic Molecules: Synthesis, Self-Association, and Antitumor Activity.

In this paper, a series of thiacalix[4]arenes were synthesized as potential theranostic molecules for antitumor therapy. We propose an original strategy for the regioselective functionalization of thiacalix[4]arene with a fluorescent label to obtain antiangiogenic agent mimetics. The aggregation properties of the synthesized compounds were determined using the dynamic light scattering. The average hydrodynamic diameter of self-associates formed by the macrocycles in 1,3-alternate conformation is larger (277-323 nm) than that of the similar macrocycle in cone conformation (185-262 nm). The cytotoxic action mechanism of the obtained compounds and their ability to penetrate into of human lung adenocarcinoma and human duodenal adenocarcinoma cells were established using the MTT-test and flow cytometry. Thiacalix[4]arenes in 1,3-alternate conformation did not have a strong toxic effect. The toxicity of macrocycles in cone conformations on HuTu-80 and A549 cells (IC50 = 21.83-49.11 µg/mL) is shown. The resulting macrocycles are potential theranostic molecules that combine both the pharmacophore fragment for neoplasmas treatment and the fluorescent fragment for monitoring the delivery and biodistribution of nanomedicines.

Albumin/Thiacalix[4]arene Nanoparticles as Potential Therapeutic Systems: Role of the Macrocycle for Stabilization of Monomeric Protein and Self-Assembly with Ciprofloxacin.

The therapeutic application of serum albumin is determined by the relative content of the monomeric form compared to dimers, tetramers, hexamers, etc. In this paper, we propose and develop an approach to synthesize the cone stereoisomer of p-tert-butylthiacalix[4]arene with sulfobetaine fragments stabilization of monomeric bovine serum albumin and preventing aggregation. Spectral methods (UV-vis, CD, fluorescent spectroscopy, and dynamic light scattering) established the influence of the synthesized compounds on the content of monomeric and aggregated forms of BSA even without the formation of stable thiacalixarene/protein associates. The effect of thiacalixarenes on the efficiency of protein binding with the antibiotic ciprofloxacin was shown by fluorescence spectroscopy. The binding constant increases in the presence of the macrocycles, likely due to the stabilization of monomeric forms of BSA. Our study clearly shows the potential of this macrocycle design as a platform for the development of the fundamentally new approaches for preventing aggregation.

Towards Asymmetrical Methylene Blue Analogues: Synthesis and Reactivity of 3-N'-Arylaminophenothiazines.

The search for new ways to obtain analogues of the well-known Methylene Blue dye is an important synthetic task. Herein, we proposed and developed an approach to the synthesis of 3-N'-arylaminophenothiazines and asymmetrical 3,7-di(N'-arylamino)phenothiazines. This approach included the optimization of synthetic strategy by quantification analysis of the positive charge distribution in the cation of 3-N'-arylaminophenothiazine derivative. The obtained experimental data are confirmed by DFT studies. Two synthetic routes for asymmetrical phenothiazine diarylamino derivatives were suggested and verified. The developed convenient and versatile synthetic approach makes it easy to obtain aromatic Methylene Blue isostructural analogues with various substituents. As a result, a series of novel 3-N'-arylaminophenothiazines and asymmetrical 3,7-di(N'-arylamino)phenothiazines containing ester, tert-butoxycarbonyl, sulfonic acid, hydroxyl and amine groups were obtained in high yields.

Electrochemical DNA Sensor Based on Carbon Black-Poly(Methylene Blue)-Poly(Neutral Red) Composite.

The detection of small molecules interacting with DNA is important for the assessment of potential hazards related to the application of rather toxic antitumor drugs, and for distinguishing the factors related to thermal and oxidative DNA damage. In this work, a novel electrochemical DNA sensor has been proposed for the determination of antitumor drugs. For DNA sensor assembling, a glassy carbon electrode was modified with carbon black dispersed in DMF. After that, pillar [5]arene was adsorbed and Methylene blue and Neutral red were consecutively electropolymerized onto the carbon black layer. To increase sensitivity of intercalator detection, DNA was first mixed with water-soluble thiacalixarene bearing quaternary ammonium groups in the substituents at the lower rim. The deposition of the mixture on the electropolymerized dyes made it possible to detect doxorubicin as model intercalator by suppression of the redox activity of the polymerization products. The DNA sensor made it possible to determine 0.5 pM-1.0 nM doxorubicin (limit of detection 0.13 pM) with 20 min of incubation. The DNA sensor was successfully tested on spiked samples of human plasma and doxorubicin medication.

Design of Micro- and Nanoparticles: Self-Assembly and Application.

The modern world throws down an increasing number of challenges to humanity [...].

Structure-Activity Relationship of the Thiacalix[4]arenes Family with Sulfobetaine Fragments: Self-Assembly and Cytotoxic Effect against Cancer Cell Lines.

Regulating the structure of macrocyclic host molecules and supramolecular assemblies is crucial because the structure-activity relationship often plays a role in governing the properties of these systems. Herein, we propose and develop an approach to the synthesis of the family of sulfobetaine functionalized thiacalix[4]arenes with regulation of the self-assembly and cytotoxic effect against cancer cell lines. The dynamic light scattering method showed that the synthesized macrocycles in cone, partial cone and 1,3-alternate conformations form submicron-sized particles with Ag+ in water, but the particle size and polydispersity of the systems studied depend on the macrocycle conformation. Based on the results obtained by 1H and 1H-1H NOESY NMR spectroscopy and transmission electron microscopy for the macrocycles and their aggregates with Ag+, a coordination scheme for the Ag+ and different conformations of p-tert-butylthiacalix[4]arene functionalized with sulfobetaine fragments was proposed. The type of coordination determines the different shapes of the associates. Cytotoxic properties are shown to be controlled by the shape of associates, with the highest activity demonstrated by thiacalix[4]arenes in partial cone conformation. This complex partial cone/Ag+ is two times higher than the reference drug imatinib mesylate. High selectivity against cervical carcinoma cell line indicates the prospect of their using as components of new anticancer system.

PAMAM-calix-dendrimers: Synthesis and Thiacalixarene Conformation Effect on DNA Binding.

A convenient method for the synthesis of the first generation PAMAM dendrimers based on the thiacalix[4]arene has been developed for the first time. Three new PAMAM-calix-dendrimers with the macrocyclic core in cone, partial cone, and 1,3-alternate conformations were obtained with high yields. The interaction of the obtained compounds with salmon sperm DNA resulted in the formation of the associates of the size up to 200 nm, as shown by the UV-Vis spectroscopy, DLS, and TEM. It was demonstrated by the CD method that the structure of the DNA did not undergo significant changes upon binding. The PAMAM-calix-dendrimer based on the macrocycle in cone conformation stabilized DNA and prevented its degradation.

Supramolecular Amphiphiles Based on Pillar[5]arene and Meroterpenoids: Synthesis, Self-Association and Interaction with Floxuridine.

In recent years, meroterpenoids have found wide biomedical application due to their synthetic availability, low toxicity, and biocompatibility. However, these compounds are not used in targeted drug delivery systems due to their high affinity for cell membranes, both healthy and in cancer cells. Using the approach of creating supramolecular amphiphiles, we have developed self-assembling systems based on water-soluble pillar[5]arene and synthetic meroterpenoids containing geraniol, myrtenol, farnesol, and phytol fragments. The resulting systems can be used as universal drug delivery systems. It was shown by turbidimetry that the obtained pillar[5]arene/synthetic meroterpenoid systems do not interact with the model cell membrane at pH = 7.4, but the associates are destroyed at pH = 4.1. In this case, the synthetic meroterpenoid is incorporated into the lipid bilayer of the model membrane. The characteristics of supramolecular self-assembly, association constants and stoichiometry of the most stable pillar[5]arene/synthetic meroterpenoid complexes were established by UV-vis spectroscopy and dynamic light scattering (DLS). It was shown that supramolecular amphiphiles based on pillar[5]arene/synthetic meroterpenoid systems form monodisperse associates in a wide range of concentrations. The inclusion of the antitumor drug 5-fluoro-2'-deoxyuridine (floxuridine) into the structure of the supramolecular associate was demonstrated by DLS, 19F, 2D DOSY NMR spectroscopy.