Annotation of the results obtained in 2021
All goals specified in the Project for the third year have been fully reached. New results were obtained in all the main directions of the Project fulfilment. A method for the synthesis of mono- and disubstituted phenothiazine derivatives based on two synthetic routes, i.e. (1) the reaction of 3-(phenylamino)phenothiazine-5-ium triiodide with a series of arylamines and (2) the interaction of various 3-N'-arylaminophenothiazines with aniline, was developed. It has been shown that the first synthetic route is characterized by higher yields, while the second synthetic route can be used for the synthesis of 3,7-di(N'-arylamino)phenothiazines with sterically loaded arylamine fragments. It has been established that the main factors affecting the reactivity of 3-N'-arylaminophenothiazines in reactions with arylamines are the conjugation between aromatic fragments and the mesomeric effect of the substituent. A series of phenothiazine derivatives containing ester, tert-butoxycarbonyl, sulfonic acid, hydroxyl, and amine groups were obtained in high yields. The characteristics (size, shape) of particles formed during the self-assembly of two- and three-component supramolecular systems based on thiacalixarene derivatives, disubstituted aryliminophenothiazine derivatives, and biomolecules in aqueous and aqueous-organic media were determined by dynamic light scattering, UV spectroscopy, transmission and scanning electron microscopy. The formation of various forms of “carboxylated thiacalixarene:lysozyme” associates depending on the configuration of the macrocycle was shown. A monodisperse system was formed in the case of a mixture of “carboxylated thiacalixarene (partial cone):lysozyme” (1:1) one day after mixing the components. The interaction of carboxylated thiacalixarenes with ammonium aryliminophenothiazine was shown by UV spectroscopy and dynamic light scattering, and while there was no interaction of ammonium derivatives of thiacalixarene with a carboxylated phenothiazine derivative. It was found that the stability of the two-component systems "thiacalixarene: aryliminophenothiazine" depended on the charge of the fragments in their structures. Dynamic light scattering was used to show the formation of commutative three-component supramolecular systems "carboxylated thiacalixarene (partial cone): ammonium aryliminophenothiazine: lysozyme" at a component ratio of 1:2:1, respectively, which consisted of submicron and micron particles. In the reporting period, an exhaustive investigation of the electrochemical behavior of new monosubstituted phenothiazine derivatives bearing nitro and amino groups has been performed. All the compounds showed electrochemical activity in a wide pH range. According to the cyclic voltammograms, the contribution of the substituent nature to the position of the oxidation/reduction peaks was revealed. The electron withdrawing nitro group led to the positive shift of the equilibrium redox potential, while the amino group demonstrated the opposite effect. Transformations of the arylamine was for all the studied monosubstituted compounds. The introduction of additional groups into the phenothiazine molecule, as well as the synthesis of symmetrical disubstituted derivatives, led to disturbance of the electrochemical reversibility according to the criteria of peak potential difference and the peak currents ratio. The рН influence on the electrochemistry of new compounds was demonstrated. The pH ranges corresponding to the maximum currents of the studied phenothiazines were determined. For the aminated and unsubstituted derivatives, this range covered 5.5–6.0 pH; for the nitro derivative, a consistent decrease in currents with increasing pH was registered. By analyzing the dependences of the peak currents on the potential scan rate, sorption on the surface of glassy carbon electrode was indicated. This is most pronounced for the nitro derivative. Based on the dependencies of the equilibrium redox potential on the pH, a reaction scheme was proposed for transitions between the redox states of new phenothiazine derivatives assuming a two-electron transfer. The kinetic parameters of the electrode reactions of new compounds were calculated. The values of the transfer coefficient indicate higher overvoltage of electroreduction for the unsubstituted compound (α = 0.37) and reversible electrooxidation for the nitro derivative (α = 0.60). The highest value of the heterogeneous rate constant of the electron transfer was obtained for the nitro derivative (3×10-4 cm/s), which may be due to its more intense sorption. The conditions for the single-stage transfer of three-component systems “phenothiazine derivative : thiacalix[4]arene : biocomponent” to the glassy carbon electrode by electropolymerization were determined. The most pronounced current-voltage signals were obtained for the system at pH = 8.2, for 1,3-alternate macrocycle configuration bearing ammonium or carboxyl fragments, and at the phenothiazine : macrocycle molar ratio of 4:1. DNA-sensor, which involved the preliminary interaction of the ammonium macrocycle and DNA molecules has been developed. This approach allowed improving operational and analytical characteristics of the DNA-sensor. The 1.5 times greater signal change toward intercalator was recorded compared to the sensor without thiacalix[4]arene. The detection limit achieved for the cytostatic drug doxorubicin was diminished by about two orders of the magnitude to 1.3 pM.

 

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Annotation of the results obtained in 2019
All goals specified in the Project for the first year have been fully reached. New results were obtained in all the main directions of the Project fulfilment. A step-by-step method for the synthesis of a number of cationic macrocycles containing positively charged quaternary ammonium and imidazolium fragments based on the p-tert-butylthiacalix[4]arene with high yields was developed. The developed method consists in the aminolysis of thiacalixarene ester derivatives with amines containing a tertiary amine or imidazole groups, followed by alkylation of the compounds with halogen-containing reagents with different lengths of the alkyl substituent, and the replacement of bromide and iodide anions to chloride ions in the synthesized quaternary ammonium and imidazolium salts using ion exchange resins. Synthetic strategy for the synthesis of new water-soluble carboxylate derivatives of thiacalix[4]arene in cone, partial cone and 1,3-alternate conformations was developed for the first time. The developed strategy consists in the alkylation of macrocyclic tertiary amines with reagents containing the highly reactive Br-CH2-CO-R (R = OEt or N(CH2COOEt)2) fragments with the following chemoselective hydrolysis of ester fragments. A review article was published [1] based on the results of the analysis and systematization of literature data and the experience of the scientific team in the field of self-assembly of macrocycles and silver ions. The article systematizes the current practical experience of researchers in the synthesis and properties study of supramolecular systems of macrocyclic compounds (including thiacalixarenes) and silver ions. Generalized data allow to develop approaches to the creation of materials - sensor modifiers, including non-conductive molecules of macrocycles and silver nanoparticles. For the first time, new phenothiazine derivatives containing ester, carboxyl, hydroxyl, amide, amino groups and quaternary ammonium fragments were obtained. For the synthesis of phenothiazine with amide and hydroxyl groups, divergent (direct aminolysis of ester derivative of phenothiazine by an aminoethanol) and convergent (aminolysis of ethyl ester of 4-aminobenzoic acid with aminoethanol followed by reaction of the product with phenothiazine) synthetic approaches were used. It was shown that the final yield of the target compound with the convergent approach was 38.4%, which is significantly higher than with the divergent approach (15%). It was shown that oligomerization occurs when tertiary amines are added to phenothiazine in alcohols; as a result, oligo(3.10)phenothiazines were obtained for the first time. Obtaining polymeric forms of phenothiazines in a chemical way is not a trivial task, and the chemical preparation of (3.10)oligomers consisting 3-6 phenothiazine fragments is a breakthrough result, the development of which opens up the possibility of polyphenothiazines synthesis. The self-assembly of the obtained thiacalix[4]arene derivatives in water was investigated by dynamic light scattering and transmission electron microscopy. The studied compounds form nanoparticles with a low polydispersity index in water. It was shown by dynamic light scattering that the obtained thiacalix[4]arenes are able to form two-component supramolecular systems with phenothiazine derivatives in water, and submicron-sized particles are formed. Establishing the electrochemical characteristics of synthesized materials is an essential part of any research devoted to the electrochemical (bio)sensors designing. Investigation of the electrochemical activity of new compounds allows predicting the characteristics of the developed devices, screening the most optimal candidates for the role of a transducer modifier, and identifying patterns that relate the chemical structure of new compounds and their redox activity on the electrode surface. As the first phase of the Project, the phenothiazine derivative N-phenyl-3-(phenylimino)-3H-phenothiazin-7-amine (PhTz) was subjected to an electrochemical study on the surface of a glassy carbon electrode [2]. The results of the study revealed the features of the PhTz electrode reaction on the surface of a glassy carbon electrode: a mixed electron transfer regime, i.e. the presence of adsorption processes, the electrochemical quasi-reversibility of the reaction, and the strong influence of the pH of the medium on the formal redox potential (E0’) of the PhTz(ox)/PhTz(red) pair. The scheme of electrochemical transformations of a new compound on the surface of a glassy carbon electrode, involving the transfer of different amounts of hydrogen protons and electron in reactions carried out in acidic (pH <7) and alkaline (pH> 7) media was proposed. During the first phase of the project, protocol for the electropolymerization of a new phenothiazine derivative PhTz was developed. The optimal conditions for the electrosynthesis of polyPhTz were established, including the range of potential scanning, the composition of the background electrolyte, the ratio of the aqueous and organic parts of the working solution and its pH. The formation of a film of a new material on the surface of a glassy carbon electrode is confirmed by electron microscopy and electrochemical impedance spectroscopy. The parameters of electron transfer and its reversibility using an electrode coated with a new electropolymerized material based on an aryliminophenothiazine derivative were determined. Based on the obtained values, the polyPhTz film can be attributed to redox-active non-conducting polymers. The electrosynthesized coatings were tested as a modifier of the indicator electrode during potentiometric titration of organic acids and in the determination of COD (chemical oxygen demand). The results of this work allowed us to propose a protocol for the development of a DNA sensor based on non-covalent interactions of a new polymer material and double-stranded DNA molecules. The possibility of discrimination of native and damaged (thermal denaturation and chemical oxidation) DNA molecules according to electrochemical impedance spectroscopy using the DNA sensor developed is demonstrated. 1. https://www.mdpi.com/1422-0067/21/4/1425 2. https://www.sciencedirect.com/science/article/pii/S0013468620305879

 

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Annotation of the results obtained in 2020
All goals specified in the Project for the second year have been fully reached. New results were obtained in all the main directions of the Project fulfilment. A method for the synthesis of new derivatives of p-tert-butylthiacalix[4]arene containing simultaneously quaternary ammonium, amide and amino acid fragments in cone, partial cone, and 1,3-alternate conformations was developed. The developed method consists in aminolysis of thiacalixarene ester derivatives with amines containing tertiary amino groups, followed by alkylation of the resulting compounds with reagents containing ester groups and amino acid fragments. Further chemoselective hydrolysis of ester fragments in the obtained compounds gave the corresponding carboxylate derivatives based on thiacalix[4]arene in three conformations - cone, partial cone, and 1,3-alternate, in zwitterion form. A series of macrocyclic ammonium salts and betaines have been obtained in high yields. For the first time, a series of catechol-containing Schiff bases based on tetrasubstituted at the lower rim of thiacalix[4]arene derivatives in three stereoisomeric forms - cone, partial cone, and 1,3-alternate were synthesized. The selective recognition of copper (II) cations in a series of d-metal cations (copper (II), nickel (II), cobalt (II), and zinc (II)) was shown by UV-vis spectroscopy. A number of physical methods have shown that copper (II) ions are coordinated to the nitrogen atom of the imine group and the nearest oxygen atom of the catechol fragment in thiacalixarene derivatives. High thermal stable organic-inorganic copper-containing materials were obtained on base of 1,3-alternate + Cu (II) complexes. The antibacterial activity and cytotoxicity of macrocyclic quaternary ammonium salts obtained in the first year of the Project's implementation in relation to a number of gram-positive and gram-negative bacteria were studied. The antibacterial activity and cytotoxicity of macrocyclic quaternary ammonium salts obtained in the first year of the Project's implementation in relation to a number of Gram-positive and Gram-negative bacteria were studied. The obtained compounds have a high antibacterial effect against Gram-positive (S. aureus, S. epidermidis, B. subtilis) bacteria, comparable to chlorhexidine, miramistin and benzalkonium chloride. It was found that most of the quaternary ammonium derivatives of thiacalix[4]arene in 1,3-alternate conformation more effectively inhibit the growth of the studied bacterial strains in comparison with compounds in cone conformation. The study of the cytotoxicity of human skin fibroblast cells have shown that all compounds are less toxic than the control drugs and have a large therapeutic window. When establishing the mechanism of biological activity, it was shown that compounds in cone conformation are adsorbed on the membrane surface of model vesicles, while the inclusion of lipophilic alkyl fragments of macrocycles in 1,3-alternate conformation into the membrane leads to vesicle “clumping”. The interaction of compounds containing quaternary ammonium and carboxyl fragments with amino acids and a series of model proteins - lysozyme, bovine serum albumin, hemoglobin in water was studied. It was shown that the obtained ammonium derivatives do not interact with amino acids and proteins. Thiacalix[4]arenes in cone, partial cone, and 1,3-alternate conformations, containing iminodipropionic acid fragments along the lower rim, interact with lysozyme and hemoglobin to form nano- and submicron associates. Stoichiometry (1: 1) and protein binding constants (LogK = 3.36-5.10) were determined. Electrochemical investigations in the framework of the second year of the Project was mainly devoted to assessing how introduction of carboxylic (PhTz-(COOH)2) and amino (PhTz-(NH2)2) groups into the structure of N-phenyl-3-(phenylimino)-3H-phenothiazine-7-amine (PhTz) affected their electrochemical behavior. For correct comparison, the experimental conditions (concentration, potential range, pH, nature of the background electrolyte) were specified to be the same for the synthesized PhTz-(NH2)2 and PhTz-(COOH)2 molecules and PhTz. The presence of new electron-donating amino groups in the structure of PhTz-(NH2)2 facilitated the process of electrooxidation against that of PhTz. In the case of PhTz-(COOH)2, on the contrary, the oxidation peaks on voltammograms shifted to the positive potentials demonstrating lower reactivity of the molecule. The electrochemical behavior of the new investigated compounds was strongly influenced by the pH. For the carboxylic derivative, the electrode reaction was accompanied by the transfer of 2 electrons and 2 hydrogen ions in the whole range of the pH of working solution. For PhTz-(NH2)2, the pH region with a half as many hydrogen ions transferred has been found. Moreover, transition between the protonated oxidized and protonated reduced forms, absent in the case of PhTz appeared for the aminated derivative. Thus, positive effect of amino groups on the stability of the reduced protonated form of the compound in the acidic pH range has been established. According to the results, experimentally confirmed scheme of transitions between redox states was proposed for both PhTz-(NH2)2 and PhTz-(COOH)2. Comparison of the kinetic parameters of reactions of PhTz-(NH2)2, PhTz-(COOH)2, and PhTz indicated insignificant influence of additional functional groups on the rate of the electrode reaction (k0 of 11×10-4 cm×s-1 for PhTz and 8×10-4 cm×s-1 for both PhTz-(NH2)2 and PhTz-(COOH)2). New investigated compounds showed different ability of electropolymerization. In general, the quantities of electrodeposited product decreased as follows from the quartz crystal microbalance and electrochemical impedance spectroscopy data. According to the electropolymerization efficiency, the following range of the compounds has been obtained: PhTz > PhTz-(NH2)2 > PhTz-(COOH)2. Strong negative effect of carboxyl groups on the electropolymerization of the PhTz-(COOH)2 was mentioned. Electrochemical study of two-component systems "phenothiazine derivative: macrocycle" made it possible to reveal positive contribution of two macrocycles (carboxylated thiacalix[4]arene in 1,3-alternate conformation and ammonium thiacalix[4]arene in cone conformation) to the kinetic characteristics of the PhTz electrode reaction. Their inclusion provided the best characteristics of the permeability toward low molecular compounds. The synthesized materials showed a significant change in the charge transfer resistance after the DNA immobilization via physical adsorption. This was used to construct the DNA sensor for the quantitative determination of the anticancer drug doxorubicin in the concentration range from 0.003 nM to 1 nM.

 

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