Annotation of the results obtained in 2022
In accordance with the intended goal, taking into account that the fluorophore largely determines the properties of the resulting delivery system as a whole, we continued research aimed at the design and synthesis of new fluorophores and fluorescent organic nanoparticles, improvement (tuning) of photophysical properties of previously synthesized compounds. As a result of research conducted in 2022, new heterocyclic fluorophores and fluorescent organic nanoparticles based on 5-amino-2-aryl-2H-1,2,3-triazole-4-carboxamides, 2-arylidene-4,5-diarylthiazoles, 4-aryl-2-(2H-1,2,3-triazol-4-yl)thiazoles, 4-(chloromethyl)-(2H-1,2,3-triazol-4-yl)thiazoles, ((2H- 1,2,3-triazol-4-yl)thiazol-5-yl)ethanones, (9H-carbazol-3-yl)-2H-1,2,3-triazol-4-yl)thiazole, 1H-thieno[ 3,4-d]-1,2,3-triazolium-olates, as well as boron complexes with arylaminopropenones — 2,2-difluoro-3,6-diaryl-2,3-dihydro-1,3,2-oxazaborinines were synthesized, their structure was proved using spectral methods. Specific features of their structure, intermolecular and intramolecular interactions were studied, the nature of these interactions was considered with 1Н and 13С, 11B, 19F NMR spectra, UV spectra and emission spectra, X-ray diffraction data and quantum chemical calculations. An important achievement is the discovery of the unique photophysical properties of highly functionalized 2-aryl-1,2,3-triazoles containing carboxamide and amino groups (CAT). A change in substituents in the carboxamide fragment or in the amino group causes a potent change in the fluorescence quantum yield (from 10 to 96%) and a change in the fluorescence lifetime (from 0.691 to 6.884 ns). CAT showed a strong AIEE effect in DMSO-water and 1,4-dioxane-water (v/v, 1:9) mixtures. The reason for such a strong effect may be not only due to their limited solubility in a mixture of organic solvents and water, but also the unique architecture of nanoparticles. The balance between numerous intermolecular contacts and a strong protic solvent such as water weakens these interactions and reduces nonradiative energy losses. The formation of nanoparticles was recorded by the dynamic light scattering method (DLS). It should be noted that CATs have a good ability to detect Hg2+ ions, and are also able to displace ions from complexes with receptors and eliminate the toxic effects of this metal. At the end of these experiments, we showed the possibility of using test strips treated with a solution of CAT for the determination of Hg2+ ions. Intensive spectral studies of fluorophore solutions were carried out and their photophysical characteristics were determined, which show that the absorption maxima of the obtained substances are located at the visible light border (380–390 nm) or in the visible region (501–530 nm). They are characterized by good to excellent molar extinction coefficients (10400-73500 M-1 cm-1), quantum yields (0.5 to 95%), and good to excellent Stokes shifts (74 to 173 nm). The results of quantum mechanical calculations show high values of the oscillator strength, both during excitation and radiation. Thus, the photophysical characteristics of the obtained fluorophores correspond to the recommendations necessary for operation in biological systems. Methods for modifying fluorophores (acylation, bromination, hydrolysis, Suzuki-Miyaura reaction) were studied both for introducing active centers into their molecule to form hybrid systems using various linkers, and for modeling or tuning the electronic system (lengthening the conjugation chain, varying the nature of substituents) , as well as to change the physico-chemical properties (solubility) and biological behavior (introduction of fragments of biomolecules). For various fluorophores, the features of the implementation of reactions, the area of distribution of the studied transformations in a particular series, and the limits of application were determined. The possibility of implementing the AIE/AIEE effect was studied for all synthesized fluorophores in a mixture of an organic solvent (DMSO, 1,4-dioxane, tetrahydrofuran, alcohol) and water or a buffer (pH 7.4). The influence of various factors on the formation of excimers and nanoparticles was determined (an increase in the concentration of a fluorophore, the addition of water or a buffer, and the addition of a solvent with a high viscosity (ethylene glycol)). It was shown that, in addition to CAT, 2,2-difluoro-3,6-diaryl-2,3-dihydro-1,3,2-oxazaborinines also have the AIEE effect. The formation of particles was registered using the DLS method, their size was determined, and the chemical and photostability were studied. The possibility of obtaining new 5-, 6- and 7-membered spirocyclic complexes of arylaminopropenones with derivatives of ortho-hydroxybenzoic acids (citric acid, mandelic acid), 1,2-hydroxydihydroxy compounds (dopamine, dihydroxyphenylalanine, caffeic acid, dopamine or N-acetyl-, N-Boc-dopamine, dihydroxyphenylalanine, ethyl 2-acetamido-3-(3,4-dihydroxyphenyl)propanoate) and boric acid were studied. It was shown that the obtained compounds have a high extinction coefficient (up to 45400 M-1 cm-1) and significant fluorescence in the crystalline form with a quantum yield from 2.6 to 65%, AIEE genic behaviour and their solutions in DMSO showed an increase in quantum yield by 1.5 - 7.5 times with addition of water. The analysis of the obtained spectral data, quantum-chemical characteristics and geometry of the complexes was carried out, recommendations for tuning the photophysical properties of spiroborates were formulated. The possibility of their modification by means of an acylation reaction was demonstrated and the new complexes containing a fragment of a biomolecule (glycine ester) or fluorophores containing two spirocyclic oxazaborinine cycles were obtained. New hybrids of 2-arylidenthiazoles containing a carboxylic acid residue (chlorambucil, caffeic acid, benzoic acids with substituents of different nature), amino acids (lysine, phenylalanine and tryptophan, di- and tripeptides), a biologically active RGD tripeptide, as well as dimethylaniline and hydroxy compounds were obtained. Synthesis of hybrids of (2H-1,2,3-triazolyl)thiazoles with biomolecules and their models (chlorambucil and methoxybenzoic acid) has been carried out. Their photophysical properties were studied in DMSO, and mixtures of DMSO-water, DMSO-buffer (pH 7,4). It was shown that the optical characteristics are in good agreement with the requirements allowing their use in biological systems (absorption at the visible region border, contrast fluorescence, large Stokes shift (up to 130 nm)). It should be noted that for these hybrid structures, a shift of the absorption and emission bands to the long wavelength region is observed, as well as an increase in the quantum yield in the DMSO-buffer mixture. Conjugates containing two biomolecules (chlorambucil, methoxybenzoic acid) were obtained, which is one of the rareast examples in the synthesis of photocourier systems. Photophysical properties and structure of these conjugates were studied. Photodissociation of hybrids was studied using chromato-mass-spectrometric studies and NMR control of the reaction mixture composition. Kinetics of dissociation was studied by spectrophotometric methods. Chromatography-mass study of the resulting mixture composition was carried out at different radiation power (20% and 50%), excitation wavelengths (λexc = 250 nm, and 360 nm) and in different solvents (in a mixture of acetonyl-water in density (9:1 , 7:3) and MeOH. As a result of these studies, the structures of the main photodissociation products were established, and its features were determined depending on the structure of the fluorophore and the biomolecule. The data obtained made it possible to compare the influence of the structures of the fluorophore, the biomolecule, and the linker on the rate of photodissociation. The formation of products of the transformation was demonstrated using the RGD-peptide hybrid as an example. The advantages and disadvantages of various hybrid systems were considered and compared with the known photosensitive carriers of biomolecules, the problems of further development of research and ways to overcome them were identified. A qualitative assessment of the phototoxic effects of the studied substances in situ was carried out using MTT test and a confocal microscope with a culture of epithelial cells of the African green monkey (Vero), MIA PaCa-2 (human pancreatic cancer cell line) and a fibroblast cell line. It was shown that the synthesized hybrids do not possess cytotoxicity. The penetration of hybrids and fluorophores into the cell has been demonstrated, depending on the structure of the hybrid molecule, they selectively accumulated in the ER, the Golgi apparatus, lysosomes, and in the nucleus (nucleolus). A good contrasting ability of the studied compounds was shown, with quick penetration into the cell (30 min) and sufficiently bright images. Experiments were carried out to assess the penetration of the studied substances into cellular spheroids as well as their phototoxic effect. The obtained results were published in 3 articles, 2 of them in Q1 journals. Currently, one article is under consideration in the journal Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy and experimental material has been prepared for future publications. The results were delivered as oral and poster presentations at international and Russian conferences on organic synthesis, heterocytclic chemistry and chemistry of materials (16 theses), PhD dissertation and 3 master's dissertations were defended.

 

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Annotation of the results obtained in 2020
The main aim of the project is the design and synthesis of new luminescent molecular couriers and organic fluorescent nanoparticles for targeted delivery in the treatment and diagnosis of socially significant diseases and the study of cellular processes. Targeted delivery of an biological active compound (it may be a drug, genes, natural compounds, a reporter molecule, etc.) involves the point delivery of a biomolecule, diagnostics or sensor and its release from this complex system at a strictly defined time, suitable for the study of biological systems or treatment of pathologies in medicine. Light is currently one of the attractive exogenous (external) factors for the release of a biomolecule, since it is not invasive and its effect can be carried out with high spatial and temporal precision, i.e. direction of action. The greatest problem at present in the implementation of such targeted photo delivery is the choice of a phototrigger (fluorophore) compound and the process of releasing a drug or diagnostic molecule from a photocourier using remote control. An important factor in the implementation of the intended aims is heterocyclic fluorophores, which must have certain photophysical characteristics, physicochemical properties, and must also have substituents or functional groups that allow not only adjusting the optical properties, but contribute to the formation of hybrids that are stable to photoradiation or capable of releasing a biologically active molecule, medicine or natural compound under the action of light. A distinctive feature of our research is the use of original effective fluorophores, synthesized over the past years in our laboratory. The photophysical properties of these fluorophores, and for some of them, their behavior in biological media (cytotoxicity, penetration into cells and cell organelles, the possibility of using them in flow cytometry) have already been studied. One of the disadvantages of many fluorophores and chromophores when used for photolysis of courier molecules in biological conditions in vitro is the need to use radiation in the ultraviolet region of the spectrum. Such radiation is undesirable for living cells. The solution to this problem can be the synthesis and use of new fluorophores with lower excitation energy, which are less dangerous for living organisms and the ability to penetrate deeper into tissues. At the first stage of work under the grant, we synthesized a number of such fluorophores, studied their behavior in biological media and showed that they penetrate the cell membrane and are selectively localized in cell organelles. New unique heterocyclic fluorophores with different photophysical characteristics have been synthesized, providing the possibility of obtaining photo couriers with good photophysical properties: high quantum yield, absorption wavelength in both the ultraviolet and visible regions of the spectrum. The maximum absorption spectra provide excitation in both UV and visible spectral ranges (340-462 nm) with a molar extinction coefficient of up to 43000 M-1 cm-1. The emission of the obtained compounds has a maximum located in the region from blue to orange-red spectral range (423-565 nm). Fluorophores show a significant Stokes shift (up to 174 nm), which is an important property for their use in biological research. The fluorescence quantum yield varies significantly depending on the structure of the compounds (up to 95%). The design and synthesis of fluorophores was carried out in such a way that they contained the necessary functional groups and substituents (COOH, OH, NH2, COMe, Me, OMe), convenient for their further modification in order to form a linker system and bind to biomolecules. Among the fluorophores, which we propose to use as a phototrigger component, the new spirocyclic azaborinine complexes with bright green fluorescence are noteworthy. The complexes are highly stable, which will make it possible to carry out various chemical transformations to obtain photocouriers that absorb in the visible region and have intense fluorescence. In addition, the method of their synthesis allows one to introduce the necessary functional groups to obtain phototriggers with biomolecules. All this makes it possible to recommend the obtained complexes as promising candidate compounds for the creation of new conjugates with a biomolecule. The structure of all fluorophores was studied using 1H and 13C NMR spectra, EI-TOF-MS spectra and FTIR spectra. Spatial structure, formation of intramolecular and intermolecular interactions (analysis of XRD data). This made it possible to explain some of the features of absorption and fluorescence of the substances studied. We studied the optical properties of fluorophores in solvents of different polarity, as well as in a mixture of solvents to determine their sensitivity to external influences that can change their optical properties in biological media. On the basis of solvatochromic studies, correlations of the Stokes shift and the characteristics of solvents were carried out according to the Lippert-Mataga and Reinhardt equation, which made it possible to quantitatively compare the same type of series of fluorophores in their sensitivity to the nature of solvents, as well as to quantitatively estimate the effect of changing the polarity of molecules in an excited state as compared to the ground state. The results obtained were confirmed and explained using quantum mechanical calculations carried out by Dr. E. Benassi. (Shihezi University, China). The ground state molecular geometry of the compounds under investigation was fully optimized at density functional theory (DFT) level, both in vacuo and in solvents (Toluene, 1,4-Dioxane, THF, DCM, DMSO, MeCN). Solvent effects were taken into account via the implicit polarizable continuum model in its integral equation formalism (IEF−PCM). The photophysical characteristics were determined in the ground (GS, S1) and excited states (ES, S1v and S1r) using quantum mechanical calculations. The vibrational frequencies and thermochemicals were computed in harmonic approximation at T = 298.15 K and p = 1 atm, and no imaginary frequencies were found. Vertically excited electronic states were also investigated at Time-Dependent DFT (TD-DFT) level. The long-range corrected CAM-B3LYP functional was used, coupled with the triple-ζ 6-311++G** basis set. UV-Vis absorption spectra were simulated accounting for the first ten singlet excited states. Studies have been carried out to evaluate and confirm the implementation of various optical phenomena (ICT, AIE, AIEE). In this case, both experimental data, (spectral) and theoretical studies (quantum mechanical calculations) were used. Thus, the change in the physicochemical characteristics with a change in the microenvironment (various solvents, their mixtures, the presence of analytes) was reliably explained and evaluated by means of a change in the electronic distribution of HOMO and LUMO in the excited and ground state, and the analysis and assessment of the degree of intramolecular charge transfer (ICT) in different series of fluorophores. The change in fluorescence upon aggregation (AIEE) was carefully studied for each group of substances using solvents with different nature (DMSO-water, 1,4-dioxane-water), as well as in solutions with different fluorophore concentrations. We studied the possibility of introducing functional groups to obtain photocourier systems with different photoresistance. First of all, we tested the possibility of designing new photocourier systems using bromoacetyl and benzyl linkers. We tested several approaches to the introduction of a bromoacetyl group into a fluorophore molecule and determined the optimal variant for different types of heterocyclic fluorophores. That opened the way for the development of research and the production of fluorophore-biomolecule hybrids by the alkylation reaction of the hydroxy group of alcohols and acids (including amino acids). The reaction of bromination of the side methyl group in the aromatic fragment of the fluorophore was studied. New bromomethyl derivatives of fluorophores have been obtained, most of which do not exhibit fluorescence. It should be noted that this modification option plays a special role, since the bromobenzyl fragment can be used independently for the synthesis of a hybrid compound with a biomolecule. Another option involves hydrolysis and the formation of an OH group, which will then be used to form a carbonate or carbamate bridge connecting the fluorophore molecule and the biomolecule. A biological screening of fluorophores, the excitation of which occurs in the visible region (dihydrothiazolopyrimidines, 4H-thieno[3,4-d]-1,2,3-triazole-2-ethanolates, 2,2-difluoro-1,3,2-oxazaborinin glycinates and spiro [benzo [d][1,3,2] dioxaborinin-2,2'-[1,3,2]oxaazaborinin]-6'-yl)thiophenes. It was found that all the investigated fluorophores perfectly penetrate into the Vero cell, staining it yellow, green, orange-red and selectively accumulate in lysosomes, endoplasmic reticulum and the cell membrane. There is no phototoxic effect on living cells and no photodegradation of the dye.

 

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Annotation of the results obtained in 2021
To fulfill the aim set and followed in 2021, we carried out research according to the following scheme: the synthesis of new luminophores based on the heterocyclic fluorophores obtained in our laboratory, the synthesis of new photocouriers using various linkers and biomolecules, the study of the mechanism and kinetics of their photodissociation, the study of biological behavior through in vitro experiments. The main disadvantage of phototrigger systems proposed for the creation of photo couriers for targeted drug delivery, is high activation energy, which may be unsafe for living systems. We corrected the photophysical properties of the fluorophores and photo couriers and moved their absorption wavelength it in the visible region. Following the previously obtained patterns of dependence between photophysical properties and structure of the compound, we obtained a series of various heterocyclic fluorophores with an absorption maxima from 400 nm to 465 nm with blue, green-orange fluorescence: Amidst the photophysical studies of the newly synthesized 1,2,3-triazolo[4,5-d]pyrimidines bearing the CCl3 group, we found that they have been transformed upon UV irradiation, which led to significant increase in the emission intensity up to 90,1–98.6%. Mass spectrometric experiments and quantum chemical calculations have confirmed that these compounds undergo phototransformation due to the homolytic cleavage of the C–Cl bond and the formation of 2H-1,2,3-triazolo[4,5-d]pyrimidine-5-carbonylchlorides, which are readily reacting with N-, S- and O-nucleophiles, including derivatives of amino acids (for example, glycine and cysteine) under mild conditions. Some of these compounds were isolated and characterized by a complex of spectral methods. Kinetic studies determined the reaction rate constants of the phototransformation of these fluorophores depending on the structure and the solvent used. In vitro biological studies using a confocal laser microscope have established that 2H-1,2,3-triazolo[4,5-d]pyrimidines penetrate the cell and selectively accumulate in the cell membrane (presumably in the bilipid layer), as well as in the Golgi apparatus and endoplasmic reticulum (ER). Generation of an active intermediate (2H-1,2,3-triazolo[4,5-d]pyrimidine-5-carbonyl chloride) upon irradiation of 2H-1,2,3-triazolo[4,5-d]pyrimidines in cell compartments can result in the interaction of a fluorophore and a biomolecule (peptide, amino acid, etc.) and the formation of a new hybrid structure. Such processes are certainly interesting and require further research, since they can be used to study biological systems or to influence pathogenic cells. In addition, 2H-1,2,3-triazolo[4,5-d]pyrimidines can be used as fluorescent dyes for biology, since Golgi markers and ER markers are usually expensive or difficult to obtain due to their complex structure. During the implementation of the project in 2021, we developed and synthesized salicyl-spiroborate complexes (SSBs) based on β-ketoiminate with various substituents in aromatic rings. We have investigated the geometric, electronic and spectral properties of new complexes in the ground (GS) and excited electronic states (ES) and demonstrated their excellent photophysical characteristics. In 2021, we expanded the range of reactions and conducted more intensive research on the synthesis of hybrid structures containing a "benzyl", "carbamate" and "carbonate" linker. Since the carboxyl group is one of the most general functions used for photoregulated delivery of drugs and diagnostics, we carried out more detailed studies related to the production of a fluorophore containing a “benzyl” linker and obtained a series of photocouriers based on various aromatic carboxylic acids, biomolecules containing a carboxyl group. as well as amino acids. To study this type of the binding of a fluorophore and a biocomponent, we used a series of model compounds (benzoic acid and its 4-nitro-, 4-chloro- and 4-methoxy-substituted), as well as caffeic, salicylic acid and Boc-protected amino acids (glycine, GABA) The photophysical properties of the obtained photocourier systems were studied in organic solvents of various nature, as well as in acetonitrile-water mixtures (λmax = 362-384 nm molar extinction coefficient ε = 17500-25300, λem = 472-521, QY up to 29% and a significant Stokes shift SS = 100-142 nm). Thus, the absorption maximum of the obtained ethers located in the region close to the visible region and their excitation is possible when using irradiation of about 400 nm and more. Moreover, the extinction coefficients have rather high values, so excitation intensity provides high brightness. The emission maxima located from 472 to 521 nm and are shifted bathochromically in polar DMSO by 31-43 nm in comparison with non-polar CH2Cl2. Although the quantum yield of ethers decreases compared to the initial fluorophore, they remain at a fairly good level in a mixture of acetonitrile and water (7.4-8.3%). The characteristics of the GSs and ESs upon absorption of a quantum of light and the transition to the vertical state of photocourier molecules, as well as their subsequent geometric relaxation and emission, were calculated using the density functional theory ((TD)-DFT) taking solvent into account (in the framework of the polarizable continuum model)). It is shown that photocourier molecules are characterized by a high oscillator strength both during absorption (fw ~ 1.02-1.24) and emission (fw ~ 1.02). The dipole moment is large enough in the ground state (10.9-11.4 D) and increases upon transition to an excited state up to 14.6-16.3 D. Photodissociation of ethers was studied using a photoreactor equipped with two mercury lamps (P = 26 W, λex = 385 nm). Based on the results obtained, a photodissociation mechanism was proposed. Using quantum mechanical calculations, as well as HPLC/HRMS experiments, we found that the molecule of the biocomponent and the fluorophore exist in the form of a molecular complex at the first stage of photodissociation. We carried out kinetic studies of photodissociation and determined the rate constant and half-life of the photo couriers. The processes of photo- and hydrolytic dissociation of N,O-salicylspiroborates were studied by a HPLC/HRMS method and 1H and 11B NMR spectroscopy. Release of the initial organic ligand, salicylic acid and boric acid was shown during photodissociation in protic solvents (MeOH, EtOH), and in mixtures of ethanol-water, DMSO-water. This finding suggested the possibility of using these complexes as a potential light-sensitive component for an intracellular drug delivery system. Further studies established that photodissociation proceeds faster in polar and protic EtOH than in polar DMSO. The addition of water to DMSO and EtOH solutions significantly increased the photodissociation rate, even for the more stable NO-SCB complexes. The kinetics of photodissociation of NO-SSB in EtOH was studied using absorption spectra at λ = 465 nm. Photodissociation of NO-SSB with electron-withdrawing substituents in the aromatic ring proceeds faster than for complexes containing electron-donor substituents. Thus, we have found that it is possible to manage the process of photodissociation of SSB with the help of a solvent and the structure of the complex. To assess biological behavior (penetration through the cell membrane, distribution in the cell, phototoxicity), studies were performed using a confocal laser microscope. The results obtained show that all synthesized fluorophores penetrate well into cells and give good images when irradiated with a laser at different excitation wavelengths. It should be noted that no toxic effects caused by fluorophores were observed during the experiment. An important result of the study is the detection of selective accumulation of the investigated substances in the endoplasmic reticulum, the Golgi apparatus or at the cell membrane. NO-SSB stains the reticular structure, in the endoplasmic reticulum (ER) and the Golgi apparatus. Moreover, the complex penetrated equally well into mesenchymal and muscle cells, as well as fixed cells (see figure). It should be noted that no staining of the outer cell membrane, mitochondria, or nuclear membrane was observed, which indicates the specificity of the dye for ER. In contrast, commercial glibenclamide-based dyes do not work in fixed cells because the target enzyme loses its configuration. It is known that the properties of real tumors differ from two-dimensional samples, and a more realistic response to the effect of drugs is provided by three-dimensional cell structures, among which the spheroid model is the closest to real properties. The ability of SSB complexes to penetrate cell spheroids was investigated. The series of photos above shows confocal sections of a cell spheroid at different heights from the base. The images obtained demonstrate that the substances under study penetrate the cells of the spheroid as efficiently as in a monolayer culture, which opens up prospects for it in this area of biological research.

 

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