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Project titleNovel approaches to reducing the microbial resistance to antibiotics in mixed infections: screening of new antimicrobial semi-synthetic thioterpenoids, the characterization of molecular targets and mechanisms, the development of an effective delivery system for antimicrobials and its visualization by using BODIPY-luminophore conjugates

AffiliationG.A. Krestov Institute of Solution Chemistry of the Russian Academy of Sciences,

Research area 03 - CHEMISTRY AND MATERIAL SCIENCES, 03-203 - Chemistry of coordination compounds

KeywordsLuminophores, BODIPY, biomarkers, conjugates, synthesis, identification, molecular structure, spectral properties, lipo- and hydrophilicity, photo and thermal stability, quantum chemical analysis, spectrophotometry, spectrofluorimetry, thermogravimetry

Annotation
The vast majority of infectious diseases of human and animals is associated with the formation of biofilms, that is, microbial consortia embedded in self-produced high-molecular matrix consisting of proteins, nucleotides and polysaccharides. While in biofilms, bacteria become extremely resistant to antibiotics and the immune system of a host. The biofilm formation by pathogenic bacteria significantly reduces the efficacy of antimicrobial therapy, leads to chronic diseases and slows down the wounds healing. Therefore, the development of new approaches to increase the biofilm-embedded bacteria susceptibility to antimicrobials is the great challenge for modern pharmaceutics. One of the classes of membranotropic substances capable of changing the properties of cell membranes, in particular their permeability, is natural terpenes, especially sulfur-containing derivatives of monoterpenoids. Thioterpenoids have been shown to be able to embed into the cell membrane by immersion in it with its hydrophobic terpene fragment, and to bind to the hydrocarbon chains of phospholipids. Therefore, the introduction of a compact lipophilic monoterpene fragment into a molecule facilitates its transmembrane transport and binding to the cell membrane and organelles, which allows expecting an increase in the biological activity of compounds as well as a multi-target effect. Taking into account low toxicity of thioterpenoids (LD50 ranges from 2000 to 10000 mg/kg, chronic toxicity and genotoxicity are not detected), these compounds look like promising agents for developing new antimicrobials. Recently, it has also been shown that many infectious processes are caused rather by consortium of bacteria including the resident flora than by one microorganism. As a result, mixed bacterial and fungal-bacterial biofilms are formed on open wounds, mucous membranes, body tissues, surfaces of implanted devices. In mixed biofilms, synergistic, mutualistic and antagonistic interactions are observed between bacteria, so the treatment of mixed infections is much more difficult and longer. One of the most dangerous consequences of synergy between microorganisms is an increased resistance to antimicrobials due to the survival of some microorganisms in the biofilm of resistant strains and the transition to an uncultivated state (persistence). On the other hand, in case of mixed S. aureus - P. aeruginosa biofilms, 10 times lower concentrations of ciprofloxacin lead to the same reduction in the number of living bacteria as in monocultures. Undoubtedly, such differences in the effectiveness of antibiotics against mono-and mixed cultures should be taken into account when treating polymicrobial infections. However, there are almost no data on the behaviour and interaction of S. aureus, K. pneumonia, E. coli, S. mutans, C. albicans and some other species in mixed biofilms. Respectively, the molecular mechanisms and tools of microorganisms which give rise to the complex nature of interactions in the polymicrobial community also remain uncharacterized. Any data on the efficacy of even widely used conventional antibiotics against such biofilms are also missing. The project is devoted to in vitro modeling of various mixed bacterial and fungal-bacterial biofilms in order to characterize the interaction types between microorganisms inside them and to develop new approaches to improve the efficiency of their eradication based on a combination of natural bacterial antagonistic factors and the thioterpenoids-based drug delivery systems. Since low molecular weight hydrophobic molecules of BODIPY dyes are able to easily penetrate into the lipid layers of cell membranes, bind to hydrophobic protein fragments, the BODIPYs fragments will be used to label individual biomolecules, determine their localization as well as reveal its mechanism of action. The use of BODIPY dyes conjugated with biologically active thioterpenoids and antimicrobials will allow the process of penetration of the latter into the cell to be visualized in real time and characterized at the molecular level. Searching for new substances on the basis of semi-synthetic diterpenoids and antibiotics/antifungals of different types (nitrofurans, beta-lactams, fluoroquinolones, azoles, etc.), identifying molecular mechanisms of resistance, developing effective delivery systems of antimicrobial agents and its imaging with application of conjugates with BODIPY biomarkers pose a particular challenge and can be performed within the framework of interdisciplinary project joining scientists from the various fields such as organic and inorganic synthesis, coordination chemistry, biochemistry, microbiology, genetics and medicine. During project implementation, the following objectives will be fulfilled: 1) Characterizing the microbial community of the oral mucosa formed under conditions of infectious lesions caused by bacterial microflora and Candida sp fungi. Based on these data, the models of mixed bacterial and fungal-bacterial biofilms will be developed. 2) Evaluating the efficacy of various antibiotics for the treatment of the obtained consortia. The interaction character between different types of bacteria in the mixed biofilms will be characterized including the molecular mechanisms of these interactions, which determine the change in the efficiency of antimicrobial drugs for the treatment of mixed infections (the scientific group of Doctor of Biology, Prof. Kayumov A.R.); 3) Developing methods for synthesis of sulfur-containing terpenoids based on monoterpene mentane-, bornane-, carane- and pinane-type thiols, sulfochlorides, tosylates (mesylates, halides), ketones, alcohols coupled with heterocyclic thiols, amines, cabonyl compounds, alcohols, including penicillin, cephalosporin, fluoroquinolone, triazole, nitrofurane, pyrimidine, imidazole, hexethydine, amphenicol and cycloserine fragments. The antimicrobial activity of the compounds synthesyzed will be evaluated; the substances having the potential to be used in medicine, animal and plant protection from pathogenic microbial and micromycete infections associated with the biofilm formation will be identified (the scientific group of Doctor of Chemistry, Prof. Rubtsova S. A.); 4) Developing methods for synthesis of BODIPY luminophores containing alkyl (-CH2-)n substituents with reactive terminal either ether or carboxyl groups in the meso-spacer of the indacene skelet for conjugation with semisynthetic thioterpenoids and their derivatives. The effect of structure features and properties of a medium, including model physiological solutions, on spectral-luminescent characteristics, lipo- and hydrophilicity, photo- and thermal stability of synthesized BODIPY luminophores, as well as their conjugates with thioterpinoids will be studied (the scientific group of Prof. Antina E. V.); 5) Obtaining thioterpenoids fused with BODIPY lumonophores, which will allow the process of penetration of compounds into the cell and into the biofilm to be traced and described in real time, as well as to characterize the factors affecting this process, their significance and contribution. The received information will allow us to adjust recommendations on rational use of conventional and novel antimicrobials depending on microbial structure of infection and to increase efficiency of its therapy.

Expected results
The study results are of both fundamental and practical interest. Within the framework of this project, as part of an interdisciplinary study, one of the main fundamental interests will be the results aimed at creating new fluorescent biomarkers based on alkyl-substituted BODIPYs containing alkyl (-CH2-)n substituents with reactive terminal ether groups or carboxylic acids residues in the meso-spacer , in order to visualize and elucidate the molecular mechanisms of action of sulfur-containing terpenoids and their derivatives, for which a high antimycotic and antibacterial activity for has established due to the phenomenon of synergism. The influence of structural factors and media properties, including model physiological solutions, on the spectral and luminescent characteristics, lipo and hydrophilicity, photo and thermal stability of both the synthesized BODIPY luminophores and their conjugates with thioterpene derivatives will be studied during in the implementation of the interdisciplinary project. The process of penetration of substances into the cell will be visualized and characterized in real-time by thioterpenoids and their derivatives conjugated with various BODIPYs luminophores. Scientific group under the leadership of Dr. Sc. Kayumov Airat Rashitovich (Kazan (Volga region) Federal University, Kazan University, KFU. Republic of Tatarstan, Kazan) will receive laboratory models of polymicrobial biofilms from the most common conditionally pathogenic microorganisms that form mono- and polymicrobial biofilms on the mucous membranes of the oral cavity under the conditions of an infectious process caused by bacterial and fungal agents. In the obtained mixed cultures, the nature of the interaction between microorganisms, the pattern of gene expression and the structure of the biofilm matrix itself will be determined depending on the types of microorganisms forming the consortium. Based on the obtained data, conclusions will be made about the patterns governing the switch of bacteria from an antagonistic strategy to commensalism and changes in the antibiotic resistance of microorganisms in mixed communities, including the molecular mechanisms of these processes. This will allow further development of drugs to suppress these processes and reduce the resistance of the consortium. With the participation of a scientific group under the leadership of Dr. Rubtsova Svetlana Albertovna from the Institute of Chemistry, Komi Science Center UB RAS (Komi Republic, Syktyvkar) will be synthesized a number of biologically active sulfur-containing terpenoids based on monoterpene thiols, sulfonyl chlorides, tosylates (mesylates, halides), ketones, alcohols, menthana, boronova, structures with heterocyclic thiols, amines, cabonyl compounds, alcohols, including those containing penicillic, cephalosporin, fluoroquinolone, triazole, nitrofuran, pyrimidine, imidazole, hexetidine, amphenicol and cycloserine fragments. This will allow for easier penetration of antimicrobial agents into the biofilm and the microbial cell itself. The characteristic of the sensitivity of microbial strains and consortia to the action of various groups of thioterpenoids and their conjugates with antibiotics will be given, and the possibility of increasing the effectiveness of antibiotics by introducing them in the form of conjugates with thio derivatives of terpenes will be evaluated. Wherein, the molecular mechanism of the terpenes derivatives effect on the susceptibility of microorganisms in the composition of biofilms to antimicrobial agents will be characterized. The data of the increased (or vice versa reduced) bacteria resistance to various antibiotics in mixed infections is of interest to infectious disease doctors, resuscitators and surgeons for the correct choice of antibiotics for antimicrobial therapy depending on the composition of the microbial contamination of the focus of inflammation and is significant at the global level. In addition, in view of recent trends in the combination of antibiotic therapy with substances that increase their effectiveness, the membrane permeability properties of terpenes obtained during the project will be useful for further development of new delivery vehicles and increase the effectiveness of antimicrobial agents for the treatment of mixed infections, including microbial and fungal-bacterial biofilms. The proposed results can be achieved within the framework of an interdisciplinary project and only with the joint work of scientists from the fields of organic and inorganic synthesis, coordination chemistry, biochemistry, microbiology, genetics and medicine.

Annotation of the results obtained in 2023
Within the framework of an interdisciplinary project, together with a scientific group led by Dr. Kayumov A.R. (KFU, Kazan), the synthesis of water-soluble conjugate forms based on meso-butane and meso-pentane BODIPY acids and myrtenol with a miramistin charged quaternary ammonium group was performed for the first time. The obtaining conjugates of a new type, the studing their spectral and biological properties, a scientific group led by Dr. Antina E.V. (ISC RAS, Ivanovo) synthesized the necessary working amount of BODIPY dyes containing butane or pentane acid residues and their esters in the meso-spacer, guided by the methods developed and tested in previous reporting periods of Project. The scientific groups carried out a cycle of joint synthetic work on the production of the myrtenol and thioterpenoid conjugates with alpha-propanoic acid of the BODIPY. For the synthesis of the corresponding conjugates, a scientific group from Ivanovo developed a working amount of the alpha-propane acid of the BODIPY. In order to develop new effective photosensitizers based on halogen-substituted BODIPY esters for antibacterial and antimycotic photodynamic therapy, a scientific group led by Dr. Antina E.V. (ISC RAS, Ivanovo) tested synthesis methods during the reporting period and obtained working quantities of 4,4'-dibromo- and 4,4'–diiodisubstituted BODIPY phosphors with ester (СН2)3СООСН3 group in the meso-spacer. Identification of all synthesized compounds for the fourth reporting period of the Project was carried out using H1, C13 NMR, mass and electron spectroscopy, as well as elemental analysis. The results of molecular docking and spectral studies being obtained during the reporting period by a scientific group led by Doctor of Chemical Sciences Antina E.V. (ISC RAS, Ivanovo) are summarized and analyzed. The process of molecular complexes formation of alpha- and meso-functionalized BODIPY and their terpene conjugates with serum albumins was established to be spontaneous and proceeds due to hydrogen and Van der Waals interactions. At the same time, alpha substitution of hydrogen atoms in the pyrrole nuclei of BODIPY by ester groups with terpenes and beta halogenation of the dipyrromethene backbone increases the affinity of phosphors to serum blood proteins. The results of spectral studies showed the introduction of carboxylic acid residues and their ester groups with myrtenol and thioterpene, including the miramistin charged quaternary ammonium group, into the alpha and meso positions of the dipyrromethene backbone of BODIPY doesn't to reduce the fluorescent characteristics of luminophores (up to φ ~96%) by comparison with the source BODIPY precursors. The introduction of propanoic acid residues into the alpha position of pyrrole nuclei and, especially, beta halogenation of luminophores significantly (up to ~50 nm) shifts the maxima of absorption bands and fluorescence to the red region compared with BODIPY meso-carboxylic acids and their esters. The most noticeable decrease in fluorescence is observed for dibromo- and diiodisubstituted BODIPY esters. The quantum yields of fluorescence of halogenated dyes are no more than ~2-32%, depending on the molecular structure and properties of the medium, which is due to the manifestation of the "heavy" atom effect. An important advantage of dibromo- and diiodisubstituted BODIPY esters is the high quantum yield of singlet oxygen generation: ΦΔ = 50-61% and 65-78%, respectively, which is of interest in the development of new theranostics based on them with the functions of PDT, APDT agents and fluorescent dyes. It is important to note that, unlike most hydrophobic BODIPY esters with alcohols, conjugates containing meso- or alpha-ester substituents, including along with the terpene radical a positively charged quaternary ammonium group, demonstrate satisfactory solubility for biological studies (up to c ~10-5 mol/l) in water and buffer solutions with pH from 1.65 to 9.18. However, dyes are chemically photostable in both acidic and alkaline aqueous media and retain high fluorescence (up to φ ~83%). The meso-substitution of the proton of the BODIPY methine bridge with residues of carboxylic acids and their esters was noted to increases the photostability of the chromophore system of dyes by almost ~2 times compared with meso-unsubstituted analogues. A marked decrease (up to ~ 2-4 times) in the photostability of dyes is observed in 2,6-dibromo- and diiodipyrromethene esters of BODIPY compared with non-halogenated analogues. An increase in the rate of photodestruction of halogenated chelates under UV irradiation may be facilitated by the effective generation of singlet oxygen by dyes due to the effect of a "heavy" atom. The introduction of a positively charged ammonium group into the meso-substituent of BODIPY conjugates was found to increased the affinity of the dye to hydrophilic media by almost ~9-14 times compared with uncharged analogues, as a result of increased in the hydration contribution to luminophore solvation. However, the combination of halogenation and meso-substitution increases the affinity of BODIPY esters to lipid biostructures by ~1.3 times and promotes the efficient transport of phosphors through the cell membrane layer compared with carboxylic acids of boron(III) dipyrromethenates. A scientific group led by Dr. Kayumov A.R. (KFU, Kazan) conducted in vitro biological studies to study the effectiveness of transport and localization of BODIPY conjugates in cellular organelles of pathogenic microorganisms. The conjugation of BODIPY fluorophore and methanol containing a miramistin fragment was found to favored the penetration of dyes into mycelial fungi Fusarium solani. However, it worsened their binding to S. aureus, K. pneumoniae and P. aeruginosa. The charged group of quaternary ammonium introduced between the myrtenol and fluorophore fragments was discovered to restored the staining of bacterial cells, but does not to affected the staining of fungi. It has been shown that alpha-functionalized BODIPY conjugates with methanol and triterpenoid, unlike structurally related carboxylic acid, are able to effectively penetrate the cytoplasm of C. albicans fungal cells. In this case, the conjugate with a triterpene residue most intensively stains the membrane structures of organelles (membranes of nuclei and mitochondria), in comparison with the myrtenol conjugate. As a result of determining the sensitivity of planktonic cells of the reference and clinical strains of yeast fungi to antifungal drugs, the alpha-substituted monoterpene conjugates of BODIPY was found to exhibiting effective antifungal activity, unlike carboxylic acid. The set of the results of theoretical (molecular docking) and experimental (in vitro) studies showed that conjugation of BODIPY with monoterpenoids is a promising way to increase the affinity of dyes to biostructures. In addition, BODIPY conjugates with monoterpenes are of interest using as fluorescent markers in the assessment of membrane structures of pathogen cells, as well as in determining effectors and modulating in vitro metabolic pathways of a fungal cell. It is relevant in studying of drug transport in vivo. The results of scientific research being obtained within the framework of the interdisciplinary Project for the fourth reporting period with the participation of two scientific groups (leader Dr. Antina E.V. (ISC RAS, Ivanovo) and leader Dr. Kayumov A.R. (KFU, Kazan)), are reflected in nine articles published in co-authorship and separately.

Publications

1. Ekaterina N. Nuraneeva, Galina B. Guseva , Elena V. Antina , Anatoly I. V’yugin The influence of structural effects and the solvent properties on spectral, generation characteristics, photostability and lipophilicity of 1,3,5,7-tetra-methyl-BODIPY and its alkylated and iodinated derivatives Elsevier, 439 (2023) 114611 (year - 2023) https://doi.org/10.1016/j.jphotochem.2023.114611

2. Galina B. Guseva, Michail M. Lukanov, Alexander A. Ksenofontov, Elena V. Antina, Svetlana Lisovskaya, Liliya E. Nikitina, Aigul R. Galembikova, Sergey V. Boichuk Carboxyl-BODIPY based fluorescent biomarkers: Spectral characteristics, photostability and possibilities for practical application Elsevier, 444 (2023) 114926 (year - 2023) https://doi.org/10.1016/j.jphotochem.2023.114926

3. Galina B. Guseva, Yuliya V. Eremeeva, Elena V. Antina, Ilmir R. Gilfanov, Svetlana A. Lisovskaya, Olga V. Ostolopovskay, Elena Y. Triznac, Airat R. Kayumov and Liliya E. Nikitina Effect of meso-substituents and medium properties on the photo- and рН-stability, penetration efficiency into bacterial and microscopic fungi cells of terpene-BODIPY conjugates Elsevier, 123701 (year - 2023) https://doi.org/10.1016/j.saa.2023.123701

4. Antina E.V. ХИМИЯ И МОЛЕКУЛЯРНАЯ ФОТОНИКА ДИПИРРОМЕТЕНОВЫХ КРАСИТЕЛЕЙ И ЛЮМИНОФОРОВ Казанский государственный медицинский университет, Казань, С. 11-13 (year - 2023)

5. Dudina V.S., Nuraneeva E.N. ВЛИЯНИЕ СТРУКТУРНЫХ И СОЛЬВАТАЦИОННЫХ ФАКТОРОВ НА СПЕКТРАЛЬНЫЕ, ГЕНЕРАЦИОННЫЕ ХАРАКТЕРИСТИКИ, ФОТОСТАБИЛЬНОСТЬ И ЛИПОФИЛЬНОСТЬ 1,3,5,7- ТЕТРАМЕТИЛ-BODIPY КРАСИТЕЛЯ И ЕГО АЛКИЛ-, ИОДЗАМЕЩЕННЫХ ПРОИЗВОДНЫХ Ивановский государственный химико-технологический университет, Иваново, 242 (year - 2023)

6. Dudina V.S., Nuraneeva E.N., Guseva G.B. ЛЮМИНОФОРЫ НА ОСНОВЕ АЛКИЛ- И ИОДЗАМЕЩЕННЫХ BODIPY: СПЕКТРАЛЬНЫЕ, ГЕНЕРАЦИОННЫЕ ХАРАКТЕРИСТИКИ, ФОТОСТАБИЛЬНОСТЬ И ЛИПОФИЛЬНОСТЬ ООО «МЕСОЛ», Россия, Москва, С. 250 (year - 2023)

7. Guseva G.B. BODIPY КРАСИТЕЛИ КАК ЭФФЕКТИВНЫЕ БИОМАРКЕРЫ ДЛЯ ФЛУОРЕСЦЕНТНОЙ ДИАГНОСТИКИ Казанский государственный медицинский университет, Казань, С. 17-19. (year - 2023)

8. Nuraneeva E.N., Guseva G.B., Antina E.V. ГАЛОГЕНЗАМЕЩЕННЫЕ ДИПИРРОМЕТЕНАТЫ БОРА(III) И ЦИНКА(II) КАК ПОТЕНЦИАЛЬНЫЕ СЕНСИБИЛИЗАТОРЫ ДЛЯ ФОТОДИНАМИЧЕСКОЙ ИНАКТИВАЦИИ МИКРООРГАНИЗМОВ Казанский государственный медицинский университет, Казань, С. 74-75 (year - 2023)

Annotation of the results obtained in 2020
In order to create new fluorescent biomarkers for visualization and elucidation of the action mechanisms of sulfur-containing terpenoids and their derivatives, a research team led by Dr. E.V. Antina (G.A. Krestov Institute of Solution Chemistry of Russian Academy of Sciences, Ivanovo) developed the synthesis methods and obtained 3,3',5,5'-tetramethyl-dipyrromethenates of boron (III) with meso-(CH2)3СООСН3 and meso-(CH2)3СООН substituents. The molecular structures of the synthesized luminophores were identified using mass spectrometry, X-ray diffraction, 1H NMR and elemental analysis. Geometric optimization (wB97X-D/def2TZVP) and TDDFT analysis (CAMB3LYP/def2-TZVP (PCM, DMSO)) of individual luminophores molecules were performed. The research results made it possible to analyze the influence of structural and conformational factors on the properties of the synthesized complexes. With the aim of the possible practical use of luminophores as fluorescent markers, the results of molecular docking were obtained, which made it possible to determine the most probable binding sites and types of interaction of BODIPY luminophores with transport blood proteins using the example of bovine and human serum albumin. The influence of structural and solvation factors on practically important spectral-luminescent characteristics of synthesized boron(III) dipyrromethenates in organic/inorganic media of different nature has been studied by the methods of spectrophotometry and spectrofluorimetry. It was found that the complexes absorb with high extinction coefficients in the blue-green region of the visible spectrum (496-503 nm) and emit (at 512-520 nm) with a high fluorescence quantum yield (~70-100%), which makes them easily detectable fluorescent markers. The fluorescence quantum yield of luminophores is maximum (~100%) in non-polar and weakly polar media (cyclohexane, benzene, toluene). The fluorescence decreases slightly (to ~ 80–97%) in proton-donor chloroform and alcohols. The increasing of the fluorescence quantum yield of BODIPY in the sequence of alcohols: ethanol, propanol, butanol-1, octanol-1, is apparently caused by a decrease in the mobility of the extended meso-substituent with increasing viscosity of the medium. The most noticeable fluorescence quenching of luminophores is observed in strong electron-donor media (up to ~73%), which is probably due to the high polarity and solvating ability of the medium. It has been shown that the luminophore substituted in the meso-position by butanoic acid is soluble in water up to ~5 ∙ 10-6 mol/l, which makes it promising for biological research. In order to predict the efficiency of the transport of markers across lipid or protein fragments of cell membranes, the lipophilicity of synthesized boron(III) dipyrromethenates was studied by determining the values of the distribution coefficient of luminophores in the two-phase model system water–octanol-1. Studies have shown that the introduction of an extended substituent (-(CH2)3-СООСН3) into the meso-position of the BODIPY nucleus leads to an increase in the hydrophobicity of the luminophore by almost ~1.3 times compared to the meso-unsubstituted analog and by ~5.1 times compared to the chelate substituted in the meso-position by the butanoic acid remainder. The practically important characteristics of the obtained luminophores have been studied, in particular, their photo- and thermal stability. It was found that the replacement of a hydrogen atom in the methine meso-spacer of BODIPY by extended -(CH2)3СООСН3 and -(CH2)3СООН groups noticeably increases the photostability of the synthesized luminophores in comparison with the meso-unsubstituted complex. This is probably due to the effect of steric shielding of the meso-spacer carbon atom, as well as the redistribution of electron density in the luminophores molecule due to the manifestation of the electronic effects of the meso-substituents. The results of the development and research of the physicochemical properties of new meso-modified fluorescent markers based on boron(III) dipyrromethenates were used in the framework of an interdisciplinary Project together with a scientific group led by Dr. Kayumov A.R. (Kazan Federal University, Kazan) in vitro biological research and analysis of the possibilities of practical application of the obtained BODIPY luminophores as fluorescent markers. The results of biological studies have shown that boron(III) dipyrromethenate with a meso-(CH2)3СООСН3 substituent exhibits the ability to efficiently penetrate cell membranes and can be proposed as a marker for the identification of pathogens of fungal infections, as well as for visualization of structural changes in the plasma membrane, which is important for purification of mammalian cells during apoptotic death. The results obtained for the reporting period of the Project are reflected in two articles published in the journals of the first quartile: 1) Galina Guseva, Elena Antina, Mikhail Berezin, Svetlana Lisovskaya, Roman Pavelyev, Airat Kayumov, Olga Lodochnikova, Daut Islamov, Konstantin Usachev, Sergei Boichuk and Liliya Nikitina. Spectroscopic and in vitro investigations of boron(III) complex with meso-4-methoxycarbonylpropylsubstituted dipyrromethene for fluorescence bioimaging applications. Molecules, 2020, V. 25, 4541, doi:10.3390/molecules25194541. 2) Galina. B. Guseva, Elena V. Antina, Mikhail B. Berezin, Roman S. Pavelyev, Airat R. Kayumov, Irshad S. Sharafutdinov, Svetlana А. Lisovskaya, Olga A. Lodochnikova, Daut R. Islamov, Konstantin S. Usachev, Sergei V. Boichuk, Liliya E. Nikitina. Meso-substituted-BODIPY based fluorescent biomarker: Spectral characteristics, photostability and possibilities for practical application. Journal of Photochemistry & Photobiology A: Chemistry 401 (2020) 112783. doi.org/10.1016/j.jphotochem.2020.112783.

Publications

1. Galina Guseva, Elena Antina, Mikhail Berezin, Svetlana Lisovskaya, Roman Pavelyev, Airat Kayumov, Olga Lodochnikova, Daut Islamov, Konstantin Usachev, Sergei Boichuk and Liliya Nikitina Spectroscopic and in vitro investigation of boron(III) complex witch meso-4-methoxycarbonylpropylsubstituted dipyrromethene for fluorescence bioimaging applications. Molecules, 25, 19, 4541, 1-15 (year - 2020) https://doi.org/10.3390/molecules25194541

2. Galina. B. Guseva, Elena V. Antina, Mikhail B. Berezin, Roman S. Pavelyev, Airat R. Kayumov, Irshad S. Sharafutdinov, Svetlana А. Lisovskaya, Olga A. Lodochnikova, Daut R. Islamov, Konstantin S. Usachev, Sergei V. Boichuk, Liliya E. Nikitina Meso-substituted-BODIPY based fluorescent biomarker: Spectral characteristics, photostability and possibilities for practical application Journal of Photochemistry & Photobiology A: Chemistry, 401, 112783 (year - 2020) https://doi.org/10.1016/j.jphotochem.2020.112783

3. Guseva G.B., Antina E.V., Nuraneeva E.N. ВЛИЯНИЕ МЕЗО-ЗАМЕСТИТЕЛЯ И ПРИРОДЫ СРЕДЫ НА СПЕКТРАЛЬНО-ЛЮМИНЕСЦЕНТНЫЕ СВОЙСТВА И ПРОЦЕССЫ ФОТОХИМИЧЕСКИХ ПРЕВРАЩЕНИЙ BODIPY КРАСИТЕЛЕЙ Сборник научных трудов XVII Международной конференции "Спектроскопия координационных соединений", С. 187-188 (year - 2020)

4. Guseva G.B., Antina E.V., Nuraneeva E.N. МЕЗО-ЗАМЕЩЕННЫЕ ДИПИРРОМЕТЕНАТЫ БОРА(III) КАК НОВЫЕ ФЛУОРЕСЦЕНТНЫЕ МАРКЕРЫ ДЛЯ БИОХИМИЧЕСКИХ ИССЛЕДОВАНИЙ VIII Международная конференция по физической химии краун-соединений, порфиринов и фталоцианин, с. 97 (year - 2020)

Annotation of the results obtained in 2021
In order to develop, on the basis of BODIPY, effective fluorescent biomarkers for visualizing metabolic pathways and elucidating the molecular mechanisms of monoterpenoids action and their derivatives exhibiting antimycotic and antibacterial activity, a scientific group led by Dr. E.V. Antinoy (ISC RAS, Ivanovo) at the second stage of the interdisciplinary Project, synthesis methods were developed and tetramethyl-substituted BODIPY luminophores with meso-(CH2)nCOOH and meso-(CH2)nCOOCH3 (n = 3, 4) substituents were obtained in working quantities. As part of an interdisciplinary project, together with a scientific group led by Dr. Kayumova A.R. (KFU, Kazan) BODIPY conjugates with (+)-myrtenol and its thioanalog were synthesized. The identification of the molecular structure of the synthesized compounds was carried out using the methods of X-ray diffraction, 1H, and 13C NMR, elemental analysis, and mass spectrometry. Geometric optimization of luminophores molecules (wB97X-D/def2 TZVP), TDDFT analysis (CAMB3LYP/def2-TZVP (PCM, ДМСО)) and molecular docking were carried out. A comparative analysis of the obtained results at the first and second stages of the Project showed that the introduction of meso-substituents and an increase in the length of their hydrocarbon chain causes a slight blue shift in the maximum of the characteristic intense S0-S1 band compared to the meso-unsubstituted analog due to the manifestation of the total electronic effect of oxygen-containing substituents and enhancement of the distortion of the plane of the aromatic indacene structure of the chromophore. Based on the molecular docking results, it was found that the most stable complexes with BSA form luminophores with carboxylic acid residues. The BODIPY conjugate with myrtenol, due to its greater hydrophobicity, has a higher affinity to the HSA hybrid pockets, which contributes to the formation of stable supramolecular conjugate – HSA systems. During the reporting period, we studied the influence of structural and solvation effects on the spectral properties of boron(III) dipyrromethenates substituted at the meso-position by the pentanoic acid residue -(CH2)4СООН or its esters with methanol -(CH2)4СООСН3 and myrtenol in a solvents series of various nature: non-polar and aromatic hydrocarbons (cyclohexane, toluene, benzene), proton-donating (chloroform, ethanol, propanol, 1-butanol, 1-octanol) and polar (N,N-dimethylformamide, dimethyl sulfoxide, pyridine, water). It was found that, in contrast to completely hydrophobic forms of BODIPY esters, the carboxylic acid derivatives are slightly soluble in water with an approximate solubility index of up to ~ 5∙10-6 mol/L, which is of interest for the use of these compounds in biochemistry and medicine. It was found that meso-substitution does not reduce the high fluorescence quantum yield of the compounds. A more noticeable effect on the fluorescence of meso-functionalized dyes is exerted by the medium properties. It was found that the values of the fluorescence quantum yield of luminophores are maximum (almost ~100%) in non-polar and aromatic media (cyclohexane, benzene, toluene), but the values decrease to ~80% in proton-donating chloroform and alcohols. The flare-up of BODIPY fluorescence in the sequence of alcohols: ethanol, propanol, 1-butanol, 1-octanol can be caused by a mobility decrease of the extended meso-substituent with an increase in the medium viscosity, which requires special further studies. A more noticeable fluorescence quenching (up to values of φfl = ~ 60–70%) of luminophores is observed in highly polar electron-donor media. The ability of hydrophobic meso-substituted BODIPY luminophores to associative processes in water was studied. It was found that the introduction of an extended alkyl substituent with an ether group into the BODIPY meso-spacer prevents the formation of aggregates in aqueous solutions, while the characteristics of the intensely emitting form of the monomer are retained, which makes it possible to use this type of luminophores in bioimaging. The transport capabilities of the synthesized luminopores across the double lipid layer of cell membranes were assessed based on the results of the analysis of their lipophilicity. Analysis of the distribution coefficients of luminophores in model two-phase media 1-octanol – water and HEPES – water indicates a higher (~1.2–1.4 times) lipophilicity of the BODIPY esters and the corresponding conjugate with myrtenol as compared to the meso-unsubstituted analog, which is of interest for bioimaging. An analysis of the BODIPY fluorescent sensors' sensitivity to hydrophobic/hydrophilic zones of transport blood albumin (BSA, HSA) was carried out based on the results of spectrophotometric and fluorometric titration in model physiological media (phosphate buffer, DMSO). The research results showed that an increase in the concentration of SA is accompanied by a significant flare-up of fluorescence (in ~ 2–18 times) and a blue shift of the emission maximum band of luminophores (up to 7 nm). The observed nonlinear character of the Stern-Volmer dependence indicates that the fluorescence flare-up of meso-substituted BODIPY luminophores and the corresponding conjugate with mertinol is due to a mixed mechanism, including both dynamic and static components. An analysis of the thermodynamic parameters (Kb and ∆G) of the binding efficiency of BODIPY – SA showed that the conjugate with myrtenol forms the most stable supramolecular systems with HSA as compared to BODIPY luminophores containing pentanoic acid or its ester residue. Practically important characteristics of luminophores, in particular, their photo- and thermal stability, have been studied. Comparative analysis of research results for the first and second reporting periods of the Project made it possible to conclude that the replacement of both carboxylic acid residues by ester groups, and lengthening by one СН2-fragment of the hydrocarbon chain of the meso-substituent have practically no significant effect on the photostability of BODIPY luminophores. A practically important result is the revealed tendency of an almost twofold increase in the photostability of meso-substituted BODIPY luminophores in comparison with the meso-unsubstituted analog, which is retained in all studied organic solvents. The observed differences may be due to the manifestation of the steric shielding effect of conjugated π-bonds of the meso-methine group as the most chemically active fragment of the aromatic chromophore system of BODIPY luminophores. The medium nature has a noticeable effect on the photodestruction of the studied luminophores. It was found that the photooxidation process of dyes in solutions is more efficient (~2–3 times) in aromatic toluene in comparison with cyclohexane or 1-octanol. The observed differences can be caused by an increase in the polarization of the aromatic system of the dipyrromethene ligand due to π–π stacking with molecules of the aromatic solvent. A more efficient (by ~10 times) course of the photodegradation processes of luminophores was observed in DMSO, which is probably caused by the high polarity and solvating ability of the solvent. Thermogravimetric analysis of crystalline samples of meso-substituted boron(III) dipyrromethenates in an inert argon atmosphere showed that the destruction of the complexes proceeds due to intramolecular oxidation-reduction processes. The beginning destruction temperature of chelates is in the range of 259–287° C. Moreover, in comparison with the meso-unsubstituted BODIPY, the thermal stability of a luminophore with with an esterified substituent increases by almost 30 degrees. Within the framework of an interdisciplinary Project, together with colleagues from Kazan, biological research was carried out and the possibilities of the practical application of meso-substituted luminophores for bioimaging were assessed. The research results showed that BODIPY luminophores, substituted at the meso-position by the methyl ester residues of pentanoic or butanoic acids, are able to efficiently penetrate cell membranes and intensively stain the membrane structures of organelles, which can be used in the study of pathogens of bacterial and fungal infections. In addition, it was found that the meso-substituted BODIPY esters penetrate much faster into the cells of gram-positive bacteria compared to gram-negative microorganisms, which is of interest in the development of new fluorescent markers for differential staining of gram-positive and gram-negative bacteria. The conducted biological studies suggested that the conjugation of BODIPY with myrtenol changes the preferred target for staining, which determines the specificity of its bio transport and localization in cell structures. During the reporting period, the obtained research results by the team under the leadership of Dr. E.V. Antinoy (ISC RAS) within the framework of an interdisciplinary Project are reflected in three articles, of which two articles were published in the journals of the first quartile and one in the journal of the second quartile, as well as in the abstracts of six reports presented at conferences at the International and All-Russian levels.

Publications

1. Galina B. Guseva, Elena V. Antina, Mikhail B. Berezin, Roman S. Pavelyev, Airat R. Kayumov, Olga V. Ostolopovskaya, Ilmir R. Gilfanov, Larisa L. Frolova, Alexander V. Kutchin, Rustem F. Akhverdiev, Svetlana A. Lisovskaya, Elena Y. Trizna, etl. Design, Spectral Characteristics, and Possibilities for Practical Application of BODIPY FL-Labeled Monoterpenoid ACS Applied Bio Materials, 4, 8, 6227–6235 (year - 2021) https://doi.org/10.1021/acsabm.1c00550

2. Galina B.Guseva, Ekaterina N.Nuraneeva, Mikhail B.Berezin, Elena V.Antina Effect of meso-substituents and solvent on the photo- and thermal stability of BODIPY dyes Journal of Photochemistry and Photobiology A: Chemistry, 423, 113620 (year - 2022) https://doi.org/10.1016/j.jphotochem.2021.113620

3. Lubov A. Antina, Alexander A. Ksenofontov, Alexander V. Kazak, Nadezhda V. Usol’tseva, Elena V. Antina , Mikhail B. Berezin Effect of ms-substitution on aggregation behavior and spectroscopic properties of BODIPY dyes in aqueous solution, Langmuir-Schaefer and poly(methyl methacrylate) thin films Colloids and Surfaces A: Physicochemical and Engineering Aspects, 618, 126449 (year - 2021) https://doi.org/10.1016/j.colsurfa.2021.126449

4. Guseva G.B., Antina E.V. Влияние мезо-замещения на термостабильность дипиррометенатов бора(III) Кластер конференций 2021: XIV Международная научная конференция «Проблемы сольватации и комплексообразования в растворах», 2021, С. 52 (year - 2021)

5. Guseva G.B., Antina E.V., Berezin M.B. Мезо-функционализированные bodipy люминофоры как новые биомаркеры: синтез, структура, спектральные свойства и фотостабильность Кластер конференций 2021: XIV Международная научная конференция «Проблемы сольватации и комплексообразования в растворах», 406 (year - 2021)

6. Smirnova A.S., Nuraneeva E.N. Мезо-замещенные дипиррометенаты бора(III) и конъюгат с тиотерпеноидом: синтез, спектральные и фотохимические свойства Всероссийская школа-конференция молодых ученых «Фундаментальные науки – специалисту нового времени», С. 311 (year - 2021)

7. Smirnova A.S., Nuraneeva E.N., Guseva G.B., Antina E.V. Мезо-карбокси- и мезо-метоксикарбонилбутилзамещенные дипиррометенаты бора(III) и конъюгат с тиотерпеноидом: синтез, спектральные свойства, фотостабильность XI Конференция молодых ученых по общей и неорганической химии, 2021, 61-62 (year - 2021)

8. Smirnova U. V., Nuraneeva E. N. Мезо-замещенные BODIPY люминофоры: структура, спектральные свойства и фотостабильность Всероссийская школа-конференция молодых ученых «Фундаментальные науки – специалисту нового времени», С. 312 (year - 2021)

9. Smirnova U.V., Guseva G.B., Nuraneeva E.N. Люминофоры на основе мезо-замещенных дипиррометенатов бора(III) как флуоресцентные маркеры при биовизуализации XI Конференция молодых ученых по общей и неорганической химии, 2021, С. 140 (year - 2021)

Annotation of the results obtained in 2022
In order to create effective fluorescent biomarkers based on BODIPY dyes for bioimaging of monoterpenoids and their derivatives that exhibit antimycotic and antibacterial activity, a scientific group led by Dr. Antina E.V. (G.A. Krestov Institute of Solution Chemistry of the Russian Academy of Sciences, Ivanovo) at the third stage of the interdisciplinary Project, synthesis methods were developed and boron(III) dipyrromethenates were obtained in working quantities, in the molecules of which asymmetric substitution on pyrrole nuclei is realized: the alpha-position of one of the pyrrole nuclei contains a propanoic acid residue, while the alpha position of the second nucleus contains a methyl group or a thiophene residue, respectively. Within the framework of an interdisciplinary project, together with a scientific group led by Dr. Kayumov A.R. (KFU, Kazan) synthesis of BODIPY conjugates with titerpenoid was performed. The molecular structure of the synthesized compounds was identified using X-ray diffraction, 1H and 13C NMR, elemental analysis, and mass spectrometry. Geometrical optimization of BODIPY luminophore molecules containing propanoic acid and/or thiophene residues was carried out using DFT and TDDFT analysis. It has been established that one of the probable causes of BODIPY fluorescence quenching in the presence of electron-donating solvents may be the formation of BODIPY∙Solv molecular complexes that implement photoinduced electron transfer (PeT). To screen functionalized BODIPY as promising luminescent probes for blood transport proteins, molecular docking for dyes with bovine serum albumin and human serum albumin was performed. The results of a computer study showed that the process of formation of BODIPY–SA molecular complexes is spontaneous and proceeds due to the prevailing hydrophobic and specific interactions. Moreover, the most stable complexes with serum albumins are formed by luminophores substituted in the meso position of the dipyrromethene core by pentanoic and butanoic acid residues and, especially, BODIPY conjugates with a thiotherpene fragment. During the reporting period, the spectral and luminescent characteristics of alpha-substituted carboxylic acids BODIPY and conjugates of meso-substituted BODIPY with thiotherpenoid were studied. It was found that the introduction of a propanoic acid residue into the alpha-position of one of the pyrrole cores of BODIPY gives a slight red shift (by 1–3 nm) of the absorption and fluorescence maximum bands compared to the tetramethyl-substituted analogue. The replacement of the alpha-CH3 group in the second pyrrole core of boron(III) dipyrromethanate with a thiophene residue leads to a more significant (by ~60 nm) red shift of the absorption and fluorescence bands. It has been established that α-substituted carboxylic acids BODIPY have the highest fluorescence quantum yield (from ~83 to ~91%) in aromatic benzene and toluene, as well as in proton-donor chloroform and alcohols. Significant quenching (up to ~43–54%) of dye fluorescence, especially in the case of the carboxylic acid BODIPY with a thiophene substituent, is recorded in solvents with pronounced electron donor properties (DMF, DMSO, Py). The observed fluorescence quenching of BODIPY carboxylic acids is due to the formation of BODIPY∙Solv molecular complexes via the formation of a strong hydrogen bond between the OH group of the BODIPY carboxyl fragment and the solvent heteroatom by the PeT mechanism. Functionalization of the BODIPY dipyrromethene core by covalent binding of a thiotherpenoid to a butanoic or pentanoic acid residue leads to a hypsochromic shift of the absorption band maxima up to ~7 nm and a noticeable (up to ~2-fold) increase in the Stokes shift compared to the meso-unsubstituted analogue. The observed effect can be caused by structural differences between the ground and excited states of the luminophores due to the conformational mobility of the meso-substituent. The results of a comparative analysis of the obtained data for the first, second, and third reporting periods showed that differences in the nature of the meso-substituent have an insignificant effect on the position of the absorption (or emission) band maxima and the dye fluorescence quantum yield. In the general case, irrespective of the medium properties, the fluorescence quantum yields of BODIPY conjugates with thiterpenoid decrease within the error (by ~2–10%) compared to meso-substituted BODIPY carboxylic acids, their esters, and myrtenol conjugate. The medium nature has a more noticeable effect on the spectral characteristics of dyes. BODIPY conjugates with thiotherpenoid exhibit maximum fluorescence (φ = ~83–98%) in nonpolar, aromatic, and proton donor solvents (cyclohexane, benzene, toluene, chloroform, and alcohols). A more noticeable fluorescence quenching (up to φ = ~72%) of the compounds is observed in strong electron-donating media (DMSO, DMF, pyridine), which may be due to an increase in the efficiency of the universal solvation of luminophores by polar electron-donating solvent molecules. The results of spectral studies have shown that both BODIPY carboxylic acids and BODIPY conjugates with a thiotherpenoid are very stable in both acidic and alkaline media, which is important in the development of new fluorescent biomarkers. The affinity for blood transport proteins of BODIPY luminophores containing butanoic acid residue, its ester and the corresponding BODIPY conjugate with thiotherpenoid in the meso-spacer was studied. The results of fluorescent titration showed that an increase in protein concentration is accompanied by a significant increase in fluorescence (1.2–6 times for BSA, 1.1–5.5 times for HSA) and a blue shift in the dye emission maximum (by 1–2 nm for BSA and 1–8 nm for HSA). The analysis of the thermodynamic parameters (Kb and ∆G) characterizing the efficiency of BODIPY–SA binding showed that the conjugate of BODIPY with HSA forms the most stable supramolecular systems compared to BODIPY luminophores containing a butanoic acid residue and its ester. The studied BODIPY dyes can be proposed as fluorescent probes for hydrophilic and hydrophobic regions of blood transport proteins. The stability of alpha-substituted carboxylic acids BODIPY and conjugates of BODIPY with a thiotherpenoid under the action of UV irradiation in different nature solvents, including those simulating biological media, was evaluated. It has been established that the introduction of a propanoic acid residue into the alpha-position of one of the pyrrole nuclei almost does not reduce the photostability of luminophores compared to the symmetrically substituted tetramethylated analog. A tendency for an almost twofold increase in the photostability of BODIPY conjugates with a thiotherpenoid was revealed, which is retained in all the studied media. The observed differences may be due to the manifestation of the effect of steric screening of the conjugated π-bonds of the meso-methine group as the most reactive fragment of the aromatic chromophore system of BODIPY luminophores. The photooxidation of dyes in solutions proceeds more efficiently (~2–3 times) in aromatic toluene than in cyclohexane or 1-octanol, which can be caused by an increase in the polarization of the aromatic system of the dipyrromethene ligand due to π–π stacking with aromatic solvent molecules. The occurrence of luminophores photodegradation processes is enhanced by almost ~6–10 times in DMSO, especially in water, which is probably caused by the formation of radical products due to solvent photolysis. The results of biological studies have shown that dyes individually penetrate and specifically stain cells of Candida albicans and filamentous fungi Fusarium solani and their structural organelles, which is of interest in studying the differences in a wide range of mycotic infections. It has been demonstrated that conjugation of BODIPY with a thiotherpenoid is an excellent way to increase the affinity of dyes for biostructures, including blood components. During the reporting period, the results of obtained research by a team led by Dr. Antina E.V. are reflected in two articles, one of which was published in the journal Q1 in the form of a Review, the second article in the journal Q2, as well as in the abstracts of three reports presented at the conferences of the International and All-Russian levels.

Publications

1. Galina B. Guseva, Elena V. Antina, Mikhail B. Berezin, Anastassia S. Smirnova, Roman S. Pavelyev, Ilmir R. Gilfanov, Oksana G. Shevchenko, Svetlana V. Pestova, Evgeny S. Izmest’ev, Svetlana A. Rubtsova, Olga V. Ostolopovskaya, Sergey V. Efimov et al. Design, Spectral Characteristics, Photostability, and Possibilities for Practical Application of BODIPY FL-Labeled Thioterpenoid MDPI AG, 9, 5, 210 (year - 2022) https://doi.org/10.3390/bioengineering9050210

2. Elena Antina, Natalia Bumagina, Yuriy Marfin, Galina Guseva, Liliya Nikitina, Dmitry Sbytov and Felix Telegin BODIPY Conjugates as Functional Compounds for Medical Diagnostics and Treatment MDPI, 27, 4, 1396 (year - 2022) https://doi.org/10.3390/molecules27041396

3. E.N. Nuraneeva, G.B. Guseva, E.V. Antina Люминофоры на основе алкил-, эфир- и галогензамещенных дипиррометенатов бора(III): спектральные свойства, фотостабильность и липофильность Кубанский гос. ун-т, Краснодар, 84 (year - 2022)

4. Smirnova A.S. Мезо-замещенные дипиррометенаты бора(III) и их конъюгаты с монотерпеновыми производными: структура, спектральные характеристики, липофильность, фотостабильность, области практического применения Ивановский государственный химико-технологический университет, 156 (year - 2022)

5. Smirnova U.V., Nuraneeva E.N. Влияние структурных и сольватационных факторов на спектральные свойства, липофильность и фотостабильность BODIPY люминофоров, содержащих метильные заместители, остатки карбоновых кислот и их сложные эфиры Ивановский государственный химико-технологический университет, Иваново, 157 (year - 2022)