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Objective: Fluorogens are synthetic molecules that become fluorescent in specific environments, i.e. in pockets of fluorogen-activating protein (FAP). In pursuit of increased diversity of fluorescent labeling methods, our group is interested in developing new FAP-fluorogen pairs with unique features. Methods: In this study, we focused on synthesis of prospective fluorogens for NanoLuc luciferase used as FAP. A row of orto-substituted aryliden-imidazolones and their derivatives containing styrene moiety was synthesized and tested in vitro with purified NanoLuc protein. The selected dye was used as fluorogen in widefield microscopy of live HEK 293 cells transiently transfected with NanoLuc-H2B coding plasmid. Results and Discussion: The structure of the GFP and Kaede proteins chromophores is very similar to the structure of the luciferins of the NanoLuc luciferase. In this regards, we synthesized a series of such arylidene-imidazolones and tested them with NanoLuc protein. It was found that some the created compounds are able to bind to the NanoLuc protein and form fluorescent complexes. One of them had a high affinity for NanoLuc protein and was used as a successful fluorogen in pair with NanoLuc in live cell microscopy. Conclusions: We report a series of orto-substituted aryliden-imidazolones and their derivatives containing styrene moiety. These compounds can be used as ligands of NanoLuc protein. Together with NanoLuc these fluorogens can be used for genetically encoded labeling in fluorescence microscopy, as demonstrated by staining HEK293 cells. [ABSTRACT FROM AUTHOR]

Objective: Lipid droplets (adiposomes) are cellular organelles with distinguish structure and functions that play significant role in energy homeostasis and lipotoxicity and oxidative stress prevention, therefore represent a desirable target for fluorescent labeling. In this work, we proposed a number of new fluorogenic compounds—arylidene-rhodanines and their acyclic analogues – substances that label lipid droplets due to structural fragments responsible for the appearance of fluorescence in a lipophilic environment. Methods: The target arylidene-rhodanines and their acyclic analogues were obtained using modified literature methods of Knoevenagel condensation, the optical properties of the resulting compounds in a standard set of solvents were studied and then the most promising substances were tested as fluorescent dyes on Hela Kyoto cells using widefield microscopy. Discussion: We discovered that the derivatives containing a 2,5-dimethoxybenzylidene fragment or similar groups are characterized by a noticeable variation in fluorescence quantum yield depending on the environment properties. Cellular labeling of such compounds was studied. We showed that incubation of Hela Kyoto cells with substances (In) and (III) led to fluorescence appearance in the BFP channel, associated with the location of lipid droplets detected in the cells when visualized in transmitted light. Conclusions: As a result, we found that two obtained compounds—dimethoxybenzylidene-rhodanine and dimethoxybenzylidene-malononitrile – can be used as selective fluorogenic dyes for lipid droplets labeling in live Hela Kyoto cells under widefield fluorescent microscopy conditions. [ABSTRACT FROM AUTHOR]

Objective: The phenomenon of chemiluminescence - the emission of light by a substance after oxidation - has been known for a very long time. Substances capable of chemiluminescence are actively used for the detection and quantitative determination of a wide variety of analytes. In this work, we studied the possibility of using 3-amino-coumaranones for the detection of hydrogen peroxide and urease activity study. Methods: In this work, we synthesized a series of 3-amino-coumaran-2-one derivatives, which can be divided into two groups: amide derivatives and urea derivatives. The optical and chemiluminescent properties of all the obtained compounds were studied under various conditions. Results and Discussion: In this work, a series of 3-amino-coumaranones was synthesized. It has been found that amide derivatives and derivatives of asymmetrically substituted ureas have significant chemiluminescence under various conditions. The introduction of the electron-donor groups in the coumaranone moiety and increasing of the conjugated π-system of coumaranones allow to shift 30–40 nm of the chemiluminescence maxima to the long-wavelength region. For N,N-disubstituted unsymmetrical urea derivatives it is shown that the luminescence intensity can be different under various conditions. It has been shown that derivatives of N,N-disubstituted unsymmetrical ureas can be used for the determination of hydrogen peroxide, and some compounds, due to their bright luminescence in the presence of urease, can be used as indicators of its enzymatic activity. Conclusions: We showed that some 3-amino-coumaranones can be used for detecting the urease and hydrogen peroxide. [ABSTRACT FROM AUTHOR]

We report a novel mutant of FAST–FAST T70V. The fluorescent properties of the complex were studied to show that the T70V mutation only slightly affects the fluorescent properties but has no effect on the photostability of the protein–fluorogen complex. Thus, evidence was obtained showing that the threonine alcohol group is not involved in the irreversible photodegradation of the complex, despite the possible contact of the amino acid with the fluorogen. [ABSTRACT FROM AUTHOR]