In this work, we have shown that the introduction of a trifluoromethyl group into the me-ta-position of arylidene imidazolones (GFP chromophore core) leads to a dramatic increase in their fluorescence in nonpolar and aprotic media. The presence of a pronounced solvent-dependent gradation of fluorescence intensity makes it possible to use these substances as fluorescent polarity sensors. In particular, we showed that one of the created compounds could be used for selective labeling of the endoplasmic reticulum of living cells.

2-Acyloxybenzaldehydes are converted into 2-hydroxybenzofuranones in good to excellent yields (60–99%). The reaction proceeds at room temperature in DMSO upon 365 nm LED irradiation under photocatalyst-free conditions. The present atom-economical synthetic approach represents the aldehyde group umpolung reactivity.

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.

Objective: A pair of fluorogenic dyes with three electron-donating groups, based on arylideneimidazolones, is presented. Methods: The formation of imines of the corresponding aldehydes with the subsequent [3 + 2] cycloaddition was used for the synthesis of arylidene-imidazolones. The optical properties of the new compounds were studied. The fluorescence microscopy was used for imaging lipid droplets of the living cells, labeled by the dyes. Results and Discussion: We discovered that a pair of the proposed substances is characterized by a significant bathochromic shift of the absorption and emission maxima, as well as by a noticeable variation of the position of the emission maximum depending on the properties of the environment. We demonstrated that (Z)-5-(3,5-bis(dimethylamino)-4-(ethylamino)benzylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one can stain lipid droplets in HeLa Kyoto and Huh 7.5 cell lines. Conclusions: We designed a new selective fluorogenic dye based on arylidene-imidazolones and showed that it can be applicable for lipid droplets labeling in living cells.

Objective: The design of a new fluorogen-activating protein nano-frFAST and a series of fluorogens with an extended π-system are presented. Methods: The formation of imines of the corresponding cinnamaldehydes followed by [2 + 3]-cycloaddition was used for the synthesis of arylallylidene-imidazolones. The arylallylidene-rhodanine synthesis was performed by Knoevenagel condensation of the corresponding cinnamaldehydes with rhodanine. The other compounds were synthesized by condensation of arylidene-imidazolone analogues with isonicotinaldehyde. The new protein was screened in vitro and its optical properties were studied. Spectrofluorimetric titration was performed to evaluate the binding constant. Imaging of the [nano-frFAST–HPAR-DOM]-labeled cellular structures was performed by the wide-field fluorescence microscopy. Results and Discussion: We have shown that (Z)-5-((E)-3-(4-hydroxy-2,5-dimethoxyphenyl)allylidene)-2-thioxothiazolidin-4-one (hereafter, HPAR-DOM) can act as an excellent fluorogen for the nano-frFAST protein. We have demonstrated that the nano-frFAST–HPAR-DOM complex can be used in fluorescent labeling of individual compartments of the living HeLa Kyoto cells in the far-red region of the spectrum. Conclusions: We have developed the novel fluorogen-activating protein nano-frFAST and the fluorogen HPAR-DOM for this protein and demonstrated that this pair can be used as a genetically encoded far-red fluorescent label in living cells.