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.

In this paper, we propose a fluorescence-lifetime imaging microscopy (FLIM) multiplexing system based on the fluorogen-activating protein FAST. This genetically encoded fluorescent labeling platform employs FAST mutants that activate the same fluorogen but provide different fluorescence lifetimes for each specific protein-dye pair. All the proposed probes with varying lifetimes possess nearly identical and the smallest-in-class size, along with quite similar steady-state optical properties. In live mammalian cells, we target these chemogenetic tags to two intracellular structures simultaneously, where their fluorescence signals are clearly distinguished by FLIM. Due to the unique structure of certain fluorogens under study, their complexes with FAST mutants display a monophasic fluorescence decay, which may facilitate enhanced multiplexing efficiency by reducing signal cross-talks and providing optimal prerequisites for signal separation upon co-localized and/or spatially overlapped labeling. A genetically encoded labeling system uses smallest-in-class fluorogen-activating protein tags for time-resolved fluorescence multiplexed cellular imaging, offering monoexponential decay and potential for sophisticated fluorescence lifetime analysis.

In the present study, we demonstrated that the introduction of a 1,4-diethyl-1,2,3,4-tetrahydroquinoxalin moiety into the arylidene part of GFP chromophore-derived compounds results in the formation of environment-sensitive fluorogens. The rationally designed and synthesized compounds exhibit remarkable solvent- and pH-dependence in fluorescence intensity. The solvent-dependent variation in fluorescence quantum yield makes it possible to use some of the proposed compounds as polarity sensors suitable for selective endoplasmic reticulum fluorescent labeling in living cells. Moreover, the pH-dependent emission intensity variation of other fluorogens makes them selective fluorescent labels for the lysosomes in living cells.

NanoFAST is the smallest fluorogen-activating protein, consisting of only 98 amino acids, used as a genetically encoded fluorescent tag. Previously, only a single fluorogen with an orange color was revealed for this protein. In the present paper, using rational mutagenesis and in vitro screening of fluorogens libraries, we expanded the color palette of this tag. We discovered that E46Q is one of the key substitutions enabling the range of possible fluorogens to be expanded. The introduction of this and several other substitutions has made it possible to use not only orange but also red and green fluorogens with the modified protein.

We report a series of orto-substituted aryliden-imidazolones and their derivatives containing styrene moiety. These compounds can be used like ligands of NanoLuc protein. Together with NanoLuc this fluorogens can be used for genetically encoded labeling in fluorescence microscopy, as demonstrated by staining HEK293 cells.

We report a series of 3-aminocoumaranones possess significant chemiluminescence under various conditions. The introduce 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. We showed that some 3-aminocoumaranones can be used for detecting of the urease and hydrogen peroxide.

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.

Solvatochromic compounds have emerged as valuable environment-sensitive probes for biological research. Here we used thiol-reactive solvatochromic analogs of the green fluorescent protein (GFP) chromophore to track conformational changes in two proteins, recoverin and the A2A adenosine receptor (A2AAR). Two dyes showed Ca2+-induced fluorescence changes when attached to recoverin. Our best-performing dye, DyeC, exhibited agonist-induced changes in both intensity and shape of its fluorescence spectrum when attached to A2AAR; none of these effects were observed with other common environment-sensitive dyes. Molecular dynamics simulations showed that activation of the A2AAR led to a more confined and hydrophilic environment for DyeC. Additionally, an allosteric modulator of A2AAR induced distinct fluorescence changes in the DyeC spectrum, indicating a unique receptor conformation. Our study demonstrated that GFP-inspired dyes are effective for detecting structural changes in G protein-coupled receptors (GPCRs), offering advantages such as intensity-based and ratiometric tracking, redshifted fluorescence spectra, and sensitivity to allosteric modulation.

A new operationally simple single step method for synthesis indole-2-carboxylates from 2-dimethylaminobenzaldehydes and nitroacetic acid esters in 22-59 % yields has been developed. A new simple single step method for synthesis indole-2-carboxylates from 2-dimethylaminobenzaldehydes and nitroacetic acid esters are presented.image

ortho -Dialkylaminoarylidene malonates undergo hydrogen transfer mediated cyclization in the absence of a catalyst under irradiation. The process suits Green chemistry principles: it does not need any toxic solvents and requires only light to proceed.

A simple procedure was developed for the synthesis of 2H-pyrido[3,4-c][1,2]benzoxazine-2,4(3H)-diones by the treatment of o-fluorophenyl-substituted 6-oxo-6H-1,2-oxazine-3-carboxylates with primary amines. The reaction proceeded according to the mechanism previously described for the formation of 3-(hydroxyimino)pyridine-2,6-diones followed by intramolecular nucleophilic substitution involving the hydroxyimino group to form the 1,2-oxazine ring.

The study of structure–function relationships between a chromophore and its protein environment plays a key role in photophysical engineering of fluorescent proteins (FPs), specifically, in the guided design of their new variants with a higher fluorescence quantum yield (FQY). The known approaches to FQY increasing mostly rely on suppression of the excited state nonradiative processes, but no tools have been suggested for the tuning of the radiative rate constant (kr), which is also a potentially “adjustable” value. Here, we propose an experimental approach, where the synthetic chromophore of FP models the “fixation” of the most important radiationless constants and allows monitoring of the fluorescence lifetime flexibility (as an indicator of the kr value). As a proof-of-concept, we studied the time-resolved fluorescence behavior of the green and blue FP chromophore analogs in diverse chemical environments. The conformationally locked analog of the GFP chromophore in most cases showed monophasic fluorescence decay kinetics with a lifetime of 2.7–3.0 ns, thus adequately modeling the typical behavior of GFPs with the highest FQYs. Under the conditions of stimulated ionization of this chromophore, we observed the increased (up to 4.3–4.6 ns) fluorescence lifetimes, which can be interpreted in terms of an increase in the radiative constant (kr). The conformationally locked analog of the Sirius chromophore showed biexponential fluorescence decay kinetics, partly simulating the properties of the blue FPs. In an acetic acid solution, this compound exhibited distinct fluorescent properties (elevated fluorescence intensity with a major lifetime population of 4 ns), which can be interpreted as emission of an unusual cationic form of the chromophore.

Interaction of imidazol-5-one-based donor–acceptor cyclopropanes with thiourea and thioacetamide was studied. Previously undescribed spirocyclic 2-amino- and 2-iminotetrahydrothiophenes are formed in the reaction.

In this work, we showed that the well-known NanoLuc luciferase can act as a fluorogen activating protein for various arylidene-imidazolones structurally similar to the Kaede protein chromophore. We showed that such compounds can be used as fluorescent sensors for this protein and can also be used in pairs with it in fluorescent microscopy as a genetically encoded tag.

A methodology was developed for the synthesis of 1-ethoxy-6-methyl-2H-[1,2]oxazino[3,4-c]quinoline-2,5(6H)-diones from methyl 4-[2-(dimethylamino)phenyl]-5-ethoxy-6-oxo-6H-1,2-oxazine-3-carboxylates by the action of titanium tetrachloride under inert atmosphere. This interaction provides a rare example of demethylation followed by cyclization, yielding several new compounds.

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.

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.

(1-Alkyltetrahydroquinolin-8-yl)methylidene-1H-imidazol-5(4H)-ones undergo spirocyclization by the action of SnCl4 via 1,5-hydride shift to form spirocyclic julolidine derivatives in 68–97% yields.

Pyridineacetic acids undergo dimerization under action of the activating agents, giving rise to 2-hydroxy-3-(pyridin-2-yl)-4H-quinolizin-4-ones. The introduction of a difluoroboryl bridge into such derivatives leads to novel highly fluorescent compounds.

In present paper, we showed that phosphazenes formed from azidoacetamides and its derivatives can be successfully used for variously substituted 3-alkyl-2-thiohydantoins creation. The presented reaction with carbon disulfide is characterized by high yields and good functional group tolerance.