A Combination of Library Screening and Rational Mutagenesis Expands the Available Color Palette of the Smallest Fluorogen-Activating Protein Tag nanoFAST.

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.

Synthesis of chroman-annulated cyclopropanols via photoinduced intramolecular [2 + 1]-cycloaddition of 2-allyloxybenzaldehydes.

2-Allyloxybenzaldehydes undergo [2 + 1] cycloadditions under 365 nm LED irradiation to form the corresponding chroman-fused cyclopropanols. The reaction proceeds easily without any catalysts or additives in dimethyl sulfoxide.

Meta-CF3-Substituted Analogues of the GFP Chromophore with Remarkable Solvatochromism.

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.

NanoLuc Luciferase as a Fluorogen-Activating Protein for GFP Chromophore Based Fluorogens.

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.

[1,5]-Hydride Shift Triggered N-Dealkylative Cyclization into 2-Oxo-1,2,3,4-tetrahydroquinoline-3-carboxylates via Boronate Complexes.

A new simple one-pot two-step protocol for the synthesis of 2-oxo-1,2,3,4-tetrahydroquinoline-3-carboxylate from 2-(2-(benzylamino)benzylidene)malonate under the action of BF3·Et2O was developed. It was shown that the reaction proceeds through the formation of a stable iminium intermediate containing a difluoroboryl bridge in the dicarbonyl fragment of the molecule.

Micro-Raman Characterization of Structural Features of High-k Stack Layer of SOI Nanowire Chip, Designed to Detect Circular RNA Associated with the Development of Glioma.

The application of micro-Raman spectroscopy was used for characterization of structural features of the high-k stack (h-k) layer of "silicon-on-insulator" (SOI) nanowire (NW) chip (h-k-SOI-NW chip), including Al2O3 and HfO2 in various combinations after heat treatment from 425 to 1000 °C. After that, the NW structures h-k-SOI-NW chip was created using gas plasma etching optical lithography. The stability of the signals from the monocrine phase of HfO2 was shown. Significant differences were found in the elastic stresses of the silicon layers for very thick (>200 nm) Al2O3 layers. In the UV spectra of SOI layers of a silicon substrate with HfO2, shoulders in the Raman spectrum were observed at 480-490 cm-1 of single-phonon scattering. The h-k-SOI-NW chip created in this way has been used for the detection of DNA-oligonucleotide sequences (oDNA), that became a synthetic analog of circular RNA-circ-SHKBP1 associated with the development of glioma at a concentration of 1.1 × 10-16 M. The possibility of using such h-k-SOI NW chips for the detection of circ-SHKBP1 in blood plasma of patients diagnosed with neoplasm of uncertain nature of the brain and central nervous system was shown.

NanoFAST: structure-based design of a small fluorogen-activating protein with only 98 amino acids.

One of the essential characteristics of any tag used in bioscience and medical applications is its size. The larger the label, the more it may affect the studied object, and the more it may distort its behavior. In this paper, using NMR spectroscopy and X-ray crystallography, we have studied the structure of fluorogen-activating protein FAST both in the apo form and in complex with the fluorogen. We showed that significant change in the protein occurs upon interaction with the ligand. While the protein is completely ordered in the complex, its apo form is characterized by higher mobility and disordering of its N-terminus. We used structural information to design the shortened FAST (which we named nanoFAST) by truncating 26 N-terminal residues. Thus, we created the shortest genetically encoded tag among all known fluorescent and fluorogen-activating proteins, which is composed of only 98 amino acids.

Color Tuning of Fluorogens for FAST Fluorogen-Activating Protein.

Using benzylidene imidazolone core, we created a panel of color-shifted fluorogenic ligands for FAST protein without compromise to the binding efficiency and the utility for live-cell protein labeling. This study highlights the potential of benzylidene imidazolones derivatives for rapid expansion of a pallet of live-cell fluorogenic labeling tools.

Nitroacetic Esters in the Regioselective Synthesis of Isoxazole-3,5-dicarboxylic Acid Derivatives.

An efficient and high-yielding strategy to prepare "unsymmetrical" 4-aryl-isoxazol-3,5-dicarboxylic acid derivatives from nitroacetic esters and aromatic aldehydes has been developed. The strategy is based on the isolation and usage of the previously missed intermediate of the Dornow reaction-5-hydroxy-6-oxo-4-aryl-6H-1,2-oxazine-3-carboxylates. In addition, the mechanism of the Dornow reaction was partially revised.

Red-Shifted Substrates for FAST Fluorogen-Activating Protein Based on the GFP-Like Chromophores.

A genetically encoded fluorescent tag for live cell microscopy is presented. This tag is composed of previously published fluorogen-activating protein FAST and a novel fluorogenic derivative of green fluorescent protein (GFP)-like chromophore with red fluorescence. The reversible binding of the novel fluorogen and FAST is accompanied by three orders of magnitude increase in red fluorescence (580-650 nm). The proposed dye instantly stains target cellular proteins fused with FAST, washes out in a minute timescale, and exhibits higher photostability of the fluorescence signal in confocal and widefield microscopy, in contrast with previously published fluorogen:FAST complexes.

Photoinduced Proton Transfer of GFP-Inspired Fluorescent Superphotoacids: Principles and Design.

Proton transfer remains one of the most fundamental processes in chemistry and biology. Superphotoacids provide an excellent platform to delineate the excited-state proton transfer (ESPT) mechanism on ultrafast time scales and enable one to precisely control photoacidity and other pertinent functionalities such as fluorescence. We modified the GFP core ( p-HBDI chromophore) into two series of highly fluorescent photoacids by fluorinating the phenolic ring and conformationally locking the backbone (i.e., biomimetics). The trifluorinated derivatives, M3F and P3F, represent two of the strongest superphotoacids with p Ka* values of -5.0 and -5.5, respectively, and they can efficiently transfer a proton to organic solvents like methanol. Tunable femtosecond stimulated Raman spectroscopy (FSRS) and femtosecond transient absorption (fs-TA) were employed to dissect the ESPT of M3F and P3F in methanol, particularly with structural dynamics information. By virtue of resonantly enhanced FSRS signal and global analysis of fs-TA spectra, we revealed an inhomogeneous ESPT mechanism consisting of three parallel routes following the initial small-scale proton motion and contact ion-pair formation within ∼300 fs: The first route consists of ultrafast protolytic dissociation facilitated by the pre-existing, largely optimized H-bonding chain; the second route is limited by solvent reorientation that establishes a suitable H-bonding wire for proton separation; the third route is controlled by rotational diffusion that requires rotation of the anisotropically reactive photoacid in a bulky solvent with a complex H-bonding structure over larger distances. Furthermore, we provided new design principles of enhancing photoacidity in a synergistic manner: incorporating electron-withdrawing groups into proximal (often as "donor") and distal (often as "acceptor") ring moieties of the dissociative hydroxyl group to lower the ground-state p Ka and increase the Δp Ka, respectively.

Designing redder and brighter fluorophores by synergistic tuning of ground and excited states.

We strategically modified the GFP core via chemical synthesis to make redder and brighter biomimetic fluorophores. Based on quantum calculations, solvatochromism analysis, and femtosecond Raman, we unveiled the additive effect of tuning the electronic ground and excited states, respectively, to achieve a dramatic emission redshift with a "double-donor-one-acceptor" structure.

Excited-state locked amino analogues of the green fluorescent protein chromophore with a giant Stokes shift.

We design a novel class of excited-state locked GFP chromophores by introducing an amine group at the double exo-bond and a difluoroboryl bridge. We show that these chromophores intrinsically exhibit a very large Stokes shift of 1 eV. Further tuning through chemical modifications of their aryl substituents makes them environmentally sensitive.

Pyridinium Analogues of Green Fluorescent Protein Chromophore: Fluorogenic Dyes with Large Solvent-Dependent Stokes Shift.

Novel fluorogenic dyes based on the GFP chromophore are developed. The compounds contain a pyridinium ring instead of phenolate and feature large Stokes shifts and solvent-dependent variations in the fluorescence quantum yield. Electronic structure calculations explain the trends in solvatochromic behavior in terms of the increase of the dipole moment upon excited-state relaxation in polar solvents associated with the changes in bonding pattern in the excited state. A unique combination of such optical characteristics and lipophilic properties enables using one of the new dyes for imaging the membrane structure of endoplasmic reticulum. An extremely high photostability (due to a dynamic exchange between the free and absorbed states) and selectivity make this compound a promising label for this type of cellular organelles.