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We showcase the successful combination of photochemistry and kinetic target‐guided synthesis (KTGS) for rapidly pinpointing enzyme inhibitors. KTGS is a fragment‐based drug discovery (FBDD) methodology in which the biological target (BT) orchestrates the construction of its own ligand from fragments featuring complementary reactive functionalities. Notably, fragments interacting with the protein binding sites leverage their spatial proximity, facilitating a preferential reaction. Consequently, the resulting bivalent ligand exhibits heightened affinity. Within the realm of KTGS strategies, in situ click chemistry stands out as the most widely used to identify potent protein binders. This approach requires significant protein contributions, such as binding interactions and appropriate orientations of fragments, to overcome high activation barriers. This leads to prolonged incubation times and the potential for generating false negatives, thereby limiting this strategy to proteins that are stable enough in buffer. We herein unveil the possibility to integrate photochemistry into the realm of KTGS, accelerating the ligation reaction between fragments to a time scale of minutes. This approach should significantly expand the narrow reactivity window of traditional KTGS reactions, paving the way for the exploration and development of novel photo‐KTGS reactions. [ABSTRACT FROM AUTHOR]

Coumarins still remain one of the most widely explored fluorescent dyes, with a broad spectrum of applications spanning various fields, such as molecular imaging, bioorganic chemistry, materials chemistry, or medical sciences. Their fluorescence is strongly based on a push‐pull mechanism involving an electron‐donating group (EDG), mainly located at the C7 or C8 positions of the dye core. Unfortunately, up to now, these positions have been very limited to hydroxyl or amino groups. In this study, we present in detail the synthesis of the first series of coumarins bearing a vinyl sulfide as the EDG at the C7 position. These derivatives were prepared by thiol‐yne reaction, promoted by ruthenium‐ or porphyrin‐based photoredox catalysis, enabling rapid late‐stage diversification. We also functionalized coumarins with short peptides, and BSA protein as a proof‐of‐concept study, in a single‐step process. This strategy, capable of proceeding under aqueous conditions, overcomes the protection/deprotection steps usually required by traditional methods, which also use strong bases and organic solvents. Moreover, the photophysical properties such as absorption and emission of obtained coumarins (for 3‐CF3, 3‐benzothiazole, 6–8‐difluoro derivatives), predominantly exhibited large Stokes shifts (up to 204 nm) and maintained intramolecular charge transfer (ICT) characteristics. [ABSTRACT FROM AUTHOR]

The constant need for high‐performance aniline‐ or phenol‐based fluorophores suitable for the construction of activity‐based fluorescent probes, led us to study both synthesis and photophysics of C3‐N‐methylpyridinium‐4‐yl substituted 7‐(dialkylamino)/7‐hydroxycoumarins. Indeed, in the field of photoactive organic molecules, the positively charged N‐alkylpyridinium‐4‐yl groups are often used as acceptor units to dramatically impact spectral features through promoting intramolecular charge transfer (ICT) processes. They are also known as effective water‐solubilizing and mitochondria targeting moieties. The poor fluorescence efficiency of cationic 7‐hydroxycoumarin derivatives in aqueous physiological conditions was highlighted and rationalized by the predominance of a neutral quinonoid form in such buffer medium. The ability of the excited singlet state (S1) of this neutral species to undergo intersystem crossing (ISC) to triplet state (T1) was partly supported by phosphorescence measurements of singlet oxygen. We also took advantage of green‐emissive properties of 7‐(diethylamino)‐3‐(N‐methylpyridinium‐4‐yl)coumarin to successfully design and validate a novel small‐molecule fluorescent probe for the detection of alkaline phosphatase (ALP), based on the "covalent‐assembly" principle. A practical use of ortho‐formylated 7‐hydroxy‐3‐(N‐methylpyridinium‐4‐yl)coumarin was next considered with the synthesis of a Fe(III)‐salen complex whose the potential as a "molecular disassembly" probe for fluorogenic sensing of pyrophosphate (PPi) anion was assessed. [ABSTRACT FROM AUTHOR]

Chemiluminescent probes are being considered as a convenient option for optical imaging. Several strategies were reported to increase the probe chemiluminescence efficiency. In this study, a series of chemiluminescent cassettes based on adamantyl stabilized 1,2‐dioxetanes ("Schaap's dioxetane") linked to a fluorophore (BODIPY or dicyanoisophorone fluorophore) by a conjugated linker have been synthetized. Their chemiluminescent decomposition and the photoluminescence properties of their respective emissive species were investigated. [ABSTRACT FROM AUTHOR]

Photophysical properties of quinoxalin‐2(1H)‐ones have long been neglected, in particular compared with those of coumarins, their oxygenated counterparts, despite their complementarity in terms of absorbance and fluorescence windows. This review provides first an overview of the synthesis of quinoxalin‐2(1H)‐ones, focusing on the most recent contributions. Fluorescence properties of quinoxalin‐2(1H)‐ones and of their π‐extended derivatives are tackled through the description of a series of examples and applications, including chemosensing and biosensing. [ABSTRACT FROM AUTHOR]

Proteins are able to irreversibly assemble biologically active ligands from building blocks bearing complementary reactive functions due their spatial proximity, through a kinetic target‐guided synthetic process (also named in situ click chemistry). Although linkages thus formed are mostly passive, some of them have shown to significantly contribute to the protein binding through for instance hydrogen bonding and stacking interactions. Biocompatible reactions and click chemistry are a formidable source of inspiration for developing such new protein‐directed ligations. This study reports a proximity‐induced thiol‐yne synthesis of carbonic anhydrase inhibitors. Not only this example widens the arsenal of kinetic target‐guided synthesis (KTGS) eligible reactions, but the obtained product displayed unsuspected photophysical properties. The corresponding vinyl sulfide linkage conjugated to a coumarin core proved to be engaged in a monodirectional Z to E photoisomerization process. Further investigations guided by theoretical calculations showed that fine‐tuning of the nature of the substituents on the coumarin moiety allows to obtain a bidirectional photochemical process, thus discovering a new photoswitching moiety, displaying moreover fluorescence properties. Due to the spectral tunability of coumarin derivatives, this work should open new opportunities for the design of vinyl sulfide‐based photoswitch systems with modular photophysical properties. [ABSTRACT FROM AUTHOR]

Broad scope one‐pot diazirine synthesis strategies have been developed using two different oxidants depending on the nature of the starting material. In all cases, an inexpensive commercial solution of ammonia (NH3) in methanol (MeOH) was employed, avoiding the difficult use of liquid ammonia. With aliphatic ketones, t‐butyl hypochlorite (t‐BuOCl) was found to be the best oxidant whereas it is preferable to use phenyliodine diacetate (PIDA) with aromatic ketones, aldehydes and imines. The nature of the imine‐protecting group is essential and only t‐butyl imine allowed the synthesis of 15N2‐diazirine with complete 15N incorporation, emphasizing a key trans‐imination step in the reaction mechanism. These methods are operationally simple, and tolerant to most functional groups, providing diazirines with yields ranging from 20 to 99%. [ABSTRACT FROM AUTHOR]

A method for regioselective solid‐phase synthesis of disubstituted peptidoisoxazoles is reported. Improved conditions for the synthesis of 3,5‐disubstituted isoxazoles (mimicking s‐trans peptide bond) without metal catalysts were implemented. New conditions enabling the synthesis of 3,4‐disubstituted isoxazoles (mimicking s‐cis peptide bond) using ruthenium catalysts were developed. This study provides a means for the regioselective preparation of new libraries of peptidomimetic molecules containing an isoxazole moiety within the backbone. [ABSTRACT FROM AUTHOR]

Non‐protein thiols such as GSH, Hcy, or Cys, are present at different concentration levels in the body depending on the thiol species and their localization (plasma, cell, urine etc···). They play vital roles in controlling redox‐mediated biological processes, and abnormal levels of biothiols are related to a variety of syndromes. In few years, fluorometric methods have gain increasing attention enabling a rapid, sensitive and spatiotemporal detection of biothiols. Herein, is reported a novel two‐step access to a water‐soluble fluorogenic probe based on the 7‐aminocoumarin dye conjugated to a pyrazolone moiety. Upon the addition of biothiols, the fluorescence is turned‐on at 485 nm by a reversible thio‐Michael addition process, which enables the detection of biothiols in the low nanomolar range. A novel and water‐soluble pyrazolone‐coumarin‐based probe for the fluorogenic detection of thiols is reported. The sensing mechanism of this probe involves a rapid and reversible thiol‐Michael addition. [ABSTRACT FROM AUTHOR]

Abstract: A new series of 3‐hydroxy‐2‐pyridine aldoxime compounds have been designed, synthesised and tested in vitro, in silico, and ex vivo as reactivators of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) inhibited by organophosphates (OPs), for example, VX, sarin, cyclosarin, tabun, and paraoxon. The reactivation rates of three oximes (16–18) were determined to be greater than that of 2‐PAM and comparable to that of HI‐6, two pyridinium aldoximes currently used by the armies of several countries. The interactions important for a productive orientation of the oxime group within the OP‐inhibited enzyme have been clarified by molecular‐modelling studies, and by the resolution of the crystal structure of the complex of oxime 17 with Torpedo californica AChE. Blood–brain barrier penetration was predicted for oximes 15–18 based on their physicochemical properties and an in vitro brain membrane permeation assay. Among the evaluated compounds, two morpholine‐3‐hydroxypyridine aldoxime conjugates proved to be promising reactivators of OP‐inhibited cholinesterases. Moreover, efficient ex vivo reactivation of phosphylated native cholinesterases by selected oximes enabled significant hydrolysis of VX, sarin, paraoxon, and cyclosarin in whole human blood, which indicates that the oximes have scavenging potential. [ABSTRACT FROM AUTHOR]

The use of nonfluorescent azo dyes as dark quenchers in activatable optical bioprobes based on the Förster resonance energy transfer (FRET) mechanism and designed to target a wide range of enzymes has been established for over two decades. The key value of the azo moiety (−N=N−) to act as an efficient 'ON-OFF' switch of fluorescence once introduced within the core structure of conventional organic-based fluorophores (mainly fluorescent aniline derivatives) has recently been exploited in the development of alternative reaction-based small-molecule probes based on the 'profluorescence' concept. These unprecedented 'azobenzene-caged' fluorophores are valuable tools for the detection of a wide range of reactive (bio)analytes. This review highlights the most recent and relevant advances made in the design and biosensing/bioimaging applications of azo-based fluorogenic probes. Emphasis is also placed on relevant achievements in the synthesis of bioconjugatable/biocompatible azo dyes used as starting building blocks in the rational and rapid construction of these fluorescent chemodosimeters. Finally, a brief glimpse of possible future biomedical applications (theranostics) of these 'smart' azobenzene-based molecular systems is presented. [ABSTRACT FROM AUTHOR]

Fluorogenic reactions are largely underrepresented in the toolbox of chemoselective ligations despite their tremendous potential, particularly in chemical biology and biochemistry. In this respect, we have investigated in full detail the fluorescence behaviour of the azaphthalamide, a scaffold which is generated through a hetero-Diels-Alder reaction of 5-alkoxyoxazole and maleimide derivatives under mild conditions that are compatible with, among others, peptide chemistry. The scope and limitations of such a fluorogenic labelling strategy were examined through four distinct applications, which target enzymatic activities or bioorthogonal reactions. [ABSTRACT FROM AUTHOR]

The 5-alkoxyoxazole ring system has proved to be a useful and versatile building block that is involved in a variety of chemical transformations (e.g., hetero-Diels-Alder reactions, formal 1,3-dipolar cycloadditions, photochemical [2+2] cycloadditions, Claisen rearrangements, and ring-opening processes). 5-Alkoxyoxazoles have provided synthetic access to heteroaromatic scaffolds such as pyridines, furans, oxadiazolines, pyrrolidines, and significant linear chemical patterns, including amino acid derivatives. This review focuses on the broad range of reactivity of 5-alkoxyoxazoles and their subsequent applications to the syntheses of naturally occurring compounds and biologically active molecules. [ABSTRACT FROM AUTHOR]

Herein, we present an improved synthetic pathway to huprine derivatives (the most potent family of non-covalent acetylcholinesterase inhibitors described to date) with different functional groups at the C9-position of the scaffold. Our approach enables selection of the desired terminal function prior to construction of the huprine scaffold and consists of three main steps: enol formation from a bicyclic ketone, Suzuki-Miyaura cross coupling with different borane derivatives and a Friedländer condensation to access to the quinoline moiety. This synthetic route was found particularly useful for construction of huprine scaffolds equipped with methylene chains bearing different terminal groups such as hydroxy, azide, nitro, amino and carboxylic groups. These functionalized huprines are used as key intermediates in the preparation of multi-target directed ligands for the treatment of Alzheimer's disease or in the preparation of resins for purification of cholinesterases by affinity chromatography. [ABSTRACT FROM AUTHOR]

Since the recent discovery that hydrogen sulfide plays a key role in many biological and physiological processes, the development of novel methods to efficiently detect this molecule has become an intensive field of research. In this context, fluorimetric strategies have become one of the methods of choice. However, very few different reducible chemical functionalities have been successfully used in the design of reduction-based probes for the fluorescence detection of hydrogen sulfide. Reported herein is the first nitroso-based fluorescence turn-on probe that displays an almost instantaneous and strong fluorescence response towards hydrogen sulfide. A comparative study carried out with the hydroxylamino-based probe analogue showed a significant difference in fluorescence response. [ABSTRACT FROM AUTHOR]

Members of a series of boron difluoride complexes with 3-(heteroaryl)-2-iminocoumarin ligands bearing both a phenolic hydroxyl group (acting as a fluorogenic center) and an N-aryl substituent (acting as a stabilizing moiety) have been synthesized in good yields by applying a straightforward two-step method. These novel fluorogenic dyes belong to the family of 'Boricos' (D. Frath et al., Chem. Commun.­ 2013, 49, 4908-4910) and are the first examples of phenol-based fluorophores of which the photophysical properties in the green-yellow spectral range are dramatically improved by N, N-chelation of a boron atom. Modulation of their fluorescence properties through reversible chemical modification of their phenol moieties has been demonstrated by the preparation of the corresponding 2,4-dinitrophenyl (DNP) ethers, which led to a dramatic 'OFF-ON' fluorescence response upon reaction with thiols. Additionally, to expand the scope of these '7-hydroxy-Borico' derivatives, particularly in biolabeling, amine or carboxylic acid functionalities amenable to (bio)conjugation have been introduced within their scaffold. Their utility has been demonstrated in the preparation of fluorescent bovine serum albumin (BSA) conjugates and 'Borico'-DOTA-like scaffolds in an effort to design novel monomolecular multimodal fluorescence- radioisotope imaging agents. [ABSTRACT FROM AUTHOR]

The isoindolone scaffold is present in many biologically active compounds. Here, we have developed a shorter and more efficient synthesis of tetrahydropyrido[2,1- a]isoindolone. The key step of this approach is a cyclization to form the γ-lactam ring under Shibasaki's conditions. Thus, tetrahydropyrido[2,1- a]isoindolone and superior analogues, namely hexahydroazepino[2,1- a]isoindolone and hexahydroazocino[2,1- a]isoindolone, have been prepared in only three steps in 39, 25, and 19 % overall yields, respectively. This novel strategy offers a shorter alternative to existing procedures. [ABSTRACT FROM AUTHOR]

Derivatives of 2-(2′-hydroxy-5′-dimethylaminobenzyl)-4,6-dimethylamino-1,3,5-triazine were synthesized by an original multistep protocol that afforded, after quaternization of the N, N-dimethylaminobenzyl moiety, water-soluble fluorescent dyes. These fluorophores exhibited excited-state intramolecular proton transfer and large Stokes shifts (ΔSS > 10000 cm-1). The phenol residue was masked by an enzyme-labile protecting group based on self-immolative para-hydroxybenzyl alcohol. Lipases were used to unveil the fluorescence under physiological conditions (phosphate-buffered saline, pH 7.2) without aggregation or precipitation of the fluorescent dyes. [ABSTRACT FROM AUTHOR]

A transition-metal-free method for the synthesis of N-substituted unsymmetrical sulforhodamine fluorophores from an unusual monobrominated sulfoxanthene dye and primary or secondary amines by direct SNAr-type reactions is presented. The simplicity and effectiveness of this 'postamination' procedure were demonstrated through the rapid preparation of a library of multifunctional red-emitting rhodamine analogues. Some of these analogues are equipped with a reactive handle and retain the two water-solubilizing sulfonic acid moieties of the starting halogenated derivative; this makes them ideal candidates for biolabeling applications. The potential utility of this expeditious strategy to finely tune the photophysical (Stokes shift) and physicochemical properties (amphiphilic character and water solubility) of the sulforhodamine scaffold was also demonstrated through the appendage of a fluorescent amine to create a fluorescence resonance energy transfer (FRET) pair, a long-chain amine, and tetraazamacrocycles derived from cyclen. [ABSTRACT FROM AUTHOR]

A straightforward method to synthesize 3-(2-benzimidazolyl)-7-hydroxycoumarins, based on the condensation reaction of 7-acetoxy-3-formylcoumarin with various C- and/or N-substituted ortho-phenylenediamine derivatives is presented. This unusual approach proved particularly effective for introducing different hydrophilic groups (carboxylic or sulfonic acids or trimethylalkylammonium moieties) onto the heteroaryl scaffold, leading to cyan-green emitting coumarins that were both water-soluble and strongly fluorescent under physiological conditions. The further extension of this condensation reaction to bis(2-aminophenyl)diselenide enabled the first synthesis of 3-(2-benzoselenazolyl)-7-hydroxycoumarin. The potential utility of these new 7-hydroxycoumarins was demonstrated through the synthesis and spectroscopic and analyte-responsive behavior of fluorogenic probes suitable for sensing biologically relevant thiols and urokinase, a protease that plays a key role in cancer invasion and metastasis. [ABSTRACT FROM AUTHOR]

An efficient synthesis of water-soluble unsymmetrical sulforhodamine/sulforhodol fluorophores containing a single julolidine fragment is presented. Owing to their valuable spectral properties in aqueous buffers, these dyes, especially those bearing a free aniline or phenol moiety, are valuable components of fluorogenic probes for a variety of biosensing applications. A further extension of this synthetic methodology to unusual phenols, namely 7- N, N-dialkylamino-4-hydroxy coumarins has enabled us to provide a new family water-soluble dyes of large Stokes' shift with far-red spectral features. [ABSTRACT FROM AUTHOR]

We describe the efficient synthesis and one-step derivatization of novel, nonfluorescent azo dyes based on the Black Hole Quencher-3 (BHQ-3) scaffold. These dyes were equipped with various reactive and/or bioconjugatable groups (azido, α-iodoacetyl, ketone, terminal alkyne, vicinal diol). The azido derivative was found to be highly reactive in the context of copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions and allowed easy synthetic access to the first water-soluble (sulfonated derivative) and aldehyde-modified BHQ-3 dyes, the direct preparation of which failed by means of conventional azo-coupling reactions. The aldehyde- and α-iodoacetyl-containing fluorescence quenchers were readily conjugated to aminooxy- and cysteine-containing peptides by the formation of a stable oxime or thioether linkage, respectively. Further fluorescent labeling of the resultant peptide conjugates with red- or far-red-emitting rhodamine or cyanine dyes through sequential and/or one-pot bioconjugations, led to novel Förster resonance energy transfer (FRET) based probes suitable for the in vivo detection and imaging of urokinase plasminogen activator, a key protease in cancer invasion and metastasis. [ABSTRACT FROM AUTHOR]

Butyrylcholinesterase (BChE) is a serine hydrolase that is present in all mammalian tissues. It can accommodate larger substrates or inhibitors than acetylcholinesterase (AChE), the enzyme responsible for hydrolysis of the neurotransmitter acetylcholine in the central nervous system and neuromuscular junctions. AChE is the specific target of organophosphorous pesticides and warfare nerve agents, and BChE is a stoichiometric bioscavenger. Conversion of BChE into a catalytic bioscavenger by rational design or designing reactivators specific to BChE required structural data obtained using a recombinant low-glycosylated human BChE expressed in Chinese hamster ovary cells. This expression system yields ∼ 1 mg of pure enzyme per litre of cell culture. Here, we report an improved expression system using insect cells with a fourfold higher yield for truncated human BChE with all glycosylation sites present. We developed a fast purification protocol for the recombinant protein using huprine-based affinity chromatography, which is superior to the classical procainamide-based affinity. The purified BChE crystallized under different conditions and space group than the recombinant low-glycosylated protein produced in Chinese hamster ovary cells. The crystals diffracted to 2.5 Å. The overall monomer structure is similar to the low-glycosylated structure except for the presence of the additional glycans. Remarkably, the carboxylic acid molecule systematically bound to the catalytic serine in the low-glycosylated structure is also present in this new structure, despite the different expression system, purification protocol and crystallization conditions. Database Structural data have been submitted to the Protein Data Bank under accession number pdb . DNA sequence data for the synthetic gene have been submitted to GenBank under accession number . Structured digital abstract and by () [ABSTRACT FROM AUTHOR]

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