Azo-Sulforhodamine Dyes: A Novel Class of Broad Spectrum Dark Quenchers

A rapid access to a novel class of water-soluble dark quencher dyes was achieved using an azo-coupling reaction between a fluorescent primary arylamine derived from a sulforhodamine 101 scaffold and a tertiary aniline equipped with different bioconjugatable groups. The thus obtained nonfluorescent azo-sulforhodamine hybrids display a broad quenching range spanning the visible to NIR regions. This was demonstrated through the preparation and enzymatic activation of FRET-based fluorogenic substrates of urokinase

Expeditious Microwave-Assisted Synthesis of 5‑Alkoxyoxazoles from α‑Triflyloxy Esters and Nitriles

A rapid and general access to diversely substituted 5-alkoxyoxazoles <b>2</b> (i.e., R¹, R² = alkyl, phenyl) from easily accessible α-triflyloxy/hydroxy esters <b>1</b> and nitriles with good yields (41–76%) is reported. The versatility of the cyclization is shown for a range of substrates with high selectivity toward triflates over mesylates and proved to be compatible with sensitive functional groups. As an illustration of this transformation, the first synthesis of the recently isolated hydroxypyridine methyl multijuguinate <b>4</b> was achieved in four steps through a hetero Diels–Alder reaction of the 5-alkoxyoxazole and acrylic acid, followed by a protodecarboxylation reaction

Universal Dark Quencher Based on “Clicked” Spectrally Distinct Azo Dyes

The first synthesis of an heterotrifunctional molecular scaffold derived from the popular DABCYL azo dye quencher has been achieved. The sequential derviatization of this trivalent azobenzene derivative with two other nonfluorescent azo dyes (Black Hole Quencher BHQ-1 and BHQ-3) and through effective reactions from the “bioconjugation chemistry” repertoire has led to an universal dark quencher (UDQ). This “clicked” poly azo dye is able to turn off an array of fluorophores covering the UV/NIR (300–750 nm) spectral range

Thermally Controlled Decarboxylative [4 + 2] Cycloaddition between Alkoxyoxazoles and Acrylic Acid: Expedient Access to 3‑Hydroxypyridines

A modified Kondrat’eva cycloaddition involving an unprecedented thermally controlled metal-free decarboxylative aromatization affords an expedient access to natural 3-hydroxypyridine/piperidine systems

Metal-Free Decarboxylative Hetero-Diels–Alder Synthesis of 3‑Hydroxypyridines: A Rapid Access to <i>N</i>‑Fused Bicyclic Hydroxypiperidine Scaffolds

A complete experimental and theoretical study of the thermally controlled metal-free decarboxylative hetero-Diels–Alder (HDA) reaction of 5-alkoxyoxazoles with acrylic acid is reported. This strategy offers a new entry to valuable 2,6-difunctionalized 3-hydroxypyridines from readily available 2- and 4-disubstituted 5-alkoxyoxazoles. The reaction conditions proved compatible with, among others, ketone, amide, ester, ether, and nitrile groups. The broad functional group tolerance of the protocol allows a rapid and versatile access to both hydroxyindolizidine and hydroxyquinolizidine derivatives via a pyridine dearomatization strategy

A Novel Bio-Orthogonal Cross-Linker for Improved Protein/Protein Interaction Analysis

The variety of protein cross-linkers developed in recent years illustrates the current requirement for efficient reagents optimized for mass spectrometry (MS) analysis. To date, the most widely used strategy relies on commercial cross-linkers that bear an isotopically labeled tag and <i>N</i>-hydroxysuccinimid-ester (NHS-ester) moieties. Moreover, an enrichment step using liquid chromatography is usually performed after enzymatic digestion of the cross-linked proteins. Unfortunately, this approach suffers from several limitations. First, it requires large amounts of proteins. Second, NHS-ester cross-linkers are poorly efficient because of their fast hydrolysis in water. Finally, data analysis is complicated because of uneven fragmentation of complex isotopic cross-linked peptide mixtures. We therefore synthesized a new type of trifunctional cross-linker to overrule these limitations. This reagent, named NNP9, comprises a rigid core and bears two activated carbamate moieties and an azido group. NNP9 was used to establish intra- and intermolecular cross-links within creatine kinase, then to map the interaction surfaces between α-Synuclein (α-Syn), the aggregation of which leads to Parkinson’s disease, and the molecular chaperone Hsc70. We show that NNP9 cross-linking efficiency is significantly higher than that of NHS-ester commercial cross-linkers. The number of cross-linked peptides identified was increased, and a high quality of MS/MS spectra leading to high sequence coverage was observed. Our data demonstrate the potential of NNP9 for an efficient and straightforward characterization of protein–protein interfaces and illustrate the power of using different cross-linkers to map thoroughly the surface interfaces within protein complexes

Kondrat’eva Ligation: Diels–Alder-Based Irreversible Reaction for Bioconjugation

Diversification of existing chemoselective ligations is required to efficiently access complex and well-defined biomolecular assemblies with unique and valuable properties. The development and bioconjugation applications of a novel Diels–Alder-based irreversible site-specific ligation are reported. The strategy is based on a Kondrat’eva cycloaddition between bioinert and readily functionalizable 5-alkoxyoxazoles and maleimides that readily react together under mild and easily tunable reaction conditions to afford a fully stable pyridine scaffold. The potential of this novel bioconjugation is demonstrated through the preparation of fluorescent conjugates of biomolecules and a novel Förster resonance energy transfer (FRET)-based probe suitable for the in vivo detection and imaging of urokinase-like plasminogen activator (uPA), which is a key protease involved in cancer invasion and metastasis

Synthesis, Biological Evaluation, and <i>in Vivo</i> Imaging of the first Camptothecin–Fluorescein Conjugate

The first synthesis and photophysical properties of a fluorecently labeled camptothecin derivative, namely, camptothecin-FI (CPT-FI), an antitumoral agent that targets topoisomerase I, are reported. The preparation of this fluorescent conjugate is based on a highly convergent and flexible approach which enables the rapid chemical modification of the AB ring system of this fragile pentacyclic alkaloid, aimed at introducing an anchoring point to graft the fluorophore. The selection of a fluorescein analogue as the reporter group has enabled us to get the first green-emitting CPT conjugate exhibiting valuable spectral properties and retaining biological properties of native CPT. Indeed, in biological models, i.e., glioma cell lines U87 and/or T98, the kinetics of cell endocytosis, as well as the efficacy of CPT-FI were compared to those of CPT. CPT-FI fluorescence was measured in the cytosolic compartment of T98 glioma cells from 5 min treatment and remained detectable until 48 h. As CPT, CPT-FI drastically inhibited glioma growth and cell cycle but exhibited a reduced affinity as compared to the native CPT. <i>In vivo</i> and <i>ex vivo</i> imaging studies of CPT-FI intratumoraly injected into a model of NIH-3T3 murine tumor xenografts in nude mice, showed accumulation around the injected site area, which is very promising to target tumors and follow biodistribution <i>in vivo</i>

Phenyltetrahydroisoquinoline–Pyridinaldoxime Conjugates as Efficient Uncharged Reactivators for the Dephosphylation of Inhibited Human Acetylcholinesterase

Pyridinium and bis-pyridinium aldoximes are used as antidotes to reactivate acetylcholinesterase (AChE) inhibited by organophosphorus nerve agents. Herein, we described a series of nine nonquaternary phenyltetrahydroisoquinoline–pyridinaldoxime conjugates more efficient than or as efficient as pyridinium oximes to reactivate VX-, tabun- and ethyl paraoxon-inhibited human AChE. This study explores the structure–activity relationships of this new family of reactivators and shows that <b>1b</b>–<b>d</b> are uncharged hAChE reactivators with a broad spectrum