Dark fluorescence quenchers are nonfluorescent dyes that can modulate the fluorescence signal of an appropriate fluorophore donor in a distance-dependent manner. Dark quenchers are extensively used in many biomolecular analytical applications, such as studies with fluorogenic protease substrates or nucleic acids probes. A very popular dark fluorescence quencher is dabcyl, which is a hydrophobic azobenzene derivative. However, its insolubility in water may constitute a major drawback, especially during the investigation of biochemical systems whose natural solvent is water. We designed and synthesized a new azobenzene-based dark quencher with excellent solubility in aqueous media, which represents a superior alternative to the much-used dabcyl. The advantage of hydrodabcyl over dabcyl is exemplarily demonstrated for the cleavage of the fluorogenic substrate hydrodabcyl-Ser-Phe-EDANS by the proteases thermolysin and papain.

The depolymerization of the biopolymer lignin can give pure aromatic monomers but selective catalytic approaches remain scarce. Here, an approach was rerouted to deliver an unusual phenolic monomer. This monomer's instability proved challenging, but a degradation study identified strategies to overcome this. Heterocycles and a useful synthetic intermediate were prepared. The range of aromatics available from the β-O-4 unit in lignin was extended.

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Spiroscytalin, a natural 3-spirotetramic acid of hitherto uncertain absolute configuration, was synthesized for the first time by a one-pot Knoevenagel-IMDA reaction of an l-phenylalanine-derived tetramic acid and (R)-2-methyl-deca-6E,8E-dienal. Its absolute configuration was assigned by the known configurations of the starting compounds and by NOESY correlations. Its identity with the natural isolate was proved by the comparison of the NMR and circular dichroism spectra and of the specific optical rotations. Its absolute configuration (3R,5S,6S,7R,11S,14R) is enantiomeric to that originally proposed by the isolating group. This natural isomer of spiroscytalin showed moderate activity against Candida albicans and good activity against an export-deficient mutant of Escherichia coli.

Phytochelatins (PCs) are nonribosomal thiol-rich oligopeptides synthetized from glutathione (GSH) in a γ-glutamylcysteinyl transpeptidation reaction catalyzed by PC synthases (PCSs). Ubiquitous in plant and present in some invertebrates, PCSs are involved in metal detoxification and homeostasis. The PCS-like enzyme from the cyanobacterium Nostoc sp. (NsPCS) is considered to be an evolutionary precursor enzyme of genuine PCSs because it shows sufficient sequence similarity for homology to the catalytic domain of the eukaryotic PCSs and shares the peptidase activity consisting in the deglycination of GSH. In this work, we investigate the catalytic mechanism of NsPCS by combining structural, spectroscopic, thermodynamic, and theoretical techniques. We report several crystal structures of NsPCS capturing different states of the catalyzed chemical reaction: (i) the structure of the wild-type enzyme (wt-NsPCS); (ii) the high-resolution structure of the γ-glutamyl-cysteine acyl-enzyme intermediate (acyl-NsPCS); and (iii) the structure of an inactive variant of NsPCS, with the catalytic cysteine mutated into serine (C70S-NsPCS). We characterize NsPCS as a relatively slow enzyme whose activity is sensitive to the redox state of the substrate. Namely, NsPCS is active with reduced glutathione (GSH), but is inhibited by oxidized glutathione (GSSG) because the cleavage product is not released from the enzyme. Our biophysical analysis led us to suggest that the biological function of NsPCS is being a part of a redox sensing system. In addition, we propose a mechanism how PCS-like enzymes may have evolved toward genuine PCS enzymes.

The water-soluble quencher hydrodabcyl can be activated as an N-succinimidyl ester that is readily accessible from crude hydrodabcyl and storable for a long time. With primary and secondary amines, it reacts swiftly and chemoselectively, even in the presence of other competing nucleophiles such as those typically present in natural peptides. One of the three phenolic OH groups of hydrodabcyl is amenable to selective mono-Boc protection resulting in reduced polarity, advantageous to its further use in organic synthesis. The advantages of hydrodabcyl over dabcyl in spectrometric applications are exemplified by the pH dependence of its absorbance spectra.

This study aimed at searching for the enzymes that are responsible for the higher hydroxylation of flavonols serving as UV-honey guides for pollinating insects on the petals of Asteraceae flowers. To achieve this aim, an affinity-based chemical proteomic approach was developed by relying on the use of quercetin-bearing biotinylated probes, which were thus designed and synthesized to selectively and covalently capture relevant flavonoid enzymes. Proteomic and bioinformatic analyses of proteins captured from petal microsomes of two Asteraceae species (Rudbeckia hirta and Tagetes erecta) revealed the presence of two flavonol 6-hydroxylases and several additional not fully characterized proteins as candidates for the identification of novel flavonol 8-hydroxylases, as well as relevant flavonol methyl- and glycosyltransferases. Generally speaking, this substrate-based proteome profiling methodology constitutes a powerful tool for the search for unknown (flavonoid) enzymes in plant protein extracts.