Boris Vauzeilles

Click-mediated labeling of bacterial membranes through metabolic modification of the lipopolysaccharide inner core.

Metabolic glycan labeling has recently emerged as a very powerful method for studying cell-surface glycans, which has applications that range from imaging glycans in living multicellular organisms, such as zebrafish or mice, to the identification of metastasis-associated cell-surface sialoglycoproteins. This strategy relies on the cellular biosynthetic machinery assimilating a modified monosaccharide that contains a bioorthogonal chemical reporter. The metabolic incorporation of this reporter into glycans can be further visualized by chemical ligation with a label, such as a fluorescent probe. Somewhat surprisingly, previous studies have mainly focused on the labeling of vertebrate glycans by using derivatives of common monosaccharides, such as Nacetyl neuraminic acid (or its N-acetylmannosamine precursor), N-acetylglucosamine, N-acetylgalactosamine, and fucose. In spite of a much higher degree of diversity in their monosaccharide building blocks as well as an essential role in bacterium–host interactions and bacterial virulence, bacterial polysaccharides have been poorly explored in terms of in vivo structural modifications. Bacteria are divided into Grampositive and Gram-negative bacteria. Whereas Gram-positive bacteria are surrounded by a peptidoglycan cell wall, Gramnegative bacteria are covered by a dense layer of lipopolysaccharides that are embedded in their outer membrane. These lipopolysaccharides are involved in the structural integrity of the cell and are often considered as determinants of pathogenicity. Although lipopolysaccharides appear to be an interesting target for specific and well-defined glycan metabolic labeling in Gram-negative bacteria, attempts to achieve this goal have been limited to the introduction of modified l-fucose derivatives into a customized, genetically engineered strain of Escherichia coli. Although it is a very interesting proof of concept, this l-fucose-based approach has some limitations as l-fucose is not generally present within the lipopolysaccharides of all Gram-negative bacteria, but is found in the O-antigens of specific strains. Secondly, free lfucose is not an intermediate in the normal E. coli “de novo” pathway and, therefore, should not be directly activable into a nucleotide-sugar donor without the introduction of an alternative pathway, known as the “salvage pathway”, into the organism of interest by genetic engineering (metabolic pathway engineering). Furthermore, once activated in the form of a modified guanosine-5’-diphosphate–fucose (GDP– Fuc), the l-fucose analogue might be transformed into a correspondingly modified GDP–mannose (GDP–Man) by the reverse de novo pathway, and potentially further metabolized into various other compounds, a process which could result in the chemical reporter being spread through other pathways of sugar metabolism or beyond. As a result of all of these limitations, and as our goal was labeling the lipopolysaccharides of bacteria with no genetic modification, we investigated whether another sugar could be used as a target for the metabolic modification of glycans. From all of the potential targets, 3-deoxy-d-mannooctulosonic acid (KDO) appears to be a very attractive candidate. Indeed, KDO is a specific and essential component of the inner core of lipopolysaccharides, and has long been considered as being present in the lipopolysaccharides of almost all Gram-negative species (as well as higher plants and algae), in which at least one residue is directly connected to lipid A (Scheme 1a). Because of its vital importance, KDO has been considered as a determinant for the characterization of Gram-negative bacteria, and the KDO pathway as a potential target for the development of new antibacterial compounds. In the KDO pathway (Scheme 1b), arabinose5-phosphate (arabinose-5-P) is condensed with phosphoenolpyruvate (PEP) to give KDO-8-phosphate (KDO-8-P), which is then transformed into free KDO, and further activated to form the cytidine monophosphate (CMP)–KDO donor prior to lipopolysaccharide elaboration. For all of these reasons, we hypothesized that the KDO pathway, as a lipopolysaccharidespecific pathway, may be tolerant enough to incorporate a modified analogue of KDO, such as 8-azido-8-deoxy-KDO (1, Scheme 2), into the core of E. coli lipopolysaccharides, and potentially other Gram-negative bacteria. Given the presence of free KDO as an intermediate in the pathway, we postulated that if the cell penetration of this analogue of KDO was sufficient, it could then be directly activated, partially replace endogenous KDO in lipopolysaccharides, and be detected on the cell surface by azide–alkyne click chemistry (Figure S1 in the Supporting Information). Moreover, modification of the C8-position of KDOwith a bioorthogonal azido group should prevent reverse metabolism by KDO-8-P [*] Dr. A. Dumont, Dr. S. Dukan Aix Marseille Universit , Laboratoire de Chimie Bact rienne (UMR 7283), Institut de Microbiologie de la M diterran e (IMM), CNRS, 31 Chemin Joseph Aiguier 13402 Marseille (France) E-mail: sdukan@imm.cnrs.fr

Lipo-chitooligosaccharidic Symbiotic Signals Are Recognized by LysM Receptor-Like Kinase LYR3 in the Legume Medicago truncatula

While chitooligosaccharides (COs) derived from fungal chitin are potent elicitors of defense reactions, structurally related signals produced by certain bacteria and fungi, called lipo-chitooligosaccharides (LCOs), play important roles in the establishment of symbioses with plants. Understanding how plants distinguish between friend and foe through the perception of these signals is a major challenge. We report the synthesis of a range of COs and LCOs, including photoactivatable probes, to characterize a membrane protein from the legume Medicago truncatula. By coupling photoaffinity labeling experiments with proteomics and transcriptomics, we identified the likely LCO-binding protein as LYR3, a lysin motif receptor-like kinase (LysM-RLK). LYR3, expressed heterologously, exhibits high-affinity binding to LCOs but not COs. Homology modeling, based on the Arabidopsis CO-binding LysM-RLK AtCERK1, suggests that LYR3 could accommodate the LCO in a conserved binding site. The identification of LYR3 opens up ways for the molecular characterization of LCO/CO discrimination.

Click chemistry on a ruthenium polypyridine complex. An efficient and versatile synthetic route for the synthesis of photoactive modular assemblies.

In this Communication, we present the synthesis and use of [Ru(bpy)(2)(bpy-CCH)](2+), a versatile synthon for the construction of more sophisticated dyads by means of click chemistry. The resulting chromophore-acceptor or -donor complexes have been studied by flash photolysis and are shown to undergo efficient electron transfer to/from the chromophore. Additionally, the photophysical and chemical properties of the original chromophore remain intact, making it a very useful component for the preparation of visible-light-active dyads.

Selection of the biological activity of DNJ neoglycoconjugates through click length variation of the side chain.

A series of neoglycoconjugates derived from deoxynojirimycin has been prepared by click connection with functionalised adamantanes. They have been assayed as glycosidase inhibitors, as inhibitors of the glycoenzymes relevant to the treatment of Gaucher disease, as well as correctors of the defective ion-transport protein involved in cystic fibrosis. We have demonstrated that it is possible to selectively either strongly inhibit ER-α-glucosidases and ceramide glucosyltransferase or restore the activity of CFTR in CF-KM4 cells by varying the length of the alkyl chain linking DNJ and adamantane.

Phenylenediamine catalysis of “click glycosylations” in water: practical and direct access to unprotected neoglycoconjugates

Phenylenediamine-catalyzed click chemistry leads to the efficient, practical, and column-free preparation of neoglycoconjugates from unprotected glucosyl azide, in pure water when aglycon solubility permits.

Efficient electron transfer through a triazole link in ruthenium(II) polypyridine type complexes

Spectroscopic, electrochemical and theoretical characterisations of photoactive systems readily assembled via click-chemistry show an efficient bi-directional charge shift through the triazole link.

A one-step β-selective glycosylation of N-acetyl glucosamine and recombinant chitooligosaccharides ☆

Abstract N-Acetyl glucosamine and chitooligosaccharides are selectively converted into β-glycosides without protection of the other hydroxyl groups by alkylation of the anomeric alkoxides in N,N-dimethylformamide containing lithium bromide. Addition of the lithium salt notably improves the stereoselectivity of the glycosylation of the monomer and the efficiency of the process with higher oligomers.

Carbon dioxide reduction via light activation of a ruthenium–Ni(cyclam) complex

In this paper we report the synthesis of a chromophore-catalyst assembly designed for the photoreduction of carbon dioxide. The chromophore unit is made up of a ruthenium trisbipyridyl-like unit covalently attached to a nickel cyclam (cyclam = 1,4,8,11-tetraazacyclotetradecane) via a triazole ring. The intramolecular electron transfer activation of the catalyst unit by visible light was studied by nanosecond flash photolysis and EPR spectroscopy. In aqueous solutions (pH = 6.5), activation of the Ru(II)-Ni(II) modular assembly with 450 nm visible light in the presence of a sacrificial electron donor accomplishes the reduction of CO2 into CO and H2 in a ratio of 2.7 to 1.

Identification of Living Legionella pneumophila Using Species‐Specific Metabolic Lipopolysaccharide Labeling

Legionella pneumophila is a pathogenic bacterium involved in regular outbreaks characterized by a relatively high fatality rate and an important societal impact. Frequent monitoring of the presence of this bacterium in environmental water samples is necessary to prevent these epidemic events, but the traditional culture-based detection and identification method requires up to 10 days. Reported herein is a method allowing identification of Legionella pneumophila by metabolic lipopolysaccharide labeling which targets, for the first time, a precursor to monosaccharides that are specifically present within the O-antigen of the bacterium. This new approach allows easy detection of living Legionella pneumophila, while other Legionella species are not labeled.

Novel imino sugar α-glucosidase inhibitors as antiviral compounds.

Deoxynojirimycin (DNJ) based imino sugars display antiviral activity in the tissue culture surrogate model of Hepatitis C (HCV), bovine viral diarrhoea virus (BVDV), mediated by inhibition of ER α-glucosidases. Here, the antiviral activities of neoglycoconjugates derived from deoxynojirimycin, and a novel compound derived from deoxygalactonojirimycin, by click chemistry with functionalised adamantanes are presented. Their antiviral potency, in terms of both viral infectivity and virion secretion, with respect to their effect on α-glucosidase inhibition, are reported. The distinct correlation between the ability of long alkyl chain derivatives to inhibit ER α-glucosidases and their anti-viral effect is demonstrated. Increasing alkyl linker length between DNJ and triazole groups increases α-glucosidase inhibition and reduces the production of viral progeny RNA and the maturation of the envelope polypeptide. Disruption to viral glycoprotein processing, with increased glucosylation on BVDV E2 species, is representative of α-glucosidase inhibition, whilst derivatives with longer alkyl linkers also show a further decrease in infectivity of secreted virions, an effect proposed to be distinct from α-glucosidase inhibition.