Environmentally benign glycosylation of aryl pyranosides and aryl/alkyl furanosides demonstrating the versatility of thermostable CGTase from Thermoanaerobacterium sp.

International audienceAn extensive study on the specificity of transglycosylation and disproportionation of Thermoanaerobacterium sp. cyclodextrin glucosyltranferases against aryl glucopyranosides or furanosides was achieved. While a mixture of maltoside and isomaltoside was obtained respectively using p-nitrophenyl glucopyranoside as acceptor, only one regioisomer, namely the p-nitrophenyl α-D-Glcp-(1,3)-α-L-Araf was isolated using p-nitrophenyl arabinofuranoside as acceptor. Interestingly, similar outcomes were found when using p-nitrophenyl galactofuranoside. Furthermore, activation by microwave irradiation resulted in faster reaction times and higher yields and led to glucosidic oligosaccharides with up to 70% conversion. The influence of the anomeric and C-4 configurations of the glycosidic acceptors on the transglycosylation, previously stated for the CGTase family, was not observed and unconventional substrate specificity towards alkyl furanosides was highlighted

Araf51 with improved transglycosylation activities: One engineered biocatalyst for one specific acceptor

International audienceA random mutagenesis of the arabinofuranosyl hydrolase Araf51 has been run in order to access to efficient biocatalysts for the synthesis of alkyl arabinofuranosides. The mutants were selected on their ability to catalyze the transglycosylation reaction of p-nitrophenyl α-L-arabinofuranoside (pNP-Araf) used as a donor and various aliphatic alcohols as acceptors. This screening strategy underlined 5 interesting clones, each one corresponding to one acceptor. They appeared to be much more efficient in the transglycosylation reaction compared to the wild type enzyme whereas no self-condensation or hydrolysis products could be detected. Moreover, the high specificity of the mutants towards the alcohols for which they have been selected validates the screening process. Sequence analysis of the mutated enzymes revealed that, despite their location far from the active site, the mutations affect significantly the kinetics properties as well as the substrate affinity of these mutants towards the alcoholacceptors in the transglycosylation reaction

4-Nitro­phenyl α-l-rhamnopyran­oside hemihydrate

In the title compound, C12H15NO7·0.5H2O, there are two independent mol­ecules in the asymmetric unit, together with one water molecule. The pyran­oside rings each have close to a 1 C 4 chair conformation and the nitro groups are almost coplanar with the benzene rings. The water mol­ecule links the two independent mol­ecules through O—H⋯O hydrogen bonds. All the hydroxyl groups are involved in hydrogen-bond inter­actions, giving rise to a three-dimensional network

Graphical Abstract Biocatalyzed synthesis of difuranosides and their ability to trigger production of TNF-D D Biocatalyzed synthesis of difuranosides and their ability to trigger production of TNF-D D

International audienceTransglycosylation reactions biocatalyzed by the native arabinofuranosidase Araf51 and using D-galactosyl, D-fucosyl and 6-deoxy-6-fluoro-D-galactosyl derivatives as donors and acceptors provided di-to pentahexofuranosides. The immunostimulatory potency of these compounds, and more especially their ability to induce production of TNF-α, was evaluated on the murine macrophage cell line, Raw 264.7. The results obtained showed concentration-dependent and most importantly, structure-dependent responses. Interestingly, oligoarabinofuranosides belonging to the oligopentafuranoside family displayed concentration-, chain length and aglycon-dependent bioactivities irrespective of their fine chemical variations. Thus, neo-oligofuranosides in D-Galf series, as well as their D-Fucf and 6-fluorinated counterparts are indeed potential sources of immunostimulating agents

The versatile enzyme Araf51 allowed efficient synthesis of rare pathogen-related β-d-galactofuranosyl-pyranoside disaccharides.

International audienceThe preparation of galactofuranosyl-containing disaccharidic parts of natural glycoconjugates was performed according to a chemo-enzymatic synthesis. Our goals were firstly to develop an alternative approach to standard chemical strategies by limiting the number of reaction and purification steps, and secondly to evaluate the scope of the Araf51 biocatalyst to transfer a galactofuranosyl moiety to a set of pyranosidic acceptors differing from each other by the series, the anomeric configuration as well as the conformation. The study of binding mode of the resulting disaccharides was also performed by molecular modeling and showed significant differences between (1→2)- and (1→6)-linked disaccharides

Exploring the synthetic potency of the first furanothioglycoligase through original remote activation.

International audienceThioglycosidic bonds are of utmost importance in biomolecules as their incorporation led to more stable glycomimetics with potential drug activities. Until now only chemical methods were available for their incorporation into glycofuranosyl conjugates. Herein, we wish to describe the use of the first furanothioglycoligase for the preparation of a great variety of thioaryl derivatives with moderate to excellent yields. Of great interest, a stable 1-thioimidoyl arabinofuranose, classically used in chemical glycosylation, was able to efficiently act as a donor through an original enzymatic remote activation mechanism. Study of the chemical structure as well as the nucleophilicity of the thiol allowed us to optimize this biocatalyzed process. As a consequence, this mutated enzyme constitutes an original, mild and eco-friendly method of thioligation

Contribution à l'étude de l'a-L-arabinofuranosidase AbfD3 (synthèse chimique de nouveaux donneurs de glycofuranosyle activés et transglycosylation enzymatique)

Les sucres sous forme furanosidique représentent des cibles particulièrement intéressantes dans la mesure où ils ont été mis en évidence au niveau de la paroi cellulaire de certains microorganismes pathogènes. Le Chapitre I de cette thèse décrit les principaux glycofuranoconjugués naturels ainsi que les principales méthodologies de synthèse chimique permettant de préparer de telles structures. Le Chapitre II expose les différents outils enzymatiques utilisés en synthèse glycosidique, en mettant particulièrement l accent sur les glycosyl transférases et les glycosidases. La première partie du travail décrit (Chapitre III), a permis de contribuer à l étude de la versatilité d une -L-arabinofuranosidase, l AbfD3, grâce à la synthèse de nouveaux substrats glycofuranosidiques activés. La seconde partie du travail (Chapitre IV) a été consacrée à la synthèse et à l étude de substrats enzymatiques d intérêts: les fluorures de glycofuranosyle.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Synthèse innovante de thiofuranosides (développement d'une méthode de ligation biocatalysée)

Les hexofuranosides comme les D-galactofuranosides (D-Galf), absents chez les mammifères, sont particulièrement présents dans de nombreux micro-organismes souvent pathogènes. Ces oligosaccharides et leurs analogues sont donc de bons candidats pour (i) améliorer nos connaissances sur leurs rôles biologiques et physico-chimiques, (ii) définir de nouveaux traitements antiparasitaires. Dans ce contexte, notre objectif consiste aux mutations des enzymes conçues pour l amélioration des activités de transglycosylation, ainsi que la synthèse chimique de substrats innovants. La formation de structures a-L-arabinofuranosidiques, analogues 6-déoxy du Galf, contenant une liaison S-glycosidique inclue (i) les production, purification et étude d arabinofuranosidases Araf51 de Clostridium thermocellum recombinées dans E.coli BL21 et mutées (thioligases), (ii) la préparation chimique des donneurs et accepteurs et (iii) la synthèse biocatalysée de thioglycosides dont un glycodendrimère.Hexofuranosides, such as D-Galactofuranoside (D-Galf), are particularly spread in numerous pathogenic microorganisms but unknown in mammals. Thus, such oligosaccharides and their analogues are promising candidates (i) to improve our knowledge about their biological and physico-chemical roles and (ii) to define new antiparasitic treatments. In this context, our aim relies on mutations of enzymes for thiotransglycosylation, as well as chemical synthesis of innovating substrates. These lead to the preparation of a-L-arabinofuranosides, 6-deoxy-Galf analogues, containing S-glycosidic linkage. This study includes (i) the recombinant production of Clostridium thermocellum arabinofuranosidase Araf51, as well as its mutants (thioligases), in BL21 E.coli expression system, followed by the purification and the activity studies of both enzymes, (ii) chemical preparation of donors and acceptors and (iii) biocatalytic synthesis of thioglycosides including a glycodendrimer.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Nucléotides-sucrés furaniques (synthèses et activités anti-mycobactériennes)

Les sucres sous forme furanose sont complètement absents chez les mammifères mais présents dans de nombreux micro-organismes souvent pathogènes et responsables de maladies graves telles que la tuberculose, la lèpre, la maladie de Chagas Les enzymes responsables de leur biosynthèse sont peu ou pas connues mais représentent de nouvelles cibles thérapeutiques intéressantes. Au cours de cette thèse, nous avons souhaité développer de nouvelles méthodes de synthèses permettant d obtenir des sucres rares et plus particulièrement des nucléotides-sucres furaniques. Ce mémoire présente dans un premier temps, une approche chimique permettant un accès direct aux cibles visées. Dans un second temps, ce travail met en évidence le potentiel biocatalytique de nucléotidyltransféases pour la préparation de nucléotides-sucres furaniques non naturels. Enfin, les activités biologiques des composés préparés ont été évaluées sur les enzymes intervenants dans la biosynthèse de l arabinogalactane mycobactérien.Furanosides are original glycosides which are presents in several micro-organisms, particulary in pathogenic species, such as those responsible for tuberculosis, leprae, Chagas diseases... The fact that those glycoconjugates are absent in mammal makes those five-membered ring sugars, valuable targets to design new pharmacophores and/or treatments. Therefore, a better understanding of furanoconjugates 's biosynthetic pathways is a crucial step of this new therapeutic strategy. This report presents a straightforward chemical synthesis of nucleotides-sugars from thioimidoyl furanosides. Then, this work underlines also the biocatalytic behavior of nucleotidylyltransferases in order to synthesize non-natural furanosyl nucleotides. At last, activities of those new compounds were evaluated on the enzymes involved in the biosynthesis of the arabinogalactan of Mycobacterium tuberculosis.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Versatile Synthesis of Rare Nucleotide Furanoses

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