New biocatalyst for C-glycosylation: Tailored hydrolases to access modular C-glycoside scaffolds with industrial potential – C-linkAse

Glycoconjugates are key molecules involved in many biological phenomena and more particularly in cellular communication and molecular recognition through their saccharidic part. Their natural occurrence and relevant biological role contributed to the strong emergence of glycosciences in the last 20 years. Nevertheless, these highly complex structures showed often drawbacks associated with the instability of the O-glycosidic bond. To overcome this problem, chemists managed to swap the O-glycosidic bond with more stable mimetic like C-glycosidic bond. C-glycosides, extremely rare in Nature, are free from anomeric effect, stable towards chemical and enzymatic hydrolysis and exhibit biological properties like antitumor, antiviral or glycosidase inhibitory activities. In addition stable alkyl C-glycosides showed surfactant properties and found some applications in the domain of detergence or cosmetics.
The synthesis of such scaffolds often involved nucleophilic attack on the activated anomeric carbon, transition-metal mediated C-glycosylation, anomeric radical reaction or sugar ring formation. Such strategies used toxic catalysts, multisteps synthesis, protecting group manipulations and time consuming purifications. Recently, new model reactions emerged using unprotected carbohydrates that can react with either ß-diketone or phosphonate under mild conditions. While highly powerful, such alternatives lead often to a mixture of anomers. There is so an urgent need for a selective synthesis of industrially relevant C-glycosides using procedures that respect the environment.
The development of synthetic steps catalyzed by enzymes is gaining momentum nowadays. Molecular biology allowed in particular to tailor the activity of proteins like enzymes and to widen either the scope of the reaction or the substrates. Major advantages of such process are the high selectivity of the enzyme towards the substrate and the clean conversion to one and only one product. As many enzymes work in aqueous media under mild condition, often at room temperature, the fingerprint on the environment is low and the amount of energy used is minimum. Based on these observations, we wish in the C-linkAse project to develop a one pot synthesis of C-glycoside scaffolds thanks to a biocatalyzed C-glycosylation in aqueous media. In this context, a new donor of glycosyl will be conceived. Such donor will be grafted with a function that can be activated only in the catalytic site of the enzyme. It will be obtained thanks to a two steps procedure starting from renewable carbohydrate using efficient and selective protocol.
Owing to our expertise in the mutation, production and use of furanosidases, we decided to develop first an innovative C-furanosylation with L-arabinofuranose as donor using a tailored arabinofuranosidase. As the enzyme chosen is a retaining glycosidase, it is able to cleave or produce only one anomer. Thus it will be a real asset in order to obtain one diastereoisomer of the resulting C-furanoside. Then this strategy will be extended first to mimics of L-arabinofuranose (D-galactofuranose and derivatives). The resulting scaffolds would bear reactive functions that could be used as a platform for further modulation. It should be able to accommodate various substituents of different nature like alkyl chains (surfactant, antiparasitic application), dendrimers (vaccine application), or amino-acids (C-glycoproteins).
While bio-catalyzed O- and S-glycosylations have been reported elsewhere, C-glycosylation constitutes the real challenge of the project. No example in the literature exists on such evolved biocatalysis process. The success of this idea will open a complete new area of research towards carbone-carbone bond formation.