Asking more from metabolic oligosaccharide engineering

Abstract

Glycans form one of the four classes of biomolecules, are found in every living system and present a huge structural and functional diversity. As an illustration of this diversity, it has been reported that more than 50% of the human proteome is glycosylated and that 2% of the human genome is dedicated to glycosylation processes. Glycans are involved in many biological processes such as signalization, cell-cell or host pathogen interactions, immunity, etc. However, fundamental processes associated with glycans are not yet fully understood and the development of glycobiology is relatively recent compared to the study of genes or proteins. Approximately 25 years ago, the studies of Bertozzi's and Reutter's groups paved the way for metabolic oligosaccharide engineering (MOE), a strategy which consists in the use of modified sugar analogs which are taken up into the cells, metabolized, incorporated into glycoconjugates, and finally detected in a specific manner. This groundbreaking strategy has been widely used during the last few decades and the concomitant development of new bioorthogonal ligation reactions has allowed many advances in the field. Typically, MOE has been used to either visualize glycans or identify different classes of glycoproteins. The present review aims to highlight recent studies that lie somewhat outside of these more traditional approaches and that are pushing the boundaries of MOE applications.

Fig. 1. Common bioorthogonal reactions that have been applied to metabolic oligosaccharide engineering. The Staudinger ligation occurs between an azide-modified glycan and a modified triarylphosphine to yield an amide bond. The Cu(i)-catalyzed azide–alkyne cycloaddition (CuAAC) yields a stable triazole. This versatile reaction can be performed in either orientation, but azido-tags result in a lower background signal. As an alternative to CuAAC, the formation of triazoles can be promoted through ring strain in the strain-promoted azide–alkyne cycloaddition. Finally, tetrazine tags will rapidly undergo an inverse electron-demand Diels–Alder reaction with activated alkynes, like cyclopropenes.

Fig. 2. Glyco-motif targeting. Examples of chemo-enzymatic labelling using a given glycosyltransferase (named in red), its associated nucleotide sugar, and the corresponding acceptor glycol-motif. Step 1: an unnatural monosaccharide is transferred from its activated form onto the targeted glycan through the action of a glycosyltransferase. Step 2: the introduced reporter (x) is reacted with a probe bearing a complementary bioorthogonal function (y) allowing its detection.

Fig. 3. Cell/tissue targeting strategies. (A) The chemically modified monosaccharide (blue hexagon) bears a moiety able to be selectively cleaved off by enzymes secreted only by targeted cells. In normal cells, the chemical reporter does not enter the cell whereas in the target cells, the unnatural monosaccharide does go through the plasma membrane upon cleavage of the moiety. (B) The unnatural monosaccharide is encapsulated into a ligand functionalized liposome. Liposomes' ligands are designed to interact only with receptors specific of the targeted cells, allowing the selective delivery of the monosaccharide into desired cells. (C) Entry of an unnatural monosaccharide functionalized with a metabolite which has a specific carrier. Once into the cytosol, the metabolite is cleaved and the modified sugar can be metabolized.

Fig. 4. Cross-linking reporters. Structures of reported analogs of sialic acid used for the cross-linking strategy. (1) N-Acetyl neuraminic acid, (2) 9-phenylazido-neuraminic acid, SiaAAz, (3) SiaDAz, and (4) 9-azido-SiaDAz.

Pierre-André Gilormini

Anna R. Batt

Matthew R. Pratt

Christophe Biot