Development of bio-orthogonal fluorescent tags for carbohydrate analysis and functional assays

Abstract

Heterogeneous populations of structurally complex carbohydrates mediate many biological important processes, such as cell-cell adhesion, signal transduction and pathogen recognition events. However, due to the non-template driven nature of glycan biosynthesis and very often challenging synthetic routs, the accessibility of these information rich structures is limited and, consequently, their study presents significant challenges limiting progress in glycomics. Here, we will present our recent efforts to develop novel fluorescent tags bearing bio-orthogonal modifications enabling chromatographic separation of glycans obtained from natural sources and their downstream uses in functional assays, like glycan microarrays. These labels will be based upon 2-anthranilic acid and 2-aminobenzamide employed in industrial glycan analysis. Our tags will carry additional ‘clickable’ alkyne, norbornene or tetrazine modifications enabling the conjugation with appropriately functionalised ligation partners (e.g. lipids) via copper catalysed alkyne-azide cycloaddition or via catalyst-free inverse electron demand Diels-Alder reactions. Finally, we will show labelling of commercially available carbohydrates and a mixture of linear glucose oligomers prepared by the acidic hydrolysis of dextran with the novel labels and test their performance, effectiveness, and stability compared to commercial tags. The selected labels will also be used to tag glycans mixtures of N-linked carbohydrates cleaved enzymatically from the model glycoproteins. We believe that developed fluorescent bio-orthogonal tags will provide exciting methodology for utilisation of natural glycan resources, generation of complex neo-glycoconjugates and functional assays, making carbohydrates more accessible to the wider community