Exploration of specific carbohydrate epitopes in their native habitat with the Staudinger ligation

Abstract

Glycans modulate biological processes and play a vital role in the function of receptors. However, the structure and functions of glycans are often obscured due to the heterogeneity of these structures. We hypothesize that the function of a specific carbohydrate epitope can be elucidated by incorporating an epitope of interest onto an existing glycan and monitoring its interactions within its native habitat. To explore this hypothesis, we have introduced the epitope of interest by adapting the Staudinger ligation strategy. In this strategy a nucleophilic phosphane reagent linked to an epitope reacts bioorthogonally with an electrophilic azide containing molecule and appends the epitope to the desired molecule. An azide can be incorporated into the glycan by the cell’s biosynthetic machinery. We synthesized galactose-, lactose- and N-acetyl lactosamine (LacNAc)-phosphane reagents and succesfully demonstrated the use of this strategy to make defined glycoproteins. We also demonstrated that this method has utility for immobilization of carbohydrates onto azide derivitized surfaces under mild conditions using very low concentrations of the reagents without altering their binding affinity for lectins. The binding interactions of the carbohydrate epitopes with the lectins, jacalin, and wheat-germ agglutinin (WGA) were examined using surface plasmon resonance. We also designed and synthesized a tri-galactose-phosphane reagent to study multivalent interactions usually observed in biological systems.