Chemoenzymatically Synthesized O ‐Acetylated GD3 Gangliosides to Examine Viral Receptor Specificities in a Cellular Context

Gangliosides are a class of sialic acid‐containing glycosphingolipids involved in a wide range of biological processes. The terminal sialic acid of gangliosides can be O ‐acetylated at C7 and/or C9 hydroxyl, contributing to ganglioside structural complexity and function. It has been difficult to obtain panels of structurally well‐defined O ‐acetylated gangliosides for binding and functional studies. We describe here a chemoenzymatic strategy that can provide, for the first time, 7‐ O ‐, 9‐ O ‐, and 7,9‐di‐ O ‐acetylated GD3 gangliosides. It is based on the chemical assembly of a common tetrasaccharide precursor as α‐glycosyl fluoride that is coupled to sphingosine by a glycosynthase, followed by O ‐acetyl editing by coronaviral hemagglutinin‐esterases (HEs). The resulting synthetic glycosphingolipids have been employed for cell surface remodeling of erythrocytes. Analysis by liquid chromatography and ion mobility mass spectrometry (LC‐IM‐MS) demonstrated successful integration of the glycosphingolipids into the plasma membrane with preservation of acetyl ester patterns. Using human coronavirus HKU1 spike‐functionalized virus‐like particles, we demonstrate that the resulting glycan‐remodeled erythrocytes can be utilized in hemagglutination (HA) studies as a label‐free method to investigate viral protein binding to individual glycoforms in a cellular environment.