Chemoenzymatically Synthesized <i>O</i> ‐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.