Identification of an Unnatural Sulfated Monosaccharide as a High-Affinity Ligand for Pan-Variant Targeting of SARS-CoV-2 Spike Glycoprotein.

Identifying smaller sulfated glycan fragments that recognize target proteins with high affinity is highly challenging. In this work, we show that microarray screening of 53 small glycan fragments helped identify distinct sulfated monosaccharide to tetrasaccharide fragments that bind to multiple isoforms of SARS-CoV-2 spike glycoprotein (SgP) with high affinity. Our library consisted of natural and unnatural glycan sequences with a wide range of sulfation levels. The unnatural features arose from the presence of phosphate or fluoro groups on the natural sulfated GAG scaffold as well as sulfate modification of idose fragments that were monomer to tetramer long. None of the natural glycans yielded much promise, which probably conveys the importance of the polymeric glycosaminoglycan chain in SgP biology. However, the unnatural idose fragments with sulfation at the 2, 3, 4, and 6 positions displayed high affinities (100-500 nM) for wild-type, Delta, and Omicron variants of SgP. The unnatural sulfated idose monosaccharide is the smallest molecule known to date that can be classified as a high-affinity, pan-variant fragment. This fragment is expected to serve as the lead for the design of pan-variant ligands with sub-nM inhibition potency.