Chemoenzymatic Synthesis and Comprehensive Characterization of Keratan Sulfate and 6-Sulfo-SLex

Abstract

Keratan sulfate (KS) is a proteoglycan present in the cornea, bone, cartilage, brain, and epithelial cells, where it plays essential roles in corneal transparency, wound healing, cell adhesion, and inflammation regulation. KS has potential for treating conditions such as rheumatoid arthritis, asthma, and chronic obstructive pulmonary disease, and disruptions in its production are associated with diseases like macular degeneration, keratoconus, ALS, Alzheimer’s, mucopolysaccharidosis IV, and poor prognosis in various cancers. Compositional and structural analyses have identified three distinct types of KS: KS-I (corneal KS), which is attached to a protein via an N-linked bond; KS-II (skeletal KS), which is linked to a protein through an O-linkage to serine or threonine and features varying amounts of α-L-fucose and N-acetylneuraminic acid capping residues; and KS-III, which contains an additional O-linked region through D-mannose. Although these types differ in chain length, sulfation, and protein linkages, they all share common structural elements, such as repeating disaccharide units, non-sulfated poly-N-acetyllactosamine, and variable sulfated regions with 6-sulfated N-acetylglucosamine and D-galactose. Additionally, the structural diversity of KS is further increased by α-L-fucosylation at C3 of GlcNAc and the addition of monosaccharide capping residues, including α-Neu5Ac, α-Neu5Gc, α-Gal, β- GlcNAc6S, and β-GalNAc6S. Sharing the characteristic 6-O-sulfation of GlcNAc with KS, 6-sulfo-SLex is a terminal epitope on O-glycans, defined by a sialyl LacNAc motif bearing an O-sulfate at carbon-6 and an α-linked L-fucose at carbon-3 of the GlcNAc residue. This structure is important for binding to L-selectin, human sialic acid-binding immunoglobulin-like lectin (Siglec), and viruses such as influenza and coronaviruses. The sulfation patterns at C-6 of Gal and C-6 of GlcNAc, along with α-fucosylation, are key components of the sulfation code that mediate carbohydrate-protein interactions. Deciphering this code, using a wide range of structurally well-defined complex KS and 6-sulfo-SLex glycans, could greatly improve both therapeutic and biological outcomes. However, further research on these glycans is limited by the challenges of synthesizing their structures, due to their distribution, diversity, and complexity. This thesis presents a series of efficient and concise methods for the synthesis of a diverse library of KS and 6- sulfo-SLex glycans, enabling systematic exploration of their structure–function relationships in distinct glycan-mediated recognition events.