Chemical Synthesis and Functional Characterization of Fluorinated Heparan Sulfate Fragments and Fondaparinux-Based Glycomimetics.

Abstract

The glycocalyx, a dense layer of glycans on the cell surface, plays a fundamental role in mediating cellular communication, adhesion, and signal transduction. Among its key constituents, heparan sulfate (HS) is a highly anionic, linear glycosaminoglycan that binds to a broad array of proteins, including growth factors, chemokines, and microbial virulence factors. Its biological activity is governed by structural heterogeneity arising from differential sulfation patterns, uronic acid composition, and the conformational flexibility of L-iduronic acid. While considerable progress has been made in understanding the role of sulfation motifs and uronic acid composition in HS–protein interactions, the contribution of individual hydroxyl groups to molecular recognition and biological function remains largely unexplored. In this thesis, we report the design, synthesis, and biological evaluation of a suite of fluorinated HS ligands ranging from disaccharides to pentasaccharides. Employing a divergent synthetic strategy, we successfully constructed seven structurally defined HS disaccharide analogues incorporating site-specific fluorine substitutions at key glucuronic acid 3-hydroxyl positions. Conformational analysis via NMR spectroscopy revealed that all glucuronic acid residues maintained a stable 4C1 conformation, irrespective of fluorine incorporation. Functional interrogation through glycan microarray and cellular assays demonstrated that fluorination at the N-acetylated glucosamine unit enhanced fibroblast growth factor 2 (FGF2) binding affinity and promoted mitogenic signalling in vitro. When conjugated to synthetic neoproteoglycan scaffolds, these fluorinated HS ligands modulated FGF2 signalling cascades with comparable efficacy to their N-sulfated counterparts, highlighting their potential as functional mimetics.