Synthetic Approaches and Biological Functions of N-sulfated Heparan Sulfate Oligosaccharides and Proteoglycan Mimetics

Abstract

Heparan Sulfate (HS), a member of the glycosaminoglycan family, is composed of repeating units of α (1-4) linked D-glucosamine and uronic acid residues (L-iduronic acid and D-glucuronic acid) with diverse sulfation patterns. For instance, HS disaccharides can arrange themselves into 48 distinct possibilities, and this structural diversity enables binding to numerous proteins, thereby modulating a wide array of biological processes. Despite significant progress in the synthesis of structurally defined HS oligosaccharides, the importance of N-sulfated HS ligands remains underexplored. In my thesis, I synthesized a wide range of N-sulfated HS ligands and examined their structure-activity relationships. Chapter 1 outlines the divergent approach employed for synthesizing HS oligosaccharides. It includes a comprehensive review of the synthesis of disaccharide building blocks with various orthogonal protecting groups, promoters, and optimized conditions to improve glycosylation yields. It also discusses the synthesis of universal disaccharide building blocks, one-pot synthesis, and automated solid-phase synthesis of HS oligosaccharides. Finally, the chapter highlights the current understanding of the structure-activity relationships of heparan sulfate-binding proteins (HSBPs). Chapter 2 focuses on the synthesis of N-sulfated HS disaccharides using a divergent strategy and their subsequent characterization. Employing a panel of 14 disaccharides, we conducted high-throughput microarray analyses involving 9 growth factors and 14 chemokines. Our findings revealed that highly sulfated N-sulfated L-iduronic acid ligands exhibit a strong tendency to bind a broad range of growth factors compared to their D-glucuronic acid counterparts. Furthermore, we observed that 3-O-sulfation of HS disaccharides plays a pivotal role in fine-tuning the binding interactions with several growth factors and chemokines. Chapter 3 describes the synthesis of 19F-NMR-sensitive fondaparinux to monitor blood-fondaparinux interactions. To achieve this, trifluoromethyl phenyl glycosylated fondaparinux was synthesized using a [2+3] glycosylation strategy. The 19F-NMR analysis of the conjugate exhibited a strong T1 value and detectable 19F-NMR signals at micromoles concentrations. When treated with blood, a 0.2 ppm shift in the NMR signal of fondaparinux was observed. Further in vivo activity studies and 19F-NMR analysis of the blood microenvironment behavior of the complex are currently underway. xv Chapter 4 outlines a continuous flow strategy for the insertion of sulfation groups onto HS disaccharides and tetrasaccharides. Using flow chemistry, conditions critical for 6-O-sulphation and 3-O-sulphation with SO3.salts complex at varying temperatures were optimized. These optimied conditions were then employed to synthesie a broad range of sulfation patterns on HS disaccharides and tetrasaccharides. The results demonstrated that flow chemistry is an efficient approach to sulfate HS in a short timeframe with high yields. Chapter 5 details the synthesis of HS hexasaccharides containing L-iduronic acid and L-idose. When these two hexasaccharides were functionalized on a proteoglycan backbone and expressed on cancer cell surfaces, the L-idose-based hexasaccharide underwent endocytosis significantly faster than the L-iduronic acid-based variant. This highlights the importance of L-iduronic acid in maintaining HS oligosaccharides on the cell membrane. Furthermore, L-idose-based proteoglycan mimetics were observed to localize in the nuclear region after 1–2 hours, suggesting their potential as markers for targeting the nucleus of cells using proteoglycan mimetics.