Amide Couplings of Sugar Amino Acids and their Sulfates: Toward Heparan Sulfate Mimetics

<p><strong>Heparan sulfate (HS) is a sulfated glycosaminoglycan with huge therapeutic potential. It is produced by all animal cells and plays roles in many cell signalling, regulatory, and proliferation processes. Nature produces HS as heterogenous populations with glycans of various lengths and sulfation patterns. This makes characterisation of HS and determination of structure-activity relationships extremely difficult. Unlike the widely used therapeutic heparin, HS is available in only limited supply, generally from commercial heparin side streams. Homogenous HS populations can be provided by synthesis but due to the complexity of these structures, preparation of even small oligomers takes much effort and resource. Synthetically more accessible HS mimetics can help bridge the gap.</strong></p><p>Linking carbohydrate moieties using amide linkages rather than glycosyl linkages is one way to simplify oligomer synthesis. Amide bonds are routinely generated in peptide synthesis through reactions that are high yielding and well established. Employing sugar amino acids (SAAs) as starting components can facilitate the assembly of oligomeric HS mimetics. In this way, oligomers with defined sulfation patterns can be prepared and used to discern structure-activity relationships.</p><p>Sulfated SAAs are highly polar compounds which are difficult to handle and manipulate and there are limited examples of amide couplings of sulfated SAAs in the literature.</p><p>In this work, sulfated SAA monomer building blocks suitable for participating in amide couplings were prepared. Synthetic routes to these materials were established, including the reliable installation of sulfate groups. Then, three approaches to amide bond formation between sulfated SAAs were explored: couplings in the solution phase, couplings on the solid phase, and solution phase polymerisations.</p><p>Methodology was developed which enabled the solution phase coupling of sulfated SAAs with unsulfated SAAs. Key to these reactions were the choice of coupling reagents and the salt form of the SAA sulfates. A range of techniques were investigated for the purification of the sulfated materials from other highly polar reaction components including silica gel chromatography, dialysis, acidic resin work-up, and chemical derivatisations. The limit of the coupling technology was determined where amide formation could not be effected between two sulfated SAAs in solution.</p><p>Next, coupling reactions of sulfated SAAs on the solid phase were investigated. This involved extensive resin loading studies of SAAs, in which the stability of sulfated SAAs to solid phase conditions was demonstrated through loading and recovery assessments. Coupling reactions were realised, but again amide bonds between sulfated coupling partners remained elusive.</p><p>Sulfated SAA polymers were synthesised using an undirected solution phase coupling process and a post-polymerisation sulfation strategy. Characterisation of these sulfated polymers was undertaken, and their degrees of sulfation and polymerisation assessed. Pre-polymerisation sulfation strategies once again illustrated the reluctance of sulfated SAAs to form amide bonds with one another.</p>