IGF::OT::IGF CHEMOENZYMATIC SYNTHESIS OF HEPARAN SULFATE OLIGOSACCHARIDES

Glycan Therapeutics Corporation, Raleigh NC

Investigators

Abstract

The goal of this contract is to synthesize a heparan sulfate (HS) library consisting of structurally homogeneous oligosaccharides, in response to an SBIR contract call from NCl-321 Chemically Defined Glycan Libraries. The Contractor specializes in the synthesis of structurally diverse HS oligosaccharides. The Contractor has built an innovative chemoenzymatic method for Glycan synthesis. Polymeric HS contains the disaccharide repeating units of glucuronic or iduronic acid linked to glucosamine that carry sulfo groups. Chemical synthesis of structurally homogeneous HS oligosaccharides is extremely difficult. The chemoenzymatic method reduces synthetic complexity, providing a cost-effective approach to prepare a diverse collection of HS oligosaccharides. A library of 77 oligosaccharides with different sizes and sulfation patterns shall be constructed using this method. The library represents the basic structural elements found in naturally occurring HS. Synthesis shall be carried out under good laboratory practice (GLP) at a scale of 3 mg for each compound, which amount is sufficient for structural analysis by NMR and mass spectrometry as well as the purity analysis by anion-exchange high performance liquid chromatography (HPLC). Four milestones for sample preparations and deliveries are clearly marked.Heparan sulfate (HS) is a member of glycosaminoglycan family that is present in large quantities on the cell surface and in the extracellular matrix. HS is a highly sulfated polysaccharide with a disaccharide repeating unit of glucuronic acid (GLcA) or iduronic acid (ldoA) linked to glucosamine. Both ldoA and glucosamine residues carry sulfo groups. HS participates in numerous biological processes, including embryonic development, assisting viral and bacterial infections and regulating blood coagulation. The sulfation patterns and the location of ldoA residues, as well as the size of HS molecules play critical roles in determining the functions HS. Unfortunately, knowledge of HS is far less developed than knowledge of proteins and nucleic acids. One grand challenge in HS studies is to make HS oligosaccharides widely available to the scientific research community. Chemical synthesis of HS has been taken to respond to this challenge. However, the facts that complicated synthesis and low yield of products led to the unsuitableness for oligosaccharide library construction and for HS market. The objective of this contract is to employ a state-of-the-art chemoenzymatic approach to synthesize HS oligosaccharides with different structures under GLP conditions. A total of 77 oligosaccharides in the library have been selected based on the natural structures of HS and shall be synthesized. The oligosaccharides cover the range of tetra- to nona-saccharides.consisting of N-, 6-0-, 3-0-, and 2-0-, sulfation as well as ldoA residues. None of these compounds in the library is currently commercially available. Each compound has an aldehyde moiety at its reducing end and has a unique chromophoric group for easy detection. The purity of each compound is more than 98%. The structures of the newly synthesized compounds shall be proven by NMR and Mass spectrometry analyses. All 77 oligosaccharides at 50 ug of each compounds along with their NMR spectra shall be delivered to NIH by the end of Phase II. The targeted oligosaccharides can be divided into six groups, structurally differing in the size, sulfation patterns and with or without ldoA residues. Each compound has a pAHP (para-azido hexanamido) phenyl) capped GlcA end, which exhibits similar chemical reactivity to that of the reducing end of a given sugar residue ..