Design and synthesis of hyaluronan based glycopolymers for self-assembly with hyaluronan binding peptides

摘要

Introduction Hyaluronan (HA) is a highly abundant anionic polysaccharide found throughout mammalian connective tissues. It has a simple linear structure alternating the units of N-acetyl-glucosamine (GlcNAc) and glucuronic acid (GlcA). Despite its simple structure, HA is capable of an amazing variety of conformations ranging from extended chains to relaxed coils, which are transient and rapidly interchanging in solution. HA is involved in the extracellular matrix organization and many aspects of cell behaviour, essential to a wide range of biological processes (e.g. morphogenesis, tumorigenesis and inflammation). Varying chain lengths of HA affect its biological functions and it has been suggested that the interaction of HA with specific proteins (CD44, RHAMM, TSG-6), known as HA-binding proteins, is responsible for stabilizing particular conformations of the polysaccharide, leading to HA-protein complexes with distinct architectures and biological properties. Thus, this work aims to design and synthesize glycopolymers mimicking the composition and structure of HA (defined length and identical conformation) and combine these glycopolymers with HA-binding peptides, such as Pep-1 identified by phage display, to develop innovative supramolecular biomaterials. Material and Methods 4,6,11,13 were produced adapting previous literature, 1 ,3-Dipolar cycloaddition general procedure: Sugar (1 equiv), Monomer (1.2 Equiv), Na-Ascorbate (0.1 equiv), CuSO_4 (0.05 equiv) purified by flash chromatography (CH_2Cl_2:CH_3OH 4:1). Polymerisation general procedure: Monomers (100 equiv), RAFT agent (1 equiv), Initiator (0.1 equiv) in butan-2-one at 70 °C. Pep-1 was synthesised by microwave assisted solid phase synthesis and purified on a reverse phase HPLC. Results The HA repeating unit presents a synthetic challenge to make a controlled sequence glycopolymer. Styrene-maleic anhydride (Fig 2A-C) produce highly alternating polymers which are being expanded to use more complex maleimide and styrene's.7 was synthesised in 24% across 4 steps (Fig. 1B) confirmed by NMR results. Unprotected GlcA favours the internal cyclisation catalysed by SnCl_4 rather than the azide substitution at the 1' position. Acylation followed by methylation (Fig. 1C) prevented the internal cyclisation to catalyse the substitution to form 11 (Fig 1D) with the corresponding alkyne 14 was prepared over 2 steps.Pep-1 was successfully synthesized (Fig. 2D/E). Its binding affinity to HA glycopolymers will be investigated and their interactions tuned to create new biomaterials with dynamic properties. Conclusion The HA sugars have been modified to posse click functional groups to form the glycomonomer which can be polymerised to generate glycopolymers mimics of the natural HA. In addition, HA glycopolymers with different architectures (e.g. branched, star-like) can also be synthesized, expanding the capacity to develop new and diverse peptide-HA glycopolymer hybrid biomaterials.