Abstract
Hyaluronic acid (HA), the only non-sulphated glycosaminoglycan, serves numerous structural and biological functions in the human body, from providing viscoelasticity in tissues to creating hydrated environments for cell migration and proliferation. HA is also involved in the regulation of morphogenesis, inflammation and tumorigenesis through interactions with specific HA-binding proteins. Whilst the physicochemical and biological properties of HA have been widely studied for decades, the exact mechanisms by which HA exerts its multiple functions are not completely understood. Glycopolymers offer a simple and precise synthetic platform for the preparation of glycan analogues, being an alternative to the demanding synthetic chemical glycosylation. A library of homo, statistical and alternating HA glycopolymers were synthesised by reversible addition–fragmentation chain transfer polymerisation and post-modification utilising copper alkyne–azide cycloaddition to graft orthogonal pendant HA monosaccharides (N-acetyl glucosamine: GlcNAc and glucuronic acid: GlcA) onto the polymer. Using surface plasmon resonance, the binding of the glycopolymers to known HA-binding peptides and proteins (CD44, hyaluronidase) was assessed and compared to carbohydrate-binding proteins (lectins). These studies revealed potential structure-binding relationships between HA monosaccharides and HA receptors and novel HA binders, such as Dectin-1 and DEC-205 lectins. The inhibitory effect of HA glycopolymers on hyaluronidase (HAase) activity was also investigated suggesting GlcNAc- and GlcA-based glycopolymers as potential HAase inhibitors.
Highlights
Glycopolymers provide a simpler alternative method to the total chemical synthesis of glycans
For glucuronic acid (GlcA), the monosaccharide requires acetylation followed by esterification to inhibit the formation of a bicyclic lactone,[28] before using BF3ÁOEt2 as a catalyst to form the glycosidic bond with the alcohol donor (Scheme S2, Electronic supplementary information (ESI)†)
The glycopolymers were synthesised by first producing a well-defined polymer backbone of poly(4vinylbenzyl chloride) (P1) using reversible addition–fragmentation chain transfer (RAFT) polymerisation to give P1 with a dispersity (Ð) of 1.13 (Scheme 1 and Table 1)
Summary
Glycopolymers provide a simpler alternative method to the total chemical synthesis of glycans (e.g. synthesis of precisely defined oligosaccharides by chemical glycosylation). HA interactions with toll-like receptors (TRLs), immune receptors that participate in the innate defence against bacterial infection, have been described in the literature.[46] For example, HA oligosaccharides were shown to activate dendritic cells via TRL-4.47 we have extended our binding studies to lectins, carbohydrate-binding proteins involved in pathogen invasion and immune system activation.[48] C-type lectins, such as DC-SIGN, MBL and SP-D, predominantly bind monosaccharides through the C3 and C4 hydroxides, often mediated by a metal ion such as calcium.[49] as lectins are found as clusters on the cell surface, glycopolymers may be able to bind across multiple sites and produce an amplified lectin signal.[8] Using a series of these C-type lectins and our SPR assay, HA showed no specific binding to DC-SIGN, MBL or SP-D, whilst the various glycopolymers display differing binding specificities for each lectin (Fig. S21–S23, ESI†).
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