Coupling of substances containing a primary amine to hyaluronan via carbodiimide-mediated amidation

Abstract: The purpose of this study was to investigate the carbodiimide-mediated amidation of hyaluronan (HA). The carbodiimide-mediated amidation includes the formation of a urea derivative, O-acylisourea, between the carbodiimide and a carboxylic group of HA, which a primary amine can displace, resulting in an amide bond. Reaction conditions were investigated and optimized, the molecular weights Mn and Mw were determined with size-exclusion chromatography and by-products were analysed with 1H NMR. The reaction is done at room temperature in slightly acidic pH, giving a degree of substitution between 5 to 15 %. A catalyst, N-hydroxysuccinimide, was needed for the coupling to be successful, since O-acylisourea was shown not to be reactive enough towards primary amines. It was found out that dissociated primary amines successfully couple to HA, contrary to what has been suggested before. 1H NMR revealed that O-acylisourea readily forms a by-product, which is covalently attached to HA, through the means of rearrangement. Also, 1H NMR showed that the carbodiimide reacts with phenols. An increase of Mn and Mw compared with native HA was observed and attributed to ester bond formation between a hydroxyl- and carboxylic group of HA polysaccharides. To conclude, the carbodiimide-mediated amidation is an unspecific reaction which is not suited for the coupling of primary amines to HA.