Abstract

In hydrogels of cross-linked polysaccharides, the total amount of cross-linker and the degree of cross-linking influence the properties of the hydrogel. The substitution position of the cross-linker on the polysaccharide is another parameter that can influence hydrogel properties; hence methods for detailed structural analysis of the substitution pattern are required.NMR and LC-MS methods were developed to determine the positions and amounts of substitution of 1,4-butanediol diglycidyl ether (BDDE) on hyaluronic acid (HA), and for the first time it is shown that BDDE can react with any of the four available hydroxyl groups of the HA disaccharide repeating unit. This was achieved by studying di-, tetra-, and hexasaccharides obtained from degradation of BDDE cross-linked HA hydrogel by chondroitinase. Furthermore, amount of linker substitution at each position was shown to be dependent on the size of the oligosaccharides. For the disaccharide, substitutions were predominantly at Delta G1cA-OH2 and G1cNAc-OH6 while in the tetra- and hexasaccharides, it was mainly at the reducing end G1cNAc-OH4. In the disaccharide there was no substitution at this position. Since chondroitinase is able to completely hydrolyse non-substituted HA into unsaturated disaccharides, these results indicate that the enzyme is prevented to cleave on the non-reducing side of an oligosaccharide substituted at the reducing end G1cNAc-OH4. The procedure can be adopted for the determination of substitution positions in other types of polymers. (c) 2015 The Authors. Published by Elsevier Ltd.

Keywords

Hyaluronic acid; Hydrogel; Cross linked; Substitution; NMR; MS

Published in

Carbohydrate Polymers
2016, volume: 136, pages: 1348-1357
Publisher: ELSEVIER SCI LTD

Authors' information