Structure characterization of a heavily fucosylated chondroitin sulfate from sea cucumber (H. leucospilota) with bottom-up strategies

Two bottom-up strategies, disaccharide and oligosaccharide analyses, were applied to elucidate the structure of a fucosylated chondroitin sulfate (FCS). The FCS was hydrolyzed with mild acid. The remained part was digested with CS lyase for disaccharide analysis. The products from each step were analyzed and the results revealed that mild sulfuric acid mainly released sulfated fucose branches, but also affected some residues and sulfo-groups on the backbone. Over 140 oligosaccharide fragments were generated by catalytic oxidation and identified by HPSEC-MS, including sulfated fucose oligosaccharides exclusively from branches, sulfated backbone fragments, and junctional fragments. Based on the results provided by these two methods, the proposed backbone of the FCS is mainly composed of GlcA -> GalNAc4S6S and GlcA -> GalNAc6S, and the branch is mainly located at GalNAc. The longest branch observed is nonasaccharide, and most of the fucose on the branches are mono and/or disulfated. NMR results supported the conclusion.