Generation and structural characterization of a range of unmodified chondroitin sulfate oligosaccharide fragments

Chondroitin sulfate C has been used to demonstrate an approach of generating a range of unmodified glycosaminoglycan oligosaccharide fragments. This involves cleavage by oxymercuration treatment of the nonreducing terminal 4,5-unsaturated uronic acid (DeltaUA) residues from the fragments produced by enzymatic digestion of chondroitin sulfate with chondroitinase ABC. Carrying out the reaction on the unfractionated digestion mixture produces a range of mono- to tridecasaccharides, the compositions of which were established by liquid secondary ion mass spectrometry (LSIMS) and their chromatographic patterns compared with oligosaccharides in the untreated digest. Ten of the main sequences, tri- to octasaccharides, isolated by HPLC from the treated and untreated digests were fully characterized by a combination of LSIMS and 1H NMR. Of these, 6 are homologs of the series with structures DeltaUA1-[3GalNAc(6S)beta1- 4GlcAbeta1]n-3Gal-NAc(6S) and [GalNAc(6S)beta1-4GlcAbeta1]n- 3GalNAc(6S), where n = 1-3. The other 4 sequences, DeltaUA1-[3Gal-NAc(6S)beta1-4GlcAbeta1]n-3GalNAc(4S) and [GalNAc(6S)beta1-4GlcAbeta1]n-3GalNAc(4S), where n = 1 and 2, contain the alternative 4-sulfated GalNAc at the reducing terminal. These results establish that oligosaccharides generated by oxymercuration treatment retain their integrity and only lack the terminal DeltaUA residue.