The disaccharides formed by deaminative cleavage of N-deacetylated glycosaminoglycans

Abstract

Chondroitin 4-sulphate, chondroitin 6-sulphate, dermatan sulphate and keratan sulphate were N-deacetylated by treatment with hydrazine and then cleaved with HNO2 at pH 4.0, and the resulting products were reduced with NaB3H4. This reaction sequence cleaved the glycosaminoglycans at their N-acetyl-D-glucosamine or N-acetyl-D-galactosamine residues, which were converted into 3H-labelled 2,5-anhydro-D-mannitol (AManR) or 2,5-anhydro-D-talitol (ATalR) residues respectively. The end-labelled disaccharides, composed of D-glucuronic acid (GlcA), L-iduronic acid (IdoA) or D-galactose (Gal) and one of the anhydrohexitols, were identified as follows: both chondroitin 4-sulphate and chondroitin 6-sulphate gave GlcA→ATalR(4-SO4), GlcA→ATalR(6-SO4), IdoA→ATalR (4-SO4) and GlcA(2-SO4)→ATalR(6-SO4); dermatan sulphate gave IdoA→ATalR(4-SO4), GlcA→ATalR(4-SO4), GlcA→ATalR(6-SO4)→IdoA(2-SO4)ATalR(4-SO4) and IdoA→ATalR (4,6-diSO4); keratan sulphate gave Gal(6-SO4)→AManR(6-SO4), Gal→AManR(6-SO4), Gal(6-SO4)→AManR and Gal→AManR. Several additional disaccharides were generated by treatment of the uronic acid-containing disaccharides with hydrazine to epimerize their uronic acid residues at C-5. A number of these disaccharides were found to be substrates for lysosomal sulphatases and glycuronidases. Methods were developed for the separation of all of the disaccharide products by h.p.l.c. The rate of N-deacetylation of chondroitin 4-sulphate by hydrazinolysis was significantly lower than the rate of N-deacetylation of chondroitin 6-sulphate or chondroitin. Dermatan sulphate was N-deacetylated at an intermediate rate. The relative amounts of disaccharides obtained from chondroitin 4-sulphate, chondroitin 6-sulphate and dermatan sulphate under optimum hydrazinolysis/deamination conditions were comparable with the amounts of the corresponding products released from the polymers by chondroitinase treatment.