Identification of O-sulphate substituents on D-glucuronic acid units in heparin-related glycosaminoglycans using novel synthetic disaccharide standards

The two disaccharides, methyl 4-O-(2-O-sulpho-beta-D-glucopyranosyl-uronic acid)-2-deoxy-2-amino-alpha-D-glucopyranoside and methyl 4-O-(3-O-sulpho-beta-D-glucopyranosyluronic acid)-2-deoxy-2-amino-alpha-D-glucopyranoside were prepared by de novo synthesis, and converted to the corresponding 2,5-anhydro-D-[1-H-3]mannitol derivatives by deamination with nitrous acid followed by reduction with (NaBH4)-H-3. The resultant labelled products were used as standards in the identification, by anion-exchange high-performance liquid chromatography (HPLC), of disaccharides generated by HNO2/(NaBH4)-H-3 treatment of heparan sulphate isolated from human brain, The two standards, containing 2-O- and 3-O-sulphated glucuronic acid, respectively, were clearly separated by the HPLC procedure, Comparison with the deamination products derived from heparan sulphate showed that the mono-O-sulphated disaccharide species containing a sulphated glucuronic acid unit co-eluted with the 2-O-sulphated standard, The corresponding component isolated from other heparan sulphate preparations, or from heparin, also eluted at the same position, No disaccharide derived from heparin or heparan sulphate appeared at the elution position of the 3-O-sulphated standard, It is concluded that D-glucuronic acid units in heparin-related glycosaminoglycans may be sulphated at C2, whereas no evidence has been found for sulphation at C3, By contrast, analysis of mono-O-sulphated disaccharides derived from a chemically sulphated, bacterial capsular polysaccharide (generated by Escherichia coli K5) clearly demonstrated the occurrence of O-sulphate groups at C-3 of D-glucuronic acid units.