IDENTIFICATION OF O-SULFATE 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-ur onic 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-gluc opyranoside were prepared by de novo synthesis, and converted to the c orresponding 2,5-anhydro-D-[1-H-3]mannitol derivatives by deamination with nitrous acid followed by reduction with (NaBH4)-H-3. The resultan t labelled products were used as standards in the identification, by a nion-exchange high-performance liquid chromatography (HPLC), of disacc harides generated by HNO2/(NaBH4)-H-3 treatment of heparan sulphate is olated from human brain, The two standards, containing 2-O- and 3-O-su lphated glucuronic acid, respectively, were clearly separated by the H PLC procedure, Comparison with the deamination products derived from h eparan sulphate showed that the mono-O-sulphated disaccharide species containing a sulphated glucuronic acid unit co-eluted with the 2-O-sul phated standard, The corresponding component isolated from other hepar an sulphate preparations, or from heparin, also eluted at the same pos ition, No disaccharide derived from heparin or heparan sulphate appear ed at the elution position of the 3-O-sulphated standard, It is conclu ded that D-glucuronic acid units in heparin-related glycosaminoglycans may be sulphated at C2, whereas no evidence has been found for sulpha tion at C3, By contrast, analysis of mono-O-sulphated disaccharides de rived 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.