Sulfation on the glucosamine sugar unit in heparan sulfate (HS) is linked to various biological functions, including the anticoagulant activity to treat thrombotic disorders in hospitals. The 3-O-sulfated glucosamine is biosynthesized by heparan sulfate glucosamine 3-sulfotransferases. Because of its biological significance, there is a need for 3-O-sulfated oligosaccharide standards to facilitate the compositional analysis of HS. These oligosaccharides must contain a Δ 4,5 -unsaturated uronic acid (ΔUA) residue at the nonreducing end, which is due to the depolymerization reaction catalyzed by heparin lyases used during the compositional analysis procedure. Here, we describe a protocol for the preparation of one 3-O-sulfated disaccharide (compound 4) and three 3-Osulfated tetrasaccharides (compound 1−3) in a milligram scale. The synthesis of 3-O-sulfated disaccharide and tetrasaccharide standards was completed by degrading synthetic octasaccharides using heparin lyases. Further analysis revealed that 3-O-sulfated oligosaccharide standards are labile under basic conditions, confirming the findings from a previous study. The unwanted degradation was reduced by decreasing the pH in the presence of phosphate buffer. The 3-O-sulfated oligosaccharide standards are reagents to characterize 3-O-sulfation in HS derived from biological sources.

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