Heparan sulfate is a sulfated polysaccharide that displays essential physiological functions. Here, we report a LC-MS/MS-based method for quantitatively determining the individual disaccharide composition and total amount of heparan sulfate. Using eight 13 C-labeled disaccharide calibrants and one 13 C-labeled polysaccharide calibrant, we complete the analysis in one-pot process. The method is both sensitive and has the throughput to analyze heparan sulfate from mouse tissues and plasma.

The complex sulfation motifs of heparan sulfate glycosaminoglycans (HS GAGs) play critical roles in many important biological processes. However, an understanding of their specific functions has been hampered by an inability to synthesize large numbers of diverse, yet defined, HS structures. Herein, we describe a new approach to access the four core disaccharides required for HS/heparin oligosaccharide assembly from natural polysaccharides. The use of disaccharides rather than monosaccharides as minimal precursors greatly accelerates the synthesis of HS GAGs, providing key disaccharide and tetrasaccharide intermediates in about half the number of steps compared to traditional strategies. Rapid access to such versatile intermediates will enable the generation of comprehensive libraries of sulfated oligosaccharides for unlocking the “sulfation code” and understanding the roles of specific GAG structures in physiology and disease.

The 3-O-sulfated glucosamine in heparan sulfate (HS) is a low-abundance structural component, but it is a key saccharide unit for the biological activities of HS. A method to determine the level of 3-O-sulfated HS is lacking. Here, we describe a LC–MS/MS based method to analyze the structural motifs. We determined the levels of 3-O-sulfated structural motifs from pharmaceutical heparin manufactured from bovine, porcine, and ovine. We discovered that saccharide chains carrying 3-O-sulfation from enoxaparin, an FDA-approved low-molecular weight heparin, displayed a slower clearance rate than non-3-O-sulfated sugar chains in a mouse model. Lastly, we detected the 3-O-sulfated HS from human brain. Furthermore, we found that a specific 3-O-sulfated structural motif, tetra-1, is elevated in the brain HS from Alzheimer’s disease patients (n = 5, p = 0.0020). Our method offers a practical solution to measure 3-O-sulfated HS from biological sources with the sensitivity and quantitative capability.

Chondroitin sulfate (CS) is as ulfated polysaccharide that playsessential physiological roles.Here,wereport an enzyme-based method for the synthesis of al ibrary of 15 different CS oligosaccharides.This library covers 4-O-sulfated and 6-O-sulfated oligosaccharides ranging from trisaccharides to nonasaccharides.Wealso describe the synthesis of unnatural 6-O-sulfated CS pentasaccharides containing either a6 -Osulfo-2-azidogalactosamine or a6 -O-sulfogalactosamine residue.T he availability of structurally defined CS oligosaccharides offers an ovel approach to investigate the biological functions of CS.

Heparan sulfate (HS) is a highly sulfated polysaccharide playing essential physiological and pathophysiological roles in the animal kingdom. Heparin, a highly sulfated form of HS, is a widely used anticoagulant...