Characterization of uniformly and atom-specifically (13)C-labeled heparin and heparan sulfate polysaccharide precursors using (13)C NMR spectroscopy and ESI mass spectrometry.

The biological actions of heparin and heparan sulfate, two structurally related glycosaminoglycans, depend on the organization of the complex heparanome. Due to the structural complexity of the heparanome, the sequence of variably sulfonated uronic acid and glucosamine residues is usually characterized by the analysis of smaller oligosaccharide and disaccharide fragments. Even characterization of smaller heparin and heparan sulfate oligosaccharide or disaccharide fragments using simple 1D (1)H NMR spectroscopy is often complicated by the extensive signal overlap. (13)C NMR signals, on the other hand, overlap less and therefore, (13)C NMR spectroscopy can greatly facilitate the structural elucidation of the complex heparanome and provide finer insights into the structural basis for biological functions. This is the first report of the preparation of anomeric carbon-specific (13)C-labeled heparin and heparan sulfate precursors from the Escherichia coli K5 strain. Uniformly (13)C- and (15)N-labeled precursors were also produced and characterized by (13)C NMR spectroscopy. Mass spectrometric analysis of enzymatically fragmented disaccharides revealed that anomeric carbon-specific labeling efforts resulted in a minor loss/scrambling of (13)C in the precursor backbone, whereas uniform labeling efforts resulted in greater than 95% (13)C isotope enrichment in the precursor backbone. These labeled precursors provided high-resolution NMR signals with great sensitivity and set the stage for studying the heparanome-proteome interactions.