Nitrous acid deaminative cleavage of heparins using reductive amination tagging by sulfanilic acid

Heparins and low-molecular-weight heparins are widely used anticoagulants whose structural complexity challenges complete characterization. Chromatographic building block analyses and NMR are key techniques. Depolymerization is typically performed by heparinase digestion, though deaminative cleavage with nitrous acid remains essential for preserving the C5 configuration of uronic acids. We revisited nitrous acid depolymerization and addressed detection limitations by implementing sulfanilic acid tagging via reductive amination. This enabled UV-based detection and quantification of disaccharides and acetylated tetrasaccharides in a streamlined 1.5-day workflow. Optimizing this tagging strategy allowed clear identification of major building blocks released by nitrous acid cleavage. Comparative analysis of porcine mucosal, ovine mucosal, bovine mucosal, and bovine lung heparins revealed distinct profiles, especially in acetylated fractions. Sulfated acetylated disaccharides were predominantly iduronic, while the non-sulfated acetylated disaccharide IVa displayed a mixed iduronic/glucuronic configuration across all four heparin species. IVa units were virtually absent from internal chain regions and instead detected as glycoserine-linked sequences at the non-reducing end of the linkage region. A global balance of acetylated structures, with and without glycoserine influence, was established and compared to the enzymatic building block method, used as a quantification reference. Nitrous acid depolymerization remains a key method for heparin analysis. The updated conditions improve sensitivity and chromatographic resolution, enhancing analytical potential. While the protocol remains relatively complex, the results, particularly for the acetylated fraction, offer valuable biosynthetic insights.