Structural elucidation of an amylopectin from Astragalus membranaceus by integrating selective and quantitative NMR...

Elucidating homopolysaccharide structures presents significant challenges due to pronounced NMR signal overlap. In this study, we elucidated the structure of a neutral homopolysaccharide APS-N, purified from Astragalus membranaceus by integrating high-resolution, highly selective, and quantitative NMR techniques with empirical database matching. First, eight well-resolved H1/H2 cross-peaks in the DQF-COSY spectrum were assigned to define monosaccharide residues A-H. Complete sets of ¹H and ¹³C chemical shifts for each monosaccharide spin system were obtained from TOCSY and 2D HSQC-TOCSY experiments, with proton assignments further verified by 1D DREAMTIME TOCSY and paired with their carbon counterparts in HSQC spectrum. An in-house R script was then developed to identify residues A-H by systematically comparing their ¹³C/¹H chemical shift pairs against an empirical database of 1929 fully assigned sugar residues from literatures. It revealed that APS-N consisted exclusively of glucose residues with diverse glycosidic linkage patterns. The ¹H and ¹³C assignments were further validated by intra-residue HMBC correlations. Subsequently, the glycosidic linkages were determined by mapping the assignments of the inter-residue cross-peaks in HMBC, ROESY and 2D HSQC-ROESY spectra. Finally, integration with ¹H qNMR-derived stoichiometric ratios enabled complete structural elucidation of APS-N. In summary, the proposed workflow provides an effective alternative for homopolysaccharide structural characterization.