Isolation and structure elucidation of a highly haemolytic saponin from the Merck saponin extract using high-field gradient-enhanced NMR techniques

Saponins SAPO50 and SAPO30, of which SAPO50 is highly haemolytic, have been isolated from the commercial Merck Saponin. Their structures have been determined exclusively by high-field gradient-enhanced NMR methods. The 1H and 13C NMR spectra of these saponins in pyridine-deuterium oxide have been assigned by homonuclear and heteronuclear correlation experiments. Anomeric configurations were obtained by combined use of JCH, 3JH-1,H-2, and 1D-NOESY data. Sugar residues were identified by use of 3JHH values obtained from their subspectra recorded using an optimized 1D-z-TOCSY sequence. Linkage assignments were made using the ge-HMBC and 1D-NOESY spectra. This study shows that SAPO50 represents a hitherto undescribed saponin with the following structure: 3-O-β-d-xylopyranosyl-(1 → 3)-[β-d-galactopyranosyl-(1 → 2)]-β-d-glucuronopyranosyl gypsogenin 28-O-(6-deoxy-β-d-glucopyranosyl)-(1 → 4)-[β-d-xylopyranosyl-(1 → 3)-β-d-xylopyranosyl(1 → 4)]-α-l-rhamnopyranosyl-(1 → 2)-β-d-fucopyranoside. SAPO30, however, corresponds to a saponin previously described [D. Frechet, B. Christ, B. Monegier du Sorbier, H. Fischer, and M. Vuilhorgne, Phytochemistry, 30 (1991) 927–931.