Sulfated fuco-oligosaccharides from Acaudina leucoprocta: Structural characterization and anticoagulant activity

Sulfated fucans from sea cucumbers are potent anticoagulants, but their structural complexity has hindered understanding of their structure-activity relationship at the oligosaccharide level. Herein, we report the preparation, structural characterization, and anticoagulant evaluation of structurally defined sulfated fuco-oligosaccharides from the sea cucumber, Acaudina leucoprocta. Crude polysaccharides were fractionated by anion-exchange chromatography to obtain a fucan sulfate fraction (WS-0). Mild acid hydrolysis of WS-0 generated an oligosaccharide mixture, which was purified by sequential Bio-Gel P-2 and anion-exchange chromatography, yielding a series of oligosaccharide fractions with degrees of polymerization (DP) ranging from 1 to 9. Their structures were elucidated using ESI-MS, MS/MS, and 800 MHz NMR spectroscopy. Two structurally defined fuco-disaccharides were obtained: α-L-Fucp2S4S-(1 → 3)-α/β-L-Fucp (dWS0-4-0.14) and a mixture of α-L-Fucp2S-(1 → 3)-α/β-L-Fucp and α-L-Fucp4S-(1 → 3)-α/β-L-Fucp (dWS0-4-0.05). Among the oligosaccharide fractions, dWS0-3-1 (DP 3-6) exhibited the strongest anticoagulant activity, prolonging APTT to 47.4 ± 4.7 s and TT to 43.2 ± 1.0 s at 400 μg/mL, while showing minimal effect on PT. Comparative analysis revealed that both chain length and sulfation pattern co-determine anticoagulant potency, with 2,4-di-O-sulfation enabling shorter chains to inhibit thrombin. This study provided the first structural map of anticoagulant fuco-oligosaccharides from A. leucoprocta, identifying minimal structural motifs for activity and offering promising leads for developing novel antithrombotic agents.