Effect of sequential oxidation-reduction-sulfation on the conformation and anticoagulant properties of ulvan from Ulva fasciata.

Ulvans are sulfated polysaccharides from green macroalgae of the Ulva genus, widely studied for their anticoagulant properties and potential biomedical applications. In this study, oversulfated ulvan derivatives were synthesized using two distinct strategies: (i) direct sulfation of native ulvan and (ii) a sequential process involving periodate oxidation, NaBH₄ reduction, and subsequent sulfation. These routes generated derivatives with distinct degrees of sulfation (DS), molar mass (Mw), and structural features. Structural characterization by NMR spectroscopy confirmed successful chemical modifications, while circular dichroism revealed the loss of the native exciton couplet, suggesting disruption of the ordered helical arrangement upon sulfation and partial depolymerization. Anticoagulant assays (APTT, TT, and PT) demonstrated that activity correlated positively with both DS and Mw, and remained preserved even after partial cleavage of CC bonds in the polysaccharide backbone. All sulfated derivatives exhibited stronger anticoagulant effects than native ulvan. Notably, P2r-S retained thrombin inhibition despite its lower DS (1.4) compared with UF-S (1.8) and P1r-S (1.9), suggesting that factors beyond DS, such as sulfate distribution and Mw, contribute to its activity profile. Overall, the results highlight sulfation as the primary driver of anticoagulant enhancement and demonstrate how combined variations in DS, sulfate patterning, and molar mass shape ulvan bioactivity. These findings advance the understanding of structure-activity relationships in ulvans and support their development as promising marine-derived anticoagulant agents.