Oligosaccharide-assisted resolution of holothurian fucosylated chondroitin sulfate for fine structure and P-selectin inhibition.

Fucosylated chondroitin sulfate (FCS) from Holothuria mexicana (FCS_Hm) was selected for investigation because of its intriguing branch features. Selective β-eliminative depolymerization and the bottom-up assembly were performed to unravel that FCS_Hm consisted of a {D-GlcA-β1,3-D-GalNAc_4S6S} backbone and branches of alternating Fuc_S (55 %) and D-GalNAc_S-α1,2-L-Fuc_S (45 %), the highest proportion of disaccharide branch reported to date. In branches, sulfation could occur at every free -OH site except O-3 of GalNAc, being the most complex and various structure features of natural FCS. Detailed structure-activity relationship analyses showed that FCS_Hm and its depolymerized products (>8 kDa) effectively competed with SLe^X and PSGL-1 to bind with P-sel at nano-molar level and the inhibition potency increased with Mw increasing. For the structural trisaccharide unit, di-O-sulfation of the Fuc_S (Fuc_2S4S and Fuc_3S4S) was almost 10-fold more potent than mono-O-sulfation (Fuc_4S). Unexpectedly, higher sulfation of the disaccharide-branched tetrasaccharide unit reduced inhibition. The reversal may attribute to fewer interactions with P-sel by molecular docking study. These results suggested that the specific configuration underpinned the potent inhibition, whereas the size and sulfate number of branches were not the key factors for the specific binding. dHmF4 (8.0 kDa) potently blocked the platelet-leukocyte aggregates formation, further verifying the potential value in use.