Enzymatic Synthesis and Anticoagulant Evaluation of Fucosylated Chondroitin Sulfate Polysaccharides with Defined Sulfation Patterns.

Fucosylated chondroitin sulfate (FCS), a uniquely structured glycosaminoglycan with outstanding anticoagulant activity and a lower risk of bleeding, is a promising anticoagulant candidate. However, the complex and heterogeneous structure of the natural FCS polysaccharide presents significant challenges in developing novel anticoagulant drugs and elucidating the structure-activity relationship. Herein, we developed an enzymatic system for the synthesis of FCS polysaccharides with defined sulfation and fucosylation patterns using bifunctional L-fucokinase-GDP-L-fucose pyrophosphorylase (BfFKP) and α1-3-fucosyltransferases mutant (Hpα1, 3-FucTM) and successfully synthesized FCS polysaccharides with α1-3-linked fucose (FCSA), 4-O-sulfated fucose (F4SCSA), 2, 4-O-disulfated fucose (F2S4SCSA), and natural-like mixed sulfated fucose (FNMSCSA), respectively. F2S4SCSA polysaccharide displayed excellent intrinsic anticoagulant activity, which is significantly stronger than clinical anticoagulant low molecular weight heparin and nature-like FCS. Additionally, we also elucidated the precise anticoagulant mechanism of FCS that its sulfation patterns play a crucial role in the anticoagulant activity. Specifically, FCS selectively binds to the positively charged arginine and lysine on the surface of the intrinsic factor IXa factor (K D(F2S4SCSA) = 1.05 × 10-8) predominantly by the negative-charged sulfate group of fucose on its side chain. This one-pot enzymatic cascade approach enables the high-yield synthesis of F2S4SCSA with defined structure and outstanding anticoagulant activity, thereby providing a promising route for the development of next-generation anticoagulant drugs.