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 (<i>Bf</i>FKP) and α1-3-fucosyltransferases mutant (<i>Hp</i>α1, 3-FucTM) and successfully synthesized FCS polysaccharides with α1-3-linked fucose (FCS_A), 4-O-sulfated fucose (F_4SCS_A), 2, 4-O-disulfated fucose (F_2S4SCS_A), and natural-like mixed sulfated fucose (F_NMSCS_A), respectively. F_2S4SCS_A 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 (<i>K</i> _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 F_2S4SCS_A with defined structure and outstanding anticoagulant activity, thereby providing a promising route for the development of next-generation anticoagulant drugs.