Abstract
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 (FCS(A)), 4-O-sulfated fucose (F(4S)CS(A)), 2, 4-O-disulfated fucose (F(2S4S)CS(A)), and natural-like mixed sulfated fucose (F(NMS)CS(A)), respectively. F(2S4S)CS(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 (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 F(2S4S)CS(A) with defined structure and outstanding anticoagulant activity, thereby providing a promising route for the development of next-generation anticoagulant drugs.