Combinatorial Biosynthesis of Antithrombotic Flavonoid 6-O-Glycosides From Carthamus tinctorius

Flavonoid 6-O-glycosides from Carthamus tinctorius (safflower) represent a unique class of potential antithrombotic agents. However, their pharmaceutical development has been hindered by limited natural resources. In this study, we characterized four key biosynthetic enzymes from C. tinctorius: Ct6OGT (6-O-glycosyltransferase), Ct7OGT (7-O-glycosyltransferase), Ct3OGT (3-O-glycosyltransferase), and CtFLS (flavonol synthase). Among them, Ct6OGT is the first identified high-regioselectivity flavonoid 6-O-glycosyltransferase. By combining these enzymes with upstream genes identified from safflower, we established an in vitro enzymatic approach for the synthesis of 6-hydroxykaempferol 3,6,7-tri-O-glycosides. Notably, enzymatic assays demonstrated that the 3,6,7-O-triglycosides were synthesized via a sequential linear glycosylation pathway, in the order of 6-O-, 7-O-, and finally 3-O-glycosylation. Through combinatorial biosynthesis, we further produced 26 flavonoid 6-O-glycosides, including naturally occurring compounds from C. tinctorius and novel analogs. Subsequent evaluation in a zebrafish model revealed these flavonoid 6-O-glycosides exhibited antithrombotic activities, with 6-hydroxylation and 6-O-glycosylation remarkably enhancing the potency. This work represents the first report of an enzymatic synthetic method for flavonoid 6-O-glycosides, and provides a valuable foundation for their future drug development.