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

ABSTRACT 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.