Abstract
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.