Enzymatic Synthesisof Biflavonoid Glycosides withEnhanced Antitumor Activity Using Glycosyltransferase and SucroseSynthase

Biflavonoids, a distinctive subclass of plant flavonoids, have a unique dimerized structure and possess a range of biological activities. The clinical applications of biflavonoids in human health have been impeded by challenges related to bioavailability and hydrophilicity. In contrast, biflavonoid glycosides, which demonstrate enhanced pharmacodynamic and pharmacokinetic properties compared to their aglycones, are notably limited in availability. In this work, we developed a robust enzymatic system to biosynthesize biflavonoid glycosides using <i>O</i>-glycosyltransferase UGT74AN2 and sucrose synthase <i>At</i>SuSy. This innovative system exhibited remarkable substrate promiscuity successfully, glycosylating 10 structurally diverse biflavonoids. Through purification and structural characterization, we identified four biflavonoid monoglycosides (<b>1a</b>, <b>2a</b>, <b>4a</b>, and <b>5a</b>) as well as two diglycosides (<b>1b</b> and <b>3b</b>). All synthesized products showed a significant increase in water solubility compared to their aglycones, with enhancements ranging from 20- to 980-fold. Furthermore, compound <b>1a</b> demonstrated significantly enhanced antiproliferative activity against PC-3 cells compared to its corresponding aglycones. Metabolomic and transcriptomic analyses showed that the increased antitumor activity of <b>1a</b> may be attributed to changes in the expression levels of various drug transporters, particularly within the ABC, PDE, and ATPase gene families. While compound <b>1</b> elevated the mRNA levels of several ABC transporters and ATPases, <b>1a</b> did not induce these effects, highlighting a distinct mode of action. This study established an efficient enzymatic approach for the biosynthesis of biflavonoid glycosides and underscored their potential as valuable small molecules for drug discovery.