Enzymatic Synthesis of Bufadienolide O‐Glycosides as Potent Antitumor Agents Using a Microbial Glycosyltransferase

Bufadienolides are a class of natural cardiotonic steroids and are well known for their antitumor activities. Their clinical use has been hindered by poor water solubility. Enzymatic glycosylation is a favorable approach to improve the solubility of natural products. In this work, we describe the highly efficient one-step synthesis of bufadienolide O-glucosides and O-galactosides using a microbial glycosyltransferase YjiC1, with conversion rates from 63% to 99%. Altogether 24 glycosides including 17 new compounds were obtained from 14 substrates, and their structures were identified by extensive NMR and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) analyses. These products belong to five types: glycosylation at 3 beta-OH, 7 beta-OH, 3 beta,7 beta-di-OH, 11 alpha-OH, and 12 beta-OH. The C-7 beta or C-11 alpha O-glycosides of bufadienolide are reported for the first time. Moreover, the 3-O-glycosides exhibited significant cytotoxic activities against A549 and MCF7 human cancer cell lines, and showed potent inhibitory activities against Na+/K+-ATPase with IC50 values in the range 0.053-0.76 mu M. In particular, bufalin 3-O-beta-D-glucoside showed enhanced water solubility (25-fold increase from bufalin) and potent antitumor activities (30% inhibition rate, 1.4 mg kg(-1), i.p.) on A549 human lung cancer xenograft Balb/c nude mice model, and could be a promising drug candidate.