A permissive steroid glycosyltransferase (UGT74AN1) from Asclepias curassavica exhibited robust capabilities for the regiospecific C3 glycosylation of cardiotonic steroids and C steroid precursors, and unprecedented promiscuity toward 53 structurally diverse natural and unnatural compounds to form O-, N-, and S-glycosides, along with the catalytic reversibility for a one-pot transglycosylation reaction. These findings highlight UGT74AN1 as the first regiospecific catalyst for cardiotonic steroid C3 glycosylation and exhibit significant potential for glycosylation of diverse bioactive molecules in drug discovery.

An efficient one‐pot enzymatic synthesis of cardiac glycosides with varied sugar chain lengths (average yield >80%) was carried out through sequential glycosylation using a steroid glycosyltransferase UGT74AN3 from Catharanthus roseus and a cyclodextrin glycosyltransferase from Bacillus licheniformis. A series of cardiac glycosides were obtained and showed enhanced affinity and selectivity for inhibition of the α2 isoform of Na+/K+‐ATPase. These findings demonstrate the significant potential of the one‐pot biocatalytic approach in the synthesis of diverse cardiac glycosides as potentially safer cardiotonic agents.

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Drug discovery process and biological research critically depend on the access to libraries of molecules with interesting biomolecular properties. Thus, it is extremely important to develop robust and efficient strategies to access structurally diverse druglike compound collections. We introduce here a strategy for glycosylation of the total bufadienolides in Venenum Bufonis (VB) by using a permissive glycosyltransferase YjiC1 with conversion rates up to 90%, which was more efficient than other two enzymes. Compared to the crude extract, the glycosylated VB showed lower toxicity against zebrafish and more potent inhibitory activity on Na + ,K + -ATPase. The results demonstrated the great advantages of using permissive enzymes as an alternative strategy for producing structurally diverse natural product-like libraries.

The glycosyltransferase OleD variant as a catalyst for the glycosylation of four pairs of epimers of cardiotonic steroids (CTS) was assessed.

Plant bioactive metabolites such as flavonoids are usually present in glycosylated forms by the attachment of various sugar groups. In this study, a catalytically flexible and reversible glycosyltransferase (HtUGT72AS1) was cloned and characterized from Helleborus thibetanus. HtUGT72AS1 could directly accept six sugar donors (UDP-glucose/-arabinose/-galactose/-xylose/-Nacetylglucosamine/-rhamnose) to catalyze the 3-OH glycosylation of flavonols. It also catalyzed the 4′ and 7-OH glycosylation of other types of flavonoids, which lacked the 3-OH group. Additionally, the HtUGT72AS1-catalyzed reaction was highly reversible when using 2-chloro-4-nitrophenyl glycosides as substrates, which could be used for one-pot or coupled production of bioactive glycosides. It is the first reported UGT for the synthesis of arabinosides and galactosides using a transglycosylation platform. Based on structural modeling and mutagenetic analysis, the mutation of Tyr377 to Ara enhanced the catalytic efficiency of HtUGT72AS1 toward UDP-N-acetylglucosamine, and the V146S mutant gained an improvement in the regioselectivity toward 7-OH of flavonoids.

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