An Efficient Strategy for the Glycosylation of Total Bufadienolides in Venenum Bufonis

Abstract

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 applicability of high‐speed counter‐current chromatography for preparative separation of biosynthesis products: Glycosylation products as example

“…Finally, the purified ASP and Loki glycosyltransferases were recovered or concentrated with Amicon Ultra-30 K filtration membrane at 3000 rpm for 10 min in an ice-water bath. The final concentrated target protein was stored at −80 • C after purity confirmation by 12% SDS-PAGE [31,33].…”

“…The reaction system of one-pot glycosylation was as follows: MgCl 2 (15 mL, 100 mM), Silybin B (30 mL, 40 mM), 2-chloro-4-nitrophenyl-β-D-glucopyranoside (150 mL, 25 mM), UDP (150 mL, 25 mM), Loki glycosyltransferase (150 mL), ASP glycosyltransferase (150 mL), and 50 mM Tris⋅HCl solution (2.0 L of final volume, two repeated reactions for amount accumulation). The reaction was terminated by 2.0 L of ice methanol for 24 h incubation at 37 • C. The reaction crude product was concentrated by centrifuging at 12,000 rpm for 30 min, and the supernatant was recovered in vacuum to afford crude products for subsequent HSCCC purification (1.5 g) [31,34].…”

“…Importantly, Loki and ASP glycosyltransferases could reversibly convert glycosides to aglycones in the presence of UDP. Thus, the target glycosylation products could be synthesized by developing a regeneration system without the use of expensive UDP-sugars [31,34]. The biosynthesis route for one-pot glycosylation of Silybin B is shown in Figure 1A.…”

“…Glycosylation is increasingly widespread for green and high efficiency. Targeting and water solubility could be enhanced, and side-effects, including toxicity, could be reduced through glycosylation [31][32][33][34][35]. Silybin B, originated from milk thistle (Silybum marianum, Asteraceae) known to the ancient Greeks as a medicinal plant already, is commonly used for treating liver disorders.…”

Highly Selective β-Mannosylations and β-Rhamnosylations Catalyzed by Bis-thiourea

“…Each of these strategies employs donors with specific protecting group schemes requiring multistep syntheses. Although the strongly Lewis acidic or oxidizing reaction conditions required for these couplings are generally compatible with carbohydrate substrates and can therefore be applied to oligosaccharide synthesis, they display poor tolerance toward a wide variety of common functional groups and therefore have limited utility for the glycosylation of complex acceptors. , Finally, no general methods for 1,2- cis glycosylation of less reactive nucleophiles such as neutral phenols have been identified to date. In particular, electron-rich O -aryl glycosides are difficult to access as they are prone to Fries-type rearrangement pathways to afford C -glycosides under Lewis acidic conditions …”