Reaction intensification for biocatalytic production of polyphenolic natural product di‐C‐β‐glucosides

Abstract

AbstractPolyphenolic aglycones featuring two sugars individually attached via C‐glycosidic linkage (di‐C‐glycosides) represent a rare class of plant natural products with unique physicochemical properties and biological activities. Natural scarcity of such di‐C‐glycosides limits their use‐inspired exploration as pharmaceutical ingredients. Here, we show a biocatalytic process technology for reaction‐intensified production of the di‐C‐β‐glucosides of two representative phenol substrates, phloretin (a natural flavonoid) and phenyl‐trihydroxyacetophenone (a phenolic synthon for synthesis), from sucrose. The synthesis proceeds via an iterative two‐fold C‐glycosylation of the respective aglycone, supplied as inclusion complex with 2‐hydroxypropyl β‐cyclodextrin for enhanced water solubility of up to 50 mmol/L, catalyzed by a kumquat di‐C‐glycosyltransferase (di‐CGT), and it uses UDP‐Glc provided in situ from sucrose by a soybean sucrose synthase, with catalytic amounts (≤3 mol%) of UDP added. Time course analysis reveals the second C‐glycosylation as rate‐limiting (0.4–0.5 mmol/L/min) for the di‐C‐glucoside production. With internal supply from sucrose keeping the UDP‐Glc at a constant steady‐state concentration (≥50% of the UDP added) during the reaction, the di‐C‐glycosylation is driven to completion (≥95% yield). Contrary to the mono‐C‐glucoside intermediate which is stable, the di‐C‐glucoside requires the addition of reducing agent (10 mmol/L 2‐mercaptoethanol) to prevent its decomposition during the synthesis. Both di‐C‐glucosides are isolated from the reaction mixtures in excellent purity (≥95%), and their expected structures are confirmed by NMR. Collectively, this study demonstrates efficient glycosyltransferase cascade reaction for flexible use in natural product di‐C‐β‐glucoside synthesis from expedient substrates.