One‐Pot Hetero‐Di‐C‐Glycosylation of the Natural Polyphenol Phloretin by a Single C‐Glycosyltransferase With Broad Sugar Substrate Specificity

ABSTRACT

The structural motif of hetero‐di‐C‐glycosyl compound is prominent in plant polyphenol natural products and involves two different glycosyl residues (e.g., β‐

‐glucosyl, β‐‐xylosyl) attached to carbons of the same phenolic ring. Polyphenol hetero‐di‐C‐glycosides attract attention as specialized ingredients of herbal medicines and their tailored synthesis by enzymatic C‐glycosylation is promising to overcome limitations of low natural availability and to expand molecular diversity to new‐to‐nature glycoside structures. However, installing these di‐C‐glycoside structures with synthetic precision and efficiency is challenging. Here we have characterized the syntheses of C‐β‐galactosyl‐C‐β‐glucosyl and C‐β‐glucosyl‐C‐β‐xylosyl structures on the phloroglucinol ring of the natural polyphenol phloretin, using kumquat (Fortunella crassifolia) C‐glycosyltransferase (FcCGT). The FcCGT uses uridine 5'‐diphosphate (UDP)‐galactose (5 mU/mg) and UDP‐xylose (0.3 U/mg) at lower activity than UDP‐glucose (3 U/mg). The 3'‐C‐β‐glucoside (nothofagin) is ~10‐fold less reactive than non‐glycosylated phloretin with all UDP‐sugars, suggesting the practical order of hetero‐di‐C‐glycosylation as C‐galactosylation or C‐xylosylation of phloretin followed by C‐glucosylation of the resulting mono‐C‐glycoside. Each C‐glycosylation performed in the presence of twofold excess of UDP‐sugar proceeds to completion and appears to be effectively irreversible, as evidenced by the absence of glycosyl residue exchange at extended reaction times. Synthesis of C‐β‐glucosyl‐C‐β‐xylosyl phloretin is shown at 10 mM concentration in quantitative conversion using cascade reaction of FcCGT and UDP‐xylose synthase, allowing for in situ formation of UDP‐xylose from the more expedient donor substrate UDP‐glucuronic acid. The desired di‐C‐glycoside with Xyl or Gal was obtained as a single product of the synthesis and its structure was confirmed by NMR.