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-d-glucosyl, β-d-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.