Hidden O ‐Deglycosylation Activity Triggers O → C Rearrangement for Aryl Di‐ C ‐Glucoside Formation by the C ‐Glycosyltransferase From Fortunella crassifolia

C-Glycosyl flavonoids are structural isomers and metabolically stable analogues of the corresponding flavonoid O-glycosides. O → C rearrangement of O-glycoside substrates offers a promising route to C-glycosyl compounds, but its synthetic exploitation requires efficient (bio)catalytic methods. Here, we uncover activity of the C-glycosyltransferase from Fortunella crassifolia (FcCGT) for uridine 5'-diphosphate (UDP)-dependent deglycosylation of phlorizin (phloretin 2'-β-O-glucoside). This activity is undetectable in the reverse direction (O-glycosylation of phloretin) due to the enzyme's strong preference for C-glycosylation. Phlorizin deglycosylation releases UDP-glucose and phloretin, which are subsequently used by the enzyme in a C-glycosylation step to selectively form nothofagin (phloretin 3'-β-C-glucoside). O → C rearrangement of phlorizin in the presence of additional UDP-glucose affords phloretin 3',5'-di-β-C-glucoside as a single product in quantitative yield at a concentration of up to 25 mM (~15 g/L). Trilobatin (phloretin 4'-β-O-glucoside) is not accepted by FcCGT for O → C rearrangement, but can undergo C-glycosylation to yield a mixed O,C-diglucoside of phloretin. O → C rearrangement provides excellent atom economy for C-glucoside synthesis. Owing to the higher solubility of the O-glycosylated substrate compared to the free aglycone, C-glucoside synthesis via O → C rearrangement eliminates the need for solubilization strategies such as organic cosolvents or inclusion complexation.