β-Cyclodextrin Improves Solubility and Enzymatic C-Glucosylation of the Flavonoid Phloretin

Nothofagin is a prominent bioactive ingredient of rooibos tea. We recently reported its synthesis through a glucosyltransferase cascade reaction involving 3'-C-beta-D-glucosylation of the dihydrochalcone phloretin from uridine 5'-diphosphate (UDP)-glucose and in situ formation of UDP-glucose from sucrose and catalytic amounts of UDP. Here we show that the limitation in process efficiency caused by the vanishingly low water solubility of phloretin - a major problem for biocatalytic modifications of hydrophobic natural products in general - was overcome effectively using phloretin inclusion complexation with beta-cyclodextrin. Unlike operating in a two-phase system containing uncomplexed insoluble phloretin or using organic cosolvents, the addition of beta-cyclodextrin inclusion complexes was well tolerated regarding enzyme activity and stability. Besides enhancing the effective phloretin concentration in water (similar to 0.2 mM) to about 50 mm, inclusion complexation offered the additional advantage of overcoming the complex inhibition/inactivation effect of the free/microaggregated dihydrochalcone acceptor. Thus oversaturated phloretin solution was transformed in a single batch reaction in excellent conversion (99% in solution; 88% overall) and isolated yield (78%; 17.0 g L-1). The UDPglucose was regenerated up to similar to 90 times and the nothofagin space-time yield of 2.4 mM h(-1) presented an eight-fold improvement compared to a reference reaction using 20% DMSO (dimethyl sulfoxide) and requiring controlled phloretin feed. We thus demonstrate the high potential of inclusion complexation by cyclodextrins for boosting the glycosylation of hydrophobic flavonoid-like natural products.