Leloir Glycosyltransferases and Natural Product Glycosylation: Biocatalytic Synthesis of the <i>C</i>‐Glucoside Nothofagin, a Major Antioxidant of Redbush Herbal Tea

Abstract

Nothofagin is a major antioxidant of redbush herbal tea and represents a class of bioactive flavonoid-like <i>C</i>-glycosidic natural products. We developed an efficient enzymatic synthesis of nothofagin based on a one-pot coupled glycosyltransferase-catalyzed transformation that involves perfectly selective 3'-<i>C</i>-β-d-glucosylation of naturally abundant phloretin and applies sucrose as expedient glucosyl donor. <i>C</i>-Glucosyltransferase from <i>Oryza sativa</i> (rice) was used for phloretin <i>C</i>-glucosylation from uridine 5'-diphosphate (UDP)-glucose, which was supplied continuously <i>in situ</i> through conversion of sucrose and UDP catalyzed by sucrose synthase from <i>Glycine max</i> (soybean). In an evaluation of thermodynamic, kinetic, and stability parameters of the coupled enzymatic reactions, poor water solubility of the phloretin acceptor substrate was revealed as a major bottleneck of conversion efficiency. Using periodic feed of phloretin controlled by reaction progress, nothofagin concentrations (45 mM; 20 g l^-1) were obtained that vastly exceed the phloretin solubility limit (5-10 mM). The intermediate UDP-glucose was produced from catalytic amounts of UDP (1.0 mM) and was thus recycled 45 times in the process. Benchmarked against comparable glycosyltransferase-catalyzed transformations (e.g., on quercetin), the synthesis of nothofagin has achieved intensification in glycosidic product formation by up to three orders of magnitude (μM→mM range). It thus makes a strong case for the application of Leloir glycosyltransferases in biocatalytic syntheses of glycosylated natural products as fine chemicals.