Reaction intensification for biocatalytic production of polyphenolic natural product di‐C‐β‐glucosides

Abstract

Polyphenolic aglycones featuring two sugars individually attached via C-glycosidic linkage (di-C-glycosides) represent a rare class of plant natural products with unique physicochemical properties and biological activities. Natural scarcity of such di-C-glycosides limits their use-inspired exploration as pharmaceutical ingredients. Here, we show a biocatalytic process technology for reaction-intensified production of the di-C-β-glucosides of two representative phenol substrates, phloretin (a natural flavonoid) and phenyl-trihydroxyacetophenone (a phenolic synthon for synthesis), from sucrose. The synthesis proceeds via an iterative two-fold C-glycosylation of the respective aglycone, supplied as inclusion complex with 2-hydroxypropyl β-cyclodextrin for enhanced water solubility of up to 50 mmol/L, catalyzed by a kumquat di-C-glycosyltransferase (di-CGT), and it uses UDP-Glc provided in situ from sucrose by a soybean sucrose synthase, with catalytic amounts (≤3 mol%) of UDP added. Time course analysis reveals the second C-glycosylation as rate-limiting (0.4-0.5 mmol/L/min) for the di-C-glucoside production. With internal supply from sucrose keeping the UDP-Glc at a constant steady-state concentration (≥50% of the UDP added) during the reaction, the di-C-glycosylation is driven to completion (≥95% yield). Contrary to the mono-C-glucoside intermediate which is stable, the di-C-glucoside requires the addition of reducing agent (10 mmol/L 2-mercaptoethanol) to prevent its decomposition during the synthesis. Both di-C-glucosides are isolated from the reaction mixtures in excellent purity (≥95%), and their expected structures are confirmed by NMR. Collectively, this study demonstrates efficient glycosyltransferase cascade reaction for flexible use in natural product di-C-β-glucoside synthesis from expedient substrates.

Solvent Engineering for Nonpolar Substrate Glycosylation Catalyzed by the UDP-Glucose-Dependent Glycosyltransferase UGT71E5: Intensification of the Synthesis of 15-Hydroxy Cinmethylin β-d-Glucoside

“…Our earlier studies on GT-catalyzed C -glycosylation of flavonoid acceptors have noted the requirement of substrate solubility enhancement for synthetic reaction at high efficiency. ,– Unlike here, solvent engineering approaches have not been investigated in detail for C -glycosylation. By applying the encapsulation of phloretin (a flavonoid) in HPβCD, enzymatic syntheses of the phloretin 3′-β- d -glucoside (nothofagin) ,, and the phloretin 3′,5′-di-β- d -glucoside were shown at concentrations of up to 50 mM and higher, exceeding the water solubility of the noncomplexed phloretin by about 10 2 -fold. Conversion of the phloretin acceptor was excellent as well (≥90%). ,, Solubilization by inclusion complex formation with HPβCD, clearly, increases the complexity of the overall synthetic procedure compared to the use of a standard organic (co)-solvent, ,, and it would also add to the production costs of a biocatalytic process.…”

“…By applying the encapsulation of phloretin (a flavonoid) in HPβCD, enzymatic syntheses of the phloretin 3′-β- d -glucoside (nothofagin) ,, and the phloretin 3′,5′-di-β- d -glucoside were shown at concentrations of up to 50 mM and higher, exceeding the water solubility of the noncomplexed phloretin by about 10 2 -fold. Conversion of the phloretin acceptor was excellent as well (≥90%). ,, Solubilization by inclusion complex formation with HPβCD, clearly, increases the complexity of the overall synthetic procedure compared to the use of a standard organic (co)-solvent, ,, and it would also add to the production costs of a biocatalytic process. Given the evidence from previous research that (a) HPβCD was well tolerated by different GT enzymes (including the sucrose synthase used here for UDP-glucose regeneration ,, ) and (b) it was removed relatively easily during the product isolation, , we considered the strategy of complexation by HPβCD to be promising for reaction intensification in 15HCM β- d -glucoside synthesis.…”

“…In summary, we have performed a systematic study of 15HCM solubility enhancement for UGT71E5 reaction intensification, with the aim of making 15HCM β- d -glucoside synthesis more efficient. Strategies of solvent engineering for improved solubilization of hydrophobic substrate have not been assessed broadly for biotransformations by enzymes of the Leloir glycosyltransferase class. ,,– Here, approaches based on organic solvents miscible or immiscible with the aqueous buffer failed due to limited enzyme robustness. Inclusion complexation with HPβCD represented a mild, enzyme-compatible approach to 15HCM solubility enhancement.…”

“…36,43,44 Moreover, HPβCD was readily removed during the product isolation. 36,46 We find here that organic (co)solvents were not very compatible with UGT71E5 activity and that enzyme stability was also low under the alternative solvent conditions. Applied as an inclusion complex with 2-hydroxypropyl-βcyclodextrin (HPβCD), 15HCM was dissolved to ∼100 mM in a single aqueous phase, and the UGT71E5 activity was largely unimpaired by the conditions used.…”

“…Last, we use inclusion complexation , in cyclodextrin to enhance solubilization. Earlier works from this laboratory have shown the complexation of phenolic acceptors in 2-hydroxypropyl-β-cyclodextrin (HPβCD) in order to achieve intensification of GT-catalyzed C -glycosylation reactions. ,– HPβCD was revealed to be well tolerated by different C -glycosylating GT enzymes. ,, Moreover, HPβCD was readily removed during the product isolation. , We find here that organic (co)solvents were not very compatible with UGT71E5 activity and that enzyme stability was also low under the alternative solvent conditions. Applied as an inclusion complex with 2-hydroxypropyl-β-cyclodextrin (HPβCD), 15HCM was dissolved to ∼100 mM in a single aqueous phase, and the UGT71E5 activity was largely unimpaired by the conditions used.…”