Synthesis and Characterization of a Novel Resveratrol Xylobioside Obtained Using a Mutagenic Variant of a GH10 Endoxylanase

Abstract

Resveratrol is a natural polyphenol with antioxidant activity and numerous health benefits. However, in vivo application of this compound is still a challenge due to its poor aqueous solubility and rapid metabolism, which leads to an extremely low bioavailability in the target tissues. In this work, rXynSOS-E236G glycosynthase, designed from a GH10 endoxylanase of the fungus Talaromyces amestolkiae, was used to glycosylate resveratrol by using xylobiosyl-fluoride as a sugar donor. The major product from this reaction was identified by NMR as 3-O-ꞵ-d-xylobiosyl resveratrol, together with other glycosides produced in a lower amount as 4'-O-ꞵ-d-xylobiosyl resveratrol and 3-O-ꞵ-d-xylotetraosyl resveratrol. The application of response surface methodology made it possible to optimize the reaction, producing 35% of 3-O-ꞵ-d-xylobiosyl resveratrol. Since other minor glycosides are obtained in addition to this compound, the transformation of the phenolic substrate amounted to 70%. Xylobiosylation decreased the antioxidant capacity of resveratrol by 2.21-fold, but, in return, produced a staggering 4,866-fold improvement in solubility, facilitating the delivery of large amounts of the molecule and its transit to the colon. A preliminary study has also shown that the colonic microbiota is capable of releasing resveratrol from 3-O-ꞵ-d-xylobiosyl resveratrol. These results support the potential of mutagenic variants of glycosyl hydrolases to synthesize highly soluble resveratrol glycosides, which could, in turn, improve the bioavailability and bioactive properties of this polyphenol.

Keywords: antioxidants; colonic fermentation; fungal enzyme; glycoconjugates; glycoside hydrolases; glycosynthase; polyphenols; solubility; xylobiose.

Figure 1

Analysis of the standard resveratrol glycosylation reaction catalyzed by rXynSOS-E236G glycosynthase using X₂F as the donor. (A) Thin layer chromatography (TLC). Lane 1: sample of the glycosylation mixture, containing resveratrol, X₂F, the catalyst and the reaction products. Lane 2: negative control with resveratrol and X₂F and no catalyst. Lane 3: negative control containing only the resveratrol acceptor. Lane 4: negative control consisting of only the X₂F donor. Lane 5: standards’ mixture with xylose, xylobiose, xylotriose and xylotetraose. (B) ESI-MS spectrum (positive mode). The m/z of ions corresponding to the Na⁺ adducts of the resveratrol glycosides, xylobiose and xylotetraose are indicated with blue arrows. Resveratrol was not detected in the positive mode spectrum but was clearly visible in the negative mode spectrum (Figure S1).

Figure 2

HPLC chromatogram (λ = 270 nm) of the standard resveratrol glycosylation reaction catalyzed by rXynSOS-E236G glycosynthase using X₂F as the donor. The resveratrol used as the acceptor eluted at 5.71 min, the major peak (2.38 min) corresponding to a reaction product was labeled as glycoside 1 and the minor one (1.97 min) as glycoside 2. A mixture of potential resveratrol glycosides eluted earlier.

Figure 3

Structures deduced from NMR analysis of the purified glycosides 1 and 2. (A) Glycoside 1 is the major product of the glycosylation reaction and was identified as 3-O-ꞵ-d-xylobiosyl resveratrol. (B,C) Glycoside 2 contains a mixture of minor reaction products resulting from the glycosylation of resveratrol, identified as 4′-O-ꞵ-d-xylobiosyl resveratrol (B) and 3-O-ꞵ-d-xylotetraosyl resveratrol (C) in a 3:1 ratio. Every C atom and associated proton in the molecules are numbered to clarify the identification of the signals (xylobiose C atoms are represented in blue, resveratrol C atoms of 3-O-ꞵ-d-xylobiosyl resveratrol and 3-O-ꞵ-d-xylotetraosyl resveratrol in red and resveratrol C atoms of 4′-O-ꞵ-d-xylobiosyl resveratrol in green) in the NMR spectra (Figures S2 and S3).

Figure 4

3D representation of the optimized reactions for the synthesis of 3-O-ꞵ-d-xylobiosyl resveratrol catalyzed by rXynSOS-E236G glycosynthase. The reaction conditions were predicted by a multiparametric model obtained following a response surface method. (A) Maximum production of 3-O-ꞵ-d-xylobiosyl resveratrol. (B) Maximum conversion (%) of resveratrol to 3-O-ꞵ-d-xylobiosyl resveratrol. The color code represents the production/conversion (%) range from minimum (blue) to maximum (red).

Figure 5

Kinetics of in vitro conversion under anaerobic conditions of 3-O-ꞵ-d-xylobiosyl resveratrol (white dots) into trans-resveratrol (RSV, black dots) by fecal microbiota from two different healthy volunteers.