Enzymatic Synthesis of Phloretin α‐Glucosides Using a Sucrose Phosphorylase Mutant and its Effect on Solubility, Antioxidant Properties and Skin Absorption

Abstract

Glycosylation of polyphenols may increase their aqueous solubility, stability, bioavailability and pharmacological activity. Herein, we used a mutant of sucrose phosphorylase from Thermoanaerobacterium thermosaccharolyticum engineered to accept large polyphenols (variant TtSPP_R134A) to produce phloretin glucosides. The reaction was performed using 10% (v/v) acetone as cosolvent. The selective formation of a monoglucoside or a diglucoside (53% and 73% maximum conversion percentage, respectively) can be kinetically controlled. MS and 2D‐NMR determined that the monoglucoside was phloretin 4’‐O‐α‐D‐glucopyranoside and the diglucoside phloretin‐4’‐O‐[α‐D‐glucopyranosyl‐(1→3)‐O‐α‐D‐glucopyranoside], a novel compound. The molecular features that determine the specificity of this enzyme for 4’‐OH phenolic group were analysed by induced‐fit docking analysis of each putative derivative, using the crystal structure of TtSPP and changing the mutated residue. The mono‐ and diglucoside were, respectively, 71‐ and 1200‐fold more soluble in water than phloretin at room temperature. The α‐glucosylation decreased the antioxidant capacity of phloretin, measured by DPPH and ABTS assays; however, this loss was moderate and the activity could be recovered upon deglycosylation in vivo. Since phloretin attracts a great interest in dermocosmetic applications, we analyzed the percutaneous absorption of glucosides and the aglycon employing a pig skin model. Although the three compounds were detected in all skin layers (except the fluid receptor), the diglucoside was present mainly on superficial layers.

Glycosylation of Epigallocatechin Gallate by Engineered Glycoside Hydrolases from Talaromyces amestolkiae: Potential Antiproliferative and Neuroprotective Effect of These Molecules

“…Therefore, it is expected that the glycosylation of these compounds causes a worsening of their antioxidant capacity, since some of the OH of the aglycon have disappeared when the glycosidic bonds are formed [28,29]. This activity loss was also reported in the glycosides of other phenolic compounds such as phloretin [30], resveratrol [31], and gallic acid [32], as well as EGCG [14]. In this sense, using the same method of radical scavenging capacity with ABTS •+ , it has been reported that EGCG 3′-O-α-D-glucopyranoside and EGCG 7-O-α-D-glucopyranoside experimented a similar reduction of antioxidant activity The use of this multiparametric approach was successful since EGCG conversion for all novel glycosides increased approximately twofold with respect to previous nonoptimized conditions (48.8% to 90% for EGCG using BGL-1-E521G and 30% to 60% with BxTW1-E495A) [18,19].…”

Enzymatic bioconversion of beechwood xylan into the antioxidant 2′-O-α-(4-O-methyl-D-glucuronosyl)-xylobiose

“…In the present work, we have analyzed the antioxidant activity of acidic XOS containing majorly one single product (X2_MeGlcA). The selected method was the ABTS •+ radical discoloration assay adapted to 96-well plates [55]. Results were compared with those obtained with beechwood xylan and a commercially available mixture of neutral XOS.…”

A Fungal Versatile GH10 Endoxylanase and Its Glycosynthase Variant: Synthesis of Xylooligosaccharides and Glycosides of Bioactive Phenolic Compounds

“…In this sense, previous studies have described the glucosylation of some of the phenolic compounds tested in this work and its positive effect on their properties. For instance, the glucoside of pterostilbene was proven to improve the solubility of the original aglycone and to reduce its toxicity for several human cell lines [49], while the glucosylation of phloretin also led to a higher solubility and a lower skin penetrability, which could favor a prolonged protection of the external skin layers by cosmetic preparations [55]. Nevertheless, very few cellobiosides of phenolic compounds obtained through enzymatic synthesis can be found.…”

“…The transglycosylation capability of rXynSOS and its glycosynthase variants was evaluated by setting up reactions with different donors and acceptors. The list of acceptors studied included pNPX 2 , vanillyl alcohol, 2-hydroxybenzyl alcohol, hydroquinone, gallic acid, and rosmarinic acid, which were purchased from Sigma-Aldrich, and epigallocatechin gallate (EGCG) [73], phloretin [55], and pterostilbene [49], which were provided by Prof. Francisco J. Plou (ICP-CSIC). For the native rXynSOS, 0.1% (w/v) pNPX 2 was used as xylobiose donor together with 4.6 mg/L of the enzyme and 20 mM of the acceptors.…”