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Review
. 2023 Oct 18;71(41):14890-14910.
doi: 10.1021/acs.jafc.3c04389. Epub 2023 Oct 6.

Flavonoids as Aglycones in Retaining Glycosidase-Catalyzed Reactions: Prospects for Green Chemistry

Michael Kotik  1 , Natalia Kulik  1 , Kateřina Valentová  1
Affiliations
Review

Flavonoids as Aglycones in Retaining Glycosidase-Catalyzed Reactions: Prospects for Green Chemistry

Michael Kotik et al. J Agric Food Chem. .

Abstract

Flavonoids and their glycosides are abundant in many plant-based foods. The (de)glycosylation of flavonoids by retaining glycoside hydrolases has recently attracted much interest in basic and applied research, including the possibility of altering the glycosylation pattern of flavonoids. Research in this area is driven by significant differences in physicochemical, organoleptic, and bioactive properties between flavonoid aglycones and their glycosylated counterparts. While many flavonoid glycosides are present in nature at low levels, some occur in substantial quantities, making them readily available low-cost glycosyl donors for transglycosylations. Retaining glycosidases can be used to synthesize natural and novel glycosides, which serve as standards for bioactivity experiments and analyses, using flavonoid glycosides as glycosyl donors. Engineered glycosidases also prove valuable for the synthesis of flavonoid glycosides using chemically synthesized activated glycosyl donors. This review outlines the bioactivities of flavonoids and their glycosides and highlights the applications of retaining glycosidases in the context of flavonoid glycosides, acting as substrates, products, or glycosyl donors in deglycosylation or transglycosylation reactions.

Keywords: Glucosidase; Glycoside hydrolase; Glycosyl donor; Glycosynthase; Hydrolysis; Rutinosidase; Transglycosylation.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Structures of flavonoids discussed in this review. The structures of rutinose (6-O-α-l-rhamnopyranosyl-β-d-glucopyranose) and 2-O-β-d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxaxin-3-one are also depicted. Gall, galloyl; Glc, β-d-glucosyl; GlcA, glucuronosyl; Rha, α-l-rhamnosyl; Rut, rutinosyl; Xyl, β-d-xylosyl.
Figure 2
Figure 2
Nomenclature describing the binding subsites of flavonoid-accepting β-glucosidases and β-rutinosidases. The former enzymes possess one negative subsite, whereas the latter enzymes appear to have two negative subsites, which bind the glycone moieties of, e.g., rutin, with the nonreducing end being positioned at the −2 subsite. Cleavage occurs between subsites −1 and +1, where the catalytic acid/base and nucleophile are located. The aglycone binding site is located at the +1 subsite.
Figure 3
Figure 3
Active site of AnRut with bound rutin. (A) Interactions between hydrophobic (magenta) and aromatic residues (green) in the +1 subsite of AnRut and docked rutin. The aromatic side chains of F221, F261, F284, and F286 clamp the aglycone moiety by π–π stacking interactions shown as yellow dotted lines., (B) Superposition of common residues in the −1 subsites of AnRut (carbon atoms in green, oxygen in red, and nitrogen in blue) and CaExg (magenta), depicting rutin (green-red) modeled into the active site of AnRut, and laminaritriose (β-d-Glc-(1 → 3)-β-d-Glc-(1 → 3)-Glc, magenta) cocrystallized with CaExg (PDBs 3N9K and 1EQC for the ligand and CaExg, respectively). The acid/base catalysts Glu210 and Glu192 are also shown.
Figure 4
Figure 4
Schematic representation of the substrate binding pose in the active site of PD. Areas that hold the substrate with hydrogen bonds are marked in gray. Active site areas with hydrophobic interactions are highlighted in green. The β-1,6-Xyl-β-Glc moiety of the substrate 2-phenylethyl β-primeveroside is recognized by subsites −2 and −1 and fixed by hydrogen bonds and a few hydrophobic interactions. Subsite +1 is spacious and its interactions with the aglycone have been considered far less important for substrate binding. It binds the various apolar aglycones mainly nonspecifically through hydrophobic contacts with hydrophobic residues (green dashed line) and the hydrophobic plane of the β-1,6-Xyl pyranose ring. The catalytic nucleophile (E416) and acid/base (E203) are also shown.
Figure 5
Figure 5
View of the active site of TnBgl1A with docked quercetin 4′-O-glucoside. In the +1 subsite, hydrogen bonds and hydrophobic interactions with aromatic residues (green) were involved in the binding of the substrate; π–π stacking interactions are shown as yellow dotted lines.
Figure 6
Figure 6
Structures of selected transglycosylation products and sugar acceptors. (A) Acceptors used in transglycosylations using flavonoid-based glycosyl donors and retaining glycosidases. (B) Selected products generated in AnRut-mediated transglycosylation reactions with rutin as glycosyl donor and subsequent derhamnosylation using α-l-rhamnosidase.
Figure 7
Figure 7
Active site of αRβG II with bound rutin and hesperidin as good substrates. Bound rutin (A) or hesperidin (B) were modeled into the active site, highlighting the hydrophobic residues in the +1 subsite (magenta) that participated in the binding. The presumable catalytic nucleophile Asp250 and acid/base catalyst Glu472 are also depicted. The structure of αRβG II was obtained by homology modeling using MODELER and the PDB 4I8D. Docking was performed using Autodock4.
Figure 8
Figure 8
Reaction mechanism of exo- and endo-β-glycosynthases; R1 = additional sugar in the case of endo-glycosynthases; R2 = acceptor, such as carbohydrate or alcohol. The enzyme lacks the catalytic nucleophile (Glu or Asp), which is replaced by Ala in this example.
Figure 9
Figure 9
Flavonoid glycoside products obtained in glycosynthase-mediated reactions using the E197S mutant of the Cel7B endocellulase, lactosyl fluoride as diglycosyl donor, and the flavonoid acceptors baicalein, luteolin, and quercetin.
Figure 10
Figure 10
Reaction mechanism of thioglycoligases; R1 = leaving group; −SR2 = thiol as acceptor.
Figure 11
Figure 11
Synthesis of an S-glucuronide using the E396Q mutant of the β-d-glucuronidase DtGlcA from Dictyoglomus thermophilum as a thioglycoligase, 4-chlorothiophenol as acceptor, and baicalin as a natural glucuronide donor.
Figure 12
Figure 12
Proposed reaction mechanism of a GH3-based thioglycoligase (E495A mutant of a β-xylosidase) with the third acid residue in the active site facilitating the attack of the incoming nucleophilic acceptor on the anomeric center. As the sugar donor, pNP β-d-xylopyranose is depicted.
Figure 13
Figure 13
Structures of flavonoid 7-O-α-glucoside products generated in transglycosylation reactions using α-d-glucopyranosyl fluoride as a sugar donor and the O-α-glycoligase MalA-D416A derived from an α-glucosidase of the thermophilic archeon Sulfolobus solfataricus. The left structure represents flavonol, flavanone, flavanonol, and isoflavone glucoside products obtained in these transglycosylations; the right structure represents MalA-D416A-mediated flavanol glucoside products.
Figure 14
Figure 14
Formation of quercetin 3,4′-di-O-β-glucoside by virtue of the glycosynthase activity of TnBgl1A-E349G in the presence of sodium formate as an external nucleophile using oNP β-d-glucopyranoside as the donor and isoquercitrin as acceptor.
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