Expanding Rutinosidase Versatility: Acylated Quercetin Glucopyranosides as Substrates

Rutinosidase is a diglycosidase that catalyzes the cleavage of rutinose (α‐l‐Rhap‐(1→6)‐β‐d‐Glcp) from rutin or other rutinosides. It is also able to cleave β‐glucopyranosides, e. g., isoquercitrin. This enzyme has a strong transglycosylation activity and a remarkable substrate specificity. We have shown that rutinosidase from Aspergillus niger (AnRut) is able to cleave β‐glucopyranosides acylated at C‐6 of glucose (6′‐O‐acylisoquercitrin) with acetyl, benzoyl, phenylacetyl, phenylpropanoyl, cinnamoyl, vanillyl, galloyl, 4‐hydroxybenzoyl and 3‐(4‐hydroxy‐3‐methoxyphenyl)propanoyl. The release of the respective 6‐acylglucopyranoses was confirmed by HPLC/MS and NMR methods. Selected compounds, i. e., 6′‐O‐acetyl, 6′‐O‐benzoyl, and 6′‐O‐cinnamyl derivatives of isoquercitrin, were also tested as transglycosylation substrates. Only 6′‐acetylisoquercitrin and 6′‐O‐benzoylisoquercitrin underwent transglycosylations by AnRut to produce n‐butyl 6‐acetyl‐β‐d‐glucopyranoside and n‐butyl 6‐benzoyl‐β‐d‐glucopyranoside. Isoquercitrin 6′‐O‐cinnamate yielded on hydrolytic product. Molecular modeling based on the crystal structure of AnRut showed that large aromatic moieties at C‐6′ of isoquercitrin block the side tunnel of AnRut leading into its active site and thus hinder the entry of the acceptor substrate for transglycosylation. This study demonstrates the great substrate flexibility of rutinosidase at the glycone site. Aspergillus niger rutinosidase (AnRut) efficiently cleaved a library of rutin glycomimetics ‐ isoquercitrin acylated at C‐6′ of its glucosyl moiety. The substrates tested included isoquercitrin substituted at glucosyl C‐6′ with acetyl, benzoyl, phenylacetyl, phenylpropanoyl, cinnamyl, vanillyl, galloyl, 4‐hydroxybenzoyl, and 3‐(4‐hydroxy‐3‐O‐methylphenyl)propanoyl. AnRut showed the ability to transglycosylate with the substrates 6′‐O‐acetyl isoquercitrin and 6′‐O‐benzoyl isoquercitrin substrates, affording n‐butyl 6‐acetyl‐β‐d‐glucopyranoside and n‐butyl 6‐benzoyl‐β‐d‐glucopyranoside.