Abstract:The structure of the carbohydrate moiety of a natural phenolic glycoside can have a significant effect on the molecular interactions and physicochemical and pharmacokinetic properties of the entire compound, which may include anti-inflammatory and anticancer activities. The enzyme 6-O-α-rhamnosyl-β-glucosidase (EC 3.2.1.168) has the capacity to transfer the rutinosyl moiety (6-O-α-L-rhamnopyranosylβ-D-glucopyranose) from 7-O-rutinosylated flavonoids to hydroxylated organic compounds. This transglycosylation re… Show more
“…This is probably the reason why most glycosidases do not glycosylate also phenols being weak acids. Rutinosidases seem to be rather special in their ability to glycosylate phenols …”
Section: Resultsmentioning
confidence: 99%
“…Mixture (20 mg) of 4‐ O ‐rutinosyl ( E ) ‐p ‐coumaric acid ( 3 b , 79%), 1‐ O ‐( E ) ‐p ‐coumaroyl‐β‐rutinose ( 3 a , 10%) was incubated with recombinant α‐ l ‐rhamnosidase from Aspergillus terreus (2.19 U/mL, 30.1 mg/mL) in 4 mL of 50 mM citrate‐phosphate buffer, pH 5, for 4 hours at 37 °C. Then, the reaction was terminated by enzyme denaturation (99 °C, 5 min) and the reaction mixture was analyzed by HPLC and LCMS (Figures S41 and S42, Supporting Information); a sample of the compound 3 e was isolated by preparatory HPLC to obtain clean NMR spectra (Table S13 and Figure S44, Supporting Information), compound 3 f was not isolated due to its paucity.…”
Section: Methodsmentioning
confidence: 99%
“…Rutinosidases seem to be rather special in their ability to glycosylate phenols. [25,26] It appears that for glycosylation of the carboxy group to occur, the complete structure of hydroxycinnamic acid is required. p-Hydroxyphenylacetic acid (8) lacking the conjugation of the carboxy function with the aromatic system yielded only the phenolic rutinoside 8 a.…”
Phenolic glycosides occur naturally in many plants and as such are often present in the human diet. Their isolation from natural sources is usually laborious due to their presence in complex matrices. Their chemical and enzymatic syntheses have been found complex, time-consuming, and costly, yielding only small amounts of glycosylated products. In quest of a convenient biocatalytic route to structurally complex phenolic glycosides, we discovered that the rutinosidase from Aspergillus niger not only efficiently converts hydroxylated aromatic acids (e. g. coumaric and ferulic acids) into the respective phenolic rutinosides, but surprisingly also catalyzes the formation of the respective glycosyl esters. We report here the results of a systematic study presenting the unique synthesis of naturally occurring glycosyl esters and phenolic glycosides accomplished by glycosidase catalysis. A panel of aromatic acids was tested as glycosyl acceptors and the crucial structural features required for the formation of glycosyl esters were identified. In the light of the present structure-activity relationship study, a plausible reaction mechanism was proposed. All the products were fully structurally characterized by NMR and MS. Scheme 2. Glycosylation of (E)-p-coumaric acid (3). Scheme 3. Proposed mechanism of rutinosylation of hydroxycinnamic acids at the carboxy moiety.
“…This is probably the reason why most glycosidases do not glycosylate also phenols being weak acids. Rutinosidases seem to be rather special in their ability to glycosylate phenols …”
Section: Resultsmentioning
confidence: 99%
“…Mixture (20 mg) of 4‐ O ‐rutinosyl ( E ) ‐p ‐coumaric acid ( 3 b , 79%), 1‐ O ‐( E ) ‐p ‐coumaroyl‐β‐rutinose ( 3 a , 10%) was incubated with recombinant α‐ l ‐rhamnosidase from Aspergillus terreus (2.19 U/mL, 30.1 mg/mL) in 4 mL of 50 mM citrate‐phosphate buffer, pH 5, for 4 hours at 37 °C. Then, the reaction was terminated by enzyme denaturation (99 °C, 5 min) and the reaction mixture was analyzed by HPLC and LCMS (Figures S41 and S42, Supporting Information); a sample of the compound 3 e was isolated by preparatory HPLC to obtain clean NMR spectra (Table S13 and Figure S44, Supporting Information), compound 3 f was not isolated due to its paucity.…”
Section: Methodsmentioning
confidence: 99%
“…Rutinosidases seem to be rather special in their ability to glycosylate phenols. [25,26] It appears that for glycosylation of the carboxy group to occur, the complete structure of hydroxycinnamic acid is required. p-Hydroxyphenylacetic acid (8) lacking the conjugation of the carboxy function with the aromatic system yielded only the phenolic rutinoside 8 a.…”
Phenolic glycosides occur naturally in many plants and as such are often present in the human diet. Their isolation from natural sources is usually laborious due to their presence in complex matrices. Their chemical and enzymatic syntheses have been found complex, time-consuming, and costly, yielding only small amounts of glycosylated products. In quest of a convenient biocatalytic route to structurally complex phenolic glycosides, we discovered that the rutinosidase from Aspergillus niger not only efficiently converts hydroxylated aromatic acids (e. g. coumaric and ferulic acids) into the respective phenolic rutinosides, but surprisingly also catalyzes the formation of the respective glycosyl esters. We report here the results of a systematic study presenting the unique synthesis of naturally occurring glycosyl esters and phenolic glycosides accomplished by glycosidase catalysis. A panel of aromatic acids was tested as glycosyl acceptors and the crucial structural features required for the formation of glycosyl esters were identified. In the light of the present structure-activity relationship study, a plausible reaction mechanism was proposed. All the products were fully structurally characterized by NMR and MS. Scheme 2. Glycosylation of (E)-p-coumaric acid (3). Scheme 3. Proposed mechanism of rutinosylation of hydroxycinnamic acids at the carboxy moiety.
“…The hydrolysis products have applications as nutraceuticals and dietary supplements and potentially also in cosmetics (Kiso et al 2015 ; Testai and Calderone 2017 ; Zhang et al 2018 ). The synthetic or transglycosylation activities of these enzymes can be used for the synthesis of many novel rutinosylated or glucosylated compounds, including glucosyl and rutinosyl azide (Katayama et al 2013 ; Šimčíková et al 2015 ; Mazzaferro et al 2019 ; Brodsky et al 2020 ; Karnišová Potocká et al 2021 ; Kotik et al 2021 ). Fig.…”
The glycosidases within GH5-23 cleave the glycosidic bond of β-glucosylated or rutinosylated flavonoids. Moreover, by virtue of their transglycosylation activity, glycoconjugates with glucosyl and rutinosyl moieties are accessible. Here we report the biochemical characterization and biotechnological assessment of two heterologously expressed members of GH5-23—McGlc from Mucor circinelloides and PcGlc from Penicillium chrysogenum. Both enzymes exhibited the highest hydrolytic activities with quercetin-3-β-O-glucopyranoside, whereas lower specificity constants were determined with the rutinosides narcissin, rutin and hesperidin. High stabilities against thermal, ethanol and dimethyl sulfoxide-induced inactivation, a very limited secondary hydrolysis of the formed transglycosylation products, and no detectable product inhibition were additional features appropriate for biotechnological applications. The enzymes were compared in their efficiencies to hydrolyze rutin and to synthesize 2-phenylethyl rutinoside under homogeneous and heterogeneous reaction conditions using high rutin concentrations of 100 and 300 mM. Highest transglycosylation efficiencies were achieved with fully dissolved rutin in reaction mixtures containing 25% dimethyl sulfoxide. Molecular docking and multiple sequence alignments suggest that the hydrophobic environment of aromatic residues within the + 1 subsite of GH5-23 glycosidases is very important for the binding of flavonoid glucosides and rutinosides.
“…Typical cosolvents for transglycosylation reactions include acetonitrile, DMSO, ethers, tertiary alcohols, etc. [22,23,36], and more recently, biomass-derived solvents and ionic liquids [37,38].…”
While testing the ability of cyclodextrin glucanotransferases (CGTases) to glucosylate a series of flavonoids in the presence of organic cosolvents, we found out that this enzyme was able to glycosylate a tertiary alcohol (tert-butyl alcohol). In particular, CGTases from Thermoanaerobacter sp. and Thermoanaerobacterium thermosulfurigenes EM1 gave rise to the appearance of at least two glycosylation products, which were characterized by mass spectrometry (MS) and nuclear magnetic resonance (NMR) as tert-butyl-α-D-glucoside (major product) and tert-butyl-α-D-maltoside (minor product). Using partially hydrolyzed starch as glucose donor, the yield of transglucosylation was approximately 44% (13 g/L of tert-butyl-α-D-glucoside and 4 g/L of tert-butyl-α-D-maltoside). The synthesized tert-butyl-α-D-glucoside exhibited the typical surfactant behavior (critical micellar concentration, 4.0–4.5 mM) and its properties compared well with those of the related octyl-α-D-glucoside. To the best of our knowledge, this is the first description of an enzymatic α-glucosylation of a tertiary alcohol.
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