- How Site-Directed Mutagenesis Boosted Selectivity of a Promiscuous Enzyme
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β-N-Acetylhexosaminidases (GH20; EC 3.2.1.52) are exo-glycosidases with a dual activity for cleaving both N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) units from glycostructures. This substrate promiscuity is a hurdle in the selective synthesis of N-acetylhexosamine oligosaccharides combining both GlcNAc and GalNAc units since there are hardly any GalNAc transferring enzymes available for synthetic applications. We present here site-directed mutagenesis of a synthetically potent promiscuous β-N-acetylhexosaminidase from Talaromyces flavus (TfHex), which, as a wild type, exhibits a GalNAcase/GlcNAcase ratio of 1.2. On the basis of molecular modeling, we identified crucial amino acid residues responsible for its GalNAcase/GlcNAcase selectivity. Six site-directed mutants were prepared, heterologously expressed in Pichia pastoris, purified, and kinetically characterized. As a result, novel engineered enzymes with an up to 7-times higher selectivity for either GalNAc or GlcNAc substrates were obtained, preserving the favorable properties of the wild type TfHex, mainly its transglycosylation potential and tolerance to functional groups in the substrate molecule. The substrate selectivity and transglycosylation yield were further corroborated by reaction engineering. The new selective and synthetically capable enzymes were applied in the preparation of tailored N-acetylhexosamines. (Figure presented.).
- Nekvasilová, Pavlína,Kulik, Natalia,Rychlá, Nikola,Pelantová, Helena,Petrásková, Lucie,Bosáková, Zuzana,Cva?ka, Josef,Slámová, Kristyna,K?en, Vladimír,Bojarová, Pavla
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- Applications of Shoda's reagent (DMC) and analogues for activation of the anomeric centre of unprotected carbohydrates
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2-Chloro-1,3-dimethylimidazolinium chloride (DMC, herein also referred to as Shoda's reagent) and its derivatives are useful for numerous synthetic transformations in which the anomeric centre of unprotected reducing sugars is selectively activated in aqueous solution. As such unprotected sugars can undergo anomeric substitution with a range of added nucleophiles, providing highly efficient routes to a range of glycosides and glycoconjugates without the need for traditional protecting group manipulations. This mini-review summarizes the development of DMC and some of its derivatives/analogues, and highlights recent applications for protecting group-free synthesis.
- Fairbanks, Antony J.
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- Direct Synthesis of para-Nitrophenyl Glycosides from Reducing Sugars in Water
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Reducing sugars may be directly converted into the corresponding para-nitrophenyl (pNP) glycosides using 2-chloro-1,3-dimethylimidazolinium chloride (DMC), para-nitrophenol, and a suitable base in aqueous solution. The reaction is stereoselective for sugars with either a hydroxyl or an acetamido group at position 2, yielding the 1,2-trans pNP glycosides. A judicious choice of base allows extension to di-and oligosaccharide substrates, including a complex N-glycan oligosaccharide isolated from natural sources, without the requirement of any protecting group manipulations
- Fairbanks, Antony J.,Qiu, Xin
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supporting information
(2020/03/24)
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- Scope of the DMC mediated glycosylation of unprotected sugars with phenols in aqueous solution
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Activation of reducing sugars in aqueous solution using 2-chloro-1,3-dimethylimidazolinium chloride (DMC) and triethylamine in the presence of para-nitrophenol allows direct stereoselective conversion to the corresponding 1,2-Trans para-nitrophenyl glycosides without the need for any protecting groups. The reaction is applicable to sulfated and phosphorylated sugars, but not to ketoses or uronic acids or their derivatives. When applied to other phenols the product yield was found to depend on the pKa of the added phenol, and the process was less widely applicable to 2-Acetamido sugars. For 2-Acetamido substrates an alternative procedure in which the glycosyl oxazoline was pre-formed, the reaction mixture freeze-dried, and the crude product then reacted with an added phenol in a polar aprotic solvent system with microwave irradiation proved to be a useful simplification.
- Fairbanks, Antony J.,Qiu, Xin
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p. 7355 - 7365
(2020/10/13)
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- Enzymatic synthesis of p-nitrophenyl β-chitobioside
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p-Nitrophenyl β-chitobioside (2) was prepared from p-nitrophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside (1) using β-N-acetylhexosaminidase from Aspergillus oryzae. As minor by-product a (1→6)-linked regioisomer (3a) was formed. Addition of (NH4/sub
- Kubisch, Jiri,Weignerova, Lenka,Koetter, Sven,Lindhorst, Thisbe K.,Sedmera, Petr,Kren, Vladimir
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p. 975 - 984
(2007/10/03)
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- N-ACETYLGLUCOSAMINYL DISACCHARIDE AND TRISACCHARIDE FORMATION THROUGH LYSOZYME-CATALYZED TRANSFER REACTION
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A hen egg-white lysozyme produced regioselectively 4-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-D-mannose and p-nitrophenyl 4-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-β-D-mannopyranoside through a transglycosylation reaction from N,N'-diacetylchitobiose and respectively mannose and p-nitrophenyl β-D-mannopyranoside.These enzyme reactions were efficient enough to allow the one-pot preparation of the desired disaccharide.When p-nitrophenyl 2-acetamido-2-deoxy-α-D-glucopyranoside was the acceptor, the enzyme catalyzed the formation of a β-(1-3)-linked disaccharide glycoside (p-nitrophenyl 2-acetamido-2-deoxy-3-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-α-D-glucopyranoside) with its β-(1-4)-linked isomer.This is also the case for the formation of p-nitrophenyl 3-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-α-maltoside with p-nitrophenyl α-maltoside acceptor.The results show that the anomeric configuration of the glycosidic linkage in the glycosyl acceptors had a pronounced effect on the position of transglycosylation.
- Matahira, Yoshiharu,Ohno, Kyoko,Kawaguchi, Mitsuaki,Kawagishi, Hirokazu,Usui, Taichi
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p. 213 - 226
(2007/10/02)
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- Properties and Transglycosylation Reaction of a Chitinase from Nocardia orientalis
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The hydrolytic products of a chitinase purified from Nocardia orientalis were examined on reduced (GlcNAc)n (n=2-6).The rate of hydrolysis on reduced (GlcNAc)4-6 increased with increasing chain-length of N-acetylglucosamine residues,
- Nanjo, Fumio,Sakai, Kazuo,Ishikawa, Masato,Isobe, Kiyoshi,Usui, Taichi
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p. 2189 - 2195
(2007/10/02)
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