Synthesis of Derivatized Chitooligomers using Transglycosidases Engineered from the Fungal GH20 β‐N‐Acetylhexosaminidase

Abstract

Thes ynthesis of oligosaccharidesu sing mutant glycosidases hasb een dynamically developing due to the need for novelc arbohydrate-based materials.C hitooligomers (b-1!4-linkedo ligomers of N-acetylglucosamine) are bioactive compounds applicablei nm any industrial andp harmacological areas;h owever, their accessibility is still ratherl ow. In this work, GH20 b-N-acetylhexosaminidase from the fungus Talaromyces flavus wase ngineered by site-directed mutagenesis to obtain three efficiently transglycosylating variants with ca. 200-timess uppressedh ydrolytic activity.T hus,w eh ave prepared the first GH20 transglycosidases.Inthe reactions cat-alyzed by these mutant b-N-acetylhexosaminidases we were able to easily prepare andi solate bothn atural and modified chitooligomers in sufficient amountsf or their complete spectral characterization and possible further application. Thep resented method for the synthesis of chitooligomers with aglycones suitablef or linkingt oo ther biological structures is simple and robust enough to be easily scaled up.

Glycan-decorated HPMA copolymers as high-affinity lectin ligands

“…For the preparation of 2-azidoethyl-derivatized chitooligosaccharides containing 2-6 GlcNAc moieties (5-9), we employed a one-step transglycosylation reaction catalyzed by the Tyr470Asn mutant of the β-N-acetylhexosaminidase from Talaromyces flavus, recently developed in our laboratory. 16 This mutant enzyme is the first reported transglycosidase from family 20 of glycoside hydrolases and is one of very few mutant glycosidases employing a substrate-assisted catalytic mechanism. 26 By substituting the active-site Tyr470 with asparagine, the natural hydrolytic activity was practically abolished; the enzyme almost exclusively functioned in the transglycosylation mode, producing β-1,4-linked oligosaccharides of up to six GlcNAc units with 100% selectivity using the readily available p-nitrophenyl 2-acetamido-2-deoxy β-D-glucopyranoside donor (4) (Scheme 1).…”

“…The title mutant enzyme was essentially prepared as described in our recent study. 16 In short, it was prepared by site-directed mutagenesis, extracellularly expressed in Pichia pastoris under induction by methanol, and purified in a single-step by cation-exchange chromatography. The yield of the production was ca.…”

“…The data acquired from both the ELLA and SPR assays clearly demonstrate that the degree of derivatization of the polymer chain with glycans as well as the type of spacer (its chemical character, length, and flexibility) decidedly influence the binding properties of the glycopolymers to WGA. The pronounced impact of the glycan content on the binding affinity is visible in glycopolymers tethered with single GlcNAc units (especially in the series [16][17][18], probably due to the limited flexibility and adaptability of the displayed monosaccharides to the lectin structural requirements. In the oligosaccharidedecorated glycopolymers, the contribution of the increased density of the presented glycans to the binding improvement reaches scarcely one order of magnitude.…”

“…This study describes the synthesis of thirteen HPMA copolymers decorated with chitooligosaccharide epitopes of varying lengths and their capacity to interact with WGA as a model lectin. Azide-functionalized chitooligomers of one to five GlcNAc units were prepared in a single transglycosylation reaction catalyzed by the Tyr470Asn mutant of β-N-acetylhexosaminidase from Talaromyces flavus; 16 they were conjugated in a defined density to propargyl-functionalized HPMA copolymers by the CuAAC reaction. The glycopolymers exhibited extremely strong binding to WGA, as shown in the competitive ELLA assays and in SPR studies; the affinities of the best conjugates were in the subnanomolar range, which ranks them among the strongest ligands reported to date.…”

Two‐Step Enzymatic Synthesis of β‐d‐N‐Acetylgalactosamine‐(1→4)‐d‐N‐acetylglucosamine (LacdiNAc) Chitooligomers for Deciphering Galectin Binding Behavior

“…The Tyr470 Asn variant of the β‐ N ‐acetylhexosaminidase from Talaromyces flavus ( Tf HexY470N) was produced and assayed as described previously . Human β1,4‐galactosyltransferase (His 6 ‐propeptide‐catβ4GalT‐1, β4GalT) and β4GalT variant β4GalTY284L (His 6 ‐propeptide‐catβ4GalT‐1Y284L) were produced in recombinant E. coli strains and purified by affinity chromatography as reported previously …”