Novel conjugates of N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers tethered with chitooligosaccharidic epitopes of varying lengths were shown to be potent ligands of a model lectin, wheat germ agglutinin (WGA). The azide-functionalized oligosaccharidic epitopes were prepared by the action of Tyr470Asn mutant β-N-acetylhexosaminidase from Talaromyces flavus in a single reaction step and were conjugated to HPMA copolymer precursors in a defined pattern and density through Cu + -catalyzed azide-alkyne cycloaddition. The soluble, biocompatible, and structurally flexible synthetic glycopolymers were studied for their binding to WGA in a competitive enzyme-linked lectin assay (ELLA), and the kinetics of interaction were analyzed by surface plasmon resonance (SPR). To the best of our knowledge, this study presents the first HPMA copolymers derivatized with long oligosaccharides that demonstrate high affinity to a lectin target. The binding affinities in the low nanomolar and subnanomolar ranges place the prepared glycopolymers among the best WGA ligands reported to date. This study demonstrates the targeting potential of these glycopolymers for therapeutically relevant lectins. † Electronic supplementary information (ESI) available: Structural characterization of functionalized chitooligomers 3, 5-9 (NMR data and spectra, MS spectra, HPLC chromatograms); structural identification of chitooligomer standards 26-29; synthesis of polymer precursors 12a and 12b; structural characterization of glycopolymers 13-25 (NMR spectra). See a The molecular weights (M n ) and dispersities (Đ) of the polymers were determined using GPC with MALS and RI detection. b Average number of glycans per polymer chain (glycan content, mol%); n = 1, monovalent standard. c Relative potency, i.e. IC 50 (monovalent standard)/IC 50 (multivalent glycopolymer). d Relative potency per glycan.
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.
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