Glycan-decorated HPMA copolymers as high-affinity lectin ligands

Abstract

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.

An Oxygen‐Tolerant PET‐RAFT Polymerization for Screening Structure–Activity Relationships

“…[44,45] In this assay the polymers were incubated at varying concentrations with af ixed concentration of ConAhorseradish peroxidase (ConA-HRP) conjugate,a nd then transferred onto awell plate pre-coated with ac opolymer of acrylic acid and mannose acrylamide.Free ConA-HRP,which was not bound to the functional polymer in the incubation step,w as allowed to adhere to the well, and was then quantified by development of the HRP after washing away any unbound material. [44,45] In this assay the polymers were incubated at varying concentrations with af ixed concentration of ConAhorseradish peroxidase (ConA-HRP) conjugate,a nd then transferred onto awell plate pre-coated with ac opolymer of acrylic acid and mannose acrylamide.Free ConA-HRP,which was not bound to the functional polymer in the incubation step,w as allowed to adhere to the well, and was then quantified by development of the HRP after washing away any unbound material.…”

“…[44,46,47] Our aim in this work was not primarily to find as trongly binding polymer but rather to investigate whether the method could provide asimple way to screen for fine effects of changes to the polymer size and structure on binding efficiency. [44,46,47] Our aim in this work was not primarily to find as trongly binding polymer but rather to investigate whether the method could provide asimple way to screen for fine effects of changes to the polymer size and structure on binding efficiency.…”

“…While the conversion was close to 100 %, to ensure no free mannose from unincorporated NHS acrylate (or pipetting error) remained in the sample,t he polymers were purified prior to use.W es creened the functionalised polymers for their ability to bind ConA using am odified version of an enzyme linked lectin assay reported for wheat germ lectin. [44,45] In this assay the polymers were incubated at varying concentrations with af ixed concentration of ConAhorseradish peroxidase (ConA-HRP) conjugate,a nd then transferred onto awell plate pre-coated with ac opolymer of acrylic acid and mannose acrylamide.Free ConA-HRP,which was not bound to the functional polymer in the incubation step,w as allowed to adhere to the well, and was then quantified by development of the HRP after washing away any unbound material. Figure 4shows the inhibition of ConA resulting from polymer-ConA binding against polymer concentration for the linear, 3-arm star,and 4-arm star series.At low polymer concentration, low binding to ConA was observed, and stronger binding was observed as the concentration increased.…”

“…Zuschriften 1576 www.angewandte.de of IC 50 values for other lectin binding polymers in the literature. [44,46,47] Our aim in this work was not primarily to find as trongly binding polymer but rather to investigate whether the method could provide asimple way to screen for fine effects of changes to the polymer size and structure on binding efficiency. Thes uccess of the approach is evident from the differences between the polymers observed in these data.…”

“…We chose to test the hypothesis that such am ethod would allow screening of bioactivity by screening the effect of polymer structure on binding to am odel lectin ConA. Theb inding of glycopolymers to lectins is wellstudied, [40][41][42][43][44][45][46][47] and data on the affinity of mannose star polymers to ConA exist to validate our findings. [46,47] To this end we prepared libraries of mannose functionalised linear, 3arm and 4-arm star glycopolymers by post-polymerisation modification of clickable polymer scaffolds, [48] and screened them for their binding affinity to ConA.…”