title: A benzoboroxole-based affinity ligand for glycoprotein purification at physiological pH.
abstract: Developing ligands capable of carbohydrate recognition has become increasingly important as the essential roles of glycoproteins and glycolipids in a diverse array of cellular signaling, pathophysiology, and immune response mechanisms are elucidated. Effective ligands for the glycan portion of glycoproteins and glycolipids are needed for pre-enrichment proteomics strategies, as well as for the purification of individual glycoproteins from complex biological milieu encountered both in biochemistry research and bio-pharmaceutical development. In this work, we developed a carbohydrate specific affinity ligand for glycoprotein purification using a one-pot, multi-component synthesis reaction (Ugi synthesis) and an amine-functionalized benzoboroxole moiety immobilized on agarose beads. Benzoboroxoles are unique boronic acid derivatives that have recently been found to bind specifically to the cis-diol groups of carbohydrates at physiological pH, with superior affinity to any other Wulff-type boronic acid. The solid-phase affinity ligand developed herein specifically binds the carbohydrate moiety of the glycoprotein glucose oxidase, as well as a fluorescein isothiocyanate-dextran, as shown through deglycosylation binding studies. Additionally, the ligand is able to purify glucose oxidase from crude Escherichia coli lysate, at physiological pH, equitably to commercially available boronic acid-functionalized agarose beads that required alkaline pH conditions. Thus, this affinity ligand is a marked improvement on current, commercially available boronic acid-based glycoprotein enrichment matrices and has the potential to exhibit high individual glycoprotein specificity because of the additional functional groups available for variation on the Ugi scaffold.
authors.affiliation: Institute of Biotechnology, Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, CB2 1QT, UK.|Department of Chemistry, Valparaiso University, Valparaiso, IN, 46383, USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Rowe
authors.affiliation: Institute of Biotechnology, Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, CB2 1QT, UK.
authors.forename: Graziella
authors.initials: G
authors.lastname: El Khoury
authors.affiliation: Institute of Biotechnology, Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, CB2 1QT, UK.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Lowe
mesh_terms: D001426:Bacterial Proteins / Q000302:isolation & purification; D001897:Boronic Acids / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D005949:Glucose Oxidase / Q000302:isolation & purification; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; D001897:Boronic Acids; D006023:Glycoproteins; D008024:Ligands; D005949:Glucose Oxidase
keywords: Ugi reaction; benzoboroxole; boronic acid; carbohydrate affinity ligand; carbohydrate receptor; glycoprotein ligand