title: Development of a multifunctional aminoxy-based fluorescent linker for glycan immobilization and analysis.
abstract: Glycan arrays have become a technique of choice to screen glycan-protein interactions in a high-throughput manner with high sensitivity and low sample consumption. Here, the synthesis of a new multifunctional fluorescent linker for glycan labeling via aminoxy ligation and immobilization is described; the linker features a fluorescent naphthalene group suitable for highly sensitive high-performance liquid chromatography-based purification and an azido- or amino-modified pentanoyl moiety for the immobilization onto solid supports. Several glycoconjugates displaying small sugar epitopes via chemical or chemoenzymatic synthesis were covalently attached onto a microarray support and tested with lectins of known carbohydrate binding specificity. The glycan library was extended using glycosyltransferases (e.g. galactosyl-, sialyl- and fucosyltransferases); the resulting neoglycoconjugates, which are easily detected by mass spectrometry, mimic antennal elements of N- and O-glycans, including ABH blood group epitopes and sialylated structures. Furthermore, an example natural plant N-glycan containing core α1,3-fucose and β1,2-xylose was also successfully conjugated to the fluorescent linker, immobilized and probed with lectins as well as antihorseradish peroxidase. These experiments validate our linker as being a potentially valuable tool to study glycozyme and lectin specificities, sensitive enough to allow purification of natural glycans.
authors.affiliation: Department für Chemie, Universität für Bodenkultur, 1190 Wien, Austria.
authors.forename: Carmen
authors.initials: C
authors.lastname: Jiménez-Castells
authors.affiliation: Department für Chemie, Universität für Bodenkultur, 1190 Wien, Austria.
authors.forename: Rhiannon
authors.initials: R
authors.lastname: Stanton
authors.affiliation: Department für Chemie, Universität für Bodenkultur, 1190 Wien, Austria.
authors.forename: Shi
authors.initials: S
authors.lastname: Yan
authors.affiliation: Department für Chemie, Universität für Bodenkultur, 1190 Wien, Austria.
authors.forename: Paul
authors.initials: P
authors.lastname: Kosma
authors.affiliation: Department für Chemie, Universität für Bodenkultur, 1190 Wien, Austria iain.wilson@boku.ac.at.
authors.forename: Iain Bh
authors.initials: IB
authors.lastname: Wilson
mesh_terms: D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D016695:Glycosyltransferases / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis* / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D011134:Polysaccharides; D016695:Glycosyltransferases
keywords: N-glycoside; chemo-enzymatic synthesis; fluorescent labeling; glycan arrays; natural glycans