title: Synthesis of amine-functionalized heparin oligosaccharides for the investigation of carbohydrate-protein interactions in microtiter plates.
abstract: The synthesis of well-defined oligosaccharides is crucial for the establishment of structure-activity relationships for specific sequences of heparin, contributing to the understanding of the biological role of this polysaccharide. It is highly convenient that the synthetic oligosaccharides contain an orthogonal functional group that allows selective conjugation of the probes and expands their use as chemical tools in glycobiology. We present here the synthesis of a series of amine-functionalized heparin oligosaccharides using an n+2 modular approach. The conditions of the glycosylation reactions were carefully optimized to produce efficiently the desired synthetic intermediates with an N-benzyloxycarbonyl-protected aminoethyl spacer at the reducing end. The use of microwave heating greatly facilitates O- and N-sulfation steps, avoiding experimental problems associated with these reactions. The synthesized oligosaccharides were immobilized in 384-well microtiter plates and successfully probed with a heparin-binding protein, the basic fibroblast growth factor FGF-2. The use of hexadecyltrimethylammonium bromide minimized the amount of sugar required for attachment to the solid support. Using this approach we quantified heparin-protein interactions, and surface dissociation constants for the synthetic heparin derivatives were determined.
authors.affiliation: Glycosystems Laboratory, Instituto de Investigaciones Químicas, Centro de Investigaciones Científicas Isla de La Cartuja, CSIC and Universidad de Sevilla, Américo Vespucio, 49, 41092 Sevilla, Spain.
authors.forename: Susana
authors.initials: S
authors.lastname: Maza
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Macchione
authors.forename: Rafael
authors.initials: R
authors.lastname: Ojeda
authors.forename: Javier
authors.initials: J
authors.lastname: López-Prados
authors.forename: Jesús
authors.initials: J
authors.lastname: Angulo
authors.forename: José L
authors.initials: JL
authors.lastname: de Paz
authors.forename: Pedro M
authors.initials: PM
authors.lastname: Nieto
mesh_terms: D000588:Amines / Q000737:chemistry; D060326:Chemistry Techniques, Synthetic; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D006031:Glycosylation; D006493:Heparin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism*; D006801:Humans; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding
chemical_list: D000588:Amines; D009844:Oligosaccharides; D016222:Fibroblast Growth Factor 2; D006493:Heparin