Efficient and expedient two-step pyranose-retaining fluorescein conjugation of complex reducing oligosaccharides: galectin oligosaccharide specificity studies in a fluorescence polarization assay
| Autor: | Christopher T. Öberg, Hakon Leffler, Eric Fillion, Susanne Carlsson, Ulf J. Nilsson |
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| Rok vydání: | 2003 |
| Předmět: |
Stereochemistry
Galectins Molecular Sequence Data Biomedical Engineering Carbohydrates Pharmaceutical Science Oligosaccharides Bioengineering Fluorescence Polarization Acylation Galectin Pharmacology chemistry.chemical_classification Binding Sites Molecular Structure Staining and Labeling Ligand binding assay Organic Chemistry Oligosaccharide Fluorescence chemistry Pyranose Carbohydrate Sequence Fluorescence anisotropy Biotechnology Conjugate |
| Zdroj: | Bioconjugate chemistry. 14(6) |
| ISSN: | 1043-1802 |
| Popis: | Fluorescence labeling of naturally occurring saccharides provides a tool for studying lectins. A practical and efficient two-step protocol for fluorescence labeling of reducing sugars without disrupting their pyranose structure has been developed, consisting of generation of the amino sugar using NH(4)HCO(3)(s)/NH(3)(aq, concentrated) followed by BOP-mediated acylation with derivatives of 5- or 6-carboxyfluorescein. The acylated conjugates were subsequently run against galectins-1, -3, and -8, beta-galactoside recognizing lectins of current interest, in a fluorescence polarization binding assay. Upon analyzing a collection of isomerically pure 5- and 6-carboxyfluorescein derivatives with different tether lengths, we found that conjugates based on 5-carboxyfluorescein gave significantly better results than the ones based on 6-carboxyfluorescein and that galectins-1 and -8 favored conjugates with different tether lengths than did galectin-3. The results show that fluorescence labeling can be chemically tuned to find optimal probes for individual galectins but also probes interacting well with many galectins. |
| Databáze: | OpenAIRE |
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