Efficient and expedient two-step pyranose-retaining fluorescein conjugation of complex reducing oligosaccharides: galectin oligosaccharide specificity studies in a fluorescence polarization assay.
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.
Oberg
Christopher T
CT
Department of Chemistry, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada.
Carlsson
Susanne
S
Fillion
Eric
E
Leffler
Hakon
H
Nilsson
Ulf J
UJ
D001665:Binding Sites; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry; D005454:Fluorescence Polarization / Q000379:methods*; D037161:Galectins / Q000737:chemistry*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000737:chemistry*; D013194:Staining and Labeling
D002241:Carbohydrates; D037161:Galectins; D009844:Oligosaccharides