One-pot fluorescent labeling of xyloglucan oligosaccharides with sulforhodamine

Xyloglucan oligosaccharides fluorescently labeled with sulforhodamine have proved to be a valuable tool in the assessment of transglycosylating activity of plant xyloglucan endotransglucosylase/hydrolase (XTH; EC 2.4.1.207). Here we describe a simple and fast procedure for their preparation. Accordingly, the starting xyloglucan-derived oligosaccharides are in the first step converted to their corresponding 1-amino-1-deoxyalditols (glycamines) by incubation with ammonium acetate and NaCNBH(3) at 80 degrees C for 2-4 h, and in the second step, the glycamines are reacted with Lissamine rhodamine B sulfonyl chloride to obtain fluorescently labeled derivatives of the oligosaccharide glycamines. All operations are carried out in a single centrifuge tube and the products from the individual reaction steps are isolated on the basis of their differential solubility in organic solvents. Using the described protocol, the whole procedure can be accomplished in less than 24 h. The sulforhodamine-labeled xyloglucan oligosaccharides thus obtained proved suitable as substrates for a sensitive fluorescence assay of the transglycosylating activity of XTH.