Linkage position analysis of pyridylamino-disaccharides by HPLC of fluorogenic Smith degradation products.

Oligosaccharides are often converted to fluorogenic pyridylamino-oligosaccharides (PA-oligosaccharides) to be analyzed sensitively. A method for determining the glycosidic linkage position to the PA-reducing-end residue was developed with PA-disaccharides as model compounds. Periodate oxidation of PA-disaccharides was carried out at 0 degrees C for 15 min or at 4 degrees C for 40 h, and the reaction mixtures were reduced with borohydride. The fluorogenic products obtained at 4 degrees C for 40 h were purified by reversed phase HPLC, and the fraction collected were hydrolyzed with acid. The hydrolysates were analyzed by reversed phase HPLC. PA-glyceraldehyde was formed from 2-substituted PA-disaccharides with PA-hexose, PA-threose (or PA-erythrose) from 3-substituted ones, and PA-glycolaldehyde from 4- or 6-substituted ones. HPLC analysis of the products obtained at 0 degrees C for 15 min revealed a difference between 4- and 6-substituted ones. PA-glyceraldehyde was formed from 6-substituted ones, but not from 4-substituted ones. The linkage position, therefore, can be determined by analyzing fluorogenic product(s). As for PA-disaccharides with PA-N-acetylglucosamine, the linkage position can be simply determined by analysis of 40-h oxidation-reduction mixtures. 2-Acetamido-2-deoxy derivatives of PA-threose, PA-xylose, and PA-glyceraldehyde were formed from 3-, 4-, and 6-substituted ones, respectively. The linkage position analysis was successfully applied to determination of the structures of two Fuc-Man-PAs produced through the transglycosylation action of bovine kidney alpha-L-fucosidase.