Development of Recovering Equipment of Free Oligosaccharides Chemically Converted from Fluorescent Labeled Glycans by Hydrogen Peroxide and Its Application of Two-dimensional Liquid Chromatography

本文研究了用各种芳香胺还原胺化法制备的荧光衍生物对游离低聚糖的化学再生。麦芽糖衍生物的4-氨基苯甲酸乙酯（p-ABEE）、2-氨基苯腈（o-ABN）、4-氨基苯腈（p-ABN）、7-氨基-4-甲基香豆素（AMC）、2-氨基苯甲酸（o-ABA）、2-氨基苯酰胺（o-ABAD）、2-氨基吡啶（AP）和8-氨基萘-1,3,6-三磺酸（ANTS）在30℃过氧化氢/乙酸水溶液中孵育。麦芽糖从p-ABEE， p-ABN和AMC衍生物中回收率相当好，约为90%的麦芽糖。麦芽糖从其o-取代苯胺（o-ABA， o-ABAD和o-ABN）衍生物中回收率为5-40%，但从AP和ANTS衍生物中无法回收麦芽糖。然而，事先用溴化氰处理AP衍生物可以高产地再生麦芽糖。作为一种应用，从酰胺柱上分离的一些糖蛋白中分离出p- abee标记的n -聚糖，通过游离糖将峰组分转化为AP衍生物，然后通过反相色谱法进行定位。

This study examined chemical regeneration of free oligosaccharides from their Fuorescent derivatives prepared by reductive amination with various aromatic amines. Maltose derivatives of ethyl 4-aminobenzoate (p-ABEE), 2-aminobenzonitrile (o-ABN), 4-aminobemonitrile (p-ABN), 7-amino-4-methylcoumarin (AMC), 2-aminobenzoic acid (o-ABA), 2-aminobenzamide (o-ABAD), 2-aminopyridine (AP), and 8-aminonaphthalene-1,3,6-trisulfonare (ANTS) were incubated at 30℃ with an aqueous solution of hydrogen peroxide/acetic acid. Recoveries of maltose from p-ABEE, p-ABN, and AMC derivatives were fairly good and gave approximately 90% of maltose. Recoveries of maltose from its o-substituted aniline (o-ABA, o-ABAD, and o-ABN) derivatives were 5-40%, but maltose was unrecoverable from AP and ANTS derivatives. Nevertheless, prior treatment of an AP derivative with cyanogen bromide enabled the regeneration of maltose in high yields. As an application, p-ABEE-labeled N-glycans from some glycoproteins separated on an amide column were identified by converting peak components to their AP derivatives via free saccharides and following mapping by reversed phase chromatography.