Separation of Oligosaccharide Isomers Containing Acetamido and Neutral Sugars by High-Performance Liquid Chromatography

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Abstract
High performance liquid chromatography (HPLC) was investigated for the separation of the following reduced oligosaccharides containing neutral and acetamido sugars: Gal.beta.1-3GlcNAc-ol and Gal.beta.1-4GlcNAc-ol, Gal.beta.1-3GlcNAc.beta.1-6Gal-ol, Gal.beta.1-4GlcNAc.beta.1-6Gal-ol, Gal.beta.-4GlcNAc.beta.1-3Gal-ol, Gal.beta.1-3GlcNAc.beta.1-3Gal.beta.1-4Glc-ol (LNT-ol), Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4Glc-ol (LNNT-ol), Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4GlcNAc-ol, Gal.beta.1-3[Fuc.alpha.1-4]GlcNAc.beta.1-3Gal.beta.1-4Glc-ol, Gal.beta.1-4[Fuc.alpha.1-3]GlcNAc.beta.1-3Gal.beta.1-4Glc-ol. These alditols were studied as standards for the separation of mixtures of reduced oligosaccharides obtained from glycoproteins. A combination of serveral HPLC sysems using normal and reverse phase column packings was required for separation of the isomers as follows. A novel chromatography system using acetylated alditols eluted from silica (Hypersil) with dichloromethane/hexane/isopropranol as mobile phase separated the disaccharides and the first trisaccharide from the next 2. These last 2 trisaccharides could in turn be separated as non-acetylated alditols chormatographed on Hypersil eluted with aqueous acetonitrile containing 0.05% tetraethylenepentamine (TEPA) or on silica chemically bonded with aminopropyl groups (APS-Hypersil). The pentasaccharides were resolved as acetylated alditols chromatographed on reverse phase, octodecyl silica (ODS-Hypersil). Isomeric separation of the tetrasaccharides was not achieved. However, LNT-ol could be obtained essentially free of LNNT-ol by isolation of its di-N-acetylaed product. The 3rd tetrasaccharide studied was readily separated from the other tetrasaccharides and the pentasaccharide isomers on normal or reverse phase chromatography because of its greater acetamido/netural sugar ratio. In general the varying ratios of acetamido/neutral sugars and their different glycosidic linkages conferred distinct but predictable chromatographic properties to the alditols on silica and reverse phase chromatography.

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