Characterization of lacto-N-hexaose and two fucosylated derivatives from human milk by high-performance liquid chromatography and proton NMR spectroscopy.

The branched hexasaccharide whose trivial name is lacto-N-hexaose (LNH) as well as a mono- and a difuco-derivative were isolated from human breast milk of a Lewis a positive donor by high-performance liquid chromatography (HPLC). Reversed-phase HPLC on C18 columns which have been used previously in our laboratory for successful separations of the smaller milk oligosaccharides were not effective in this system. However, normal-phase HPLC on an amino-bonded silica column (Varian AX-5) gave both analytical and preparative separations. The identity of the LNH core structure was verified both by HPLC and by NMR spectroscopy of the fucosidase digestion products of the fucosylated oligosaccharides. LNH was identified by NMR spectroscopy of the reduced and acetylated derivative whose high field spectrum has been previously reported. The positions of fucosylation of beta-GlcNAc residues were determined by the characteristic chemical shifts of the resonances assigned to alpha-fucose H1 and H5. The assignment of many of the resonances to sugar ring protons was possible using difference decoupling methods. The observation of nuclear Overhauser effects between the anomeric protons and the aglycone protons of adjacent residues confirmed the assignment of the glycosidic linkages.