Separation of human milk oligosaccharides by recycling chromatography. First isolation of lacto-N-neo-difucohexaose II and 3'-Galactosyllactose from this source.

Lacto-N-neo-difucohexaose II, beta-D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-beta-D-Glcp-NAc-(1----3)-beta-D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-D-Glc, and 3'-galactosyllactose, beta-D-Galp-(1----3)-beta-D-Galp-(1----4)-D-Glc, were isolated for the first time from human milk by means of a recycling chromatography technique. Through this method, carried out mainly on columns of K+ ion-exchange resins and either Bio-Gel P-4 or TSK 40 W(S) gel filtration media, up to one gram of an oligosaccharide mixture could be handled and lacto-N-neo-difucohexaose II separated from isomeric lacto-N-difucohexaose I, alpha-L-Fucp-(1----2)-beta-D-Galp-(1----3)-[alpha-L-Fucp-(1----4)]-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glc, and II, beta-D-Galp-(1----3)-[alpha L-Fucp-(1----4)]-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-D-Glc. This method also permitted resolution of isomeric mixtures of the trisaccharides 2'-fucosyllactose, alpha-L-Fucp-(1----2)-beta-D-Galp-(1----4)-D-Glc, and 3-fucosyllactose, beta-D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-D-Glc, the tetrasaccharides lacto-N-tetraose, beta-D-Galp-(1----3)-beta-D-GlcpNac-(1----3)-beta-D-Galp-(1----4)-D-Glc, and lacto-N-neo-tetraose, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glc, and the pentaoses lacto-N-fucopentaose I, alpha-L-Fucp-(1----2)-beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glc, II, beta-D-Galp-(1----3)-[alpha-L-Fucp-(1----4)]-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glc, and III, beta-D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glc, which have proved difficult if not impossible to separate by other means. The isolation of these and other milk oligosaccharides is described herein. The 500-MHz 1H-n.m.r. spectra of lacto-N-neo-difucohexaose II and 3'-galactosyllactose, and their alditols, are recorded. 1H-n.m.r. data on some other milk oligosaccharides, both natural and reduced, are also given.