Synthesis of selectively radiolabeled hexasaccharides for the determination of enzymatic regioselectivity

Poly-N-acetyllactosamines provide backbone structures for functional modifications such as sialyl Lewis X. To understand how the biosynthesis of poly-N-acetyllactosamines is regulated, two branched oligosaccharides of the structure Galbeta1,4GlcNAcbeta1, 6(Galbeta1,4GlcNAcbeta1,2)-Manalpha1,6Manbeta-octyl 1 and 2 were synthesized in which one of the terminal galactose units was selectively radiolabeled. Hexasaccharides 1 and 2 were assembled from the chemically synthesized pentasaccharide precursors GlcNAcbeta1,6(Galbeta1,4GlcNAcbeta1,2)-Manalpha1,6Manbeta-octyl3 and Galbeta1,4GlcNAcbeta1,6(GlcNAcbeta1,2) - Manalpha1,6 Manbeta-octyl 4 respectively, through treatment with UDP-1-[3H]-Gal and beta1,4 galactosyltransferase. Compounds 1 and 2 were subsequently incubated with UDP-GlcNAc and the UDP-GlcNAc: Galbeta1-4Glc(NAc) beta1,3-N-acetylglucosaminyltransferase (i-GlcNAc transferase) resulting in a partial conversion to a mixture of heptasaccharides which were purified by HPLC. The branch selectivity of the addition of N-acetylglucosamine to compounds 1 and 2 was then characterized by endo-beta-galactosidase digestion of the heptasaccharides, followed by isolation of the resultant pentasaccharides on C18 reverse-phase silica cartridges. Comparison of the amount of radiolabel to a control reaction lacking endo-beta-galactosidase indicated the favored site of GlcNAc addition to be the lower beta1,2-branch over the beta1,6 branch by a 3 :1 ratio.