Chemoenzymatic synthesis of spacer-linked oligosaccharides for the preparation of neoglycoproteins

In the present work, the combination of chemical and enzymatic methods to obtain neoglycoproteins is described. Three bovine serum albumin (BSA)-conjugates, BSA-[GalNAcα-], BSA-[Gal(β1-3)GalNAc(α-], and BSA-[Neu5Ac(α2-3)Gal(β1-3)GalNAc(α-], were prepared. αGalNAc derivatives were galactosylated employing crude β-galactosidase from bovine testes. The use of oversaturated donor solutions (pNPβGal) enhanced the yields up to 60%. This method was verified using divalent structures as acceptors, that rendered di- and tri-galactosylated products. Further treatment of the disaccharides with CMP-Neu5Ac and α2-3 sialyltransferase from pork liver led to formation of trisaccharides. Finally, mono-, di-, and trisaccharides were coupled to BSA employing a thiolic group introduced into the protein for Michael addition to a maleinimide group in the spacer-arm of the saccharide components. The results were monitored by HPLC and MALDI-TOF.