RESUMO
In the present work, the combination of chemical and enzymatic methods to obtain neoglycoproteins is described. Three bovine serum albumin (BSA)-conjugates, BSA-[GalNAc alpha-], BSA-[Gal(beta 1-3)GalNAc(alpha-], and BSA-[Neu5Ac(alpha 2-3)Gal(beta 1-3)GalNAc(alpha-], were prepared. alpha GalNAc derivatives were galactosylated employing crude beta-galactosidase from bovine testes. The use of oversaturated donor solutions (pNP beta Gal) 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 alpha 2-3 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.
Assuntos
Dissacarídeos/síntese química , Glicoproteínas/síntese química , Oligossacarídeos/síntese química , Soroalbumina Bovina , Trissacarídeos/síntese química , beta-Galactosidase , Animais , Configuração de Carboidratos , Sequência de Carboidratos , Bovinos , Dissacarídeos/química , Galactose , Glicoproteínas/química , Indicadores e Reagentes , Dados de Sequência Molecular , Oligossacarídeos/química , Rotação Ocular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Trissacarídeos/químicaRESUMO
5-Azido-3-oxa-pentyl beta-D-galactopyranoside was prepared from diethylene glycol monochlorohydrin and used as a model of oligosaccharide hapten. After deprotection, a series of amides bearing thiophilic groups had been obtained through the terminal amino function and essayed in coupling reactions with thiolated BSA. Also several Lewis human blood group oligosaccharides had been conjugated with thiolated BSA demonstrating the usefulness of the methodology.
Assuntos
Glicosídeos/química , Proteínas/química , Compostos de Sulfidrila/química , Configuração de Carboidratos , Sequência de Carboidratos , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência MolecularRESUMO
In this study beta1-3 linked analogues of the T-antigen determinant were synthesized in preparative scale by transgalactosylation using beta-galactosidase from bovine testes to give synthetic antigens. Acceptors with modifications of the sugar residue such as alpha-glycosylated spacers, as well as GlcNAc-alphaOR- and 2dGal-alphaOR-substrates opened further possibilities for galactosylation.
