RESUMO
The synthesis of multivalent sialylated glycoclusters is herein addressed by a chemoenzymatic approach using the trans-sialidase of Trypanosoma cruzi (TcTS). Multivalent ß-thio-galactopyranosides and ß-thio-lactosides were used as acceptor substrates and 3'-sialyllactose as the sialic acid donor. High performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was shown to be an excellent technique for the analysis of the reaction products. Different eluting conditions were optimized to allow the simultaneous resolution of the sialylated species, as well as their neutral precursors. The TcTS efficiently transferred sialyl residues to di, tri, tetra and octa ß-thiogalactosides. In the case of an octavalent thiolactoside, up to six polysialylated compounds could be resolved. Preparative sialylation reactions were performed using the tetravalent and octavalent acceptor substrates. The main sialylated derivatives could be unequivocally assigned by MALDI mass spectrometry. Inhibition of the transfer to the natural substrate, N-acetyllactosamine, was also studied. The octalactoside caused 82 % inhibition of sialic acid transfer when we used equimolar concentrations of donor, acceptor and inhibitor.
Assuntos
Glicoproteínas/química , Lactose/análogos & derivados , Neuraminidase/química , Proteínas de Protozoários/química , Ácidos Siálicos/química , Tiogalactosídeos/química , Trypanosoma cruzi/enzimologia , Cromatografia Líquida de Alta Pressão , Lactose/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
A conformationally restricted 2-O-benzyl-3,5-O-di-tert-butylsilylene-ß-D-thiogalactofuranoside donor was prepared from benzyl α-D-galactofuranoside and its donor capability was studied for stereoselective 1,2-cis α-D-galactofuranosylation. An unusual chemical behavior in benzylation and hydrogenolysis reactions was observed after the introduction of the 3,5-O-di-tert-butylsilylene protecting group into the galactofuranosyl moiety. The influence of the solvent, temperature, and activating system was evaluated. The NIS/AgOTf system, widely used in 1,2-cis ß-arabinofuranosylation, was not satisfactory enough for 1,2-cis galactofuranosylation. However, moderate to high α-selectivity was obtained with all the acceptors employed when using p-NO2PhSCl/AgOTf as a promoting system, in CH2Cl2 at -78°C. The order of the addition of the reactants (premixing or preactivation) did not affect substantially the stereochemical course of the glycosylation reaction. The α-D-Galf-(1â6)-D-Man linkage was achieved with complete diastereoselectivity by preactivation of the conformationally constrained thioglycoside donor.
Assuntos
Tiogalactosídeos/química , Tioglicosídeos/química , Butanos/química , Configuração de Carboidratos , Técnicas de Química Sintética , Glicosilação , Silanos/química , Solventes , Estereoisomerismo , Tiogalactosídeos/síntese químicaRESUMO
Herein, we describe the design and synthesis of a novel family of hydrolytically stable glycoclusters bearing thiodigalactoside (TDG) analogues as recognition elements of ß-galactoside binding lectins. The TDG analogue was synthesized by thioglycosylation of a 6-S-acetyl-α-D-glucosyl bromide with the isothiouronium salt of 2,3,4,6-tetra-O-acetyl-ß-D-galactose. Further propargylation of the TDG analogue allowed the coupling to azido-functionalized oligosaccharide scaffolds through copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) under microwave activation. The final mono-, di-, and tetravalent ligands were resistant to enzymatic hydrolisis by Escherichia coli ß-galactosidase. Binding affinities to peanut agglutinin and human galectin-3 were measured by isothermal titration calorimetry which showed K(a) constants in the micromolar range as well as a multivalent effect. Monovalent ligand exhibited a binding affinity higher than that of thiodigalactoside. Docking studies performed with a model ligand on both ß-galactoside binding lectins showed additional interactions between the triazole ring and lectin amino acid residues, suggesting a positive effect of this aromatic residue on the biological activity.
Assuntos
Arachis/química , Galectina 3/química , Glicoconjugados/síntese química , Lectinas/química , Tiogalactosídeos/química , Proteínas Sanguíneas , Configuração de Carboidratos , Sequência de Carboidratos , Galectinas , Glicoconjugados/química , Humanos , Hidrólise , Ligantes , Modelos Moleculares , Dados de Sequência MolecularRESUMO
A new (1â6)-linked thiodisaccharide formed by two galactofuranosyl units has been synthesized. Methyl (methyl α,ß-D-galactofuranosid)uronate was employed as the starting compound, which was per-O-silylated with TBSCl and reduced with LiAlH4 to afford methyl 2,3,5-tri-O-tert-butyldimethylsilyl-ß-D-galactofuranoside (2ß) as a key precursor for the preparation of methyl per-O-tert-butyldimethylsilyl-6-thio-ß-D-galactofuranoside (12). The free thiol group of 12 was glycosylated and the product O-deprotected to afford the target ß-D-Galf-S-(1â6)-ß-d-Galf-OMe (14). The conformations of this thiodisaccharide were preliminarily studied using combined theoretical calculations and NMR data. Furthermore, the glycomimetic 14 showed to be a competitive inhibitor of the ß-galactofuranosidase from Penicillum fellutanum (K(i)=3.62 mM).
Assuntos
Antifúngicos/síntese química , Dissacarídeos/síntese química , Proteínas Fúngicas/antagonistas & inibidores , Galactose/química , Glicosídeo Hidrolases/antagonistas & inibidores , Penicillium/química , Tiogalactosídeos/síntese química , Antifúngicos/química , Configuração de Carboidratos , Dissacarídeos/química , Proteínas Fúngicas/química , Glicosídeo Hidrolases/química , Glicosilação , Cinética , Espectroscopia de Ressonância Magnética , Penicillium/enzimologia , Tiogalactosídeos/químicaRESUMO
In the present work, we followed a microarray approach to analyze the expression of glycosylation-related genes on different cell populations obtained from mouse thymus. Among other genes, transcription of the two-domain type galectin-8 was detected both in thymocytes and thymic epithelial cells (TECs), which was confirmed by reverse transcriptase (RT)-PCR assays independently carried out on both cell populations. Two splice variants, differing solely in the presence of a nine amino acid insertion in the linker peptide region connecting the two carbohydrate recognition domains (CRDs), were identified from purified thymocytes. Expression of galectin-8 was verified at the protein level in total organ extracts by western-blots of lactosyl-Sepharose purified binders. To explore the possible biological roles of locally produced galectin-8, both splice variants were recombinantly expressed in bacteria and assayed over cultured thymocytes. In spite of their binding to all cell populations, addition of either isoform of galectin-8 to thymocyte cultures induced apoptosis only of the CD4(high)CD8(high) cells through caspases pathway activation. All of these effects were prevented by the addition of thiodigalactoside (TDG) or lactose, thus indicating that the proapoptotic activity of galectin-8 was due to the specific interaction of its CRDs with defined cell surface glycans. Together, our results demonstrate intrathymic expression of galectin-8 in mouse, and suggest an active role for this lectin in shaping the mature T cell repertoire.