RESUMO
In contrast to the general hydrolases, trans-sialidase from Trypanosoma cruzi (TcTS) shows excellent regio- and stereoselectivity as well as high yields in transfer reactions. Discussed are the occurrence of trans-sialidases and studies on the transfer mechanism. In detail, the preparative use by chemoenzymatic syntheses with TcTS are outlined with emphasis on the design of modified donor and acceptor substrates. Another section focuses on attempts to develop inhibitors for TcTS, and these endeavors are based on donor- and acceptor-inspired modifications as well as on some completely different structures.
Assuntos
Química Farmacêutica/métodos , Inibidores Enzimáticos/síntese química , Glicoproteínas/química , Neuraminidase/química , Oligossacarídeos/síntese química , Proteínas de Protozoários/síntese química , Ácidos Siálicos/química , Sequência de Carboidratos , Inibidores Enzimáticos/farmacologia , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/metabolismo , Ligantes , Dados de Sequência Molecular , Neuraminidase/antagonistas & inibidores , Neuraminidase/metabolismo , Oligossacarídeos/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Ácidos Siálicos/metabolismo , Estereoisomerismo , Trypanosoma cruzi/química , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologiaRESUMO
Fucosylation is often the final process in glycan biosynthesis. The resulting glycans are involved in a variety of biological processes, such as cell adhesion, inflammation, or tumor metastasis. Fucosyltransferases catalyze the transfer of fucose residues from the activated donor molecule GDP-ß-L-fucose to various acceptor molecules. However, detailed information about the reaction processes is still lacking for most fucosyltransferases. In this work we have monitored α1,3-fucosyltransferase activity. For both donor and acceptor substrates, the introduction of a fluorescent ATTO dye was the last step in the synthesis. The subsequent conversion of these substrates into fluorescently labeled products by α1,3-fucosyltransferases was examined by high-performance thin-layer chromatography coupled with mass spectrometry as well as dual-color fluorescence cross-correlation spectroscopy, which revealed that both fluorescently labeled donor GDP-ß-L-fucose-ATTOâ 550 and acceptor N-acetyllactosamine-ATTOâ 647N were accepted by recombinant human fucosyltransferaseâ IX and Helicobacter pylori α1,3-fucosyltransferase, respectively. Analysis by fluorescence cross-correlation spectroscopy allowed a quick and versatile estimation of the progress of the enzymatic reaction and therefore this method can be used as an alternative method for investigating fucosyltransferase reactions.
Assuntos
Corantes Fluorescentes/química , Fucosiltransferases/metabolismo , Cromatografia em Camada Fina , Fucosiltransferases/genética , Glicosilação , Helicobacter pylori/enzimologia , Humanos , Espectrometria de Massas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Herein, we present a strategy for the glycoconjugation of nanoparticles (NPs), with a special focus on fluorescent quantum dots (QDs), recently described by us as "preassembly" approach. Therein, prior to the encapsulation of diverse nanoparticles by an amphiphilic poly(isoprene)-b-poly(ethylene glycol) diblock copolymer (PI-b-PEG), the terminal PEG appendage was modified by covalently attaching a carbohydrate moiety using Huisgen-type click-chemistry. Successful functionalization was proven by NMR spectroscopy. The terminally glycoconjugated polymers were subsequently used for the encapsulation of QDs in a phase transfer process, which fully preserved fluorescence properties. Binding of these nanoconstructs to the lectin Concanavalin A (Con A) was studied via surface plasmon resonance (SPR). Depending on the carbohydrate moiety, namely, D-manno-heptulose, D-glucose, D-galactose, 2-deoxy-2-{[methylamino)carbonyl]amino}-D-glucopyranose ("des(nitroso)-streptozotocin"), or D-maltose, the glycoconjugated QDs showed enhanced affinity constants due to multivalent binding effects. None of the constructs showed toxicity from 0.001 to 1 µM (particle concentration) using standard WST and LDH assays on A549 cells.
Assuntos
Polímeros/química , Pontos Quânticos/química , Linhagem Celular Tumoral , Química Click , Concanavalina A/química , Humanos , Espectroscopia de Ressonância Magnética , Nanopartículas/química , Ressonância de Plasmônio de SuperfícieRESUMO
Suitable analogs of d-mannoheptulose are currently considered as possible tools for the non-invasive imaging of pancreatic islet insulin-producing cells. Here, we examined whether (19)F-heptuloses could be used for non-invasive imaging of GLUT2-expressing cells. After 20 min incubation, the uptake of (19)F-heptuloses (25 mM) by rat hepatocytes, as assessed by (19)F NMR spectroscopy, ranged from 0.50 (1-deoxy-1-fluoro-d-mannoheptulose and 3-deoxy-3-fluoro-d-mannoheptulose) to 0.25 (1,3-dideoxy-1,3-difluoro-d-mannoheptulose) and 0.13 (1-deoxy-1-fluoro-d-glucoheptulose, 3-deoxy-3-fluoro-d-glucoheptulose and 1,3-dideoxy-1,3-difluoro-d-glucoheptulose) µmol per 3×10(6)cells. (19)F MRI experiments also allowed the detection of 1-deoxy-1-fluoro-d-mannoheptulose in rat hepatocytes. All three (19)F-mannoheptuloses cited above, as well as 7-deoxy-7-fluoro-d-mannoheptulose and 1-deoxy-1-fluoro-d-glucoheptulose inhibited insulin release evoked in rat isolated pancreatic islets by 10mM d-glucose to the same extent as that observed with an equivalent concentration (10mM) of d-mannoheptulose, while 3-deoxy-3-fluoro-d-glucoheptulose and 1,3-dideoxy-1,3-difluoro-d-glucoheptulose (also 10mM) were less potent than d-mannoheptulose in inhibiting insulin release. The 1-deoxy-1-fluoro-d-mannoheptulose and 3-deoxy-3-fluoro-d-mannoheptulose only marginally affected INS-1 cell viability. These findings are compatible with the view that selected (19)F-heptuloses may represent suitable tools for the non-invasive imaging of hepatocytes and insulin-producing cells by (19)F MRI.
Assuntos
Transportador de Glucose Tipo 2/metabolismo , Hepatócitos/metabolismo , Manoeptulose/análogos & derivados , Animais , Linhagem Celular , Sobrevivência Celular , Feminino , Flúor/química , Técnicas In Vitro , Insulina/biossíntese , Ilhotas Pancreáticas/metabolismo , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Manoeptulose/química , Manoeptulose/farmacocinética , Imagens de Fantasmas , Ratos , Ratos WistarRESUMO
Base-promoted glycosylation is a recently established stereoselective and regioselective approach for the assembly of di- and oligosaccharides by using partially protected acceptors and glycosyl halide donors. Initial studies were performed on partially methylated acceptor and donor moieties as a model system in order to analyze the key principles of oxyanion reactivities. In this work, extended studies on base-promoted glycosylation are presented by using benzyl protective groups in view of preparative applications. Emphases are placed on the influence of the acceptor anomeric configuration and donor reactivities.
RESUMO
By Fischer glycosylation both anomers of 4-chlorobutyl gluco-as well as galactopyranosides were obtained and transformed into the corresponding 4-acetylthio-butyl glycopyranosides. Dependent on the precursors two straightforward routes were followed to obtain the appropriate 3-O-sulfated derivatives. Unsubstituted and sulfated glucopyranosides were attached to gold surfaces a gold tips. Their interactions were studied using atomic force microscopy for simulations of intercellular glycoside-based interactions and discussed in-depth.
Assuntos
Glicosídeos , Ouro , Glicosilação , Microscopia de Força AtômicaRESUMO
Several α-configured C-sialosides were synthesised by cross metathesis and further synthetic derivatisation to obtain ligands for Trypanosoma cruzi trans-sialidase (TcTS), a key enzyme in Chagas disease. Affinities of these compounds to immobilised TcTS were measured by surface plasmon resonance (SPR). The K(D) values thus obtained are in the lower millimolar range and will be discussed. The results show the importance of addressing Tyr(119) and Trp(312) side chains of TcTS in target oriented ligand synthesis, since these amino acids constitute the acceptor binding region in the active site of TcTS. The best ligand showed a significant decrease of TcTS activity in a preliminary NMR based inhibition assay.
Assuntos
Doença de Chagas/tratamento farmacológico , Química Farmacêutica/métodos , Inibidores Enzimáticos/farmacologia , Enzimas Imobilizadas/antagonistas & inibidores , Glicoproteínas/antagonistas & inibidores , Glicosídeos/antagonistas & inibidores , Neuraminidase/antagonistas & inibidores , Ácidos Siálicos/química , Trypanosoma cruzi/efeitos dos fármacos , Sítios de Ligação , Domínio Catalítico , Doença de Chagas/parasitologia , Inibidores Enzimáticos/síntese química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosídeos/química , Glicosídeos/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Ligantes , Modelos Moleculares , Neuraminidase/química , Neuraminidase/metabolismo , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Ácidos Siálicos/metabolismo , Ressonância de Plasmônio de Superfície , Trypanosoma cruzi/enzimologiaRESUMO
In recent years the interest in tools for investigating carbohydrate-protein (CPI) and carbohydrate-carbohydrate interactions (CCI) has increased significantly. For the investigation of CPI and CCI, several techniques employing different linking methods are available. Surface plasmon resonance (SPR) imaging is a most appropriate tool for analyzing the formation of self-assembled monolayers (SAM) of carbohydrate derivatives, which can mimic the glycocalyx. In contrast to the SPR imaging methods used previously to analyze CPI and CCI, the novel approach reported herein allows a facile and rapid synthesis of linker spacers and carbohydrate derivatives and enhances the binding event by controlling the amount and orientation of ligand. For immobilization on biorepulsive amino-functionalized SPR chips by reductive amination, diverse aldehyde-functionalized glycan structures (glucose, galactose, mannose, glucosamine, cellobiose, lactose, and lactosamine) have been synthesized in several facile steps that include olefin metathesis. Effective immobilization and the first binding studies are presented for the lectin concanavalin A.
Assuntos
Benzaldeídos/síntese química , Carboidratos/química , Concanavalina A/química , Lectinas de Plantas/química , Polissacarídeos/síntese química , Benzaldeídos/química , Ligantes , Estrutura Molecular , Compostos Organometálicos/química , Lectinas de Plantas/metabolismo , Polissacarídeos/química , Estereoisomerismo , Ressonância de Plasmônio de Superfície/métodosRESUMO
For the first time, fully fledged phthalocyanines (Pcs) were ex post glycoconjugated, that is, via 1,3-dipolar cycloaddition reaction. This divergent approach gains rapid access to a broad range of highly diverse Pcs bearing chemically sensitive substituents. This will be a breakthrough in generating structure-activity relationships (SAR) for the development of novel bioactive molecules.
Assuntos
Indóis/química , Glicosilação , Isoindóis , Radiossensibilizantes , Relação Estrutura-AtividadeRESUMO
α ,2-3- and α ,2-6-sialylated lactosaminide precursor structures obtained by various enzymatic procedures could be used for glycosylations employing triflic acid/N-iodosuccinimide. Easily accessible selectively protected lactoside derivatives served as acceptor disaccharides to give the corresponding human milk pentasaccharides in good yields. These were characterized by spectroscopic means in the form of their peracetylated derivatives.
RESUMO
Oxidative cleavage of N-acetyl-neuraminic acid (Neu5Ac, 1) leads to the open chain octonic acid (ADOA, 2), which under various conditions is transformed into an acyclic acid or ester or into a six- or five-membered octonic acid lactone and lactam, respectively. All reactions proceed with high selectivity and in good yields. Strikingly, a simple switch of reaction conditions from basic to acidic catalysis leads to a complete switch in regioselectivity during a crucial cyclization step.
Assuntos
Ácido N-Acetilneuramínico , Catálise , CiclizaçãoRESUMO
Glutaric aciduria type 1 (GA1) is caused by the deficiency of glutaryl-CoA dehydrogenase (GCDH). Affected patients are prone to the development of encephalopathic crises during an early time window with destruction of striatal neurons and a subsequent irreversible movement disorder. 3-Hydroxyglutaric acid (3OHGA) accumulates in tissues and body fluids of GA1 patients and has been shown to mediate toxic effects on neuronal as well as endothelial cells. Injection of (3H)-labeled into 6 week-old Gcdh(-/-) mice, a model of GA1, revealed a low recovery in kidney, liver, or brain tissue that did not differ from control mice. Significant amounts of 3OHGA were found to be excreted via the intestinal tract. Exposure of Gcdh(-/-) mice to a high protein diet led to an encephalopathic crisis, vacuolization in the brain, and death after 4-5 days. Under these conditions, high amounts of injected 3H-3OHGA were found in kidneys of Gcdh(-/-) mice, whereas the radioactivity recovered in brain and blood was reduced. The data demonstrate that under conditions mimicking encephalopathic crises the blood-brain barrier appears to remain intact.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Encefalopatias/metabolismo , Glutaratos/metabolismo , Glutaril-CoA Desidrogenase/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/complicações , Animais , Transporte Biológico , Encéfalo/metabolismo , Encéfalo/patologia , Encefalopatias/etiologia , Dextranos/metabolismo , Modelos Animais de Doenças , Glutaril-CoA Desidrogenase/genética , Humanos , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Two different approaches were employed to study solid phase random glycosylations to obtain oligosaccharide libraries. In approach I, Wang resin esters were attached to the acceptors structures. Following their glycosylation and resin cleavage, the peracetylated components of the oligosaccharide libraries were characterized. In approach II, polymer-linked donor components could be employed and processed correspondingly. Approach I proved to be superior regarding yield and versatility of products and also allowed the formation of higher oligomers.
Assuntos
Técnicas de Química Combinatória , Glicosilação , Oligossacarídeos/síntese química , Configuração de Carboidratos , Sequência de Carboidratos , Espectroscopia de Ressonância Magnética , Oligossacarídeos/químicaAssuntos
Inibidores Enzimáticos/síntese química , Fucose/química , Glicoconjugados/síntese química , Glicosiltransferases/antagonistas & inibidores , Uridina/química , Cromatografia em Camada Fina , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glicoconjugados/química , Glicoconjugados/farmacologia , Espectroscopia de Ressonância Magnética , Estrutura MolecularRESUMO
Direct 6-thiophosphorylation of mannopyranoside gave both the wanted S- as well as the undesired O-phosphates. This required sequential protecting group syntheses to give mannopyranoside6-phosphate and 6-thiophosphate as well as 6-deoxy-6-thio-mannopyranoside6-phosphate, which were transformed into amino-linker compo-nents for affinity chromatography.
Assuntos
Cromatografia de Afinidade , Manose/química , Manose/síntese química , Fosfatos/química , Técnicas de Química Sintética , LigantesRESUMO
By sequential synthesis the four 2,6-dideoxy saccharide moieties of the kijanimicin tetrasaccharide could be stereoselectively assembled. For formation of all required 2-deoxy α-glycoside linkages various S-(hexopyranosyl)-phosphorodithioates as donor structures could be convincingly employed. The terminal 2-deoxy ß-glycoside linkage was stereoselectively formed following the dibromomethyl methyl ether approach. The target octadeoxy-tetrasaccha-ride could be obtained via nine subsequent steps in 5% overall yield.
Assuntos
Aminoglicosídeos/síntese química , Aminoglicosídeos/química , Configuração de Carboidratos , Sequência de CarboidratosRESUMO
Patients with glutaryl-CoA dehydrogenase (GCDH) deficiency accumulate glutaric acid (GA) and 3-hydroxyglutaric acid (3OH-GA) in their blood and urine. To identify the transporter mediating the translocation of 3OH-GA through membranes, kidney tissue of Gcdh-/- mice have been investigated because of its central role in urinary excretion of this metabolite. Using microarray analyses of kidney-expressed genes in Gcdh-/- mice, several differentially expressed genes encoding transporter proteins were identified. Real-time polymerase chain reaction analysis confirmed the upregulation of the sodium-dependent dicarboxylate cotransporter 3 (NaDC3) and the organic cation transporter 2 (OCT2). Expression analysis of NaDC3 in Xenopus laevis oocytes by the two-electrode-voltage-clamp technique demonstrated the sodium-dependent translocation of 3OH-GA with a K (M) value of 0.95 mM. Furthermore, tracer flux measurements in Chinese hamster ovary cells overexpressing OCT2 showed that 3OH-GA inhibited significantly the uptake of methyl-4-phenylpyridinium, whereas 3OH-GA is not transported by OCT2. The data demonstrate for the first time the membrane translocation of 3OH-GA mediated by NaDC3 and the cis-inhibitory effect on OCT2-mediated transport of cations.
Assuntos
Transportadores de Ácidos Dicarboxílicos/metabolismo , Glutaratos/metabolismo , Glutaril-CoA Desidrogenase/deficiência , Rim/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Simportadores/metabolismo , Animais , Transporte Biológico , Cricetinae , Cricetulus , Transportadores de Ácidos Dicarboxílicos/genética , Eletrofisiologia , Feminino , Perfilação da Expressão Gênica , Rim/metabolismo , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Oócitos , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportador 2 de Cátion Orgânico , Ovário , Simportadores/genética , Xenopus laevisRESUMO
Cross metathesis of both anomers of C-allyl sialoside (3alpha/3beta) with styrene catalyzed by the second generation Grubbs or Hoveyda-Grubbs catalysts gave the corresponding aryl derivatives (4alpha/4beta) in virtually quantitative yields. The products were hydrogenated to model compounds 5alpha/5beta. Similarly, reaction of the alpha-anomer 3alpha with galactose derivative 8 gave the olefin-linked disaccharide mimetic 9. Following hydrogenation and deprotection, the ethylene-bridged Neu5Acalpha(2-->6)Gal analogue 11 could be obtained.
Assuntos
Glicosídeos/síntese química , Ácido N-Acetilneuramínico/síntese química , Rutênio/química , Compostos Alílicos/química , CatáliseRESUMO
Novel aryl beta-d-galactopyranosides were synthesized employing phase-transfer catalysis, and assayed as potential galactose donors in the presence of beta-galactosidase from bovine testes using pNP-Gal as a reference. The aglycones were represented mainly by nitrophenols containing halogens, hydroxymethyl, aldehyde, carboxyl, ester or amino functions. An unusual intermolecular acetyl migration onto the benzylic alcohol group was observed during galactosylation of hydroxymethylnitrophenols. Pyridyl glycosides were obtained by reaction with the corresponding silver pyridinolates. Glycosides of halo-, hydroxymethyl- or methoxycarbonyl-nitrophenols as leaving groups gave virtually the same yields of transglycosylation products. A minor increase was achieved with nitrosalicylaldehyde as leaving group, whereas carboxy or amino derivatives gave very low or no yield of the transglycosylation product. Commercially available donors such as resorufinyl and 4-methylumbelliferyl beta-d-galactopyranosides exhibited a lower transglycosylation potential than these novel pNP-Gal derivatives.
Assuntos
Testículo/enzimologia , beta-Galactosidase/metabolismo , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Glicosilação , Masculino , Nitrofenóis/metabolismo , Nitrofenilgalactosídeos/metabolismo , Ressonância Magnética Nuclear BiomolecularRESUMO
Lactose was converted into a disaccharide precursor incorporating an allyl vinyl ether substructure within the glucose residue by vinylation, regioselective silylation, tosylation and subsequent elimination. The thermal Claisen rearrangement led to a novel galactosylated cyclooctenone exhibiting a boat-chair conformation.