RESUMEN
We introduced a strategy for preparing a carbohydrate microarray and demonstrated its utility for characterizing carbohydrate binding and activities. We isolated the lipopolysaccharide (LPS) components from different bacteria and explored the possibility of immobilizing these glycoconjugates on a high-binding polystyrene plate. Carbohydrate-specific combination was examined by observing the binding of the blood group B analogic LPS O-polysaccharide from Escherichia coli on the high-binding polystyrene plate and anti-B from a broad spectra antibody of human blood serum. Strong binding of antibodies was screened, as it was evident that relative response value is two times higher than control. The hybridization results indicated that this method is a reliable technique for the detection of human intestinal bacteria and is expected to be applied in diagnostics and seroepidemiology.
Asunto(s)
Lipopolisacáridos , Suero , Humanos , Lipopolisacáridos/química , Poliestirenos , Estudios Seroepidemiológicos , Carbohidratos/química , Escherichia coli , InmunoglobulinasRESUMEN
The penta-N-acetyl-chitopentaose 2 has been prepared by using recombinant E. coli strains harboring the nodC gene (encoding chitooligosaccharide synthase) from Azorhizobium caulinodans. Then, the deacetylase NodB removed the N-acetyl moiety from the nonreducing terminus of 2 to give tetra-N-acetyl-chitopentaose 3. N-Acylation of 3 with stearyl chloride was performed in DMF containing water and provided the corresponding lipo-chitopentaose nodulation factor 4. A binding chitinase assay indicated that 4 was much more stable than 3.
Asunto(s)
Quitinasas/química , Oligosacáridos/biosíntesis , Oligosacáridos/química , Acilación , Amidohidrolasas/química , Azorhizobium caulinodans/enzimología , Azorhizobium caulinodans/genética , Proteínas Bacterianas/química , Escherichia coli/genética , Ingeniería Genética/métodos , Glucósidos/biosíntesis , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismoRESUMEN
Fluorophore-assisted carbohydrate electrophoresis (FACE) is a straightforward, sensitive method for determining the presence and relative abundance of individual (oligo)saccharide in a(n) (oligo)saccharide mixture. The single terminal aldehydes of (oligo)saccharides were tagged with the charged fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS), and separated with high resolution on the basis of size by polyacrylamide gel electrophoresis. ANTS fluorescence labeling is not biased by (oligo)saccharide length. Therefore, band fluorescence intensity is directly related to the relative abundance of individual (oligo)saccharide moieties in heterogeneous sample. In the same time, it also indicates that FACE can be used to investigate the interactions of carbohydrates and proteins.
Asunto(s)
Electroforesis en Gel de Poliacrilamida/métodos , Oligosacáridos/análisis , Oligosacáridos/química , Proteínas/química , Concanavalina A/química , Colorantes Fluorescentes , Hidrólisis , NaftalenosRESUMEN
A new compound 2, possessing a tetra-N-acetyl-chitotetraosyl moiety as a constituent, was synthesized by bacterial fermentation, which used allosamizoline 1 as the initial acceptor. A 2-binding chitinase assay, indicated that the chitinase was inactivated by 2 with IC50 = 0.03 microg/mL.
Asunto(s)
Fermentación , Glucosamina/análogos & derivados , Conformación de Carbohidratos , Quitinasas/antagonistas & inhibidores , Quitinasas/metabolismo , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Glucosamina/biosíntesis , Glucosamina/química , Glucosamina/farmacología , Unión ProteicaRESUMEN
UDP-galactose 4-epimerase (EC 5.1.3.2, Gal E) from Escherichia coli catalyzes the reversible reaction between UDP-galactose and UDP-glucose. In this study, the Gal E gene from E. coli, coding UDP-galactose 4-epimerase, was cloned into pYD1 plasmid and then transformed into Saccharomyces cerevisiae EBY100 for expression of Gal E on the cell surface. Enzyme activity analyses with EBY100 cells showed that the enzyme displayed on the yeast cell surface was very active in the conversion between UDP-Glc and UDP-Gal. It took about 3 min to reach equilibrium from UDP-galactose to UDP-glucose.
Asunto(s)
Enzimas Inmovilizadas/metabolismo , Escherichia coli/enzimología , Saccharomyces cerevisiae/enzimología , UDPglucosa 4-Epimerasa/metabolismo , Clonación Molecular , ADN Bacteriano/química , ADN Bacteriano/genética , Electroforesis Capilar , Enzimas Inmovilizadas/genética , Escherichia coli/genética , Reacción en Cadena de la Polimerasa , Saccharomyces cerevisiae/genética , UDPglucosa 4-Epimerasa/genética , Uridina Difosfato Galactosa/metabolismo , Uridina Difosfato Glucosa/metabolismoRESUMEN
A sensitive,specific, and rapid method for the detection of carbohydrate-protein interactions is demonstrated by fluorophore-assisted carbohydrate electrophoresis (FACE). The procedure is simple and the cost is low. The advantage of this method is that carbohydrate-protein interactions can be easily displayed by FACE, and the carbohydrates do not need to be purified.
Asunto(s)
Carbohidratos , Electroforesis/métodos , Colorantes Fluorescentes/metabolismo , Naftalenos/metabolismo , Proteínas/metabolismo , Electroforesis/economía , Electroforesis/instrumentación , Sensibilidad y EspecificidadRESUMEN
The work presented herein is a new noncovalent glycoarray assembly method for microplates created by simply mixing together a carbohydrate and a tetradecylamine. alpha-D-Mannopyranoside, alpha-D-glucopyranoside, and alpha-D-galactopyranoside were utilized in model studies and product formations were detected by lectin binding. The method can be extended to study the steric hindrance effect of carbohydrate-protein interactions, namely the structure-function relations of carbohydrates.
Asunto(s)
Carbohidratos/química , Glucolípidos/química , Aminas/química , Sitios de Unión , Concanavalina A/química , Concanavalina A/metabolismo , Fluoresceína-5-Isotiocianato/química , Galactosa/química , Glucósidos/química , Glucolípidos/metabolismo , Lectinas/química , Lectinas/metabolismo , Manosa/química , Microscopía Fluorescente , Modelos Químicos , Proteínas/química , Proteínas/metabolismo , Relación Estructura-ActividadRESUMEN
A new compound 7, possessing a tetra-N-acetyl-chitotetraosyl moiety as a constituent, was synthesized by bacterial fermentation which used allosamizoline 6 as the initial acceptor.
Asunto(s)
Acetilcolinesterasa/metabolismo , Proteínas Bacterianas/metabolismo , Glucosamina/análogos & derivados , N-Acetilglucosaminiltransferasas/metabolismo , Animales , Electrophorus , Glucosamina/síntesis química , Glucosamina/química , Glucosamina/metabolismo , Estructura MolecularRESUMEN
The work presented herein is a new noncovalent glycoarray assembly method for microplates created by simply mixing together a carbohydrate and a tetradecylamine. Alpha-mannose was utilized in the model study and product formation was detected by lectin binding. The method can be further extended to array complex carbohydrates.
Asunto(s)
Glucolípidos/química , Secuencia de Carbohidratos , Concanavalina A , Fluoresceína-5-Isotiocianato , Humanos , Inmunoglobulina M/sangre , Datos de Secuencia MolecularRESUMEN
Cell density cultivation of recombinant Escherichia coli strains harboring the nodC gene (encoding chitooligosaccharide synthase) from Azorhizobium caulinodans has been previously described as a practical method for the preparation of gram-scale quantities of penta-N-acetyl-chitopentaose. We have now extended this method to the production of allylated derivative of penta-N-acetyl-chitopentaose by using allyl 2-acetamido-2-deoxy-beta-d-glucopyranoside (2) as the initial acceptor for the synthesis of target pentaoside in vivo.
Asunto(s)
Quitina/síntesis química , Ligasas/metabolismo , Secuencia de Carbohidratos , Quitina/análogos & derivados , Escherichia coli/genética , Ligasas/genética , Datos de Secuencia MolecularRESUMEN
N-acetyl glucosamine 1 is selectively converted into 2 without protection of the other hydroxyl groups by allylation of the anomeric alkoxide in N,N-dimethylformamide containing lithium bromide. We use cell density cultures to produce the allylated derivative of penta-N-acetyl-chitopentaose by using 2 as the initial acceptor for the synthesis of 3 in vivo. Upon periodate oxidation, 3 is transferred to 4. Compound 4 is quickly subjected to sodium borohydride reduction and NH3 amination, which afforded the target compound 5. In 5-binding chitinase assay, it indicates that the chitinase is obviously inactivated by 5 with IC50 = 4.7 micromol/L.
Asunto(s)
Quitinasas/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Glucósidos/síntesis química , Glucósidos/farmacología , Glicósidos/síntesis química , Glicósidos/farmacología , Oligosacáridos/síntesis química , Oligosacáridos/farmacología , Conformación de Carbohidratos , Secuencia de Carbohidratos , Células Cultivadas , Inhibidores Enzimáticos/química , Glucósidos/química , Glicósidos/química , Datos de Secuencia Molecular , Oligosacáridos/química , Relación Estructura-ActividadRESUMEN
The epoxyalkyl (1-->3)-beta-D-pentaglucosides 2 and 3 were synthesized in order by acetylation, glycosidation, oxidation, and deacetylation of 1. The immunological activities (superoxide anion production activity, phagocytic activity, and lymphocyte proliferation) and scavenging ability toward superoxide anion of (1-->3)-beta-D-pentaglucoside (1) and its epoxyalkyl derivatives (2 and 3) were investigated. Superoxide anion released from human blood monocytes was measured by the reduction of ferricytochrome c. Phagocytosis by peritoneal macrophages was detected through a teal ingesting that measured the chicken red blood cells (CRBC). Lymphocyte proliferation was determined by the MTT method. The scavenging ability of 1, 2, and 3 toward superoxide anions was evaluated by means of chemiluminescence (CL). The results showed that 2 and 3 had a little higher immunological activity and scavenging ability toward superoxide anion than 1, which indicated that the reducing end of the oligoglucosides was quite important for maximum biological activity.
Asunto(s)
Glucósidos/síntesis química , Macrófagos Peritoneales/efectos de los fármacos , Monocitos/efectos de los fármacos , Oligosacáridos/síntesis química , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Pollos , Glucósidos/inmunología , Humanos , Activación de Linfocitos/efectos de los fármacos , Monocitos/inmunología , Oligosacáridos/inmunología , Oligosacáridos/farmacología , Fagocitosis/efectos de los fármacos , Relación Estructura-Actividad , Superóxidos/metabolismoRESUMEN
Fluorophore-assisted carbohydrate electrophoresis (FACE) is a straightforward, sensitive method for determining the presence and relative abundance of individual (oligo)saccharides in a(n) (oligo)saccharide mixture. The single-terminal aldehydes of oligoglucoside residues released by acid hydrolysis of beta-1,3-D-glucan from yeast were tagged with the charged fluorophore ANTS (8-aminonaphthalene-1,3,6-trisulphonate), and separated with high resolution on the basis of size by PAGE. ANTS fluorescence labelling was not biased by oligoglucoside length; therefore band fluorescence intensity was directly related to the relative abundance of individual oligoglucoside moieties in a heterogeneous sample. FACE analysis revealed that the major oligoglucoside mixture released by acid hydrolysis from beta-1,3-D-glucan was composed of monosaccharide, disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide and octasaccharide, and the order of abundance from high to low was trisaccharide, monosaccharide, disaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide and octasaccharide respectively. In conclusion, FACE represents an accessible, sensitive and quantitative analytical tool enabling the characterization of a(n) (oligo)saccharide mixture.
Asunto(s)
Glucanos/análisis , Saccharomyces cerevisiae/química , Secuencia de Carbohidratos , Electroforesis en Gel de Poliacrilamida , Colorantes Fluorescentes , Glucanos/química , Hidrólisis , NaftalenosRESUMEN
A sensitive, specific, and rapid method for the detection of carbohydrate-protein interactions was demonstrated using quantum dots (QDs) as a fluorescence label coupled with protein. 1,3-Dipolar cycloaddition between azide and alkyne was exploited to attach alpha-d-glucopyranoside to a C(14) hydrocarbon chain that noncovalently binds to the microtiter well surface, and the product formation was detected by both electrospray ionization-mass spectrometry (ESI-MS) and QD- (or fluorescein isothiocyanate (FITC))-conjugated lectin binding. It indicated that the peak intensity of the fluorescence emission was proportional to the initial concanavalin A (Con A) concentration in the range of 2 x 10(-3) micromol/L to 2 x 10(-2)mmol/L with a detection limit at least 100 times lower than that of the FITC-based method.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Análisis por Micromatrices/métodos , Puntos Cuánticos , Coloración y Etiquetado/métodos , Adsorción , Carbohidratos/química , Concanavalina A/química , Concanavalina A/metabolismo , Fluoresceína-5-Isotiocianato , Fluorescencia , Análisis por Micromatrices/instrumentación , Estructura Molecular , Unión Proteica , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The title pentasaccharide was synthesized via a 2+3 strategy. The disaccharide donor, 3-O-acetyl-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl trichloroacetimidate (8), was obtained by selective coupling of allyl 2-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranoside with 3-O-acetyl-2-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl trichloroacetimidate (4), followed by deallylation, and trichloroacetimidation. Meanwhile, the trisaccharide acceptor, allyl 2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranoside (12), was prepared by coupling of allyl 2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranoside with 4, followed by deacetylation. Condensation of 8 with 12, followed by epoxidation, and deprotection, gave the target pentaoside.
Asunto(s)
Glucósidos/síntesis química , Oligosacáridos/síntesis química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Espectroscopía de Resonancia Magnética , Conformación Molecular , Estructura Molecular , Oligosacáridos/química , Oxidación-ReducciónRESUMEN
The (1-->3)-beta-D-pentaglucoside was synthesized as its (R)-2,3-epoxypropyl glycoside via 2+3 strategy. The disaccharide donor 8 was obtained by 3-selective coupling of 2 with 4, followed by deallylation, and trichloroacetimidation. Meanwhile, the trisaccharide acceptor 12 was prepared by coupling of 10 with 4, followed by deacetylation. Condensation of 8 with 12, followed by epoxidation, and deprotection, gave the target pentaoside. The results of these bioassays demonstrated that the (1-->3)-beta-D-glucanase was obviously inactivated by 15 with k(app)=3.79 x 10(-4) min(-1). At the same time, we found that the 15 was more active as compared to the laminaripentaose in eliciting phytoalexin accumulation in tobacco cotyledon tissue, and it could be kept longer time than laminaripentaose, which indicated it is much more stable than laminaripentaose.
Asunto(s)
Compuestos Epoxi/síntesis química , Compuestos Epoxi/farmacología , Glucano 1,3-beta-Glucosidasa/efectos de los fármacos , Glucósidos/síntesis química , Glucósidos/farmacología , Oligosacáridos/síntesis química , Oligosacáridos/farmacología , Extractos Vegetales/metabolismo , Bioensayo , Secuencia de Carbohidratos , Cotiledón/efectos de los fármacos , Cotiledón/metabolismo , Compuestos Epoxi/química , Compuestos Epoxi/metabolismo , Glucano 1,3-beta-Glucosidasa/metabolismo , Glucanos , Glucósidos/química , Datos de Secuencia Molecular , Estructura Molecular , Oligosacáridos/metabolismo , Extractos Vegetales/química , Polisacáridos/farmacología , Unión Proteica , Sesquiterpenos , Terpenos , Nicotiana/efectos de los fármacos , Nicotiana/metabolismo , FitoalexinasRESUMEN
We describe a approach for the synthesis of (1-->3)-beta-D-oligosaccharide derivatives 10-18. 1-9 were synthesized by treating peracetylated (1-->3)-beta-D-oligosaccharides with the corresponding alkenyl alcohols and Lewis acid (SnCl(4)) catalyst. Epoxidation of the corresponding alkenyl oligoglucosides took place by m-CPBA. NaOMe in dry methanol was used for the deacetylation of the blocked derivatives, to give 10-18 in an overall yields of 25-32%. In subsequent glucan-binding protein of soybean assays, we found that 16 was most active, with an IC(50) value of 9 mM. However, the activities of 17, 18, 13, 14, 15, 10, 11, and 12 were gradually decreased. At the same time, we found 16 was most active as compared to the other (1-->3)-beta-D- oligoglucoside derivatives in eliciting phytoalexin accumulation in soybean cotyledon tissue, and 16 was kept longer time than (1-->3)-beta-D-glucohexaose, which indicated 16 is much more stable than (1-->3)-beta-D-glucohexaose.
Asunto(s)
Compuestos Epoxi/síntesis química , Glucósidos/síntesis química , Oligosacáridos/síntesis química , Extractos Vegetales/biosíntesis , Proteínas de Plantas/metabolismo , Compuestos Epoxi/metabolismo , Compuestos Epoxi/farmacología , Glucósidos/metabolismo , Glucósidos/farmacología , Oligosacáridos/metabolismo , Oligosacáridos/farmacología , Sesquiterpenos , Terpenos , FitoalexinasRESUMEN
We describe a approach for the synthesis of a mixture of 3,4-epoxybutyl (1-->3)-beta-D-oligoglucosides. The particular (1-->3)-beta-D-glucan isolated from the cell walls of Saccharomyces cerevisiae was recovered from the aqueous medium as water-insoluble particles by the spray drying (GS) method, and it was characterized by FTIR spectroscopy. The acid-solubilized (1-->3)-beta-D-oligoglucosides were prepared by partial acid hydrolysis of glucan particles, which were qualitatively analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE). The peracetylated 3-butenyl (1-->3)-beta-D-oligoglucosides were synthesized by treating peracetylated (1-->3)-beta-D-oligoglucosides with the 3-butenyl alcohols and a Lewis acid (SnCl4) catalyst. Epoxidation of the peracetylated 3-butenyl oligoglucosides took place with m-chloroperoxybenzoic acid (m-CPBA). NaOMe in dry methanol was used for the deacetylation of the blocked derivatives, to give the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside mixture in an overall yield of 21%. The sample was analyzed by positive-ion electrospray ionization mass spectrometry (ESIMS). In a 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside-binding (1-->3)-beta-D-glucanase assay, we found that the (1-->3)-beta-D-glucanase was obviously inactivated by the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucosides. At the same time, we found the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside mixture was more active as compared to the underivatized oligoglucoside mixture in eliciting phytoalexin accumulation in tobacco cotyledon tissue. Furthermore, it could be kept for a longer time than a (1-->3)-beta-D-oligoglucoside mixture, which indicated it is much more stable than (1-->3)-beta-D-oligoglucosides.