RESUMO
Previously, monodisperse and sequence-controlled oligo(amidoamine) scaffolds were synthesized based on the step-wise assembly of tailor-made building blocks on a solid support that allow for the multivalent presentation of sugar ligands. Here, we extend on this concept using a split-and-combine approach to gain access to a small library of linear and branched glycomacromolecules. Azide side chains were introduced in the scaffold by the use of a novel building block allowing for copper-mediated azide-alkyne cycloaddition (CuAAC) of readily available propargyl-functionalized glycans. In the first stage, after assembly of the linear scaffold on solid support, the batch was divided into two. One part of the resin-bound oligomers was end-capped and further used as backbone and the other part was functionalized with propargylated α-d-mannopyranoside in the sidechain, end capped with an alkyne functionality and finally cleaved from solid support to give the branching arm. In the second stage, the linear, glycosylated and alkynylated arms were then coupled to the end capped backbone via CuAAC. In this way, branched glycomacromolecules with two and three branches, respectively, have been synthesized carrying from two to six sugar residues per molecule. Both, linear arms and branched glycomacromolecules were then subjected to a lectin binding assay using surface plasmon resonance (SPR) and model lectin Concanavalin A (Con A) showing the effect of branching as well as valency on the binding kinetics.
Assuntos
Lectinas/química , Manose/análogos & derivados , Manose/síntese química , Polissacarídeos/química , Bibliotecas de Moléculas Pequenas/síntese química , Alcinos/química , Azidas/química , Concanavalina A/síntese química , Cobre/química , Reação de Cicloadição , Cinética , Ligantes , Estereoisomerismo , TermodinâmicaRESUMO
We demonstrate microwave-induced heating of gold nanoparticles and nanorods. An appreciably higher and concentration-dependent microwave-induced heating rate was observed with aqueous dispersions of the nanomaterials as opposed to pure water and other controls. Grafted with the thermoresponsive polymer poly(N-isopropylacrylamide), these gold nanomaterials react to microwave-induced heating with a conformational change in the polymer shell, leading to particle aggregation. We subsequently covalently immobilize concanavalin A (Con A) on the thermoresponsive gold nanoparticles. Con A is a bioreceptor commonly used in bacterial sensors because of its affinity for carbohydrates on bacterial cell surfaces. The microwave-induced thermal transitions of the polymer reversibly switch on and off the display of Con A on the particle surface and hence the interactions of the nanomaterials with carbohydrate-functionalized surfaces. This effect was determined using linear sweep voltammetry on a methyl-α-d-mannopyranoside-functionalized electrode.
Assuntos
Resinas Acrílicas/química , Concanavalina A/química , Nanopartículas Metálicas/química , Resinas Acrílicas/síntese química , Concanavalina A/síntese química , Ouro/química , Micro-Ondas , Tamanho da Partícula , Propriedades de Superfície , TemperaturaRESUMO
A new triazatruxene-based fluorescent glycocluster has been designed, synthesized, and fully characterized by NMR spectroscopy and mass spectrometry. Furthermore, its specific and selective binding properties with concanavalin A (Con A) have been investigated by fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and turbidity assay. The obtained results showed that the multivalent mannose-modified triazatruxene exhibited specific binding with Con A, but no binding to peanut agglutinin (PNA) lectin or bovine serum albumin (BSA), corresponding to a two-orders-of-magnitude higher affinity than that of monovalent mannose ligands. Most interestingly, a fluorescence enhancement of the triazatruxene-based glycocluster was observed upon binding with Con A because of hydrophobic interactions involving sites close to the triazatruxene moiety. Furthermore, the inhibitory ability of the triazatruxene-based glycocluster against ORN178-induced haemagglutination has been investigated by haemagglutination inhibition assay. The results indicated selective binding with ORN178.
Assuntos
Carbazóis/química , Concanavalina A/química , Concanavalina A/síntese química , Escherichia coli/química , Corantes Fluorescentes/química , Aglutinação , Sítios de Ligação , Escherichia coli/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Espectroscopia de Ressonância Magnética , Espectrometria de MassasRESUMO
A virtual screening approach was used to identify new glycomimetics. The National Cancer Institute Diversity Set was docked into the carbohydrate binding site of the lectin concanavalin A (ConA). The resulting poses were analyzed and 19 molecules were tested for inhibition with an enzyme-linked lectin assay (ELLA). Eight of the 19 molecules inhibited ConA-carbohydrate binding. The two most potent inhibitors have IC(50) values that are an order of magnitude smaller than the monosaccharide methyl alpha-D-mannopyranoside.
Assuntos
Química Farmacêutica/métodos , Concanavalina A/síntese química , Concanavalina A/farmacologia , Motivos de Aminoácidos , Sítios de Ligação , Configuração de Carboidratos , Domínio Catalítico , Concanavalina A/química , Cristalografia por Raios X/métodos , Desenho de Fármacos , Fabaceae/metabolismo , Concentração Inibidora 50 , Lectinas/química , Metilmanosídeos/química , Modelos Químicos , Conformação Molecular , Ligação ProteicaRESUMO
A short and efficient strategy for the synthesis of multi-valent mannosides based on a selectively functionalized carbohydrate scaffold was reported involving (i) direct regioselective azidation of unprotected commercial saccharides, (ii) acetylation, (iii) grafting of the mannosyl ligands by click chemistry, and (iv) deacetylation. New glycoclusters with a valency ranging from 1 to 4 and different spatial arrangements of the epitopes were obtained. Binding affinities of the new glycoclusters toward concanavalin A (Con A) lectin were investigated by an enzyme-linked lectin essay (ELLA). The synthetic multi-valent compounds exhibited a remarkable cluster effect with a relative potency per mannoside residue ranging from 8.1 to 9.1 depending on the structures. ELLA experiments were in agreement with the establishment of favorable interactions between triazole ring and Con A, increasing the binding affinity. A new force field topology database was developed in agreement with the GLYCAM 2004 force field. Molecular dynamics performed on representative glyco-conjugates revealed interesting structural features such as rigidity of the scaffold for a well-defined presentation of the ligands and highly flexible mannose counterparts. The new glycoconjugates reported may be promising tools as probes or effectors of biological processes involving lectins.
Assuntos
Concanavalina A/síntese química , Lectinas/síntese química , Manosídeos/química , Acetilação , Azidas/química , Sítios de Ligação , Configuração de Carboidratos , Sequência de Carboidratos , Concanavalina A/metabolismo , Lectinas/metabolismo , Ligantes , Manosídeos/metabolismo , Modelos Químicos , Dados de Sequência Molecular , Triazóis/químicaRESUMO
The use of fluidizable affinity adsorbents for the adsorption of cells in expanded mode is investigated. Affinity adsorbents have been synthesized by immobilizing the lectin Concanavalin A onto the surface of triazine-activated perfluorocarbon-solids. The adsorbents were found to adsorb Saccharomyces cerevisiae cells from solution with adsorption capacities of up to 6.8 x 10(9) cells mL(-1). Adsorption kinetics were rapid with a time constant of =8 min. The adsorbed cells could be eluted using 500 mM methyl alpha,D-mannopyranoside, although the kinetics of release were slowed by the multipoint nature of the interaction. The dynamic capacity of the Con A PVA FEP in expanded mode was up to 4.5 x 10(9) cells mL(-1). The operating parameters of bed height, application flow rate, and adsorbent size distribution were investigated for any potential improvements in throughput, which may improve utility for more fragile cells. A decrease in settled-bed height from 20 to 5 cm resulted in a decrease in dynamic capacity of 27% from 4.5 to 3.3 x 10(9) cells mL(-1). An increase in application flow rate from 0.7 to 2.0 mL/min(-1) (resulting in an expansion increase from two- to fourfold) resulted in a 40% decrease in dynamic capacity from 4.0 to 2.4 x 10(9) cells mL(-1). An increase in the mean size distribution of the perfluorocarbon from 42 to 69 microm and therefore the flow rate needed for twofold expansion of 0.7 to 1.5 mL/min(-1) resulted in a 56% decrease in dynamic capacity from 4.0 to 1.8 x 10(9) cells mL(-1). The expanded bed, using certain combinations of the operating parameters, therefore shows significant potential for the robust, high efficiency and high capacity capture and separation of cells.
Assuntos
Aderência Bacteriana/fisiologia , Separação Celular/métodos , Cromatografia de Afinidade/métodos , Concanavalina A/análogos & derivados , Concanavalina A/farmacocinética , Saccharomyces cerevisiae/isolamento & purificação , Saccharomyces cerevisiae/metabolismo , Adsorção , Contagem de Células , Separação Celular/instrumentação , Cromatografia de Afinidade/instrumentação , Concanavalina A/síntese química , Enzimas Imobilizadas/farmacocinética , Fluorocarbonos/síntese química , Fluorocarbonos/química , Fluorocarbonos/farmacocinética , Membranas Artificiais , Saccharomyces cerevisiae/citologiaRESUMO
Concanavalin A, a specific glycoprotein probe, was optimally labelled to a maximum stoichiometry of 0.4 mol of chlorotriazinylaminofluorescein (CTAF)/mol of concanavalin A monomer under mild reaction conditions (pH 8.0, 6 h), and under these conditions the CTAF concanavalin A preparation retains its carbohydrate-binding ability and is able to penetrate SDS/7.5-15%-polyacrylamide gradient gels. CTAF-concanavalin A gives fluorescent bands for the glycoproteins transferrin, fetuin and deoxyribonuclease and shows no fluorescent response for the non-glycoproteins bovine serum albumin and soya-bean trypsin inhibitor. The detection limit of sensitivity for CTAF-concanavalin A, which is similar to that of fluorescein isothiocyanate-concanavalin A, is in the range 5-25 micrograms of glycoprotein. CTAF-concanavalin A is a suitable probe for the detection of glycoproteins in higher-percentage (greater than or equal to 10%) SDS/polyacrylamide gels, and will probably have other applications in, for example, fluorescent energy transfer and other structure-function studies.
Assuntos
Concanavalina A/síntese química , Eletroforese em Gel de Poliacrilamida/métodos , Fluoresceínas/síntese química , Corantes Fluorescentes/síntese química , Glicoproteínas/análise , Fenômenos Químicos , Química , Estabilidade de Medicamentos , EspectrofotometriaRESUMO
Concanavalin A (Con A) and trypsin inhibitor isolated from Acacia confusa were covalently linked with N-succinimidyl-3-(2-pyridyldithio)propionate. Con A-A. confusa trypsin inhibitor (ACTI) conjugate covalently bound (Con A-ACTI) retained about 42% of the trypsin inhibitory activity present in the native ACTI and had a higher hemagglutinating activity than did the native Con A. Con A-ACTI had a greater resistance to tryptic digestion than did the mixture of Con A and ACTI. The conjugate entered sarcoma 180 tumor cells, whereas the free ACTI did not. A single dose of the conjugate injected ip into noninbred N:NIH(S) white mice bearing sarcoma 180 had a remarkable effect of increasing the survival of tumor-bearing mice, while the mixture of an equivalent dose of free Con A and ACTI was not effective.
Assuntos
Concanavalina A/farmacologia , Sarcoma 180/metabolismo , Inibidores da Tripsina/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cromatografia em Gel , Concanavalina A/síntese química , Concanavalina A/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Testes de Hemaglutinação , Camundongos , Peptídeo Hidrolases/metabolismo , Ligação Proteica , Sarcoma 180/patologia , Inibidores da Tripsina/síntese química , Inibidores da Tripsina/metabolismoRESUMO
For the development of therapeutic agents that possess tissue-specific carriers, a method was devised to synthesize an artificial protein hybrid conjugate containing a moiety which binds to a cell membrane receptor and an active fragment of a toxic protein. By the introduction of an activated sulfhydryl group into concanavalin A (Con A), a conjugate of Con A and the ricin A-chain cross-linked with a disulfide linkage was synthesized. The purified conjugate was studied with regard to its inhibitory activity against protein synthesis in cell-free and cultured cell systems. The Con A-rich A-chain conjugate retained about one-third the inhibitory activity of ricin in a cell-free protein synthesis system. It also was highly toxic to cultured normal cells. These results indicate that the conjugate is a structural and functional analog of ricin and that the original membrane-binding chain (B-chain of ricin) could be replaced by Con A. Transformed cells were insensitive to this conjugate and required a longer preincubation time. The sensitivity of the normal cells was reduced in the presence of local anesthetics.