RESUMO
The first non-natural derivative of the rare d-glucose-2-phosphate (G2P), namely glucose-2-(O-lactic acid phosphate) (G2LP), has been synthesized. When used as sole carbon source, G2LP enables bacterial growth of the plant pathogenic strain Agrobacterium fabrum C58 (formerly referred to as Agrobacterium tumefaciens). X-ray crystallography and affinity measurements investigations reveal that G2LP binds the periplasmic binding protein (PBP) AccA similarly to the natural compounds and with the same affinity. Moreover, enzymatic assays show that it is able to serve as substrate of the phosphodiesterase AccF. The properties found for G2LP demonstrate that the very unusual glucose-2-phosphoryl residue, present in G2LP, can be used as structural feature for designing non-natural systems fully compatible with the Acc cascade of A. fabrum.
Assuntos
Agrobacterium/química , Proteínas de Bactérias/metabolismo , Ésteres/síntese química , Glucofosfatos/síntese química , Proteínas Periplásmicas de Ligação/metabolismo , Agrobacterium/crescimento & desenvolvimento , Cristalografia por Raios X , Ésteres/química , Ésteres/metabolismo , Glucofosfatos/química , Glucofosfatos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Especificidade por SubstratoRESUMO
Synthetic methods were investigated for the preparation of O and S-glucosyl thiophosphates and glucosyl 1C-thiophosphonate. Four protected glucosyl thiophosphate compounds were synthesized and characterized as precursors to glucose 1-thiophosphate. The effect of various reaction conditions and the nature of the carbohydrate and thiophosphate protecting groups and how they impact both the yields and α/ß diastereoselectivity of the glucosyl thiophosphate products were explored. A novel isomerization from an O-linked to S-linked glucosyl thiophosphate was observed. α-D-Glucose-1C-thiophosphonate was synthesized and evaluated as a substrate for the thymidylyltransferase, Cps2L. Tandem mass spectrometric analysis determined the position of sulfur in the sugar nucleotide product.
Assuntos
Glucose/análogos & derivados , Glucofosfatos/química , Glucofosfatos/metabolismo , Nucleotidiltransferases/metabolismo , Fosfatos/metabolismo , Nucleotídeos de Timina/biossíntese , Nucleotídeos de Timina/metabolismo , Configuração de Carboidratos , Ativação Enzimática , Glucose/biossíntese , Glucose/química , Glucose/metabolismo , Glucofosfatos/síntese química , Fosfatos/química , Espectrometria de Massas em Tandem , Nucleotídeos de Timina/químicaRESUMO
GlmM and GlmU are key enzymes in the biosynthesis of UDP-N-acetyl-d-glucosamine (UDP-GlcNAc), an essential precursor of peptidoglycan and the rhamnose-GlcNAc linker region in the mycobacterial cell wall. These enzymes are involved in the conversion of two important precursors of UDP-GlcNAc, glucosamine-6-phosphate (GlcN-6-P) and glucosamine-1-phosphate (GlcN-1-P). GlmM converts GlcN-6-P to GlcN-1-P, GlmU is a bifunctional enzyme, whereby GlmU converts GlcN-1-P to GlcNAc-1-P and then catalyzes the formation of UDP-GlcNAc from GlcNAc-1-P and uridine triphosphate. In the present study, methyl 2-amino-2-deoxyl-α-d-glucopyranoside 6-phosphate (1α), methyl 2-amino-2-deoxyl-ß-d-glucopyranoside 6-phosphate (1ß), two analogs of GlcN-6-P, were synthesized as GlmM inhibitors; 2-azido-2-deoxy-α-d-glucopyranosyl phosphate (2) and 2-amino-2,3-dideoxy-3-fluoro-α-d-glucopyranosyl phosphate (3), analogs of GlcN-1-P, were synthesized firstly as GlmU inhibitors. Compounds 1α, 1ß, 2, and 3 as possible inhibitors of mycobacterial GlmM and GlmU are reported herein. Compound 3 showed promising inhibitory activities against GlmU, whereas 1α, 1ß and 2 were inactive against GlmM and GlmU even at high concentrations.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Parede Celular/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Complexos Multienzimáticos/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Inibidores Enzimáticos/química , Glucosamina/análogos & derivados , Glucosamina/síntese química , Glucosamina/química , Glucosamina/farmacologia , Glucose-6-Fosfato/análogos & derivados , Glucose-6-Fosfato/síntese química , Glucose-6-Fosfato/química , Glucose-6-Fosfato/farmacologia , Glucofosfatos/síntese química , Glucofosfatos/química , Glucofosfatos/farmacologia , Estrutura Molecular , Complexos Multienzimáticos/metabolismoRESUMO
The isomerization of beta-glucose-1-phosphate (betaG1P) to beta-glucose-6-phosphate (G6P) catalyzed by beta-phosphoglucomutase (betaPGM) has been examined using steady- and presteady-state kinetic analysis. In the presence of low concentrations of beta-glucose-1,6-bisphosphate (betaG16BP), the reaction proceeds through a Ping Pong Bi Bi mechanism with substrate inhibition (kcat = 65 s(-1), K(betaG1P) = 15 microM, K(betaG16BP) = 0.7 microM, Ki = 122 microM). If alphaG16BP is used as a cofactor, more complex kinetic behavior is observed, but the nonlinear progress curves can be fit to reveal further catalytic parameters (kcat = 74 s(-1), K(betaG1P) = 15 microM, K(betaG16BP) = 0.8 microM, Ki = 122 microM, K(alphaG16BP) = 91 microM for productive binding, K(alphaG16BP) = 21 microM for unproductive binding). These data reveal that variations in the substrate structure affect transition-state affinity (approximately 140,000-fold in terms of rate acceleration) substantially more than ground-state binding (110-fold in terms of binding affinity). When fluoride and magnesium ions are present, time-dependent inhibition of the betaPGM is observed. The concentration dependence of the parameters obtained from fitting these progress curves shows that a betaG1P x MgF3(-) x betaPGM inhibitory complex is formed under the reaction conditions. The overall stability constant for this complex is approximately 2 x 10(-16) M(5) and suggests an affinity of the MgF3(-) moiety to this transition-state analogue (TSA) of < or = 70 nM. The detailed kinetic analysis shows how a special type of TSA that does not exist in solution is assembled in the active site of an enzyme. Further experiments show that under the conditions of previous structural studies, phosphorylated glucose only persists when bound to the enzyme as the TSA. The preference for TSA formation when fluoride is present, and the hydrolysis of substrates when it is not, rules out the formation of a stable pentavalent phosphorane intermediate in the active site of betaPGM.
Assuntos
Inibidores Enzimáticos/farmacologia , Fluoretos/farmacologia , Compostos de Magnésio/farmacologia , Fosfoglucomutase/antagonistas & inibidores , Biocatálise , Cristalografia por Raios X , Inibidores Enzimáticos/química , Fluoretos/química , Glucofosfatos/síntese química , Glucofosfatos/química , Cinética , Compostos de Magnésio/química , Estrutura Molecular , Fosfoglucomutase/metabolismo , Ligação ProteicaRESUMO
The selective synthesis of 1,2-cis-hexofuranosyl 1-phosphates was readily accomplished according to a procedure based on the 'Remote Activation Concept'. This approach required (i) the preparation of suitable 1,2-trans-hexofuranosyl donors, so that new heterocyclic thiofuranosides were designed and synthesized, (ii) the stereocontrolled phosphorylation of the corresponding unprotected donors and (iii) the simple and fast purification of the resulting anomeric phosphates. This approach showed to be equally efficient in the galactose, glucose and mannose series.
Assuntos
Fosfatos Açúcares/síntese química , Galactosefosfatos/síntese química , Glucofosfatos/síntese química , Glicosídeos/síntese química , Manosefosfatos/síntese química , Fosforilação , Estereoisomerismo , Tioglicosídeos/químicaRESUMO
All six regioisomeric monophosphates of octyl beta-D-galactopyranosyl-(1 --> 4)-2-acetamido-2-deoxy-beta-D-glucopyranoside have been chemically synthesized and characterized by high-resolution 1H, 13C and 31P NMR spectroscopy. Phosphorylation causes characteristic downfield shifts of the nucleus at the substituted site in the 1H and 13C NMR signals and resulted in a unique 31P signal for each compound.
Assuntos
Glucofosfatos/síntese química , Ressonância Magnética Nuclear Biomolecular , Amino Açúcares/química , Dissacarídeos/química , Glucofosfatos/química , FosforilaçãoRESUMO
3,5-Cyclic phosphates and phosphoramides of 6-halogenated glucofuranoses were synthesized via interaction of 3,5,6-bicyclophosphites of 1,2-O-alkylidene-alpha-D-glucofuranoses with halogens (followed by treatment with nucleophilic reagents) and N-chloroamines. 3,5-Cyclic trans-dibutylphosphoramides of 6-chloro-6-deoxy-1,2-O-isopropylidene- and 6-chloro-6-deoxy-(R)-(2,2,2)-trichloroethylidene)-alpha-D-glucofuranoses were shown to possess antiproliferative activity against CaOv human ovarian carcinoma cells in vitro (CE50 of approximately 10(-5) M). Cyclic trans-dibutylphosphoramide of 6-chloro-6-deoxy-1,2,-O-isopropylidene-alpha-D-glucofuranose also displayed marked antitumor effect on P-388 transplantable murine leukemia in vivo (the maximum increase in life span of 100% was reached at the quintuple injection of 100 mg/kg daily).
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Glucofosfatos/síntese química , Leucemia P388/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Divisão Celular/efeitos dos fármacos , Glucofosfatos/farmacologia , Glucofosfatos/uso terapêutico , Leucemia P388/mortalidade , Espectroscopia de Ressonância Magnética , Camundongos , Relação Estrutura-Atividade , Células Tumorais CultivadasAssuntos
Escherichia coli/enzimologia , Glucofosfatos/biossíntese , Glucosiltransferases/biossíntese , Proteínas Recombinantes/biossíntese , Técnicas Bacteriológicas , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Clonagem Molecular/métodos , Fermentação , Genes Bacterianos , Glucofosfatos/síntese química , Glucosiltransferases/isolamento & purificação , Glucosiltransferases/metabolismo , Cinética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Linear tetra(N-acetylglucosaminyl)triphosphate GlcNAc(alpha)-P-3GlcNAc (alpha)-P-3GlcNAc(alpha)-P-3GlcNAc(beta)-ONp, a fragment of the capsular antigen of E. coli K51, was synthesized by the step-by-step approach with the use of the H-phosphonate method, starting the chain from p-nitrophenyl 2-acetamido-4,6-di-O-benzoyl-2-deoxy-beta-D-glucopyranoside. The elongation cycle included the coupling of 2-acetamido-4,6-di-O-benzoyl-2-deoxy-3-O-p-methoxybenzyl-alpha-D-gluc opy ranosyl H-phosphonate with a hydroxyl component in the presence of Me3CCOCl followed by oxidation (I2) and de (methoxybenzylation) (Ce(NH4)2(NO3)6). 2-Acetamido-3,4,6-tri-O-benzoyl-2-deoxy-alpha-D-glucopyranosyl H-phosphonate was employed in the final step. After mild debenzoylation the title tetramer was isolated by anion-exchange chromatography. The data of 1H, 13C and 31P NMR spectra of the synthesized oligomers are discussed.
Assuntos
Acetilglucosamina/síntese química , Antígenos de Bactérias/química , Biopolímeros , Escherichia coli/imunologia , Glucofosfatos/síntese química , Oligossacarídeos , Sequência de Carboidratos , Cromatografia por Troca Iônica , Espectroscopia de Ressonância Magnética , Dados de Sequência MolecularRESUMO
New 1-thyminyl-D-glucosamine-4-phosphate and 1-thyminyl-D-glucosamine-4,6-disulfate derivatives were synthesized. The 1-thyminyl-D-glucosamine-4,6-disulfate derivative showed antiviral activity against HIV.
Assuntos
Antivirais/síntese química , Glucosamina/análogos & derivados , Glucofosfatos/farmacologia , HIV/efeitos dos fármacos , Timina/análogos & derivados , Glucosamina/síntese química , Glucosamina/farmacologia , Glucofosfatos/síntese química , Timina/síntese química , Timina/farmacologiaAssuntos
Antineoplásicos/síntese química , Glucofosfatos/síntese química , Lipídeo A/análogos & derivados , Mitógenos/síntese química , Açúcares Ácidos/síntese química , Animais , Carcinoma de Ehrlich/tratamento farmacológico , Glucofosfatos/farmacologia , Glucofosfatos/toxicidade , Lipídeo A/farmacologia , Camundongos , Camundongos Endogâmicos , Açúcares Ácidos/farmacologia , Açúcares Ácidos/toxicidadeRESUMO
The synthesis of uridine diphosphate glucose (UDPG), cytidine diphosphate choline (CDP-choline), glucose-1-phosphate (G1P) and glucose-6-phosphate (G6P) has been accomplished under simulated prebiotic conditions using urea and cyanamide, two condensing agents considered to have been present on the primitive Earth. The synthesis of UDPG was carried out by reacting G1P and UTP at 70 degrees C for 24 hours in the presence of the condensing agents in an aqueous medium. CDP-choline was obtained under the same conditions by reacting choline phosphate and CTP X G1P and G6P were synthesized from glucose and inorganic phosphate at 70 degrees C for 16 hours. Separation and identification of the reaction products have been performed by paper chromatography, thin layer chromatography, enzymatic analysis and ion pair reverse phase high performance liquid chromatography. These results suggest that metabolic intermediates could have been synthesized on the primitive Earth from simple precursors by means of prebiotic condensing agents.
Assuntos
Colina/análogos & derivados , Citidina Difosfato Colina/síntese química , Uridina Difosfato Glucose/síntese química , Açúcares de Uridina Difosfato/síntese química , Citidina Trifosfato , Glucosefosfato Desidrogenase/metabolismo , Glucofosfatos/síntese química , Indicadores e Reagentes , Diester Fosfórico Hidrolases/metabolismo , FosforilcolinaAssuntos
Acetato Quinase/metabolismo , Trifosfato de Adenosina/metabolismo , Glucofosfatos/síntese química , Hexoquinase/metabolismo , Fosfotransferases/metabolismo , Difosfato de Adenosina/metabolismo , Dextranos , Glucose-6-Fosfato , Indicadores e Reagentes , Cinética , Métodos , Polietilenoglicóis/síntese química , Sulfatos/síntese químicaRESUMO
Methyl 6-(ammonium 2-acetamido-2-deoxy-alpha-D-glucopyranosyl phosphate)-alpha-D-mannopyranoside was synthesized and identified by 1H-n.m.r. and 13C-n.m.r. data, acid hydrolysis, and elemental analysis. It was utilized for the determination of UDP-N-acetylglucosamine-1-phosphotransferase in an assay procedure that employed methyl alpha-D-mannopyranoside as an acceptor. The assay product was identified and characterized by thin-layer chromatography with the title reference compound. The present technique does not require [32P]UDP-N-acetylglucosamine, but effectively uses commercially available UDP-[14C]GlcNAc.
Assuntos
Acetilglucosamina/análogos & derivados , Glucofosfatos/síntese química , Manosefosfatos , Fosfotransferases/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos) , Animais , Radioisótopos de Carbono , Linhagem Celular , Cromatografia em Camada Fina , Fibroblastos/enzimologia , Humanos , Indicadores e Reagentes , Lisossomos/enzimologia , Microssomos Hepáticos/enzimologia , Mucolipidoses/enzimologia , Ratos , Uridina Difosfato N-AcetilglicosaminaRESUMO
Rabbit muscle phosphoglucomutase converts alpha-D-glucose 1-[(S)-16O,17O,18O]phosphate into D-glucose 6-[16O,17O,18O]phosphate, which is shown by 31P n.m.r. spectroscopy, after cyclization and methylation, to have the (S)-configuration at the phosphorus atom. Since phosphoglucomutase is known to catalyse phosphoryl transfer by way of a phospho-enzyme intermediate, and since individual phosphoryl-transfer steps appear in general to occur with inversion of configuration, this observation is most simply interpreted in terms of a double-displacement mechanism with two phosphoryl-transfer steps.