RESUMO
The human enteropathogen Campylobacter jejuni, like many bacteria, employs siderophores such as enterobactin for cellular uptake of ferric iron. This transport process has been shown to be essential for virulence and presents an attractive opportunity for further study of the permissiveness of this pathway to small-molecule intervention and as inspiration for the development of synthetic carriers that may effectively transport cargo into Gram-negative bacteria. In this work, we have developed a facile and robust microscale assay to measure growth recovery of C. jejuni NCTC 11168 in liquid culture as a result of ferric iron uptake. In parallel, we have established the solid-phase synthesis of catecholamide compounds modeled on enterobactin fragments. Applying these methodological developments, we show that small synthetic iron chelators of minimal dimensions provide ferric iron to C. jejuni with equal or greater efficiency than enterobactin.
Assuntos
Campylobacter jejuni/metabolismo , Enterobactina/análogos & derivados , Ferro/química , Campylobacter jejuni/crescimento & desenvolvimento , Enterobactina/síntese química , Humanos , Ferro/metabolismo , Sideróforos/síntese química , Sideróforos/química , Técnicas de Síntese em Fase SólidaRESUMO
Siderophores, such as enterobactin (Ent), are small molecules that can be selectively imported into bacteria along with iron by cognate transporters. Siderophore conjugates are thus a promising strategy for delivering functional reagents into bacteria. In this work, we present an easy-to-perform, one-pot chemoenzymatic synthesis of functionalized monoglucosylated enterobactin (MGE). When functionalized MGE is conjugated to a rhodamine fluorophore, which affords RhB-Glc-Ent, it can selectively label Gram-negative bacteria that utilize Ent, including some E. coli strains and P. aeruginosa. V. cholerae, a bacterium that utilizes linearized Ent, can also be weakly targeted. Moreover, the targeting is effective under iron-limiting but not iron-rich conditions. Our results suggest that the RhB-Glc-Ent probe is sensitive not only to the bacterial strain but also to the iron condition in the environment.
Assuntos
Enterobactina/análogos & derivados , Escherichia coli/isolamento & purificação , Pseudomonas aeruginosa/isolamento & purificação , Proteínas de Bactérias/metabolismo , Cromatografia Líquida de Alta Pressão , Enterobactina/síntese química , Enterobactina/metabolismo , Escherichia coli/metabolismo , Glucosiltransferases/metabolismo , Glicosilação , Glicosiltransferases/metabolismo , Espectrometria de Massas , Microscopia , Pseudomonas aeruginosa/metabolismo , Rodaminas/químicaRESUMO
The E. coli siderophore enterobactin, one of the strongest Fe(III) chelators known to date, is also capable of binding Si(IV) under physiological conditions. We report on the synthesis and structural characterization of the tris(catecholate) Si(IV) -enterobactin complex and its Ge(IV) and Ti(IV) analogues. Comparative structural analysis, supported by quantum-chemical calculations, reveals the correlation between the ionic radius and the structural changes in enterobactin upon complexation.
Assuntos
Complexos de Coordenação/síntese química , Enterobactina/síntese química , Escherichia coli/metabolismo , Germânio/química , Silício/química , Titânio/química , Complexos de Coordenação/química , Cristalografia por Raios X , Enterobactina/química , Conformação Molecular , Sideróforos/química , Sideróforos/metabolismo , TermodinâmicaRESUMO
The synthesis of two new iron chelators built on the tris-l-serine trilactone scaffold of enterobactin and bearing a 8-hydroxyquinoline (oxinobactin) or 8-hydroxyquinoline-5-sulfonate (sulfoxinobactin) unit has been described. The X-ray structure of the ferric oxinobactin has been determined, exhibiting a slightly distorted octahedral environment for Fe(III) and a Δ configuration. The Fe(III) chelating properties have been examined by potentiometric and spectrophotometric titrations in methanol-water 80/20% w/w solvent for oxinobactin and in water for sulfoxinobactin. They reveal the extraordinarily complexing ability (pFe(III) values) of oxinobactin over the p[H] range 2-9, the pFe value at p[H] 7.4 being 32.8. This was supported by spectrophotometric competition showing that oxinobactin removes Fe(III) from ferric enterobactin at p[H] 7.4. In contrast, the Fe(III) affinity of sulfoxinobactin was largely lower as compared to oxinobactin but similar to that of the ligand O-TRENSOX having a TREN backbone. These results are discussed in relation to the predisposition by the trilactone scaffold of the chelating units. Some comparisons are also made with other quinoline-based ligands and hydroxypyridinonate ligand (hopobactin).
Assuntos
Enterobactina/síntese química , Oxiquinolina/química , Compostos de Sulfidrila/síntese química , Termodinâmica , Cristalografia por Raios X , Enterobactina/química , Modelos Moleculares , Estrutura Molecular , Compostos de Sulfidrila/químicaRESUMO
Oxinobactin, a siderophore analogue to enterobactin but possessing 8-hydroxyquinoline instead of catechol complexing subunits, has been synthesized starting from L-serine and 8-hydroxyquinoline. Comparative iron binding studies showed that oxinobactin is as effective as enterobactin for the complexation of Fe(III) at physiological pH but with improved complexing ability at acidic pH.
Assuntos
Enterobactina/química , Oxiquinolina/química , Ligação Competitiva , Catecóis/química , Quelantes/química , Quelantes/farmacologia , Enterobactina/síntese química , Enterobactina/farmacologia , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Ferro/química , Ligantes , Metanol/química , Modelos Químicos , Sideróforos/química , Solventes/química , Espectrofotometria Ultravioleta/métodosRESUMO
Several analogues of vanchrobactin, a catechol siderophore isolated from the bacterial fish pathogen Vibrio anguillarum serotype O2 strain RV22, have been synthesized. The biological evaluation of these novel compounds showed that most of them are active as siderophores, as determined by growth promotion assays using the producer strain, as well as V. anguillarum serotype O1, Salmonella enterica, and Erwinia chrysanthemi. These compounds also gave a positive chrome azurol-S (CAS) test. On the basis of these results, we were able to deduce some structure-activity relationships. Furthermore, we found an analogue with siderophore activity that has appropriate functionality (an amino group) for use as an antibiotic vector to be employed in a "Trojan horse strategy".
Assuntos
Enterobactina/análogos & derivados , Sideróforos/química , Vibrio/química , Desenho de Fármacos , Enterobactina/síntese química , Enterobactina/química , Enterobactina/farmacologia , Estrutura Molecular , Peptídeos , Salmonella/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Enterobactin (Ent), a prototypic bacterial siderophore, is modified by both the C-glucosyltransferase IroB and the macrolactone hydrolase IroE in pathogenic bacteria that contain the iroA cluster. To investigate the possible effects of glucosylation and macrolactone hydrolysis on the physical properties of Ent, the membrane affinities and iron acquisition rates of Ent and Ent-derived siderophores were measured. The data obtained indicate that Ent has a high membrane affinity (K(x) = 1.5 x 10(4)) similar to that of ferric acinetoferrin, an amphiphile containing two eight-carbon hydrophobic chains. Glucosylation and macrolactone hydrolysis decrease the membrane affinity of Ent by 5-25-fold. Furthermore, in the presence of phospholipid vesicles, the iron acquisition rate is significantly increased by glucosylation and macrolactone hydrolysis, due to the resultant decrease in membrane sequestration of the siderophore. These results suggest that IroB and IroE enhance the ability of Ent-producing pathogens to acquire iron in membrane-rich microenvironments.
Assuntos
Bactérias/metabolismo , Membrana Celular/metabolismo , Enterobactina/síntese química , Enterobactina/metabolismo , Ferro/metabolismo , Proteínas de Bactérias/metabolismo , Enterobactina/química , Cinética , Modelos Moleculares , Conformação Molecular , Sideróforos/metabolismoRESUMO
The synthesis of two saccharide-based enterobactin analogues, methyl 2,3,4-tris-O[-N[2,3-di(hydroxy)benzoyl-glycyl]-aminopropyl]-alpha-D-glucopyranoside (H(6)L(A)) and methyl 2,3,4-tris-O-[N-[2,3-di(hydroxy)benzoyl]-aminopropyl]-alpha-D-glucopyranoside (H(6)L(B)), are reported along with their pK(a) values, Fe(III) binding constants, and aqueous solution speciation as determined by spectrophotometric and potentiometric titration techniques. Use of a saccharide platform to synthesize a hexadentate triscatechol chelator provides some advantages over other approaches to enterobactin models, including significant water solubility, resistance to hydrolysis, and backbone chirality which may provide favorable recognition and availability to cells. The protonation constants for the catechol ligand hydroxyl moieties were determined for both ligands and found to be significantly different, which is attributed to the differences in the spacer chain of the two triscatechols. Proton dependent Fe(III)-ligand equilibrium constants were determined using a model involving the sequential protonation of the Fe(III)-ligand complex. These results were used to calculate the formation constants, log beta(110) = 41.38 for Fe(III)-H(6)L(A) and log beta(110) = 46.38 for Fe(III)-H(6)L(B). The calculated pM values of 28.6 for H(6)L(A) and 28.3 for H(6)L(B) indicate that these ligands possess Fe(III) affinities comparable to or greater than other enterobactin models and are thermodynamically capable of removing Fe(III) from transferrin.
Assuntos
Enterobactina/análogos & derivados , Enterobactina/química , Enterobactina/síntese química , Compostos Férricos/química , Glucosídeos/síntese química , Ferro/química , Sideróforos/química , Algoritmos , Catecóis/química , Fenômenos Químicos , Físico-Química , Cromatografia Líquida de Alta Pressão , Enterobacteriaceae/química , Glucosídeos/química , Ferro/metabolismo , Ligantes , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Salicilatos/química , Sideróforos/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria UltravioletaRESUMO
The comparison of siderophore complex redox potentials with those of physiological reductants may aid in the clarification of the mechanism of iron metabolism. In this paper, a new chiral tris-catecholamide compound N,N',N''-tris-(2,3-dihydroxybenzoyl)-1,1,1-tris-(L-methioninemethyl++ +)-ethane or H6L (11) has been synthesised in nine steps, and may mimic the release of iron from enterobactin to the agents which are directly involved in cell metabolism. The choice of methionine as a constituent of the siderophore incorporates divalent sulphur which leads to the increase of the reduction potential of the siderophore, and consequently facilitates the iron release [Fe(III)/Fe(II) redox potential E(1/2)=-0.749 V vs (SCE)].
Assuntos
Enterobactina/síntese química , Metionina/análogos & derivados , Bactérias/química , Bactérias/efeitos dos fármacos , Proteínas de Bactérias/síntese química , Proteínas de Bactérias/metabolismo , Eletroquímica , Enterobactina/análogos & derivados , Enterobactina/metabolismo , Enterobactina/farmacologia , Ferro/metabolismo , Metionina/síntese química , Metionina/farmacologia , Oxirredução/efeitos dos fármacosRESUMO
Two examples of a new class of synthetic polycatecholate ligands, the carboxamido-2,3-dihydroxyterephthalate conjugates of spermine (8) and of spermidine (10), have been synthesized via the generally useful synthon methyl-2,3-dimethoxyterephthaloyl chloride (6). Initial biological evaluation reveals tetrameric terephthalate (8) to be an extremely effective agent for sequestering and removing plutonium from mice; a single 25-mumol/kg (ip) dose of 8 removed 73% of the plutonium citrate previously injected (iv, 1 h earlier). Under the same conditions, trimeric terephthalate (10) excreted only 49% of injected plutonium. In vitro kinetic experiments have shown that 10 rapidly and quantitatively removed Fe from human transferrin. These results are discussed in relation to the design of metal-ion specific sequestering agents.