RESUMO
Three artificial imine reductases, constructed via supramolecular anchoring utilising FeIII-azotochelin, a natural siderophore, to bind an iridium-containing catalyst to periplasmic siderophore-binding protein (PBP) scaffolds, have previously been synthesised and subjected to catalytic testing. Despite exhibiting high homology and possessing conserved siderophore anchor coordinating residues, the three artificial metalloenzymes (ArMs) displayed significant variability in turnover frequencies (TOFs). To further understand the catalytic properties of these ArMs, their kinetic behaviour was evaluated with respect to the reduction of three cyclic imines: dihydroisoquinoline, harmaline, and papaverine. Kinetic analyses revealed that all examined ArMs adhere to Michaelis-Menten kinetics, with the most pronounced saturation profile observed for the substrate harmaline. Additionally, molecular docking studies suggested varied hydrogen-bonding interactions between substrates and residues within the artificial binding pocket. Pi-stacking and pi-cation interactions were identified for harmaline and papaverine, corroborating the higher affinity of these substrates for the ArMs in comparison to dihydroisoquinoline. Furthermore, it was demonstrated that multiple cavities are capable of accommodating substrates in close proximity to the catalytic centre, thereby rationalising the moderate enantioselectivity conferred by the unmodified scaffolds.
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The immobilisation of artificial metalloenzymes (ArMs) holds promise for the implementation of new biocatalytic reactions. We present the synthesis of cross-linked artificial metalloenzyme aggregates (CLArMAs) with excellent recyclability, as an alternative to carrier-based immobilisation strategies. Furthermore, iron-siderophore supramolecular anchoring facilitates redox-triggered cofactor release, enabling CLArMAs to be recharged with alternative cofactors for diverse selectivity.
Assuntos
Oxirredução , Sideróforos , Sideróforos/química , Estereoisomerismo , Metaloproteínas/química , Metaloproteínas/metabolismo , Catálise , Biocatálise , Reagentes de Ligações Cruzadas/química , Ferro/químicaRESUMO
Technologies to improve the applicability of artificial metalloenzymes (ArMs) are gaining considerable interest; one such approach is the immobilization of these biohybrid catalysts on support materials to enhance stability and enable their retention, recovery, and reuse. Here, we describe the immobilization of polyhistidine-tagged ArMs that allow the redox-controlled replacement of catalytic cofactors that have lost activity, e.g., due to poisoning or decomposition, on immobilized metal affinity chromatography resins. By using periplasmic siderophore-binding protein scaffolds that originate from thermophilic bacteria (GstCeuE and PthCeuE) in combination with a siderophore-linked imine reduction catalyst, reaction rates were achieved that are about 3.5 times faster than those previously obtained with CjCeuE, the analogous protein of Campylobacter jejuni. Upon immobilization, the GstCeuE-derived ArM showed a decrease in turnover frequency in the reduction of dehydrosalsolidine by 3.4-fold, while retaining enantioselectivity (36%) and showing improved stability that allowed repeat recovery and recycling cycles. Catalytic activity was preserved over the initial four cycles. In subsequent cycles, a gradual reduction of activity was evident. Once the initial activity decreased to around 40% of the initial activity (23rd recycling cycle), the redox-triggered artificial cofactor release permitted the subsequent recharging of the immobilized protein scaffold with fresh, active cofactor, thereby restoring the initial catalytic activity of the immobilized ArM and allowing its reuse for several more cycles. Furthermore, the ArM could be assembled directly from protein present in crude cell extracts, avoiding time-consuming and costly protein purification steps. Overall, this study demonstrates that the immobilization of redox-reversible ArMs facilitates their "catch-and-release" assembly and disassembly and the recycling of their components, improving their potential commercial viability and environmental footprint.
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Transition-metal-based hydrogenation catalysts have applications ranging from high-value chemical synthesis to medicinal chemistry. A series of (pyridinylmethyl)sulfonamide ligands substituted with electron-withdrawing and -donating groups were synthesized to study the influence of the electronic contribution of the bidentate ligand in Cp*Ir piano-stool complexes. A variable-temperature NMR investigation revealed a strong correlation between the electron-donating ability of the substituent and the rate of stereoinversion of the complexes. This correlation was partially reflected in the catalytic activity of the corresponding catalysts. Complexes with electron-withdrawing substituents followed the trend observed in the variable-temperature NMR study, thereby confirming the rate-determining step to be donation of the hydride ligand. Strongly electron-donating groups, on the other hand, caused a change in the rate-determining step in the formation of the iridium-hydride species. These results demonstrate that the activity of these catalysts can be tuned systematically via changes in the electronic contribution of the bidentate (pyridinylmethyl)sulfonamide ligands.
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We report an electrochemical setup comprising a boron-doped diamond (BDD) working electrode for the electrochemical study of iron(III) catecholate siderophores. We demonstrate its successful application in the voltammetric investigation of iron(III) azotochelin, an iron complex of a bis(catecholate) siderophore. Cyclic voltammetry results, when complemented by UV-vis and native electrospray ionization-mass spectrometry (ESI-MS) characterization, reveal the formation of a coordinatively unsaturated tetracoordinate 1:1 complex of Fe:azotochelin (M1:L1) at neutral pH, contrary to iron(III) tetradentate siderophore complexes of other classes which favor the hexacoordinate environment of an M2:L3 species. A notable effect of pH and buffer composition on the reduction potential of iron(III) azotochelin is demonstrated. Lower pH values and buffers encompassing primary or secondary amines facilitate a positive potential shift of up to +290 mV and +250 mV vs Ag/AgCl 3 M NaCl, respectively. The study was extended to the investigation of the iron(III) complexes of hexadentate siderophores. For tris(catecholate) siderophores, enterobactin and protochelin, the reduction potentials were found to lie beyond the potential window accessible to the BDD electrode; however, we were successful in observing the electrochemical behavior of a tris(hydroxamate) siderophore, ferricrocin.
Assuntos
Compostos Férricos , Sideróforos , Sideróforos/química , Compostos Férricos/química , Ferro/química , LisinaRESUMO
Streptococcus pneumoniae (Spn) is an important Gram-positive human pathogen that causes millions of infections worldwide with an increasing occurrence of antibiotic resistance. Fe acquisition is a crucial virulence determinant in Spn; further, Spn relies on exogenous FeIII-siderophore scavenging to meet nutritional Fe needs. Recent studies suggest that the human catecholamine stress hormone, norepinephrine (NE), facilitates Fe acquisition in Spn under conditions of transferrin-mediated Fe starvation. Here we show that the solute binding lipoprotein PiuA from the piu Fe acquisition ABC transporter PiuBCDA, previously described as an Fe-hemin binding protein, binds tetradentate catechol FeIII complexes, including NE and the hydrolysis products of enterobactin. Two protein-derived ligands (H238, Y300) create a coordinately saturated FeIII complex, which parallel recent studies in the Gram-negative intestinal pathogen Campylobacter jejuni. Our in vitro studies using NMR spectroscopy and 54Fe LC-ICP-MS confirm the FeIII can move from transferrin to apo-PiuA in an NE-dependent manner. Structural analysis of PiuA FeIII-bis-catechol and GaIII-bis-catechol and GaIII-(NE)2 complexes by NMR spectroscopy reveals only localized structural perturbations in PiuA upon ligand binding, largely consistent with recent descriptions of other solute binding proteins of type II ABC transporters. We speculate that tetradentate FeIII complexes formed by mono- and bis-catechol species are important Fe sources in Gram-positive human pathogens, since PiuA functions in the same way as SstD from Staphylococcus aureus.
Assuntos
Catecóis/metabolismo , Compostos Férricos/metabolismo , Streptococcus pneumoniae/metabolismo , Sequência de Aminoácidos , Catecóis/química , Cristalografia por Raios X , Compostos Férricos/química , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Infecções Pneumocócicas/metabolismo , Infecções Pneumocócicas/microbiologia , Conformação Proteica , Streptococcus pneumoniae/químicaRESUMO
Streptococcus pneumoniae is a Gram-positive human pathogen that causes millions of infections worldwide with an increasing occurrence of antibiotic resistance. Iron acquisition is essential for its survival and virulence, especially under host-imposed nutritional immunity. S. pneumoniae expresses several ATP-binding cassette (ABC) transporters to facilitate acquisition under iron limitation, including PitABCD, PiaABCD, and PiuBCDA. The substrate specificity of PiuBCDA is not fully established. Herein, we report the backbone 1H, 13C and 15N resonance assignments of the 31 kDa soluble, extracellular domain of the substrate binding protein PiuA in the apo form and in complex with Ga(III) and the catechol siderophore-mimic 4-LICAM. These studies provide valuable information for further functional studies of interactions with other proteins, metals, and small molecules.
Assuntos
Transportadores de Cassetes de Ligação de ATP/análise , Apoproteínas/química , Proteínas de Bactérias/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Espectroscopia de Prótons por Ressonância Magnética , Streptococcus pneumoniae/metabolismo , Sequência de Aminoácidos , Isótopos de NitrogênioRESUMO
Anchoring a homogeneous catalyst onto a heterogeneous support facilitates separation of the product from the catalyst, and catalyst-substrate interactions can also modify reactivity. Herein we describe the synthesis of composite materials comprising carbon nitride (g-C3 N4 ) as the heterogeneous support and the well-established homogeneous catalyst moiety [Cp*IrCl]+ (where Cp*=η5 -C5 Me5 ), commonly used for catalytic hydrogenation. Coordination of [Cp*IrCl]+ to g-C3 N4 occurs directly at exposed edge sites with a κ2 N,N' binding motif, leading to a primary inner coordination sphere analogous to known homogeneous complexes of the general class [Cp*IrCl(NN-κ2 N,N')]+ (where N,N'=a bidentate nitrogen ligand). Hydrogenation of unsaturated substrates using the composite catalyst is selective for terminal alkenes, which is attributed to the restricted steric environment of the outer coordination sphere at the edge-sites of g-C3 N4 .
RESUMO
IR spectroscopy and model structural studies show binding of ReCl(CO)3-fragments to carbon nitride (g-C3N4) occurs viaκ2 N,N' bidentate coordination.
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To acquire essential Fe(III), bacteria produce and secrete siderophores with high affinity and selectivity for Fe(III) to mediate its uptake into the cell. Here, we show that the periplasmic binding protein CeuE of Campylobacter jejuni, which was previously thought to bind the Fe(III) complex of the hexadentate siderophore enterobactin (Kd â¼ 0.4 ± 0.1 µM), preferentially binds the Fe(III) complex of the tetradentate enterobactin hydrolysis product bis(2,3-dihydroxybenzoyl-l-Ser) (H5-bisDHBS) (Kd = 10.1 ± 3.8 nM). The protein selects Λ-configured [Fe(bisDHBS)](2-) from a pool of diastereomeric Fe(III)-bisDHBS species that includes complexes with metal-to-ligand ratios of 1:1 and 2:3. Cocrystal structures show that, in addition to electrostatic interactions and hydrogen bonding, [Fe(bisDHBS)](2-) binds through coordination of His227 and Tyr288 to the iron center. Similar binding is observed for the Fe(III) complex of the bidentate hydrolysis product 2,3-dihydroxybenzoyl-l-Ser, [Fe(monoDHBS)2](3-) The mutation of His227 and Tyr288 to noncoordinating residues (H227L/Y288F) resulted in a substantial loss of affinity for [Fe(bisDHBS)](2-) (Kd â¼ 0.5 ± 0.2 µM). These results suggest a previously unidentified role for CeuE within the Fe(III) uptake system of C. jejuni, provide a molecular-level understanding of the underlying binding pocket adaptations, and rationalize reports on the use of enterobactin hydrolysis products by C. jejuni, Vibrio cholerae, and other bacteria with homologous periplasmic binding proteins.
Assuntos
Proteínas de Bactérias/química , Campylobacter jejuni/metabolismo , Proteínas de Transporte/química , Complexos de Coordenação/química , Enterobactina/metabolismo , Ferro/metabolismo , Sideróforos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzoatos/química , Benzoatos/metabolismo , Campylobacter jejuni/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Complexos de Coordenação/metabolismo , Cristalografia por Raios X , Expressão Gênica , Hidrazonas/química , Hidrazonas/metabolismo , Ligação de Hidrogênio , Hidrólise , Transporte de Íons , Proteínas de Ligação ao Ferro , Ligantes , Modelos Moleculares , Mutação , Ligação Proteica , Eletricidade Estática , EstereoisomerismoRESUMO
Iron-bound structure: The ferric complex of a tetradentate siderophore mimic was synthesized and co-crystallized with the periplasmic binding protein CeuE of Campylobacterâ jejuni. In addition to electrostatic and hydrogen-bonding interactions between the binding pocket and the substrate, the structure showed direct coordination of two amino acid side chains to the Fe(III) center (orange, see figure).
Assuntos
Complexos de Coordenação/química , Proteínas Periplásmicas de Ligação/química , Sideróforos/química , Sequência de Aminoácidos , Materiais Biomiméticos/química , Modelos MolecularesRESUMO
Photocatalytic CO(2) reduction has been studied for two dyads with porphyrin covalently attached to rhenium tricarbonyl bipyridine moieties, and on separate components consisting of [Re(CO)(3)(Picoline)Bpy](+) and either zinc porphyrin or zinc chlorin. TONs decrease in the order: zinc porphyrin + Re > long spacer dyad > zinc chlorin + Re > short spacer dyad.
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A series of eight pyruvate-based aroylhydrazones was synthesised and characterised. The reaction of the sodium salts of the aroylhydrazones with one equivalent of copper(II) chloride allowed the isolation of neutral 1:1 complexes in which the hydrazones occupy three basal coordination sites of a square pyramidal Cu(II)-centre, with two solvent molecules completing the coordination sphere. Structural details were obtained through the determination of the crystal structures of two representative pyruvate-based aroylhydrazones and three Cu(II) complexes. The evaluation of the antimycobacterial activity of the sodium salts of the eight pryruvate hydrazones showed that the compounds are essentially inactive in their anionic form. The corresponding neutral Cu(II) complexes, however, exhibit promising antimycobacterial activities if tested under high iron (8 µg Fe per mL) conditions. As observed for the related antimycobacterial agent isoniazid, the activity of the complexes decreases if the M. tuberculosis cells are grown under low iron (0.02 µg Fe per mL) conditions. The Cu(II) complexes may thus have a similar mode of action and may require an iron-containing heme-dependent peroxidase for activation.
Assuntos
Antituberculosos/química , Cobre/química , Hidrazonas/química , Ácido Pirúvico/química , Antituberculosos/farmacologia , Cobre/farmacologia , Hidrazonas/farmacologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/efeitos dos fármacosRESUMO
The intermolecular Pauson-Khand (PK) reactions of sterically comparable (2-phenylethynyl)heteroaromatic compounds with norbornene, mediated by Co(2)(CO)(8) to give cyclopentenone products, were examined in this study. A synthetic protocol utilizing focused-microwave dielectric heating proved indispensable in the efficient synthesis of the PK cyclopentenone products. "π-Deficient" heteroaromatic substrates, e.g., 2-pyrones, and some "π-excessive" heteroaromatics such as 2- and 3-thiophene and 2-furan favor the ß-position in the newly formed cyclopentenone ring. Other π-excessive heteroaromatics such as 2-pyrrole or 2-indole favor the α-position. A π-excessive 3-indole derivative gave a nearly equal mixture of regioisomers. The position of the nitrogen in pyridyl-containing alkyne substrates also affects the regiochemical outcome of the PK reaction. A 2-pyridyl alkyne, possessing a proximal nitrogen, influences the regioselectivity relative to a 4-pyridyl variant quite dramatically, favoring the ß-position in the newly formed cyclopentenone ring. A 2-pyrimidylalkyne exhibits similar behavior to the 2-pyridylalkyne. Compounds that do not participate in PK reactions with norbornene include (2-phenylethynyl)imidazoles and the related benzimidazoles, which promote rapid decomposition of the in situ generated (µ(2)-alkyne)Co(2)(CO)(6) complexes. This stands in contrast with other nitrogen-containing heteroaromatics, e.g., pyrrole-, indole-, and pyrimidine-derived compounds, which effectively undergo PK reactions. Overall, the type of heteroaromatic group dramatically influences PK regioselectivity, which can in part be explained by rationalization of the current reaction mechanism, but not fully.
Assuntos
Alcinos/química , Cobalto/química , Ciclopentanos/síntese química , Norbornanos/química , Compostos Organometálicos/química , Ciclopentanos/química , Estrutura Molecular , Compostos Organometálicos/síntese química , EstereoisomerismoRESUMO
Nonpurine xanthine oxidoreductase (XOR) inhibitors represent important alternatives to the purine analogue allopurinol, which is still the most widely used drug in the treatment of conditions associated with elevated uric acid levels in the blood. By condensing mono-, di- and trihydroxybenzaldehydes with aromatic thiosemicarbazides, aryl hydrazides and dithiocarbazates, three series of structurally related Schiff bases were synthesised, characterised and tested for XOR inhibitory activity. Hydroxy substitution in the para-position of the benzaldehyde component was found to confer high inhibitory activities. Acyl hydrazones were generally less potent than thiocarbonyl-containing Schiff bases. Within the thiosemicarbazone series, chloro and cyano substituents in the para-position of the thiosemicarbazide unit increased activities further, up to potencies approximately four-times higher than that of the benchmark allopurinol, as measured under the same assay conditions. In order to illustrate the potential of the Schiff bases to bind directly to the molybdenum centre in the active site of the enzyme, a representative example (H2L) of each inhibitor series was co-ordinated to a cis-dioxomolybdenum(VI) unit, and the resulting complexes, [MoO2(L)MeOH], were structurally characterised. Subsequent steady-state kinetic investigations, however, indicated mixed-type inhibition, similar to that observed for inhibitors known to bind within the substrate access channel of the enzyme, remote from the Mo centre. Enzyme co-crystallisation studies are thus required to determine the exact binding mode. Finally, the coordination of representative inhibitors to copper(II) gave rise to significantly decreased IC50 values, revealing an additive effect that merits further investigation.
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Benzaldeídos/química , Inibidores Enzimáticos/farmacologia , Hidrazinas/farmacologia , Hidrazonas/farmacologia , Tiossemicarbazonas/farmacologia , Xantina Oxidase/antagonistas & inibidores , Cobre/química , Inibidores Enzimáticos/química , Hidrazinas/química , Hidrazonas/química , Molibdênio/química , Bases de Schiff/química , Relação Estrutura-Atividade , Tiossemicarbazonas/química , Xantina Oxidase/metabolismoAssuntos
Benzaldeídos/química , Inibidores Enzimáticos/farmacologia , Molibdênio/química , Compostos Organometálicos/farmacologia , Bases de Schiff/química , Xantina Oxidase/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Compostos Organometálicos/síntese química , Compostos Organometálicos/química , Relação Estrutura-Atividade , Ácido Úrico/metabolismoRESUMO
A heteroditopic ligand H(2)-L consisting of a dihydroxybenzene (catechol)-unit linked via an amide bond to a pyridyl-unit and its methyl-protected precursor Me(2)-L were synthesized, characterized, and their photophysical properties investigated. The three accessible protonation states of the ligand, H(3)-L(+), H(2)-L, and H-L(-), showed distinct (1)H NMR, absorption and emission spectroscopic characteristics that allow pH-sensing. The spectroscopic signatures obtained act as a guide to understand the signaling mechanism of the luminescent pH and molybdate sensor [Re(bpy)(CO)(3)(H(2)-L)](+). It was found that upon deprotonation of the 2-hydroxy group of H(2)-L, a ligand-based absorption band emerges that overlaps with the Re(dπ)âbpy metal-to-ligand charge transfer (MLCT) band of the sensor, reducing the quantum yield for emission on excitation in the 370 nm region. In addition, deprotonation of the catechol-unit leads to quenching of the emission from the Re(dπ)âbpy (3)MLCT state, consistent with photoinduced electron transfer from the electron-rich, deprotonated catecholate to the Re-based luminophore. Finally, reaction of 2 equiv of [Re(bpy)(CO)(3)(H(2)-L)](+) with molybdate was shown to give the zwitterionic Mo(VI) complex [MoO(2){Re(CO)(3)(bpy)(L)}(2)], as confirmed by electrospray ionization (ESI) mass spectrometry and X-ray crystallography. The crystal structure determination revealed that two fully deprotonated sensor molecules are bound via their oxygen-donors to a cis-dioxo-MoO(2) center.
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This perspective illustrates the principles and applications of molecular recognition directed binding of transition metal complexes to proteins. After a brief introduction into non-covalent interactions and the importance of complementarity, the focus of the first part is on biological systems that rely on non-covalent forces for metal complex binding, such as proteins involved in bacterial iron uptake and the oxygen-storage protein myoglobin. The second part of the perspective will illustrate how the replacement of native with non-native metal-centres can give rise to artificial metalloenzymes with novel catalytic properties. Subsequently, examples of spectroscopic probes that exploit the characteristic photophysical properties of metal-complexes for the non-covalent labelling, visualisation and investigation of proteins will be described. Finally, the use of kinetically inert metal complexes as scaffolds in drug design will be discussed and it will be highlighted how the binding of metal ions or organometallic fragments to existing drugs or drug candidates can improve their activity or even alter their mode of action.
Assuntos
Metaloproteínas/química , Elementos de Transição/química , Desenho de Fármacos , Metaloproteínas/síntese química , Ligação ProteicaRESUMO
An evaluation of the CO releasing ability of iron(II) and molybdenum(II) complexes has facilitated the discovery of the most rapid CO releaser, namely [Mo(CO)(3)(eta(5)-C(5)H(5))(eta(1)-{O}-C{=O}-O-CMe=CH-COMe=CBr)]BF(4) (CORM-F10), reported to date. The rate of CO release is related to the overall solution phase stability of the transition metal carbonyl complex. The cytotoxicity and vasodilatory properties of CORM-F10 have been determined.