Catch-and-Release: The Assembly, Immobilization, and Recycling of Redox-Reversible Artificial Metalloenzymes.

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.

Effect of Ligand Substituents on Spectroscopic and Catalytic Properties of Water-Compatible Cp*Ir-(pyridinylmethyl)sulfonamide-Based Transfer Hydrogenation Catalysts.

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.

Identification of a system for hydroxamate xenosiderophore-mediated iron transport in Burkholderia cenocepacia.

One of the mechanisms employed by the opportunistic pathogen Burkholderia cenocepacia to acquire the essential element iron is the production and release of two ferric iron chelating compounds (siderophores), ornibactin and pyochelin. Here we show that B. cenocepacia is also able to take advantage of a range of siderophores produced by other bacteria and fungi ('xenosiderophores') that chelate iron exclusively by means of hydroxamate groups. These include the tris-hydroxamate siderophores ferrioxamine B, ferrichrome, ferricrocin and triacetylfusarinine C, the bis-hydroxamates alcaligin and rhodotorulic acid, and the monohydroxamate siderophore cepabactin. We also show that of the 24 TonB-dependent transporters encoded by the B. cenocepacia genome, two (FhuA and FeuA) are involved in the uptake of hydroxamate xenosiderophores, with FhuA serving as the exclusive transporter of iron-loaded ferrioxamine B, triacetylfusarinine C, alcaligin and rhodotorulic acid, while both FhuA and FeuA are able to translocate ferrichrome-type siderophores across the outer membrane. Finally, we identified FhuB, a putative cytoplasmic membrane-anchored ferric-siderophore reductase, as being obligatory for utilization of all of the tested bis- and tris-hydroxamate xenosiderophores apart from alcaligin.

Thermostable homologues of the periplasmic siderophore-binding protein CeuE from Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius.

Siderophore-binding proteins from two thermophilic bacteria, Geobacillus stearothermophilus and Parageobacillus thermoglucosidasius, were identified from a search of sequence databases, cloned and overexpressed. They are homologues of the well characterized protein CjCeuE from Campylobacter jejuni. The iron-binding histidine and tyrosine residues are conserved in both thermophiles. Crystal structures were determined of the apo proteins and of their complexes with iron(III)-azotochelin and its analogue iron(III)-5-LICAM. The thermostability of both homologues was shown to be about 20°C higher than that of CjCeuE. Similarly, the tolerance of the homologues to the organic solvent dimethylformamide (DMF) was enhanced, as reflected by the respective binding constants for these ligands measured in aqueous buffer at pH 7.5 in the absence and presence of 10% and 20% DMF. Consequently, these thermophilic homologues offer advantages in the development of artificial metalloenzymes using the CeuE family.

Siderophore-Linked Ruthenium Catalysts for Targeted Allyl Ester Prodrug Activation within Bacterial Cells.

Due to rising resistance, new antibacterial strategies are needed, including methods for targeted antibiotic release. As targeting vectors, chelating molecules called siderophores that are released by bacteria to acquire iron have been investigated for conjugation to antibacterials, leading to the clinically approved drug cefiderocol. The use of small-molecule catalysts for prodrug activation within cells has shown promise in recent years, and here we investigate siderophore-linked ruthenium catalysts for the activation of antibacterial prodrugs within cells. Moxifloxacin-based prodrugs were synthesised, and their catalyst-mediated activation was demonstrated under anaerobic, biologically relevant conditions. In the absence of catalyst, decreased antibacterial activities were observed compared to moxifloxacin versus Escherichia coli K12 (BW25113). A series of siderophore-linked ruthenium catalysts were investigated for prodrug activation, all of which displayed a combinative antibacterial effect with the prodrug, whereas a representative example displayed little toxicity against mammalian cell lines. By employing complementary bacterial growth assays, conjugates containing siderophore units based on catechol and azotochelin were found to be most promising for intracellular prodrug activation.

Electrochemical and Solution Structural Characterization of Fe(III) Azotochelin Complexes: Examining the Coordination Behavior of a Tetradentate Siderophore.

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.

Antibiotic-functionalized gold nanoparticles for the detection of active ß-lactamases.

Antimicrobial resistance (AMR) continues to threaten the effective treatment and prevention of bacterial infections. The spread of resistant infections is accelerated by the lack of fast and cost-effective tests for the detection of AMR at the point-of-care. We aimed to address this challenge by developing a diagnostic tool to detect one of the major forms of AMR, the ß-lactamase enzymes. Antibiotic-functionalized gold nanoparticles (AuNPs) have been successfully developed for the detection of ß-lactamases in challenging biological media, namely undiluted urine. Furthermore, these tools are compatible with samples containing a urine sample preservative (boric acid) or hematuria (blood). The functionalized AuNPs interact with the active ß-lactamases, resulting in the hydrolysis of the surface-bound antibiotics, which then inhibits binding of the AuNPs to a capture protein (a penicillin-binding protein) to indicate the presence of active ß-lactamases. We successfully integrated the antibiotic-functionalized AuNPs into a new lateral flow assay (LFA), which can be used to detect active ß-lactamases down to the detection limit of 11 nM. While we demonstrate the use of antibiotic-functionalized AuNPs in an LFA format to provide a novel method of detecting active ß-lactamases, these functionalized AuNPs are amenable to a range of alternative diagnostic technologies and could lead to vital point-of-care diagnostics for the early detection of multi-drug resistant infections.

Synthesis and antimicrobial activity of an SO2-releasing siderophore conjugate.

A novel Trojan Horse conjugate consisting of an SO2-releasing 2,4-dinitrobenzenesulfonamide group attached to the monocatecholate siderophore aminochelin was synthesized to examine whether a bidentate catecholate siderophore unit could help potentiate the antimicrobial activity of SO2-releasing prodrugs. The conjugate obtained displays rapid SO2 release on reaction with glutathione, and proved more active against Staphylococcus aureus than a comparable SO2-releasing prodrug lacking the siderophore unit, although activity required micromolar concentrations. The conjugate was inactive against wild-type Escherichia coli, but activity was observed against an entA mutant strain that is unable to produce its major siderophores. Hence, the poor activity of the conjugate in wild-type E. coli may be due to the production of native siderophores that can compete with the conjugate for iron binding and uptake.

Unveiling the origin of photo-induced enhancement of oxidation catalysis at Mo(VI) centres of Ru(II)-Mo(VI) dyads.

Photo-induced oxidation-enhancement in biomimetic bridged Ru(ii)-Mo(vi) photo-catalyst is unexpectedly photo-activated in ps timescales. One-photon absorption generates an excited state where both photo-oxidized and photo-reduced catalytic centres are activated simultaneously and independently.

Experimental Methods for Evaluating the Bacterial Uptake of Trojan Horse Antibacterials.

The field of antibacterial siderophore conjugates, referred to as Trojan Horse antibacterials, has received increasing attention in recent years, driven by the rise of antimicrobial resistance. Trojan Horse antibacterials offer an opportunity to exploit the specific pathways present in bacteria for active iron uptake, potentially allowing the drugs to bypass membrane-associated resistance mechanisms. Hence, the Trojan Horse approach might enable the redesigning of old antibiotics and the development of antibacterials that target specific pathogens. Critical parts of evaluating such Trojan Horse antibacterials and improving their design are the quantification of their bacterial uptake and the identification of the pathways by which this occurs. In this minireview, we highlight a selection of the biological and chemical methods used to study the uptake of Trojan Horse antibacterials, exemplified with case studies, some of which have led to drug candidates in clinical development or approved antibiotics.

The Pneumococcal Iron Uptake Protein A (PiuA) Specifically Recognizes Tetradentate FeIIIbis- and Mono-Catechol Complexes.

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.

A Salmochelin S4-Inspired Ciprofloxacin Trojan Horse Conjugate.

A novel ciprofloxacin-siderophore Trojan Horse antimicrobial was prepared by incorporating key design features of salmochelin, a stealth siderophore that evades mammalian siderocalin capture via its glycosylated catechol units. Assessment of the antimicrobial activity of the conjugate revealed that attachment of the salmochelin mimic resulted in decreased potency, compared to ciprofloxacin, against two Escherichia coli strains, K12 and Nissle 1917, in both iron replete and deplete conditions. This observation could be attributed to a combination of reduced DNA gyrase inhibition, as confirmed by in vitro DNA gyrase assays, and reduced bacterial uptake. Uptake was monitored using radiolabeling with iron-mimetic 67Ga3+, which revealed limited cellular uptake in E. coli K12. In contrast, previously reported staphyloferrin-based conjugates displayed a measurable uptake in analogous 67Ga3+ labeling studies. These results suggest that, in the design of Trojan Horse antimicrobials, the choice of siderophore and the nature and length of the linker remain a significant challenge.

1H, 13C, 15N backbone resonance assignments of the apo and holo forms of the ABC transporter solute binding protein PiuA from Streptococcus pneumoniae.

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.

Carbon Nitride as a Ligand: Selective Hydrogenation of Terminal Alkenes Using [(η5 -C5 Me5 )IrCl(g-C3 N4 -κ2 N,N')]Cl.

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 .

Artificial imine reductases: developments and future directions.

Biocatalytic imine reduction has been a topic of intense research by the artificial metalloenzyme community in recent years. Artificial constructs, together with natural enzymes, have been engineered to produce chiral amines with high enantioselectivity. This review examines the design of the main classes of artificial imine reductases reported thus far and summarises approaches to enhancing their catalytic performance using complementary methods. Examples of utilising these biocatalysts in vivo or in multi-enzyme cascades have demonstrated the potential that artIREDs can offer, however, at this time their use in biocatalysis remains limited. This review explores the current scope of artIREDs and the strategies used for catalyst improvement, and examines the potential for artIREDs in the future.

Synthesis and biochemical evaluation of cephalosporin analogues equipped with chemical tethers.

Molecular probes typically require structural modifications to allow for the immobilisation or bioconjugation with a desired substrate but the effects of these changes are often not evaluated. Here, we set out to determine the effects of attaching functional handles to a first-generation cephalosporin. A series of cephalexin derivatives was prepared, equipped with chemical tethers suitable for the site-selective conjugation of antibiotics to functionalised surfaces. The tethers were positioned remotely from the ß-lactam ring to ensure minimal effect to the antibiotic's pharmacophore. Herein, the activity of the modified antibiotics was evaluated for binding to the therapeutic target, the penicillin binding proteins, and shown to maintain binding interactions. In addition, the deactivation of the modified drugs by four ß-lactamases (TEM-1, CTX-M-15, AmpC, NDM-1) was investigated and the effect of the tethers on the catalytic efficiencies determined. CTX-M-15 was found to favour hydrolysis of the parent antibiotic without a tether, whereas AmpC and NDM-1 were found to favour the modified analogues. Furthermore, the antimicrobial activity of the derivatives was evaluated to investigate the effect of the structural modifications on the antimicrobial activity of the parent drug, cephalexin.

Surface-Bound Antibiotic for the Detection of ß-Lactamases.

Antimicrobial resistance (AMR) has been identified as a major threat to public health worldwide. To ensure appropriate use of existing antibiotics, rapid and reliable tests of AMR are necessary. One of the most common and clinically important forms of bacterial resistance is to ß-lactam antibiotics (e.g., penicillin). This resistance is often caused by ß-lactamases, which hydrolyze ß-lactam drugs, rendering them ineffective. Current methods for detecting these enzymes require either time-consuming growth assays or antibiotic mimics such as nitrocefin. Here, we report the development of a surface-bound, clinically relevant ß-lactam drug that can be used to detect ß-lactamases and that is compatible with a range of high-sensitivity, low-cost, and label-free analytical techniques currently being developed for point-of-care-diagnostics. Furthermore, we demonstrate the use of these functionalized surfaces to selectively detect ß-lactamases in complex biological media, such as urine.

Carbon nitride as a ligand: edge-site coordination of ReCl(CO)3-fragments to g-C3N4.

IR spectroscopy and model structural studies show binding of ReCl(CO)3-fragments to carbon nitride (g-C3N4) occurs viaκ2 N,N' bidentate coordination.