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.

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.

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.

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 .

Bacteria in an intense competition for iron: Key component of the Campylobacter jejuni iron uptake system scavenges enterobactin hydrolysis product.

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 ∼ 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 ∼ 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.

Chemical and Biological Aspects of Nutritional Immunity-Perspectives for New Anti-Infectives that Target Iron Uptake Systems.

Upon bacterial infection, one of the defense mechanisms of the host is the withdrawal of essential metal ions, in particular iron, which leads to "nutritional immunity". However, bacteria have evolved strategies to overcome iron starvation, for example, by stealing iron from the host or other bacteria through specific iron chelators with high binding affinity. Fortunately, these complex interactions between the host and pathogen that lead to metal homeostasis provide several opportunities for interception and, thus, allow the development of novel antibacterial compounds. This Review focuses on iron, discusses recent highlights, and gives some future perspectives which are relevant in the fight against antibiotic resistance.

Light-Induced Activation of a Molybdenum Oxotransferase Model within a Ru(II)-Mo(VI) Dyad.

Nature uses molybdenum-containing enzymes to catalyze oxygen atom transfer (OAT) from water to organic substrates. In these enzymes, the two electrons that are released during the reaction are rapidly removed, one at a time, by spatially separated electron transfer units. Inspired by this design, a Ru(II)-Mo(VI) dyad was synthesized and characterized, with the aim of accelerating the rate-determining step in the cis-dioxo molybdenum-catalyzed OAT cycle, the transfer of an oxo ligand to triphenyl phosphine, via a photo-oxidation process. The dyad consists of a photoactive bis(bipyridyl)-phenanthroline ruthenium moiety that is covalently linked to a bioinspired cis-dioxo molybdenum thiosemicarbazone complex. The quantum yield and luminescence lifetimes of the dyad [Ru(bpy)2(L2)MoO2(solv)]2+ were determined. The major component of the luminescence decay in MeCN solution (τ = 1149 ± 2 ns, 67%) corresponds closely to the lifetime of excited [Ru(bpy)2(phen-NH2)]2+, while the minor component (τ = 320 ± 1 ns, 31%) matches that of [Ru(bpy)2(H2-L2)]2+. In addition, the (spectro)electrochemical properties of the system were investigated. Catalytic tests showed that the dyad-catalyzed OAT from dimethyl sulfoxide to triphenyl phosphine proceeds significantly faster upon irradiation with visible light than in the dark. Methylviologen acts as a mediator in the photoredox cycle, but it is regenerated and hence only required in stoichiometric amounts with respect to the catalyst rather than sacrificial amounts. It is proposed that oxidative quenching of the photoexcited Ru unit, followed by intramolecular electron transfer, leads to the production of a reactive one-electron oxidized catalyst, which is not accessible by electrochemical methods. A significant, but less pronounced, rate enhancement was observed when an analogous bimolecular system was tested, indicating that intramolecular electron transfer between the photosensitizer and the catalytic center is more efficient than intermolecular electron transfer between the separate components.

Probing bacterial uptake of glycosylated ciprofloxacin conjugates.

Mono- and disaccharide-functionalised conjugates of the fluoroquinolone antibiotic ciprofloxacin have been synthesised and used as chemical probes of the bacterial uptake of glycosylated ciprofloxacin. Their antimicrobial activities against a panel of clinically relevant bacteria were determined: the ability of these conjugates to inhibit their target DNA gyrase and to be transported into the bacteria was assessed by using in vivo and in vitro assays. The data suggest a lack of active uptake through sugar transporters and that although the addition of monosaccharides is compatible with the inhibition of DNA gyrase, the addition of a disaccharide results in a significant decrease in antimicrobial activity.

Probing linker design in citric acid-ciprofloxacin conjugates.

A series of structurally related citric acid-ciprofloxacin conjugates was synthesised to investigate the influence of the linker between citric acid and ciprofloxacin on antibacterial activities. Minimum inhibitory concentrations (MICs) were determined against a panel of reference strains and clinical isolates of bacteria associated with infection in humans and correlated with the DNA gyrase inhibitory activity. The observed trend was rationalised by computational modelling.

Enhancement of growth media for extreme iron limitation in Escherichia coli.

Iron is an essential nutrient for microbial growth and bacteria have evolved numerous routes to solubilize and scavenge this biometal, which is often present at very low concentrations in host tissue. We recently used a MOPS-based medium to induce iron limitation in Escherichia coli K-12 during the characterization of novel siderophore-conjugated antibiotics. In this study we confirm that growth media derived from commercially available M9 salts are unsuitable for studies of iron-limited growth, probably through the contamination of the sodium phosphate buffer components with over 100 µM iron. In contrast, MOPS-based media that are treated with metal-binding Chelex resin allow the free iron concentration to be reduced to growth-limiting levels. Despite these measures a small amount of E. coli growth is still observed in these iron-depleted media. By growing E. coli in conditions that theoretically increase the demand for iron-dependent enzymes, namely by replacing the glucose carbon source for acetate and by switching to a microaerobic atmosphere, we can reduce background growth even further. Finally, we demonstrate that by adding an exogeneous siderophore to the growth media which is poorly used by E. coli, we can completely prevent growth, perhaps mimicking the situation in host tissue. In conclusion, this short study provides practical experimental insight into low iron media and how to augment the growth conditions of E. coli for extreme iron-limited growth.

Redox-reversible siderophore-based catalyst anchoring within cross-linked artificial metalloenzyme aggregates enables enantioselectivity switching.

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.

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.

Staphyloferrin A as siderophore-component in fluoroquinolone-based Trojan horse antibiotics.

A series of fluoroquinolone conjugates was synthesised by linking the carboxylic acid functionality of the carboxylate-type siderophore staphyloferrin A and its derivatives to the piperazinyl nitrogen of ciprofloxacin and norfloxacin via amide bond formation. Four siderophore-drug conjugates were screened against a panel of bacteria associated with infection in humans. Whilst no activity was found against ciprofloxacin- or norfloxacin-resistant bacteria, one of the conjugates retained antibacterial activity against fluoroquinolone-susceptible strains although the structure of its lysine-based siderophore component differs from that of the natural siderophore staphyloferrin A. In contrast, three ornithine-based siderophore conjugates showed significantly reduced activity against strains that are susceptible to their respective parent fluoroquinolones, regardless of the type of fluoroquinolone attached or chirality at the ornithine Cα-atom. The loss of potency observed for the (R)- and (S)-ornithine-based ciprofloxacin conjugates correlates with their reduced inhibitory activity against the target enzyme DNA gyrase.

Interactions of a periplasmic binding protein with a tetradentate siderophore mimic.

Iron-bound structure: The ferric complex of a tetradentate siderophore mimic was synthesized and co-crystallized with the periplasmic binding protein CeuE of Campylobacter 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).

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.

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.

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.