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A highly enantioselective aza-Darzens reaction (up to 99% ee) catalyzed by an enantiopure supramolecular host has been discovered. To understand the role of host structure on reaction outcome, nine new gallium(III)-based enantiopure supramolecular assemblies were prepared via substitution of the external chiral amide. Despite the distal nature of the substitution in these catalysts, changes in enantioselectivity (61 to 90% ee) in the aziridine product were observed. The enantioselectivities were correlated to the flexibility of the supramolecular host scaffold as measured by the kinetics of exchange of a model cationic guest. This correlation led to the development of a best-in-class catalyst by substituting the gallium(III)-based host with one based on indium(III), which generated the most flexible and selective catalyst.

The results of quantum chemical and molecular dynamics calculations reveal that polyanionic gallium-based cages accelerate cyclization reactions of pentadienyl alcohols as a result of substrate cage interactions, preferential binding of reactive conformations of substrate/H3O+ pairs, and increased substrate basicity. However, the increase in basicity dominates. Experimental structure-activity relationship studies in which the metal vertices and overall charge of the cage are varied confirm the model derived via calculations.

Supramolecular catalysts emulate the mechanism of enzymes to achieve large rate accelerations and precise selectivity under mild and aqueous conditions. While significant strides have been made in the supramolecular host-promoted synthesis of small molecules, applications of this reactivity to chemoselective and site-selective modification of complex biomolecules remain virtually unexplored. We report here a supramolecular system where coencapsulation of pyridine-borane with a variety of molecules including enones, ketones, aldehydes, oximes, hydrazones, and imines effects efficient reductions under basic aqueous conditions. Upon subjecting unprotected lysine to the host-mediated reductive amination conditions, we observed excellent ε-selectivity, indicating that differential guest binding within the same molecule is possible without sacrificing reactivity. Inspired by the post-translational modification of complex biomolecules by enzymatic systems, we then applied this supramolecular reaction to the site-selective labeling of a single lysine residue in an 11-amino acid peptide chain and human insulin.

Since the trailblazing discoveries of Lehn, Cram and Pedersen, supramolecular chemistry has established itself as a cornerstone of organic chemistry. Supramolecular hosts offer defined microenvironments that mimic the active sites of enzymes, utilizing specific host–guest interactions to enable remarkable rate enhancements and product selectivity. The development of a diverse array of self-assembled hosts, coupled with the increased demand for shorter and greener synthetic routes, have spurred significant progress in the field of supramolecular catalysis. This Review Article covers recent advances in the field, ranging from novel organic reactivity aided by supramolecular hosts to catalytic cooperation between hosts and organometallic compounds or metal nanoparticles. Strides have also been made in the synthetic application of these hosts in site-selective substrate modifications and challenging photochemical reactions. These efforts have enabled the incorporation of non-covalent macromolecular catalysis in natural product syntheses, evidencing their unique advantages as a synthetic tool, and their powerful potential for practical applications. [Figure not available: see fulltext.].

Although most of the currently developed supramolecular catalysts that emulate enzymatic reactivity with unique selectivity and activity through specific host-guest interactions work under homogeneous conditions, enzymes in nature can operate under heterogeneous conditions as membrane-bound enzymes. In order to develop such a heterogeneous system, an immobilized chiral supramolecular cluster Ga416 (2) was introduced into cross-linked polymers with cationic functionalities. These heterogeneous supramolecular catalysts were used in aza-Prins and aza-Cope reactions and successfully applied to continuous-flow reactions. They showed high durability and maintained high turnovers for long periods of time. In sharp contrast to the majority of examples of heterogenized homogeneous catalysts, the newly developed catalysts showed enhanced activity and robustness compared to those exhibited by the corresponding soluble cluster catalyst. An enantioenriched cluster was also immobilized to enable asymmetric catalysis, and activity and enantioselectivity of the supported chiral catalyst were maintained during recovery and reuse experiments and under a continuous-flow process. Significantly, the structure of the ammonium cations in the polymers affected stability, reactivity, and enantioselectivity, which is consistent with the hypothesis that the cationic moieties in the polymer support interact with cluster as an exohedral protecting shell, thereby influencing their catalytic performance.

It has been previously demonstrated that nanovessels can be highly competent catalysts providing high rate accelerations and unique selectivity to the organic transformations which they mediate. However, for supramolecular assemblies to be considered a standard reagent in organic synthesis they must first demonstrate the ability to catalyze increasingly complex transformations. Herein, we report a three-component Aza-Darzens reaction that generates N-phenylaziridines, catalyzed by a supramolecular host, that provides the stereoisomer opposite to the one generated in bulk solution (trans vs cis). This transformation constitutes a rare catalytic three-component coupling within a supramolecular assembly, providing a supramolecular solution to a synthetically challenging transformation.

Performing selective transformations on complex substrates remains a challenge in synthetic chemistry. These difficulties often arise due to cross-reactivity, particularly in the presence of similar functional groups at multiple sites. Therefore, there is a premium on the ability to perform selective activation of these functional groups. We report here a supramolecular strategy where encapsulation of a hydrogenation catalyst enables selective olefin hydrogenation, even in the presence of multiple sites of unsaturation. While the reaction requires at least one sterically nondemanding alkene substituent, the rate of hydrogenation is not sensitive to the distance between the alkene and the functional group, including a carboxylate, on the other substituent. This observation indicates that only the double bond has to be encapsulated to effect hydrogenation. Going further, we demonstrate that this supramolecular strategy can overcome the inherent allylic alcohol selectivity of the free catalyst, achieving supramolecular catalyst-directed regioselectivity as opposed to directing-group selectivity.

Siderocalin/Lipocalin 2/Neutrophil Gelatinase Associated Lipocalin/24p3 is an innate immune system protein with bacteriostatic activity, acting by tightly binding and sequestering diverse catecholate and mixed-type ferric siderophores from enteric bacteria and mycobacteria. Bacterial virulence achieved through siderophore modifications, or utilization of alternate siderophores, can be explained by evasion of Siderocalin binding. Siderocalin has also been implicated in a wide variety of disease processes, though often in seemingly contradictory ways, and has been proposed to bind to a broader array of ligands beyond siderophores. Using structural, directed mutational, and binding studies, we have sought to rigorously test, and fully elucidate, the Siderocalin recognition mechanism. Several proposed ligands fail to meet rigorous binding criteria, including the bacterial siderophore pyochelin, the iron-chelating catecholamine hormone norepinephrine, and the bacterial second messenger cyclic diguanylate monophosphate. While possessing a remarkably rigid structure, in principle simplifying analyses of ligand recognition, understanding Scn recognition is complicated by the observed conformational and stoichiometric plasticity, and instability, of its bona fide siderophore ligands. Since the role of Siderocalin at the early host/pathogen interface is to compete for bacterial ferric siderophores, we also analyzed how bacterial siderophore binding proteins and enzymes alternately recognize siderophores that efficiently bind to, or evade, Siderocalin sequestration - including determining the crystal structure of Bacillus cereus YfiY bound to schizokinen. These studies combine to refine the potential physiological functions of Siderocalin by defining its multiplexed recognition mechanism.

Supramolecular hosts offer defined microenvironments that facilitate selective host-guest interactions, enabling reactivity that would otherwise be challenging in bulk solution. While impressive rate enhancements and selectivities have been reported, similar reactivity can often be accessed through modifications of reaction conditions even in the absence of the host. We report here an oxidative addition of aryl halides across the metal centers in Cu(I) and Pd(II) organometallics that is assisted by the presence of a supramolecular host, realized via electrostatic stabilization and increased local substrate concentrations. When reaction conditions were screened to assess background reactivity, alternative reactivity (typically decomposition) resulted, indicating that encapsulation led to host-selective reaction trajectories.

We have demonstrated that the microenvironment of a highly anionic supramolecular catalyst can mimic the active sites of enzymes and impart rate accelerations of a million-fold or more. However, these microenvironments can be challenging to study, especially in the context of understanding which specific features of the catalyst are responsible for its high performance. We report here the development of an experimental mechanistic probe consisting of two isostructural catalysts. When examined in parallel transformations, the behavior of these catalysts provides insight relevant to the importance of anionic host charge on reactivity. These two catalysts exhibit similar host-substrate interactions, but feature a significant difference in overall anionic charge (12- and 8-). Within these systems, we compare the effect of constrictive binding in a net neutral aza-Cope rearrangement. We then demonstrate how the magnitude of anionic host charge has an exceptional influence on the reaction rates for a Nazarov cyclization, evidenced by an impressive 680-fold change in reaction rate as a consequence of a 33% reduction in catalyst charge.

This study offers a detailed mechanistic investigation of host-guest encapsulation behavior in a new enzyme-mimetic metal-ligand host and provides the first observation of a conformational selection mechanism (as opposed to induced fit) in a supramolecular system. The Ga4L4 host described features a C3-symmetric ligand motif with meta-substituted phenyl spacers, which enables the host to initially self-assemble into an S4-symmetric structure and then subsequently isomerize to a T-symmetric tetrahedron for better accommodation of a sufficiently large guest. Selective inversion recovery 1H NMR studies provide structural insights into the self-exchange behaviors of the host and the guest individually in this dynamic system. Kinetic analysis of the encapsulation-isomerization event revealed that increasing the concentration of guest inhibits the rate of host-guest relaxation, a key distinguishing feature of conformational selection. A comprehensive study of this simple enzyme mimic provides insight into analogous behavior in biophysics and enzymology and aims to inform the design of efficient self-assembled microenvironment catalysts.

We stand fully behind our earlier suggestion [Raymond & Girolami (2023). Acta Cryst. C79, 445–455] that the claim by Fish and co‐workers [Chen et al. (1995). J. Am. Chem. Soc.117, 9097–9098; Smith et al. (2014). Organometallics, 33, 2389–2404] of a linear two‐coordinate rhodium(I) species is incorrect, and that the putative rhodium atom is in fact silver. [ABSTRACT FROM AUTHOR]

A tetravalent cerium macrocyclic complex (CeLK4) was prepared with an octadentate terephthalamide ligand comprised of hard catecholate donors and characterized in the solution state by spectrophotometric titrations and electrochemistry and in the crystal by X-ray diffraction. The solution-state studies showed that L exhibits a remarkably high affinity toward Ce4+, with log β110 = 61(2) and ΔG = -348 kJ/mol, compared with log β110 = 32.02(2) for the analogous Pr3+ complex. In addition, L exhibits an unusual preference for forming CeL4- relative to formation of the analogous actinide complex, ThL4-, which has β110 = 53.7(5). The extreme stabilization of tetravalent cerium relative to its trivalent state is also evidenced by the shift of 1.91 V in the redox potential of the Ce3+/Ce4+ couple of the complex (measured at -0.454 V vs SHE). The unprecedented behavior prompted an electronic structure analysis using L3- and M5,4-edge X-ray absorption near-edge structure (XANES) spectroscopies and configuration interaction calculations, which showed that 4f-orbital bonding in CeLK4 has partial covalent character due to ligand-to-metal charge transfer (LMCT) in the ground state. The experimental results are presented in the context of earlier measurements on tetravalent cerium compounds, indicating that the amount of LMCT for CeLK4 is similar to that observed for [Et4N]2[CeCl6] and CeO2 and significantly less than that for the organometallic sandwich compound cerocene, (C8H8)2Ce. A simple model to rationalize changes in 4f orbital bonding for tri- and tetravalent lanthanide and actinide compounds is also provided.

The scope and mechanism of the microenvironment-catalyzed C(sp(3))-C(sp(3)) reductive elimination from transition metal complexes [Au(III), Pt(IV)] is explored. Experiments detailing the effect of structural perturbation of neutral and anionic spectator ligands, reactive alkyl ligands, solvent, and catalyst structure are disclosed. Indirect evidence for a coordinatively unsaturated encapsulated cationic intermediate is garnered via observation of several inactive donor-arrested inclusion complexes, including a crystallographically characterized encapsulated Au(III) cation. Finally, based on stoichiometric experiments under catalytically relevant conditions, a detailed mechanism is outlined for the dual supramolecular and platinum-catalyzed C-C coupling between methyl iodide and tetramethyltin. Determination of major platinum species present under catalytic conditions and subsequent investigation of their chemistry reveals an unexpected interplay between cis-trans isomerism and the supramolecular catalyst in a Pt(II)/Pt(IV) cycle, as well as several off-cycle reactions.

A hydroxypyridinone building block, bifunctionalized with thiazoline, has been prepared from orthogonally protected 2-(3-(benzyloxy)-4-(ethoxycarbonyl)-6-methyl-2-oxopyridin-1(2H)-yl) acetic acid. The reactivity of the dithiazolide has been explored with two primary amines, leading to the synthesis and characterization of four new hexadentate ligands. Their complexes with selected hard trivalent ions pertinent to potential molecular imaging applications have been surveyed.

The mechanism of proton exchange in a metal-ligand enzyme active site mimic (compound 1) is described through amide hydrogen-deuterium exchange kinetics. The type and ratio of cationic guest to host in solution affect the rate of isotope exchange, suggesting that the rate of exchange is driven by a host whose cavity is occupied by water. Rate constants for acid-, base-, and water-mediated proton exchange vary by orders of magnitude depending on the guest, and differ by up to 200 million-fold relative to an alanine polypeptide. These results suggest that the unusual microenvironment of the cavity of 1 can dramatically alter the reactivity of associated water by magnitudes comparable to that of enzymes.

This Account focuses on the coordination chemistry of the microbial iron chelators called siderophores. The initial research (early 1970s) focused on simple analogs of siderophores, which included hydroxamate, catecholate, or hydroxycarboxylate ligands. The subsequent work increasingly focused on the transport of siderophores and their microbial iron transport. Since these are pseudo-octahedral complexes often composed of bidentate ligands, there is chirality at the metal center that in principle is independent of the ligand chirality. It has been shown in many cases that chiral recognition of the complex occurs. Many techniques have been used to elucidate the iron uptake processes in both Gram-positive (single membrane) and Gram-negative (double membrane) bacteria. These have included the use of radioactive labels (of ligand, metal, or both), kinetically inert metal complexes, and Mössbauer spectroscopy. In general, siderophore recognition and transport involves receptors that recognize the metal chelate portion of the iron-siderophore complex. A second, to date less commonly found, mechanism called the siderophore shuttle involves the receptor binding an apo-siderophore. Since one of the primary ways that microbes compete with each other for iron stores is the strength of their competing siderophore complexes, it became important early on to characterize the solution thermodynamics of these species. Since the acidity of siderophores varies significantly, just the stability constant does not give a direct measure of the relative competitive strength of binding. For this reason, the pM value is compared. The pM, like pH, is a measure of the negative log of the free metal ion concentration, typically calculated at pH 7.4, and standard total concentrations of metal and ligand. The characterization of the electronic structure of ferric siderophores has done much to help explain the high stability of these complexes. A new chapter in siderophore science has emerged with the characterization of what are now called siderocalins. Initially found as a protein of the human innate immune system, these proteins bind both ferric and apo-siderophores to inactivate the siderophore transport system and hence deny iron to an invading pathogenic microbe. Siderocalins also can play a role in iron transport of the host, particularly in the early stages of fetal development. Finally, it is speculated that the molecular targets of siderocalins in different species differ based on the siderophore structures of the most important bacterial pathogens of those species.

UnlabelledSiderophores, small iron-binding molecules secreted by many microbial species, capture environmental iron for transport back into the cell. Vibrio cholerae synthesizes and uses the catechol siderophore vibriobactin and also uses siderophores secreted by other species, including enterobactin produced by Escherichia coli. E. coli secretes both canonical cyclic enterobactin and linear enterobactin derivatives likely derived from its cleavage by the enterobactin esterase Fes. We show here that V. cholerae does not use cyclic enterobactin but instead uses its linear derivatives. V. cholerae lacked both a receptor for efficient transport of cyclic enterobactin and enterobactin esterase to promote removal of iron from the ferrisiderophore complex. To further characterize the transport of catechol siderophores, we show that the linear enterobactin derivatives were transported into V. cholerae by either of the catechol siderophore receptors IrgA and VctA, which also transported the synthetic siderophore MECAM [1,3,5-N,N',N″-tris-(2,3-dihydroxybenzoyl)-triaminomethylbenzene]. Vibriobactin is transported via the additional catechol siderophore receptor ViuA, while the Vibrio fluvialis siderophore fluvibactin was transported by all three catechol receptors. ViuB, a putative V. cholerae siderophore-interacting protein (SIP), functionally substituted for the E. coli ferric reductase YqjH, which promotes the release of iron from the siderophore in the bacterial cytoplasm. In V. cholerae, ViuB was required for the use of vibriobactin but was not required for the use of MECAM, fluvibactin, ferrichrome, or the linear derivatives of enterobactin. This suggests the presence of another protein in V. cholerae capable of promoting the release of iron from these siderophores.ImportanceVibrio cholerae is a major human pathogen and also serves as a model for the Vibrionaceae, which include other serious human and fish pathogens. The ability of these species to persist and acquire essential nutrients, including iron, in the environment is epidemiologically important but not well understood. In this work, we characterize the ability of V. cholerae to acquire iron by using siderophores produced by other organisms. We resolve confusion in the literature regarding its ability to use the Escherichia coli siderophore enterobactin and identify the receptor and TonB system used for the transport of several siderophores. The use of some siderophores did not require the ferric reductase ViuB, suggesting that an uncharacterized ferric reductase is present in V. cholerae.

The K12Ga4L6 supramolecular cage is photoactive and enables an unprecedented photoreaction not observed in bulk solution. Ga4L6(12-) cages photosensitize the 1,3-rearrangement of encapsulated cinnamylammonium cation guests from the linear isomer to the higher energy branched isomer when irradiated with UVA light. The rearrangement requires light and guest encapsulation to occur. The Ga4L6(12-) cage-mediated reaction mechanism was investigated by UV/vis absorption, fluorescence, ultrafast transient absorption, and electrochemical experiments. The results support a photoinduced electron transfer mechanism for the 1,3-rearrangement, in which the Ga4L6(12-) cage absorbs photons and transfers an electron to the encapsulated cinnamylammonium ion, which undergoes C-N bond cleavage, followed by back electron transfer to the cage and recombination of the guest fragments to form the higher energy isomer.

The K12Ga4L6 supramolecular cage is photoactive and enables an unprecedented photoreaction not observed in bulk solution. Ga4L6(12-) cages photosensitize the 1,3-rearrangement of encapsulated cinnamylammonium cation guests from the linear isomer to the higher energy branched isomer when irradiated with UVA light. The rearrangement requires light and guest encapsulation to occur. The Ga4L6(12-) cage-mediated reaction mechanism was investigated by UV/vis absorption, fluorescence, ultrafast transient absorption, and electrochemical experiments. The results support a photoinduced electron transfer mechanism for the 1,3-rearrangement, in which the Ga4L6(12-) cage absorbs photons and transfers an electron to the encapsulated cinnamylammonium ion, which undergoes C-N bond cleavage, followed by back electron transfer to the cage and recombination of the guest fragments to form the higher energy isomer.

The synthesis of a series of octadentate ligands containing the 1-hydroxypyridin-2-one (1,2-HOPO) group in complex with europium(III) is reported. Within this series, the central bridge connecting two diethylenetriamine units linked to two 1,2-HOPO chromophores at the extremities (5-LIN-1,2-HOPO) is varied from a short ethylene chain (H(2,2)-1,2-HOPO) to a long pentaethylene oxide chain (H(17O5,2)-1,2-HOPO). The thermodynamic stability of the europium complexes has been studied and reveals these complexes may be effective for biological measurements. Extension of the central bridge results in exclusion of the inner-sphere water molecule observed for [Eu(H(2,2)-1,2-HOPO)](-) going from a nonacoordinated to an octacoordinated Eu(III) ion. With the longer chain length ligands, the complexes display increased luminescence properties in aqueous medium with an optimum of 20% luminescence quantum yield for the [Eu(H(17O5,2)-1,2-HOPO)](-) complex. The luminescence properties for [Eu(H(14O4,2)-1,2-HOPO)](-) and [Eu(H(17O5,2)-1,2-HOPO)](-) are better than that of the model bis-tetradentate [Eu(5LIN(Me)-1,2-HOPO)2](-) complex, suggesting a different geometry around the metal center despite the geometric freedom allowed by the longer central chain in the H(mOn,2) scaffold. These differences are also evidenced by examining the luminescence spectra at room temperature and at 77 K and by calculating the luminescence kinetic parameters of the europium complexes.

The synthesis of a series of octadentate ligands containing the 1-hydroxypyridin-2-one (1,2-HOPO) group in complex with europium(III) is reported. Within this series, the central bridge connecting two diethylenetriamine units linked to two 1,2-HOPO chromophores at the extremities (5-LIN-1,2-HOPO) is varied from a short ethylene chain (H(2,2)-1,2-HOPO) to a long pentaethylene oxide chain (H(17O5,2)-1,2-HOPO). The thermodynamic stability of the europium complexes has been studied and reveals these complexes may be effective for biological measurements. Extension of the central bridge results in exclusion of the inner-sphere water molecule observed for [Eu(H(2,2)-1,2-HOPO)](-) going from a nonacoordinated to an octacoordinated Eu(III) ion. With the longer chain length ligands, the complexes display increased luminescence properties in aqueous medium with an optimum of 20% luminescence quantum yield for the [Eu(H(17O5,2)-1,2-HOPO)](-) complex. The luminescence properties for [Eu(H(14O4,2)-1,2-HOPO)](-) and [Eu(H(17O5,2)-1,2-HOPO)](-) are better than that of the model bis-tetradentate [Eu(5LIN(Me)-1,2-HOPO)2](-) complex, suggesting a different geometry around the metal center despite the geometric freedom allowed by the longer central chain in the H(mOn,2) scaffold. These differences are also evidenced by examining the luminescence spectra at room temperature and at 77 K and by calculating the luminescence kinetic parameters of the europium complexes.

We report the preparation and new insight into photophysical properties of luminescent hydroxypyridonate complexes [M(III)L](-) (M = Eu or Sm) of the versatile 3,4,3-LI(1,2-HOPO) ligand (L). We report the crystal structure of this ligand with Eu(III) as well as insights into the coordination behavior and geometry in solution by using magnetic circular dichroism. In addition TD-DFT calculations were used to examine the excited states of the two different chromophores present in the 3,4,3-LI(1,2-HOPO) ligand. We find that the Eu(III) and Sm(III) complexes of this ligand undergo a transformation after in situ preparation to yield complexes with higher quantum yield (QY) over time. It is proposed that the lower QY in the in situ complexes is not only due to water quenching but could also be due to a lower degree of f-orbital overlap (in a kinetic isomer) as indicated by magnetic circular dichroism measurements.

The effect of host structure on the selectivity and mechanism of intramolecular Prins reactions is evaluated using K12Ga4L6 tetrahedral catalysts. The host structure was varied by modifying the structure of the chelating moieties and the size of the aromatic spacers. While variation in chelator substituents was generally observed to affect changes in rate but not selectivity, changing the host spacer afforded differences in efficiency and product diastereoselectivity. An extremely high number of turnovers (up to 840) was observed. Maximum rate accelerations were measured to be on the order of 105, which numbers among the largest magnitudes of transition state stabilization measured with a synthetic host-catalyst. Host/guest size effects were observed to play an important role in host-mediated enantioselectivity.

3,4,3-LI(1,2-HOPO), 1,5,10,14-tetra(1-hydroxy-2-pyridon-6-oyl)-1,5,10,14-tetraazatetradecane), is a potent octadentate chelator of actinides. It is being developed as a decorporation treatment for internal contamination with radionuclides. Conventional HPLC methods exhibited speciation peaks and bridging, likely attributable to the agent's complexation with residual metallic ions in the HPLC system. Derivatization of the target ligand in situ with Fe(III) chloride, however, provided a single homogeneous iron-complex that can readily be detected and analyzed by HPLC. The HPLC method used an Agilent Eclipse XDB-C18 column (150 mm × 4.6mm, 5 μm) at 25°C with UV detection at 280 nm. A gradient elution, with acetonitrile (11% to 100%)/buffer mobile phase, was developed for impurity profiling. The buffer consisted of 0.02% formic acid and 10mM ammonium formate at pH 4.6. An Agilent 1200 LC-6530 Q-TOF/MS system was employed to characterize the [Fe(III)-3,4,3-LI(1,2-HOPO)] derivative and impurities. The proposed HPLC method was validated for specificity, linearity (concentration range 0.13-0.35 mg/mL, r = 0.9999), accuracy (recovery 98.3-103.3%), precision (RSD ≤ 1.6%) and sensitivity (LOD 0.08 μg/mL). The LC/HRMS revealed that the derivative was a complex consisting of one 3,4,3-LI(1,2-HOPO) molecule, one hydroxide ligand, and two iron atoms. Impurities were also identified with LC/HRMS. The validated HPLC method was used in shelf-life evaluation studies which showed that the API remained unchanged for one year at 25°C/60% RH.

Iron is an essential element for all organisms, and microorganisms produce small molecule iron-chelators, siderophores, to efficiently acquire Fe(III). Gram-positive bacteria possess lipoprotein siderophore-binding proteins (SBPs) on the membrane. Some of the SBPs bind both apo-siderophores (iron-free) and Fe-siderophore (iron-chelated) and only import Fe-siderophores. When the SBP initially binds an apo-siderophore, the SBP uses the Gram-positive siderophore-shuttle mechanism (the SBPs exchange Fe(III) from a Fe-siderophore to the apo-siderophore bound to the protein) and/or displacement mechanism (the apo-siderophore bound to the SBP is released and a Fe-siderophore is then bound to the protein) to import the Fe-siderophore. Previously, we reported that the Bacillus cereus SBP, YxeB, exchanges Fe(III) from a ferrioxamine B (FO) to a desferrioxamine B (DFO) bound to YxeB using the siderophore-shuttle mechanism although the iron exchange was indirectly elucidated. Synthetic Cr-DFO (inert metal FO analog) and Ga-DFO (nonreducible FO analog) are bound to YxeB and imported via YxeB and the corresponding permeases and ATPase. YxeB exchanges Fe(III) from FO and Ga(III) from Ga-DFO to DFO bound to the protein, indicating that the metal-exchange occurs without metal reduction. YxeB also binds DFO derivatives including acetylated DFO (apo-siderophore) and acetylated FO (AcFO, Fe-siderophore). The iron from AcFO is transferred to DFO when bound to YxeB, giving direct evidence of iron exchange. Moreover, YxeB also uses the displacement mechanism when ferrichrome (Fch) is added to the DFO:YxeB complex. Uptake by the displacement mechanism is a minor pathway compared to the shuttle mechanism.

Small molecule iron-chelators, siderophores, are very important in facilitating the acquisition of Fe(III), an essential element for pathogenic bacteria. Many Gram-negative outer-membrane transporters and Gram-positive lipoprotein siderophore-binding proteins have been characterized, and the binding ability of outer-membrane transporters and siderophore-binding proteins for Fe-siderophores has been determined. However, there is little information regarding the binding ability of these proteins for apo-siderophores, the iron-free chelators. Here we report that Bacillus cereus YxeB facilitates iron-exchange from Fe-siderophore to apo-siderophore bound to the protein, the first Gram-positive siderophore-shuttle system. YxeB binds ferrioxamine B (FO, Fe-siderophore)/desferrioxamine B (DFO, apo-siderophore) in vitro. Disc-diffusion assays and growth assays using the yxeB mutant reveal that YxeB is responsible for importing the FO. Cr-DFO (a FO analog) is bound by YxeB in vitro and B. cereus imports or binds Cr-DFO in vivo. In vivo uptake assays using Cr-DFO and FO and growth assays using DFO and Cr-DFO show that B. cereus selectively imports and uses FO when DFO is present. Moreover, in vitro competition assays using Cr-DFO and FO clearly demonstrate that YxeB binds only FO, not Cr-DFO, when DFO is bound to the protein. Iron-exchange from FO to DFO bound to YxeB must occur when DFO is initially bound by YxeB. Because the metal exchange rate is generally first order in replacement ligand concentration, protein binding of the apo-siderophore acts to dramatically enhance the iron exchange rate, a key component of the Gram-positive siderophore-shuttle mechanism.

Campylobacter jejuni NCTC11168 does not produce any endogenous siderophores of its own yet requires the CfrA enterobactin transporter for in vivo colonization. In addition, the genome of C. jejuni NCTC11168 contains three distinct TonB energy transduction systems, named TonB1, TonB2, and TonB3, that have not been tested for their role in siderophore uptake or their functional redundancy. We demonstrate that C. jejuni NCTC11168 transports ferric-enterobactin in an energy dependent manner that requires TonB3 for full activity with TonB1 showing partial functional redundancy. Moreover C. jejuni NCTC11168 can utilize a wide variety of structurally different catechol siderophores as sole iron sources during growth. This growth is solely dependent on the CfrA enterobactin transporter and highlights the wide range of substrates that this transporter can recognize. TonB3 is also required for growth on most catechol siderophores. Furthermore, either TonB1 or TonB3 is sufficient for growth on hemin or hemoglobin as a sole iron source demonstrating functional redundancy between TonB1 and TonB3. In vivo colonization assays with isogenic deletion mutants revealed that both TonB1 and TonB3 are required for chick colonization with TonB2 dispensable in this model. These results further highlight the importance of iron transport for efficient C. jejuni colonization.