Towards tailoring hydrophobic interaction with uranyl(VI) oxygen for C-H activation.

Bovine serum albumin (BSA) has a uranyl(VI) binding hotspot where uranium is tightly bound by three carboxylates. Uranyl oxygen is "soaked" into the hydrophobic core of BSA. Isopropyl hydrogen of Val is trapped near UO22+ and upon photoexcitation, C-H bond cleavage is initiated. A unique hydrophobic contact with "yl"-oxygen, as observed here, can be used to induce C-H activation.

Equilibrium Thermodynamics of Macropa Complexes with Selected Metal Isotopes of Radiopharmaceutical Interest.

To pursue the design of in vivo stable chelating systems for radiometals, a concise and straightforward method toolbox was developed combining NMR, isothermal titration calorimetry (ITC), and europium time-resolved laser-induced fluorescence spectroscopy (Eu-TRLFS). For this purpose, the macropa chelator was chosen, and Lu3+, La3+, Pb2+, Ra2+, and Ba2+ were chosen as radiopharmaceutically relevant metal ions. They differ in charge (2+ and 3+) and coordination properties (main group vs lanthanides). 1H NMR was used to determine four pKa values (±0.15; carboxylate functions, 2.40 and 3.13; amino functions, 6.80 and 7.73). Eu-TRLFS was used to validate the exclusive existence of the 1:1 Mn+/ligand complex in the chosen pH range at tracer level concentrations. ITC measurements were accomplished to determine the resulting stability constants of the desired complexes, with log K values ranging from 18.5 for the Pb-mcp complex to 7.3 for the Lu-mcp complex. Density-functional-theory-calculated structures nicely mirror the complexes' order of stabilities by bonding features. Radiolabeling with macropa using ligand concentrations from 10-3 to 10-6 M was accomplished by pointing out the complex formation and stability (212Pb > 133La > 131Ba ≈ 224Ra > 177Lu) by means of normal-phase thin-layer chromatography analyses.

Eu(III) and Cm(III) Complexation by the Aminocarboxylates NTA, EDTA, and EGTA Studied with NMR, TRLFS, and ITC-An Improved Approach to More Robust Thermodynamics.

The complex formation of Eu(III) and Cm(III) was studied via tetradentate, hexadentate, and octadentate coordinating ligands of the aminopolycarboxylate family, viz., nitrilotriacetate (NTA3-), ethylenediaminetetraacetate (EDTA4-), and ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetate (EGTA4-), respectively. Based on the complexones' pKa values obtained from 1H nuclear magnetic resonance (NMR) spectroscopic pH titration, complex formation constants were determined by means of the parallel-factor-analysis-assisted evaluation of Eu(III) and Cm(III) time-resolved laser-induced fluorescence spectroscopy (TRLFS). This was complemented by isothermal titration calorimetry (ITC), providing the enthalpy and entropy of the complex formation. This allowed us to obtain genuine species along with their molecular structures and corresponding reliable thermodynamic data. The three investigated complexones formed 1:1 complexes with both Eu(III) and Cm(III). Besides the established Eu(III)-NTA 1:1 and 1:2 complexes, we observed, for the first time, the existence of a Eu(III)-NTA 2:2 complex of millimolar metal and ligand concentrations. Demonstrated for thermodynamic studies on Eu(III) and Cm(III) interaction with complexones, the utilized approach is commonly applicable to many other metal-ligand systems, even to high-affinity ligands.

Halomonas gemina sp. nov. and Halomonas llamarensis sp. nov., two siderophore-producing organisms isolated from high-altitude salars of the Atacama Desert.

Introduction: This study aimed to identify and characterize novel siderophore-producing organisms capable of secreting high quantities of the iron-binding compounds. In the course of this, two not yet reported halophilic strains designated ATCHAT and ATCH28T were isolated from hypersaline, alkaline surface waters of Salar de Llamará and Laguna Lejía, respectively. The alkaline environment limits iron bioavailability, suggesting that native organisms produce abundant siderophores to sequester iron. Methods: Both strains were characterized by polyphasic approach. Comparative analysis of the 16S rRNA gene sequences revealed their affiliation with the genus Halomonas. ATCHAT showed close similarity to Halomonas salicampi and Halomonas vilamensis, while ATCH28T was related closest to Halomonas ventosae and Halomonas salina. The ability of both strains to secrete siderophores was initially assessed using the chromeazurol S (CAS) liquid assay and subsequently further investigated through genomic analysis and NMR. Furthermore, the effect of various media components on the siderophore secretion by strain ATCH28T was explored. Results: The CAS assay confirmed the ability of both strains to produce iron-binding compounds. Genomic analysis of strain ATCHAT revealed the presence of a not yet reported NRPS-dependant gene cluster responsible for the secretion of siderophore. However, as only small amounts of siderophore were secreted, further investigations did not lie within the scope of this study. Via NMR and genomic analysis, strain ATCH28T has been determined to produce desferrioxamine E (DFOE). Although this siderophore is common in various terrestrial microorganisms, it has not yet been reported to occur within Halomonas, making strain ATCH28T the first member of the genus to produce a non-amphiphilic siderophore. By means of media optimization, the produced quantity of DFOE could be increased to more than 1000 µM. Discussion: Phenotypic and genotypic characteristics clearly differentiated both strains from other members of the genus Halomonas. Average nucleotide identity (ANI) values and DNA-DNA relatedness indicated that the strains represented two novel species. Therefore, both species should be added as new representatives of the genus Halomonas, for which the designations Halomonas llamarensis sp. nov. (type strain ATCHAT = DSM 114476 = LMG 32709) and Halomonas gemina sp. nov. (type strain ATCH28T = DSM 114418 = LMG 32708) are proposed.

Europium(III) Meets Etidronic Acid (HEDP): A Coordination Study Combining Spectroscopic, Spectrometric, and Quantum Chemical Methods.

Etidronic acid (1-Hydroxyethylidene-1,1-diphosphonic acid, HEDP, H4L) is a proposed decorporation agent for U(VI). This paper studied its complex formation with Eu(III), an inactive analog of trivalent actinides, over a wide pH range, at varying metal-to-ligand ratios (M:L) and total concentrations. Combining spectroscopic, spectrometric, and quantum chemical methods, five distinct Eu(III)-HEDP complexes were found, four of which were characterized. The readily soluble EuH2L+ and Eu(H2L)2- species with log ß values of 23.7 ± 0.1 and 45.1 ± 0.9 are formed at acidic pH. At near-neutral pH, EuHL0s forms with a log ß of ~23.6 and, additionally, a most probably polynuclear complex. The readily dissolved EuL- species with a log ß of ~11.2 is formed at alkaline pH. A six-membered chelate ring is the key motif in all solution structures. The equilibrium between the Eu(III)-HEDP species is influenced by several parameters, i.e., pH, M:L, total Eu(III) and HEDP concentrations, and time. Overall, the present work sheds light on the very complex speciation in the HEDP-Eu(III) system and indicates that, for risk assessment of potential decorporation scenarios, side reactions of HEDP with trivalent actinides and lanthanides should also be taken into account.

A comprehensive study on the interaction of Eu(III) and U(VI) with plant cells (Daucus carota) in suspension.

Daucus carota suspension cells showed a high affinity towards Eu(III) and U(VI) based on a single-step bioassociation process with an equilibrium after 48-72 h. Cells responded with an increased metabolic activity towards heavy metal stress. Luminescence spectroscopy pointed to multiple species for both f-block elements in the culture media, providing initial hints of their interaction with cells and released metabolites. Using nuclear magnetic resonance spectroscopy, we could prove that malate, as an released metabolite in the culture medium, was found to complex with U. Luminescence spectroscopy also showed that Eu(III)-EDTA species are interacting with the cells. Furthermore, Eu(III) and U(VI) coordination is dominated by phosphate groups provided by the cells. We found that Ca ion channels of D. carota cells were involved in the uptake of U(VI), which led to a bioprecipitation of U(VI) in the vacuole of the cells, most probably as uranyl(VI) phosphates along with an intracellular sorption of U(VI) on biomembranes by lipid structures. Eu(III) could be found locally concentrated in the cell wall and in the cytoplasm with a co-localization with phosphorous and oxygen.

2-Phosphonobutane-1,2,4,-Tricarboxylic Acid (PBTC): pH-Dependent Behavior Studied by Means of Multinuclear NMR Spectroscopy.

Although 2-phosphonobutane-1,2,4,-tricarboxylic acid, PBTC, has manifold industrial applications, relevant and reliable data on the protonation of PBTC are poor. However, these data are critical parameters for ascertaining PBTC speciation, especially with regard to a sound structural and thermodynamic characterization of its metal ion complexes. A rigorous evaluation of pH-dependent 1H, 13C, and 31P chemical shifts along with accessible scalar spin-spin coupling constants (J) was performed in order to determine the pKa values of PBTC in 0.5 molal NaCl aqueous solution by means of nuclear magnetic resonance (NMR) spectroscopy. The phosphonate group revealed pKa values of 0.90 ± 0.02 and 9.79 ± 0.02, and the pKa values associated with the carboxylic groups are 3.92 ± 0.02, 4.76 ± 0.03, and 6.13 ± 0.03. Supported by DFT-calculated structures revealing strong intramolecular hydrogen bonding, the sequence of deprotonation could be unambiguously determined.

Studies of pyrroloquinoline quinone species in solution and in lanthanide-dependent methanol dehydrogenases.

Pyrroloquinoline quinone (PQQ) is a redox cofactor in calcium- and lanthanide-dependent alcohol dehydrogenases that has been known and studied for over 40 years. Despite its long history, many questions regarding its fluorescence properties, speciation in solution and in the active site of alcohol dehydrogenase remain open. Here we investigate the effects of pH and temperature on the distribution of different PQQ species (H3PQQ to PQQ3- in addition to water adducts and in complex with lanthanides) with NMR and UV-Vis spectroscopy as well as time-resolved laser-induced fluorescence spectroscopy (TRLFS). Using a europium derivative from a new, recently-discovered class of lanthanide-dependent methanol dehydrogenase (MDH) enzymes, we utilized two techniques to monitor Ln binding to the active sites of these enzymes. Employing TRLFS, we were able to follow Eu(III) binding directly to the active site of MDH using its luminescence and could quantify three Eu(III) states: Eu(III) in the active site of MDH, but also in solution as PQQ-bound Eu(III) and in the aquo-ion form. Additionally, we used the antenna effect to study PQQ and simultaneously Eu(III) in the active site.

Phenylarsonic acid-DMPS redox reaction and conjugation investigated by NMR spectroscopy and X-ray diffraction.

The reaction between 2,3-dimercaptopropane-1-sulfonate (DMPS, unithiol) and four phenylarsonic(V) acids, i.e. phenylarsonic acid (PAA), 4-hydroxy-3-nitrophenylarsonic acid (HNPAA), 2-aminophenylarsonic acid (o-APAA) and 4-aminophenylarsonic acid (p-APAA), is investigated in aqueous solution. The pentavalent arsenic compounds are reduced by DMPS to their trivalent analogs and instantly chelated by the vicinal dithiol, forming covalent As-S bonds within a five-membered chelate ring. The different types and positions of polar substituents at the aromatic ring of the arsonic acids influence the reaction rates in the same way as observed for reaction with glutathione (GSH), as well as the syn/anti molar ratio of the diastereomeric products, which was analyzed using time- and temperature-dependent nuclear magnetic resonance (NMR) spectroscopy. Addition of DMPS to the conjugate formed by a phenylarsonic(V) acid and the biologically relevant tripeptide GSH showed the immediate replacement of GSH by chelating DMPS, underlining the importance of dithiols as detoxifying agent.

Not just a background: pH buffers do interact with lanthanide ions-a Europium(III) case study.

The interaction between Eu(III) ion and different pH buffers, popular in biology and biochemistry, viz. HEPES, PIPES, MES, MOPS, and TRIS, has been studied by solution nuclear magnetic resonance spectroscopy (NMR) and time-resolved laser-induced fluorescence spectroscopy (TRLFS) techniques. The Good's buffers reveal non-negligible interaction with Eu(III) as determined from their complex stability constants, where the sites of interaction are the morpholine and piperazine nitrogen atoms, respectively. In contrast, TRIS buffer shows practically no affinity towards Eu(III). Therefore, when investigating lanthanides, TRIS buffer should be preferred over Good's buffers.

Dimeric and Trimeric Uranyl(VI)-Citrate Complexes in Aqueous Solution.

This research addresses a subject discussed controversially for almost 70 years. The interactions between the uranyl(VI) ion, U(VI), and citric acid, H3Cit, were examined using a multi-method approach comprising nuclear magnetic resonance (NMR), ultraviolet-visible (UV-vis), attenuated total reflectance Fourier-transform infrared (ATR FT-IR), and extended X-ray absorption fine-structure (EXAFS) spectroscopies as well as density functional theory (DFT) calculations. Combining 17O NMR spectroscopy and DFT calculation provided an unambiguous decision on complex configurations, evidencing for the first time that the dimeric complex, (UO2)2(HCit-H)22-, exists as two diastereomers with the syn-isomer in aqueous solution strongly favored over the anti-isomer. Both isomers interconvert mutually with exchange rates of ∼30 s-1 at -6 °C and ∼249 s-1 at 60 °C in acidic solution corresponding to an activation barrier of about 24 kJ mol-1. Upon increasing the pH value, ternary dimeric mono- and bis-hydroxo as well as trimeric complexes form, that is, (UO2)2(HCit-H)2(OH)3-, (UO2)2(HCit-H)2(OH)24-, (UO2)3(O)(Cit-H)38-, and (UO2)3(O)(OH)(Cit-H)25-, respectively. Stability constants were determined for all dimeric and trimeric species, with log ß° = -(8.6 ± 0.2) for the 3:3 species being unprecedented. Additionally, in the 6:6 sandwich complex, formed from two units of 3:3 species, the 17O NMR resonance of the trinuclear uranyl(VI) core bridging µ3-O is shown for the first time. Species distribution calculations suggest that the characterized polynuclear U(VI)-citrate species do not significantly increase uranium(VI) mobility in the environment. Furthermore, we revise the misconceptions in the aqueous U(VI)-citric acid solution chemistry, that is, structures proposed and repeatedly taken up, and outline generalized isostructural considerations to provide a basis for future U(VI) complexation studies.

Uranium(VI) bioassociation by different fungi - a comparative study into molecular processes.

After the Chernobyl and Fukushima incidents it has become clear that fungi can take up and accumulate large quantities of radionuclides and heavy metals, but the underlying processes are not well understood yet. For this study, the molecular interactions of uranium(VI) with the white-rot fungi, Schizophyllum commune and Pleurotus ostreatus, and the soil-living fungus, Leucoagaricus naucinus, were investigated. First, the uranium concentration in the biomass was determined by time-dependent bioassociation experiments. To characterize the molecular interactions, uranium was localized in the biomass by transmission electron microscopy analysis. Second, the formed uranyl complexes in both biomass and supernatant were determined by fluorescence spectroscopy. Additionally, possible bioligands in the supernatant were identified. The results show that the discernible interactions between metals and fungi are similar, namely biosorption, accumulation, and subsequent crystallization. But at the same time, the underlying biochemical mechanisms are different and specific to the fungal species. In addition, Schizophyllum commune was found to be the only fungus that, under the chosen experimental conditions, released tryptophan and other indole derivatives in the presence of uranium(VI) as determined by nuclear magnetic resonance spectroscopy. These released substances most likely act as messenger molecules rather than serving the direct detoxification of uranium(VI).

Trimeric uranyl(vi)-citrate forms Na+, Ca2+, and La3+ sandwich complexes in aqueous solution.

M. Basile, et al., Chem. Commun., 2015, 51, 5306-5309, showed that a sodium ion is sandwiched by uranyl(vi) oxygen atoms of two 3 : 3 uranyl(vi)-citrate complex molecules in single-crystals. By means of NMR spectroscopy supported by DFT calculations we provide unambiguous evidence for this complex to persist in aqueous solution above a critical concentration of 3 mM uranyl citrate. Unprecedented Ca2+ and La3+ coordination by a bis-(η3-uranyl(vi)-oxo) motif advances the understanding of uranium's aqueous chemistry. As determined from 17O NMR, Ca2+ and more distinctly La3+ cause strong O[double bond, length as m-dash]U[double bond, length as m-dash]O polarization, which opens up new ways for uranyl(vi)-oxygen activation and functionalization.

Uranium(VI) Complexes of Glutathione Disulfide Forming in Aqueous Solution.

The interactions between glutathione disulfide, GSSG, the redox partner and dimer of the intracellular detoxification agent glutathione, GSH, and hexavalent uranium, U(VI), were extensively studied by solution NMR (in D2O), complemented by time-resolved laser-induced fluorescence and IR spectroscopies. As expected for the hard Lewis acid U(VI), coordination facilitates by the ligands' O-donor carboxyl groups. However, owing to the adjacent cationic α-amino group, the glutamyl-COO reveal monodentate binding, while the COO of the glycyl residues show bidentate coordination. The log K value for the reaction UO22+ + H3GSSG- → UO2(H3GSSG)+ (pH 3, 0.1 M NaClO4) was determined for the first time, being 4.81 ± 0.08; extrapolation to infinite dilution gave log K⊖ = 5.24 ± 0.08. U(VI) and GSSG form precipitates in the whole pD range studied (2-8), showing least solubility for 4 < pD < 6.5. Thus, particularly GSSG, hereby representing also other peptides and small proteins, affects the mobility of U(VI), strongly depending on the speciation of either component.

Interdisciplinary Round-Robin Test on Molecular Spectroscopy of the U(VI) Acetate System.

A comprehensive molecular analysis of a simple aqueous complexing system-U(VI) acetate-selected to be independently investigated by various spectroscopic (vibrational, luminescence, X-ray absorption, and nuclear magnetic resonance spectroscopy) and quantum chemical methods was achieved by an international round-robin test (RRT). Twenty laboratories from six different countries with a focus on actinide or geochemical research participated and contributed to this scientific endeavor. The outcomes of this RRT were considered on two levels of complexity: first, within each technical discipline, conformities as well as discrepancies of the results and their sources were evaluated. The raw data from the different experimental approaches were found to be generally consistent. In particular, for complex setups such as accelerator-based X-ray absorption spectroscopy, the agreement between the raw data was high. By contrast, luminescence spectroscopic data turned out to be strongly related to the chosen acquisition parameters. Second, the potentials and limitations of coupling various spectroscopic and theoretical approaches for the comprehensive study of actinide molecular complexes were assessed. Previous spectroscopic data from the literature were revised and the benchmark data on the U(VI) acetate system provided an unambiguous molecular interpretation based on the correlation of spectroscopic and theoretical results. The multimethodologic approach and the conclusions drawn address not only important aspects of actinide spectroscopy but particularly general aspects of modern molecular analytical chemistry.

Network-like arrangement of mixed-valence uranium oxide nanoparticles after glutathione-induced reduction of uranium(vi).

Glutathione (GSH), a ubiquitous intracellular reducing tripeptide, is able to reduce hexavalent uranium, U(vi), to its tetravalent form, U(iv), in aqueous media in vitro, inducing the formation of nanocrystalline mixed-valence uranium oxide particles. After the initial reduction to U(v) and subsequent dismutation, the yielded U(iv) rapidly hydrolyses under near-neutral conditions forming 2-5 nm sized nanoparticles. The latter further aggregate to 20-40 nm chain-like building blocks that finally arrange as network-like structures.

Solution Species and Crystal Structure of Zr(IV) Acetate.

Complex formation and the coordination of zirconium with acetic acid were investigated with Zr K-edge extended X-ray absorption fine structure spectroscopy (EXAFS) and single-crystal diffraction. Zr K-edge EXAFS spectra show that a stepwise increase of acetic acid in aqueous solution with 0.1 M Zr(IV) leads to a structural rearrangement from initial tetranuclear hydrolysis species [Zr4(OH)8(OH2)16]8+ to a hexanuclear acetate species Zr6(O)4(OH)4(CH3COO)12. The solution species Zr6(O)4(OH)4(CH3COO)12 was preserved in crystals by slow evaporation of the aqueous solution. Single-crystal diffraction reveals an uncharged hexanuclear cluster in solid Zr6(µ3-O)4(µ3-OH)4(CH3COO)12·8.5H2O. EXAFS measurements show that the structures of the hexanuclear zirconium acetate cluster in solution and the solid state are identical.

The interaction of Eu(III) with organoborates ­ a further approach to understand the complexation in the An/Ln(III)-borate system.

The formation equilibria of salicylatoborate, lactatoborate and 3-hydroxybutyratoborate were studied by means of (11)B NMR spectroscopy. The smaller the pKa of the respective organic acid, the higher is the formation constant of the organoborate. The complexation of Eu(III) with salicylatoborate and lactatoborate was investigated by means of TRLFS (time-resolved laser-induced fluorescence spectroscopy) and (11)B NMR spectroscopy, yielding complexation constants lg ß11° = 2.6-3.2. A Eu(III)-3-hydroxybutyrate complex was characterized by TRLFS and (1)H NMR spectroscopy (lg ß11° = 2.89). DFT calculations of the investigated Eu(III)-organoborates and inorganic Eu(III)-(poly)borates provided information about the Eu(III) coordination (most likely chelate). They support the hypothesis that the complexation of Eu(III) with organic as well as inorganic borate structures containing the binding site "B(OR)4(-)" (R = H, threefold coordinated boron center(s), organic moiety) is comparable.

Kinetics and activation parameters of the reaction of organoarsenic(V) compounds with glutathione.

In this work the kinetics of the reaction of glutathione (GSH) with the organoarsenic(V) compounds phenylarsonic acid (PAA), 4-hydroxy-3-nitrophenylarsonic acid (HNPAA), p-aminophenylarsonic acid (p-APAA) and o-aminophenylarsonic acid (o-APAA) as well as monomethylarsonic acid (MMAA) and dimethylarsinic acid (DMAA) is investigated. The reaction progress is monitored in real time by (1)H NMR, allowing the determination of rate coefficients and half-lives as well as activation parameters. The reaction consists of two steps: redox reaction and conjugation. In all investigated systems the conjugation is fast compared to the redox reaction and, therefore, rate determining. All investigated phenylarsonic acids follow the same rate law, showing overall reaction orders of 3 and half-lives between 47.7 ± 0.2 and 71.0 ± 3.6 min. The methylated compounds react slower, showing half-lives of 76.6 ± 0.4 and 444 ± 10 min for DMAA and MMAA, respectively. Enthalpies of activation range from 20 to 36 (± 2) kJ mol(-1) and the entropies of activation are within -154 and -97(± 7)J mol(-1)K(-1). The results reveal a correlation of the toxicity of the arsenic compound and the reaction rate with GSH. This may pave the way for the estimation of the toxicity of such compounds by simple kinetic studies.

Americium(iii) and europium(iii) complex formation with lactate at elevated temperatures studied by spectroscopy and quantum chemical calculations.

Thermodynamic parameters for the complex formation of Am(iii) and Eu(iii) with lactate were determined with UV-vis and time-resolved laser-induced fluorescence spectroscopy (TRLFS) in a temperature range between 25 and 70 °C. The reaction enthalpy decreased with increasing ionic strength. ATR FT-IR and NMR spectroscopy in combination with density functional theory (DFT) calculations revealed structural details of the Eu(iii) lactate 1 : 1 complex: a chelating coordination mode of the lactate with a monodentate binding carboxylate group and the hydroxyl group being deprotonated.