Uranyl Affinity Research Articles

Amidoxime‐based Star Polymer Adsorbent with Ultra‐High Uranyl Affinity for Extremely Fast Uranium Extraction from Natural Seawater

AbstractPolymer adsorbents functionalized with amidoxime groups are widely considered to have the most industrial potential for uranium extraction from seawater. However, the entanglement upon the collapse of polymer chain in seawater greatly reduces the utilization ratio of amidoxime ligands. Herein, a novel star‐shaped molecular brush adsorbent (β‐CD‐g‐PAO) is obtained by densely grafting polyamidoxime (PAO) chains from β‐cyclodextrin (β‐CD). Experimental and theoretical results reveal that, in contrast to conventional linear polymer adsorbents, the star polymer adsorbent with a high grafting density of PAO side chains enables strong steric repulsion between the polymer chains, leading to increased persistence length and disentanglement of side chains. As a result, the star polymer adsorbent shows higher accessibility of the adsorption ligands with lower adsorption energy, achieving an adsorption capacity of up to 944.75 mg g−1 in uranium‐spiked seawater, surpassing that of conventional linear PAO by 3.2 times. Remarkably, this star polymer adsorbent demonstrates an extraordinarily adsorption capacity of 10.9 mg−1 g−1 in only 1 day in natural seawater, coupled with excellent affinity for uranyl ions (K = 0.31 pM). The unique topological structure design provides a new approach to solving the problem of low utilization ratio of functional ligands in polymer adsorbents.

Eruption pore matrix with cooperative chelating of spatially continued ligands for rapid and selective removal of uranium

A simple yet efficient gas-assisted preparation strategy was designed to prepare new kinds of porous copolymers sorbents in order to effectively avoid complex preparation procedure, undesired nonselective physical adsorption and slow permeable transport. Thus, an eruption pore, amidoxime functioned porous copolymers (PAO-PPoly(TA-ADBA-Cu2+)) was designed and synthesized for high-performance separation of uranium. The naturally existing small molecule thioctic acid (TA) was chosen as adsorption matrix owing to the supramolecular networks structure, plasticity and flexible compositions. For obtain the abundant spatially continued accessible recognizing sites of TA based copolymer, the 2-amino-benzonitrile (ADBA) was both selected as skeleton monomer and functional monomer on the TA matrix. Especially, the constructing chelating moieties of ADBA into porous TA copolymer matrix, where the binding motif’s coordinative interaction towards uranyl is enhanced by introducing an assistant amino group. In addition, the amino substituent of PAO-PPoly(TA-ADBA-Cu2+) assists in reducing the charge on uranyl in the copolymer sorbents and meanwhile serves as a hydrogen bond receptor, which is benefit of boosting the overall uranyl affinity of amidoxime. The Cu2+ could effectively improve the the chemical and mechanical properities of TA matrix mainly owing to the secondary ionic bonding of the copper(II)-carboxylate complexes. Benefiting from the porous structure and spatially continued affinity sites, the maximum uranium adsorption capacity of PAO-PPoly(TA-ADBA-Cu2+) is 515.13 mg g−1 at 318 K, which are superior to better than the previously reported copolymer sorbent. The obtained porous PAO-PPoly(TA-ADBA-Cu2+) materials have an excellent selectivity even when the competitive cations, as well as excellent recyclability toward uranium capture. More importantly, this work not only presents a new clue to fabricate porous copolymer sorbents with excellent chemical stability by gas-assisted preparation method, but also provides a novel platform for uranium extraction from an aqueous solution.

A Simple Fluorescence Affinity Assay to Decipher Uranyl-Binding to Native Proteins.

Determining the affinity of proteins for uranyl is key to understand the toxicity of this cation and to further develop decorporation strategies. However, usual techniques to achieve that goal often require specific equipment and expertise. Here, we propose a simple, efficient, fluorescence‐based method to assess the affinity of proteins and peptides for uranyl, at equilibrium and in buffered solution. We first designed and characterized an original uranyl‐binding fluorescent probe. We then built a reference scale for uranyl affinity in solution, relying on signal quenching of our fluorescent probe in presence of high‐affinity uranyl‐binding peptides. We finally validated our approach by re‐evaluating the uranyl‐binding affinity of four native proteins. We envision that this tool will facilitate the reliable and reproducible assessment of affinities of peptides and proteins for uranyl.

A Simple Fluorescence Affinity Assay to Decipher Uranyl‐Binding to Native Proteins

AbstractDetermining the affinity of proteins for uranyl is key to understand the toxicity of this cation and to further develop decorporation strategies. However, usual techniques to achieve that goal often require specific equipment and expertise. Here, we propose a simple, efficient, fluorescence‐based method to assess the affinity of proteins and peptides for uranyl, at equilibrium and in buffered solution. We first designed and characterized an original uranyl‐binding fluorescent probe. We then built a reference scale for uranyl affinity in solution, relying on signal quenching of our fluorescent probe in presence of high‐affinity uranyl‐binding peptides. We finally validated our approach by re‐evaluating the uranyl‐binding affinity of four native proteins. We envision that this tool will facilitate the reliable and reproducible assessment of affinities of peptides and proteins for uranyl.

Study of the decorporation efficacy and toxicity of tetradentate 3-hydroxy-2-pyridinone ligands at the cellular level

ObjectiveA series of experiments were carried out to study the comparative decorporation efficacy and toxicity of three tetradentate hydroxypyridinone ligands (3LI-1-Cm-3,2-HOPO, 5LIO-1-Cm-3,2-HOPO, and 7LIO-1-Cm-3,2-HOPO) with the variation of the linker length and the substituents. MethodsThree ligands were obtained from a 4-step synthesis via the amidation of HOPO unit and different backbones. Potentiometric titrations were carried out to evaluate the formation constants of their corresponding uranyl complexes. CCK-8 and colony formation assays were used to compare the cytotoxicity of the three ligands and uranyl using renal proximal tubular epithelial (NRK-52E) cells. In vitro uranium removal assays were performed to assess decorporation efficiencies of those ligands by co-incubating uranium and chelators with NRK-52E cells for 48 h. ResultsAmong the three ligands, 5LIO-1-Cm-3,2-HOPO exhibits the highest uranyl affinity (logβ110 ​= ​18.6(7)) in comparison to the other two ligands (logβ110 ​= ​14.9(2) for 3LI-1-Cm-3,2-HOPO and logβ110 ​= ​16.7(1) for 7LIO-1-Cm-3,2-HOPO). The results of CCK-8 tests and colony formation assays further elucidate that 5LIO-1-Cm-3,2-HOPO and 7LIO-1-Cm-3,2-HOPO both show a similar level of cytotoxicity compared with ZnNa3-DTPA at relatively low ligand concentrations. In contrast, in the 3LI-1-Cm-3,2-HOPO treated group, the cell viability decreased markedly with the increase of the ligand concentrations, and the colony formation capability of NRK-52E cells was inhibited. Furthermore, the comparative decorporation efficacy of the three ligands was obtained from in vitro uranium removal assays, suggesting that these ligands could significantly inhibit the cellular uptake and prompt the cellular release of uranium from NRK-52E cells by the level of 81.9%, 91.8%, and 87.1%, respectively. ConclusionOur current results demonstrate that the linker/backbone species would significantly affect the decorporation efficacy and toxicity of the tetradentate 3-hydroxy-2-pyridinone ligands, which is informative for the future design and modification of novel decorporation ligands.

Water-locking molecule-assisted fabrication of nature-inspired Mg(OH)2 for highly efficient and economical uranium capture.

With the depletion of uranium terrestrial deposits, researchers have focused on the development of adsorbents to extract radioactive uranium from seawater/wastewater. However, the artificial manipulation of adsorbents for the cost-effective extraction of radioactive uranium from large numbers of water samples is still significantly challenging. Herein, a facile yet versatile stepwise strategy has been reported for the fabrication of adsorbents. Magnesium hydroxide (Mg(OH)2) was fabricated via the in situ conversion of a natural ore powder (magnesite), whose unique internal pore structure is highly suitable for the development of highly efficient sorbents. The coordination interaction of the synthesized adsorbent with uranium was enhanced by further introducing inexpensive molecules with water-locking properties, which resulted in superior extraction capacity and low production cost. After careful calculation, the cost per kilogram of the adsorbent was found to be about $0.21. The adsorption behaviors of the synthesized adsorbent CMC-PAM/Mg(OH)2 were investigated by batch adsorption, flow-through column adsorption (in laboratory), and field adsorption experiments in natural seawater and river. Representatively, CMC-PAM/Mg(OH)2 was exceptional in extracting uranium not only at high concentrations with sufficient capacities in a wide pH range (1584.67 mg g-1 and 454.55 mg g-1 at pH = 5 and pH = 8, respectively), but also in trace quantities including uranium in a flow-through column (55.68 mg g-1), natural seawater (8.6 mg g-1), and river (6.7 mg g-1). Inspired by this excellent performance, the effects of competitive ions on the selective adsorption of uranium by CMC-PAM/Mg(OH)2 in simulated wastewater and seawater environments were further studied. Using a combination of FTIR spectroscopic and XPS studies, it was revealed that the amine and hydroxyl groups enhanced the overall uranyl affinity of the CMC-PAM/Mg(OH)2 composite.

Bio-inspired nano-traps for uranium extraction from seawater and recovery from nuclear waste

Nature can efficiently recognize specific ions by exerting second-sphere interactions onto well-folded protein scaffolds. However, a considerable challenge remains to artificially manipulate such affinity, while being cost-effective in managing immense amounts of water samples. Here, we propose an effective approach to regulate uranyl capture performance by creating bio-inspired nano-traps, illustrated by constructing chelating moieties into porous frameworks, where the binding motif’s coordinative interaction towards uranyl is enhanced by introducing an assistant group, reminiscent of biological systems. Representatively, the porous framework bearing 2-aminobenzamidoxime is exceptional in sequestering high uranium concentrations with sufficient capacities (530 mg g−1) and trace quantities, including uranium in real seawater (4.36 mg g−1, triple the benchmark). Using a combination of spectroscopic, crystallographic, and theory calculation studies, it is revealed that the amino substituent assists in lowering the charge on uranyl in the complex and serves as a hydrogen bond acceptor, boosting the overall uranyl affinity of amidoxime.

Structural Analysis of Uranyl Complexation by the EF-Hand Motif of Calmodulin: Effect of Phosphorylation.

Better understanding of uranyl-protein interactions is a prerequisite to predict uranium chemical toxicity in cells. The EF-hand motif of the calmodulin site I is about thousand times more affine for uranyl than for calcium, and threonine phosphorylation increases the uranyl affinity by two orders of magnitude at pH 7. In this study, we confront X-ray absorption spectroscopy with Fourier transform infrared (FTIR) spectroscopy, time-resolved laser-induced fluorescence spectroscopy (TRLFS), and structural models obtained by molecular dynamics simulations to analyze the uranyl coordination in the native and phosphorylated calmodulin site I. For the native site I, extended X-ray absorption fine structure (EXAFS) data evidence a short U-Oeq distance, in addition to distances compatible with mono- and bidentate coordination by carboxylate groups. Further analysis of uranyl speciation by TRLFS and thorough investigation of the fluorescence decay kinetics strongly support the presence of a hydroxide uranyl ligand. For a phosphorylated site I, the EXAFS and FTIR data support a monodentate uranyl coordination by the phosphoryl group and strong interaction with mono- and bidentate carboxylate ligands. This study confirms the important role of a phosphoryl ligand in the stability of uranyl-protein interactions. By evidencing a hydroxide uranyl ligand in calmodulin site I, this study also highlights the possible role of less studied ligands as water or hydroxide ions in the stability of protein-uranyl complexes.

Polymer-Supported Aminomethylphosphinate as a Ligand with a High Affinity for U(VI) from Phosphoric Acid Solutions: Combining Variables to Optimize Ligand–Ion Communication

ABSTRACTAminomethylphosphinic acid (AMPA) is immobilized onto cross-linked polystyrene beads and found to have a high affinity for the uranyl ion, especially from solutions as acidic as 6 M H3PO4. The trend in uranyl affinity by different ligands is AMPA > phosphinic > phosphonic > sulfonic. For example, the percent sorbed from 6 M H3PO4 is 89.5% (AMPA), 44.8% (phosphinic), 5.38% (phosphonic), and 2.60% (sulfonic). The high affinity shown by AMPA is due to the decreased hydrogen bonding in the phosphinic acid ligand as well as synergistic coordination of the uranyl ion by a proximate P=O of a second phosphinate ligand. AMPA contacted with synthetic wet process phosphoric acid solution sorbed 72.2% uranium compared to 0.14% for a commercially available sulfonic acid.

Predicting Stability Constants for Uranyl Complexes Using Density Functional Theory

The ability to predict the equilibrium constants for the formation of 1:1 uranyl/ligand complexes (log K1 values) provides the essential foundation for the rational design of ligands with enhanced uranyl affinity and selectivity. We use density functional theory (B3LYP) and the integral equation formalism polarizable continuum model (IEF-PCM) to compute aqueous stability constants for UO2(2+) complexes with 18 donor ligands. Theoretical calculations permit reasonably good estimates of relative binding strengths, while the absolute log K1 values are significantly overestimated. Accurate predictions of the absolute log K1 values (root-mean-square deviation from experiment <1.0 for log K1 values ranging from 0 to 16.8) can be obtained by fitting the experimental data for two groups of mono- and divalent negative oxygen donor ligands. The utility of correlations is demonstrated for amidoxime and imide dioxime ligands, providing a useful means of screening for new ligands with strong chelating capability to uranyl.

A Phosphorylation Tag for Uranyl Mediated Protein Purification and Photo Assisted Tag Removal

Most protein purification procedures include an affinity tag fused to either the N or C-terminal end of the protein of interest as well as a procedure for tag removal. Tag removal is not straightforward and especially tag removal from the C-terminal end is a challenge due to the characteristics of enzymes available for this purpose. In the present study, we demonstrate the utility of the divalent uranyl ion in a new procedure for protein purification and tag removal. By employment of a GFP (green florescence protein) recombinant protein we show that uranyl binding to a phosphorylated C-terminal tag enables target protein purification from an E. coli extract by immobilized uranyl affinity chromatography. Subsequently, the tag can be efficiently removed by UV-irradiation assisted uranyl photocleavage. We therefore suggest that the divalent uranyl ion (UO2 2+) may provide a dual function in protein purification and subsequent C-terminal tag removal procedures.

Modulating Uranium Binding Affinity in Engineered Calmodulin EF-Hand Peptides: Effect of Phosphorylation

To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T9TKE12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from Kd = 25±6 nM to Kd = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (Kd = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the νas(P-O) and νs(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in νas(UO2)2+ vibration (from 923 cm−1 to 908 cm−1) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH.

Hexadentate Terephthalamide(bis-hydroxypyridinone) Ligands for Uranyl Chelation: Structural and Thermodynamic Consequences of Ligand Variation

Several linear, hexa- and tetradentate ligands incorporating a combination of 2,3-dihydroxy-terephthalamide (TAM) and hydroxypyridinone-amide (HOPO) moieties have been developed as uranyl chelating agents. Crystallographic analysis of several {UO(2)[TAM(HOPO)(2)]}(2-) complexes revealed a variable and crowded coordination geometry about the uranyl center. The TAM moiety dominates the bonding in hexadenate complexes, with linker rigidity dictating the equality of equatorial U-O bonding. Hexadentate TAM(HOPO)(2) ligands demonstrated slow binding kinetics with uranyl affinities on average 6 orders of magnitude greater than those of similarly linked bis-HOPO ligands. Study of tetradentate TAM(HOPO) ligands revealed that the high uranyl affinity stems primarily from the presence of the TAM moiety and only marginally from increased ligand denticity. Uranyl affinities of TAM(HOPO)(2) ligands were within experimental error, with TAM(o-phen-1,2-HOPO)(2) exhibiting the most consistent uranyl affinity at variable pH.

The Influence of Linker Geometry in Bis(3‐hydroxy‐N‐methyl‐pyridin‐2‐one) Ligands on Solution Phase Uranyl Affinity

Seven water-soluble, tetradentate bis(3-hydroxy-N-methyl-pyridin-2-one) (bis-Me-3,2-HOPO) ligands were synthesized that vary only in linker geometry and rigidity. Solution-phase thermodynamic measurements were conducted between pH 1.6 and pH 9.0 to determine the effects of these variations on proton and uranyl cation affinity. Proton affinity decreases by introduction of the solubilizing triethylene glycol group as compared to unsubstituted reference ligands. Uranyl affinity was found to follow no discernable trends with incremental geometric modification. The butyl-linked 4 li-Me-3,2-HOPO ligand exhibited the highest uranyl affinity, consistent with prior in vivo decorporation results. Of the rigidly-linked ligands, the o-phenylene linker imparted the best uranyl affinity to the bis-Me-3,2-HOPO ligand platform.

Identification of uranyl binding proteins from human kidney-2 cell extracts by immobilized uranyl affinity chromatography and mass spectrometry

To improve our knowledge on protein targets of uranyl ion (UO 2 2+), we set up a proteomic strategy based on immobilized metal-affinity chromatography (IMAC). The successful enrichment of UO 2 2+-interacting proteins from human kidney-2 (HK-2) soluble cell extracts was obtained using an ion-exchange chromatography followed by a dedicated IMAC process previously described and designed for the uranyl ion. By mass spectrometry analysis we identified 64 proteins displaying varied functions. The use of a computational screening algorithm along with the particular ligand-based properties of the UO 2 2+ ion allowed the analysis and categorization of the protein collection. This profitable approach demonstrated that most of these proteins fulfill criteria which could rationalize their binding to the UO 2 2+-loaded phase. The obtained results enable us to focus on some targets for more in-depth studies and open new insights on its toxicity mechanisms at molecular level.

Specific capture of uranyl protein targets by metal affinity chromatography

To improve general understanding of biochemical mechanisms in the field of uranium toxicology, the identification of protein targets needs to be intensified. Immobilized metal affinity chromatography (IMAC) has been widely developed as a powerful tool for capturing metal binding proteins from biological extracts. However uranyl cations (UO 2 2+) have particular physico-chemical characteristics which prevent them from being immobilized on classical metal chelating supports. We report here on the first development of an immobilized uranyl affinity chromatography method, based on the cation-exchange properties of aminophosphonate groups for uranyl binding. The cation distribution coefficient and loading capacity on the support were determined. Then the stability of the uranyl-bonded phase under our chromatographic conditions was optimized to promote affinity mechanisms. The successful enrichment of uranyl binding proteins from human serum was then proven using proteomic and mass spectral analysis.

機能性カリックスアレーンの合成と分子認識作用

Calixarenes are cyclic oligomers synthesized from phenols and formaldehyde. We designed and synthesized a variety of new host molecules from calixarenes. They acted as cyclic ionophores with crown-like functions by introducing ionophoric groups. In particular, calix [5] arene and calix [6] arene with carboxylate groups acted as “super-uranophiles” exhibiting the record-breaking uranyl affinity (K=1018.4-19.2 M-1) and uranyl selectivity (1010.6-17). Inclusion of organic guest molecules in solution was effected for the first time by using water-soluble calixarenes which had either anionic, cationic, or neutral hydrophilic groups. Chiral, water-soluble calixarenes changed their conformation upon inclusion of guest molecules, which was conveniently detected by the CD spectra and provided several lines of important information for the host-guest-type complex formation. Through these studies, it was demonstrated that the moderate rigidity and the remaining conformational freedom are important characteristics of the complex formation with calixarene-based host-molecules.