Probing electrostatic potential by NMR with the use of a paramagnetic lanthanide(III) chelate

The paramagnetic complex [Yb(DOTA)](-) forms ion pairs in aqueous solution with cationic species such as N-monoalkyl- and N,N'-dialkyl-4,4'-bipyridinium cations. The magnitude and sign of the induced (1)H NMR pseudocontact shift values can be correlated to the electrostatic potential calculated at the MPWLYP/6-311G** level.     

Enhanced binding by dextran‐grafting to Protein A affinity chromatographic media

Dextran‐grafted Protein A affinity chromatographic medium was prepared by grafting dextran to agarose‐based matrix, followed by epoxy‐activation and Protein A coupling site‐directed to sulfhydryl groups of cysteine molecules. An enhancement of both the binding performance and the stability was achieved for this dextran‐grafted Protein A chromatographic medium. Its dynamic binding capacity was 61 mg immunoglobulin G/mL suction‐dried gel, increased by 24% compared with that of the non‐grafted medium. The binding capacity of dextran‐grafted medium decreased about 7% after 40 cleaning‐in‐place cycles, much lower than that of the non‐grafted medium as decreased about 15%. Confocal laser scanning microscopy results showed that immunoglobulin G was bound to both the outside and the inside of dextran‐grafted medium faster than that of non‐grafted one. Atomic force microscopy showed that this dextran‐grafted Protein A medium had much rougher surface with a vertical coordinate range of ±80 nm, while that of non‐grafted one was ±10 nm. Grafted dextran provided a more stereo surface morphology and immunoglobulin G molecules were more easily to be bound. This high‐performance dextran‐grafted Protein A affinity chromatographic medium has promising applications in large‐scale antibody purification.     

Covalent modification of calcium hydroxyapatite surface by grafting phenyl phosphonate moieties

The reaction between phenyl phosphonic dichloride (C₆H₅P(O)Cl₂) and synthetic calcium hydroxy- and fluorapatite has been investigated. The presence of mono- or polymeric (C₆H₅PO) fragment bound to hydroxyapatite was evidenced by IR, and solid-state ³¹P NMR spectroscopy. X-ray powder analysis has shown that the apatitic structure remains unchanged during the reaction. In contrast, no reaction was found using fluorapatite. According to the results found for these two different apatites a mechanism was proposed for the formation of covalent P-O-P bonds as the result of a reaction between the C₆H₅P(O)Cl₂ organic reagent and (HPO₄)⁻ and/or OH⁻ ions of the hydroxyapatite.     

Bifunctional magnetic-optical nanocomposites: grafting lanthanide complex onto core-shell magnetic silica nanoarchitecture

A new class of bifunctional architecture combining the useful functions of superparamagnetism and terbium complex luminescence into one material has been prepared via two main steps by a modified St?ber method and the layer-by-layer (LbL) assembly technique. The obtained bifunctional nanocomposites exhibit superparamagnetic behavior, high fluorescence intensity, and color purity. The architecture has been characterized by field-emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), UV-vis absorption and emission spectroscopy, X-ray diffraction, and superconducting quantum interference device (SQUID) magnetometry.     

A rare earth-DOTA-binding antibody: probe properties and binding affinity across the lanthanide series

An antibody that binds rare earth complexes selectively could be used as a docking station for a set of probe molecules, of particular interest for medical imaging and therapy. The rare earths are rich in probe properties, such as the paramagnetism of Gd, the luminescence of Tb and Eu, and the nuclear properties of Lu and Y. We find that antibody 2D12.5, initially developed to bind analogues of Y-DOTA (1,4,7,10-tetraazacyclododecane-N,N',N' ',N' '-tetraacetic acid) for radiotherapy, binds not only Y-DOTA analogues but also analogous DOTA complexes of all of the lanthanides. Surprisingly, chelates of some metals such as Gd3+ bind more tightly than the original Y3+ complex. When the shape of the complex is perturbed by either increasing or decreasing the radius of the lanthanide ion, the thermodynamic stability of the protein-ligand complex changes in a regular fashion. The behavior of DeltaDeltaG as a function of ionic radius fits a parabola, as might be expected for a system that behaves in a thermodynamically elastic way. The broad specificity and high affinity of this antibody for all rare earth-DOTA complexes make it particularly interesting for applications that take advantage of the unique characteristics of lanthanides. For example, UV excitation of the Tb-DOTA-2D12.5 complex leads to energy transfer from aromatic side chains of the antibody to bound Tb-DOTA, enhancing green terbium luminescence >104 relative to unbound Tb-DOTA.     

Silica-supported lanthanide silylamides for methyl methacrylate polymerisation: controlled grafting induces controlled reactivity

Lanthanide silylamides were grafted onto non-porous silica dehydroxylated at various temperatures, and the surface species nature and relative distribution were correlated with MMA polymerisation activity and selectivity.     

Optical control of calcium affinity in a spiroamido-rhodamine based calcium chelator

An optically controlled Ca(2+)-chelator 1 was developed to mimic natural calcium oscillations. Compound 1, a spiroamido-rhodamine derivative of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), underwent cycles of reversible transitions between a colorless closed state and a fluorescent open form. The closed-state exhibited a high affinity for Ca(2+) (K(d): 509 nM) with excellent selectivity over Mg(2+) (K(d): 19 mM). The open isomer had a 350-fold lower Ca(2+) affinity (K(d): 181 μM), while the Mg(2+) affinity was not significantly affected (K(d): 14 mM).     

Probing affinity and ubiquitin linkage selectivity of ubiquitin-binding domains using mass spectrometry

Non-covalent interactions between ubiquitin (Ub)-modified substrates and Ub-binding domains (UBDs) are fundamental to signal transduction by Ub receptor proteins. Poly-Ub chains, linked through isopeptide bonds between internal Lys residues and the C-terminus of Ub, can be assembled with varied topologies to mediate different cellular processes. We have developed and applied a rapid and sensitive electrospray ionization-mass spectrometry (ESI-MS) method to determine isopeptide linkage-selectivity and affinity of poly-Ub·UBD interactions. We demonstrate the technique using mono-Ub and poly-Ub complexes with a number of α-helical and zinc-finger (ZnF) UBDs from proteins with roles in neurodegenerative diseases and cancer. Affinities in the 2-200 μM range were determined to be in excellent agreement with data derived from other biophysical techniques, where available. Application of the methodology provided further insights into the poly-Ub linkage specificity of the hHR23A-UBA2 domain, confirming its role in Lys48-linked poly-Ub signaling. The ZnF UBP domain of isopeptidase-T showed no linkage specificity for poly-Ub chains, and the Rabex-5 MIU also exhibited little or no specificity. The discovery that a number of domains are able to bind cyclic Lys48 di-Ub with affinities similar to those for the acyclic form indicates that cyclic poly-Ub may be capable of playing a role in Ub-signaling. Detection of a ternary complex involving Ub interacting simultaneously with two different UBDs demonstrated the co-existence of multi-site interactions, opening the way for the study of crosstalk between individual Ub-signaling pathways.     

Probing the hydrogen adsorption affinity of Pt and Ir by surface interrogation scanning electrochemical microscopy (SI-SECM)

Scanning electrochemical microscopy in the surface interrogation mode (SI-SECM) has been used to probe the strength of metal-adsorbed H bonds at Pt and Ir. The generally accepted view is that this technique can only give meaningful results for similar probe and substrate electrode dimensions. However, it is shown that SI-SECM can also provide valuable information for H adsorption even when the substrate is much larger than the microelectrode probe (of 500 and 25 μm diameter respectively), for properly chosen substrate and mediator combinations that minimize the tip currents under substrate open circuit potential conditions. Linear sweep voltammetry at a microelectrode positioned at a very small probe–substrate distance (2.5 μm) showed remarkable positive feedback in the presence of an oxidizable mediator (TMPD) when a full or partial H monolayer was electrochemically pre-formed on the Pt or Ir substrate. The magnitude and shapes of chronoamperometric responses at the tip were interpreted in terms of variation of H coverage with time and changes in the open circuit potential of the substrate. The higher affinity of Ir than Pt for adsorbed H has been used to validate the approach.     

Evaluation of the adsorption affinity of proteins to calcium hydroxyapatites by desorption and pre-adsorption methods

The adsorption affinity of bovine serum albumin (BSA) and lysozyme (LSZ) to calcium hydroxyapatite (CaHAP) was evaluated by desorption and two step adsorption methods. These experiments were carried out at 15°C in a 1×10⁻⁴ mol dm⁻³ KCl solution of pH 6.0. BSA molecules were scarcely desorbed, exhibiting an irreversible adsorption of BSA, though LSZ slightly desorbed. This result supports our previous findings that LSZ adsorbs weakly onto phosphate ions exposed on ac or bc faces of CaHAP while BSA adsorbs strongly onto positively charged sites on ac or bc faces of CaHAP. The amount of adsorbed LSZ was markedly increased by the pre-adsorption of BSA, where LSZ was adsorbed onto BSA-covered CaHAP. On the other hand, the amount of adsorbed BSA was not changed by the pre-adsorption of LSZ. In both pre-adsorption systems it was confirmed by an HPLC method that no protein molecule pre-adsorbed was desorbed after the post-adsorption procedure. Therefore, it was interpreted that the enhancement of adsorption of positively charged LSZ is induced by an electrostatic attractive force through pre-adsorption of negatively charged BSA molecules with a high coverage. However, since the coverage of LSZ onto CaHAP is considerably low, no stimulation of BSA adsorption occurred on the LSZ-covered surface. The formation of double protein adsorbed layers consisting of pre- and post-adsorbed proteins was proposed.     

Probing site-specific 13C/15N-isotope enrichment of spider silk with liquid-state NMR spectroscopy

Solid-state nuclear magnetic resonance (NMR) has been extensively used to elucidate spider silk protein structure and dynamics. In many of these studies, site-specific isotope enrichment is critical for designing particular NMR methods for silk structure determination. The commonly used isotope analysis techniques, isotope-ratio mass spectroscopy and liquid/gas chromatography-mass spectroscopy, are typically not capable of providing the site-specific isotope information for many systems because an appropriate sample derivatization method is not available. In contrast, NMR does not require any sample derivatization or separation prior to analysis. In this article, conventional liquid-state ¹H NMR was implemented to evaluate incorporation of ¹³C/¹⁵N-labeled amino acids in hydrolyzed spider dragline silk. To determine site-specific ¹³C and ¹⁵N isotope enrichments, an analysis method was developed to fit the ¹H–¹³C and ¹H–¹⁵N J-splitting (J _CH and J _NH) ¹H NMR peak patterns of hydrolyzed silk fiber. This is demonstrated for Nephila clavipes spiders, where [U–¹³C₃,¹⁵N]-Ala and [1-¹³C,¹⁵N]-Gly were dissolved in their water supplies. Overall, contents for Ala and Gly isotopomers are extracted for these silk samples. The current methodology can be applied to many fields where site-specific tracking of isotopes is of interest.      

Preparation and characterization of hydrophobic calcium hydroxyapatite particles grafting oleylphosphate groups

Grafting of oleylphosphate (OP) molecules to the surface of calcium hydroxyapatite particles (HAP) was carried out by a coprecipitation method with a Ca(OH)₂–H₃PO₄ system in the presence of disodium oleylphosphate (DSOP). All the particles exhibited a single-crystal nature with rod-like shape and were elongated along c-axis from 36 to 122 nm in particle length with an increase in the concentration of DSOP. It was suggested that 084% of the phosphate ions exposed on the ac or bc faces of the HAP particles are exchanged with phosphate ions of DSOP molecules directing oleyl groups outward. The hydrophobicity of OP-grafted HAP particles was enhanced with an increase in the DSOP concentration. This high surface hydrophobicity was further confirmed by water adsorption experiments. The materials with the surface oleyl groups adsorbed much less water than the HAP particles produced without DSOP.     

Calcium-modulated conformational affinity chromatography. Application to the purification of calmodulin and S100 proteins.

The purification of proteins by affinity chromatography is based on their highly specific interaction with an immobilized ligand followed by elution under conditions where their affinity towards the ligand is markedly reduced. Thus, a high-degree purification by a single chromatographic step is achieved. However, when several proteins in the crude mixture share affinity to a common immobilized ligand, they may not be resolved by affinity chromatography and subsequent "real" chromatographic purification steps may be required. It is shown that by using properly selected gradient elution conditions, the affinities of the various proteins towards the immobilized ligand may be gradually modulated and their separation may be achieved. This is exemplified by the isolation and separation of a group of Ca(2+)-activated proteins, Calmodulin, S100a and S100b, from bovine brain extract, using a melittin-Eupergit C affinity column which is developed with Ca(2+)-chelator gradients. As expected, separation of the three proteins into individual peaks, eluted in order of increasing affinity to the matrix, was obtained. Sigmoid selectivity curves calculated from the elution volumes under different elution conditions for each of the proteins were obtained, illustrating the chromatographic behaviour of the gradient affinity separation system.     

GMP-quadruplex-based hydrogels stabilized by lanthanide ions

This work describes the gelation behaviors and properties of a biological molecule, guanosine 5′-monophosphate disodium salt(GMP), in the presence of trivalent lanthanide ions. Hydrogels composed of GMP-quadruplexes were prepared by adjusting p H,through which the protonation of phosphate group was controlled to tune the interactions between lanthanide ions and GMP.Within the p H region of 2–6, the electrostatic interaction between lanthanide ions and phosphate group is hindered and the cation-dipole interaction acts as the main driving force for the formation of G-quadruplexes. All the hydrogels were found consisting of three-dimensional network of the intertwining one-dimensional nanofibers formed by the stacking G-quartets induced by lanthanide ions. A significant fluorescence enhancement of thioflavin T(Th T), a fluorescent molecule, was found to be triggered by the G-quadruplex structures, for which the rotation of chromophoric groups on Th T molecules were prohibited due to the implant into the G-quadruplex structures.     

Protein assemblies by site-specific avidin-biotin interactions

Exploiting self-assembly systems with biological building blocks is of significant interest in the fabrication of advanced biomaterials. We assessed the potential use of site-specific ligand labeling of protein building blocks in designing functional protein self-assemblies by combining site-specifically biotinylated bacterial alkaline phosphatase (as a bidentate or tetradentate ligand unit) and streptavidin (as a tetrameric receptor).     

Stereoselective formation of ansa-metallocenes in reactions of radical-anionic salts of acenaphthylene with lanthanide and calcium halides

ansa-Metallocenes (⁵:⁵-C₂₄H₁₆)M(THF)₂ (M = Sm (1), Yb (2), Ca (3)) and (⁵:⁵-C₂₄H₁₆)MI(THF) (M = Dy (8), Er (9), Tm (10), Lu (11)) were prepared in 50—90% yields by the in situ reactions of two equivalents of potassium acenaphthylenide K⁺C₁₂H₈ ^– with MI₂ or MI₃, respectively. Complexes 2 and 3 were also obtained by direct reduction of acenaphthylene with ytterbium and calcium naphthalenides, respectively. An ESR signal of the acenaphthylene radical anion, which was observed upon dissolution of compound 2 in THF, indicates that the [C₂₄H₁₆]^2– ansa-ligand dissociated into two [C₁₂H₈]^·– radical anions. Hydrolysis of complex 2 in benzene afforded 1,1",3,3"-tetrahydro-3,3"-biacenaphthylene (4) and 3,3",4,4"-tetrahydro-3,3"-biacenaphthylene (5). The reaction of complex 2 with ZrCl₄ and the reaction of compound 3 with Me₃SiCl proceeded with the cleavage of the C—C bond between two acenaphthylene fragments of the [C₂₄H₁₆]^2– ansa-ligand to produce (²-C₁₂H₈)ZrCl₂(THF)₃ (6) and bis(trimethylsilyl)acenaphthene (Me₃Si)₂C₁₂H₈ (7), respectively. Compounds 1—3, 6, 7, and 11 were characterized by ¹H and ¹³C NMR spectroscopy. The temperature dependence of the ¹H NMR spectrum of compound 11 in tetrahydrofuran is indicative of the dynamic exchange of the solvent molecules in the coordination sphere of the Lu atom. After cooling of the solution to 210 K, the dynamic process was terminated as evidenced by the nonequivalence of the ¹H signals of two acenaphthylene fragments. According to the X-ray diffraction data for complex 11, dimerization of two acenaphthylene radical anions at the Lu atom gave rise to the rac-ansa-metallocene structure. In compound 11, the Lu atom is ⁵-coordinated by two five-membered rings of the acenaphthylene ligands and also by the I atom and the THF molecule. The coordination environment about the Lu atom is a distorted tetrahedron. The average distance between the lutetium atom and the carbon atoms of the five-membered rings is 2.623 .     

Binding of calcium and other metal ions to the EF-hand loops of calmodulin studied by quantum chemical calculations and molecular dynamics simulations

Calcium ion binding by the four EF-hand motifs of the protein calmodulin (CaM) is a central event in Ca2+-based cellular signaling. To understand molecular details of this complex process, isolated Ca2+-binding loops can be studied, by use of both experiments and calculations. In this work, we explore the metal specificity of the four Ca2+-binding loops of CaM using density functional theory (DFT) quantum chemical calculations and molecular dynamics simulations. We study CaM complexes with the physiologically important ions of calcium (Ca2+) and magnesium (Mg2+) and also with two other ions, strontium (Sr2+) and lanthanum (La3+). The former is of interest in the area of radioactive waste bioremediation, whereas the latter is often used as a probe of Ca2+-binding sites. We obtain intrinsic metal ion-loop binding energies as well as their components: vacuum, charge-transfer, solvation, entropy, and deformation terms. A detailed analysis of the results reveals that the total binding energy depends on a delicate balance among these energy components. They, in turn, are determined by the cation's charge and size as well as the amino acid composition and flexibility of the loops and the identity of the metal-chelating residues.     

"Asymmetric" catalysis by lanthanide complexes

Catalysis with lanthanide (Ln) complexes has been underestimated for long time, although Ln(III) complexes have great advantages as Lewis acid catalysts for "asymmetric" carbon-carbon bond-forming reactions. Lanthanide complexes are highly active in ligand-substitution reactions, especially with hard ligands. The association with substrates and dissociation of products are achieved fast enough for high catalyst efficiency. The asymmetric catalysis of organic reactions can be greatly advanced by the use of Ln complexes with chiral ligands such as binaphthol (binol). Ln(II) complexes are good reducing agents, which can be used in a wide variety of synthetically important reactions; when chiral ligands are used, many of these reactions are highly stereoselective. In the context of "green chemistry", the development of asymmetric Ln catalysts, and their recyclable use, is of increasing importance. This review gives an overview of the most recent developments in catalysis with lanthanide(II) and lanthanide(III) complexes.