Vacant Binding Sites Research Articles

Tungsten Oxo and Tungsten Imido Alkylidene N-Heterocyclic Carbene Complexes for the Visible-Light-Induced Ring-Opening Metathesis Polymerization of Dicyclopentadiene

A system for the visible-light-controlled ring-opening metathesis polymerization (ROMP) of endo,endo-dicyclopentadiene (DCPD) was developed by combining thermally latent group 6 olefin metathesis initiators, whose vacant binding site is occupied by an N-heterocyclic carbene (NHC), with a photoredox catalyst. Similar ruthenium-based systems have previously been utilized in numerous photolithographic applications. Here, we were able to adapt the approach to group 6 metathesis catalysts for the first time. Extensive screening of 31 different olefin metathesis initiators and five different photoredox catalysts revealed that both tungsten oxo and tungsten imido alkylidene NHC complexes together with 2,4,6-triphenylpyrylium tetrafluoroborate (TPPT) as a photocatalyst are most suitable for the desired application. We propose that, upon irradiation of a mixture of the corresponding olefin metathesis catalyst and TPPT with visible light (440 nm), the excited pyrylium compound oxidizes the NHC ligand and a metathesis-active Schrock-type catalyst is formed. This system can be applied to two-dimensional (2D) photolithography in the ROMP of DCPD. Photomasks were employed to produce cross-linked polymers with great spatial resolution within minutes by applying visible light as the sole stimulus giving access to polymers those compared to many other systems do not require thermal or UV post-curing. Notably, mixtures of the two catalysts and DCPD were latent for several days, and catalyst loadings as low as 100 ppm were sufficient for the desired applications proving the robustness and high activity of the developed system.

Time-dependent changes in FT4 and FT3 levels measured using mass spectrometry after an acute ingestion of excess levothyroxine in a case with hypothyroidism

BackgroundThyrotoxicosis is common disorder among endocrine dysfunctions. It is not rare that the free thyroid hormone level exceeds the measurement range of immunoassay. Such extreme high concentration of free thyroid hormone is generally considered to be impossible to measure correctly because of changes in the balance between free hormones and binding proteins by dilution of serum. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), however, higher concentrations are able to be determined.Case presentationWe present a case of a 21-year-old female with congenital hypothyroidism who had taken a total of 5 mg levothyroxine over three consecutive days following discontinuance of the medication for a month. Immunoassay performed 3 hours after the last ingestion showed that the patient’s free thyroxine (FT4) was over 100 pmol/L and her free triiodothyronine (FT3) was 24.5 pmol/L. With a temporary cessation of levothyroxine, the patient was kept for observation without any other medication. Two days after the last ingestion, FT4 was still over 100 pmol/L and FT3 was increased to 28.8 pmol/L. After an additional 4 days, both FT4 and FT3 levels decreased. Through this period, no thyrotoxic symptom or physical sign had appeared. We also measured FT4 and FT3 levels in her cryopreserved serum by ultrafiltration LC-MS/MS. Her FT4 level measured by ultrafiltration LC-MS/MS on the visiting day and 2 days later were 160.0 and 135.5 pmol/L, respectively, indicating that the toxic dose of levothyroxine was partly changed to T3 during the 2 days. The FT3/FT4 ratios were revealed to be low, accounting for the patient’s benign clinical course despite temporal toxic exposure to levothyroxine. It is implied that prior discontinuation of supplementary levothyroxine increases potential vacant binding sites for thyroid hormone as a buffer to prevent toxic T3 effect.ConclusionIt was helpful to clarify the time dependent changes in free thyroid hormone levels by ultrafiltration LC-MS/MS in discussing the clinical course in this case. Though mass spectrometry has a disadvantage in speed for routine laboratory use, its accurate measurement, particularly of levels exceeding the measurable range of the immunoassay, provides valuable information for more appropriate management of extreme thyrotoxicosis.

Calcium-dependent electrostatic control of anion access to the pore of the calcium-activated chloride channel TMEM16A.

TMEM16A is a ligand-gated anion channel that is activated by intracellular Ca2+. This channel comprises two independent pores and closely apposed Ca2+ binding sites that are contained within each subunit of a homodimeric protein. Previously we characterized the influence of positively charged pore-lining residues on anion conduction (Paulino et al., 2017a). Here, we demonstrate the electrostatic control of permeation by the bound calcium ions in mouse TMEM16A using electrophysiology and Poisson-Boltzmann calculations. The currents of constitutively active mutants lose their outward rectification as a function of Ca2+ concentration due to the alleviation of energy barriers for anion conduction. This phenomenon originates from Coulombic interactions between the bound Ca2+ and permeating anions and thus demonstrates that an electrostatic gate imposed by the vacant binding site present in the sterically open pore, is released by Ca2+ binding to enable an otherwise sub-conductive pore to conduct with full capacity.

A luminescence immunoassay test method for determining benzo[a]pyrene in natural water

A test method is developed for determining benzo[a]pyrene in natural water, based on the use of a polyethylene filter (frit) with adsorbed specific antibodies, placed within a transparent column. In passing a test solution, the analyte is adsorbed on the frit similarly to the process implemented in immunoaffinity preconcentration. The added conjugate of a labeled analyte takes the remained vacant binding sites of antibodies. Luminescent semiconductor nanoparticles (quantum dots) CdSe/ZnS, used as labels, enable visual determination under irradiation with UV light. The limit of detection for benzo[a]pyrene in water is similar to 0.5 ng/mL.

Heterometallic [MnPtn(L)2n](x+) Macrocycles from Dichloromethane-Derived Bis-2-pyridyl-1,2,3-triazole Ligands.

There is continued interest in the assembly of heterometallic metallosupramolecular assemblies due to the potential for new structural types and/or interesting chemical and physical properties. Two novel methylene-linked bis-2-pyridyl-1,2,3-triazole ligands (1a, 1b) were synthesized through CuAAC "click" chemistry between dichloromethane (DCM), sodium azide, and the appropriate ethynylpyridine, giving the shortest possible "regular" bis-pyridyl-1,2,3-triazole. The first example of bis-coordination of two 2-pyridyl-1,2,3-triazoles (Rpytri) around one Pt(II) center is reported, giving exclusive formation of head-to-tail [Pt(Rpytri)2](2+) complexes with vacant binding sites suitable for complexation with other metals and the formation of heterometallic assemblies. The concentration-dependent formation of an equilibrium mixture of a heterometallic [Pd2Pt2(L)4](8+) [4 + 4] square and [Pd3Pt3(L)6](12+) [6 + 6] hexagon was observed, and at lower concentrations ([reactants] = 1.5 mM) hexamer formation was negligible. The [Pt(L)2](2+) building block could also be utilized in the synthesis of a concentration-independent [Cu2Pt2(L)4](6+) metallomacrocyle. These compounds were characterized with (1)H, (13)C, and (1)H DOSY NMR spectroscopies, elemental analysis, mass spectrometry, and in some cases X-ray crystallography.

ABL001, a Potent Allosteric Inhibitor of BCR-ABL, Prevents Emergence of Resistant Disease When Administered in Combination with Nilotinib in an in Vivo Murine Model of Chronic Myeloid Leukemia

ABL001, a Potent Allosteric Inhibitor of BCR-ABL, Prevents Emergence of Resistant Disease When Administered in Combination with Nilotinib in an in Vivo Murine Model of Chronic Myeloid Leukemia

Stepwise Deprotonation of a Thiol-Functionalized Bis(1,2,4-triazolium) Salt as a Selective Route to Heterometallic NHC Complexes

Heterometallic NHC complexes have been selectively prepared at room temperature directly from an azolium salt in a two-step procedure. In the unsymmetrically substituted bis(1,2,4-triazolium) ligand precursor, one of the m-xylylene-bridged triazolium units features an unprotected o-thiophenol substituent. This renders possible a selective deprotonation and in situ monopalladation at the NHC–thiolato unit. The obtained palladium(II) complex possesses two pendant triazolium units as vacant binding sites. After a second deprotonation/metalation step, a heterodinuclear palladium(II) gold(I) complex and a heterotrinuclear palladium(II) dicopper(I) complex were obtained. In the latter, two metal centers are connected via a thiolato bridge.

Cell-bound IgE and increased expression of Faɛ-receptors on dendritic cells in cutaneous infiltrates of mycosis fungoides

Skin biopsies of 31 non-atopic patients, 20 with mycosis fungoides, six with psoriasis and five with contact dermatitis, and of five non-atopic healthy controls were compared for the presence of cell-bound IgE and vacant IgE binding sites. IgE+ cells were demonstrated in the cutaneous infiltrate of nine (45%) patients with mycosis fungoides, two (33%) with psoriasis and one (20%) with contact dermatitis. Following pre-incubation of skin sections with IgE myeloma protein to saturate vacant IgE-binding sites, 14 out of 16 patients (88%) with stage I mycosis fungoides, five (83%) patients with psoriasis and one (20%) with contact dermatitis showed an increase in the number of IgE+ cells. While cell-bound IgE was positively related to serum IgE levels the expression of IgE-binding sites was not. All IgE+ cells were HLA-DR+ dendritic cells identified as either macrophages (CD68+, CD14+) or Langerhans cells (CD1+). Skin biopsies of non-atopic healthy controls or clinically uninvolved skin in mycosis fungoides had neither any IgE+ cells nor any vacant binding sites. Inhibition studies with IgG1, IgG4 and IgE myeloma proteins as well as with several enzymatic fragments of IgE demonstrated that IgE interacted with Fc epsilon-receptors through isotype-specific structures on the Fc epsilon-fragment. Four anti-CD23 monoclonal antibodies, however, were unable to stain vacant Fc epsilon-receptors nor could they block IgE-binding. We hypothesize that locally-secreted lymphokines, like IL-4 or interferon-gamma, induce Fc epsilon-receptors on dendritic cells in the cutaneous infiltrate and that these receptors become occupied in parallel with elevated serum IgE levels.

Density functional theory on the larger active site models for [NiFe] hydrogenases: Two-state reactivity?

Abstract The mechanism for H 2 activation catalyzed by [NiFe] hydrogenases is investigated with a series of models for the Ni(II) and Ni(III) forms in both high-spin (HS) and low-spin (LS) states by density functional theory (DFT/B3LYP) calculations. The geometry optimizations include unconstrained models, partially constrained (to the crystal structure parameters) models and models with addition of nearby protein residues. Several uncertainties concerning the mechanism are addressed in our study: (1) the oxidation state of the active species that binds and cleaves H 2 ; (2) the structures and spin states prevalent in active site forms; (3) the influence of the surrounding protein environments on the active site. Adding the nearby protein residues to a fairly rigid active site framework stablizes the LS Ni(II) species. Although models for Ni–SI forms, with a vacant binding site, still prefer HS, addition of H 2 or CO stablizes the LS form. Thus, access to this LS state and two-state reactivity may play a role in the mechanism. Furthermore, the more complete protein models show that the energetic preference for the binding site for both H 2 and CO changes from Fe to Ni. This change brings the computational results in closer accord with the experimental ones.

Investigation of binding mechanisms of nuclear proteins using confocal scanning laser microscopy and FRAP

Fluorescence recovery after photobleaching (FRAP) measurements offer an important tool towards analysing diffusion processes within living biological cells. A model is presented that aims to provide a rigorous theoretical framework from which binding information of proteins from FRAP data can be extracted. A single binding reaction is considered and a set of mathematical equations is introduced that incorporates the concentration of free proteins, vacant binding sites and bound complexes in addition to the on- and off-rates of the proteins. To allow a realistic FRAP model, characteristics of the instruments used to perform FRAP measurements are included in the equation. The proposed model has been designed to be applied to biological samples with a confocal scanning laser microscope (CSLM) equipped with the feature to bleach regions characterised by a radially Gaussian distributed profile. Binding information emerges from FRAP simulations considering the diffusion coefficient, radial extent of the bleached volume and bleach constant as parameters derived from experimental data. The proposed model leads to FRAP curves that depend on the on- and off-rates. Analytical expressions are used to define the boundaries of on- and off-rate parameter space in simplified cases when molecules can move on an infinite domain. A similar approach is ensued when movement is restricted in a compartment with a finite size. The theoretical model can be used in conjunction to experimental data acquired by CSLM to investigate the biophysical properties of proteins in living cells.

A Fluorescence Ratiometric Protein Assay Using Light‐Harvesting Conjugated Polymers

AbstractSummary: A highly selective protein assay was created which combines the fluorescent ratiometric technique based on FRET with the light‐harvesting properties of conjugated polymers. The cationic poly[(9,9‐bis(6′‐N,N,N‐trimethylammonium)‐hexyl)‐fluorene phenylene] bromide (PFP‐NMe) and the negatively charged biotinylated fluorescein probe (Fl‐B) were used to detect the target protein streptavidin optically. The strong electrostatic interactions between PFP‐NMe and fluorescein result in efficient FRET from PFP‐NMe to fluorescein. In the presence of streptavidin, however, the biotin moiety of Fl‐B specifically associates with streptavidin and the fluorescein molecule is buried deeply in the adjacent vacant binding sites. This separates the fluorescein spatially from the PFP‐NMe moiety, resulting in inefficient FRET from PFP‐NMe to fluorescein. Although a nonspecific protein, such as BSA, shows nonspecific interactions with PFP‐NMe, it does not affect the fluorescent ratio value of PFP‐NMe to fluorescein. Hence, the charged neutral complex of two oppositely charged conjugated polymers can eliminate the nonspecific interactions, and thus optimize their application in protein assays.A schematic representation of the protein assay operation.magnified imageA schematic representation of the protein assay operation.

Oxygen Tolerance of the H2-sensing [NiFe] Hydrogenase from Ralstonia eutropha H16 Is Based on Limited Access of Oxygen to the Active Site

Hydrogenases, abundant proteins in the microbial world, catalyze cleavage of H2 into protons and electrons or the evolution of H2 by proton reduction. Hydrogen metabolism predominantly occurs in anoxic environments mediated by hydrogenases, which are sensitive to inhibition by oxygen. Those microorganisms, which thrive in oxic habitats, contain hydrogenases that operate in the presence of oxygen. We have selected the H2-sensing regulatory [NiFe] hydrogenase of Ralstonia eutropha H16 to investigate the molecular background of its oxygen tolerance. Evidence is presented that the shape and size of the intramolecular hydrophobic cavities leading to the [NiFe] active site of the regulatory hydrogenase are crucial for oxygen insensitivity. Expansion of the putative gas channel by site-directed mutagenesis yielded mutant derivatives that are sensitive to inhibition by oxygen, presumably because the active site has become accessible for oxygen. The mutant proteins revealed characteristics typical of standard [NiFe] hydrogenases as described for Desulfovibrio gigas and Allochromatium vinosum. The data offer a new strategy how to engineer oxygen-tolerant hydrogenases for biotechnological application.

Syntheses and structures of mononuclear {Re(CO)3Cl(NN)} ‘complex ligands’ with a pendant imino–pyridine binding site, and preparation of some heterodinuclear Re(I)–lanthanide(III) complexes

Abstract A series of potentially dinucleating ligands, each with two bidentate imino–pyridine compartments, has been used to prepare the mononuclear complexes [Re(CO)3Cl(Ln)] (n=1–4) which each have a vacant binding site and so constitute ‘complex ligands’ for the stepwise synthesis of heterodinuclear complexes. Three of the complexes (with L1, L2 and L4) have been structurally characterised. The mononuclear complex [Re(CO)3Cl(L5)], arising from decomposition of [Re(CO)3Cl(L3)] by hydrolysis of the non-coordinated imine unit, was also isolated and structurally characterised. Luminescence studies show that these compounds are only very weak emitters. The ‘complex ligand’ [Re(CO)3Cl(L1)] has been reacted with [Ln(hfac)3]·2H2O (Ln=Yb, Er) to prepare the heterodinuclear complexes [Re(CO)3Cl(μ-L1)Ln(hfac)3] in which the {Ln(hfac)}3 unit is attached to the second bidentate imino–pyridine site of [Re(CO)3Cl(L1)]. The crystal structures of these show that the bridging ligand has to twist to accommodate the two bulky metal fragments, and that the lanthanide units have an 8-coordinate square-antiprismatic geometry.

Quantitation of the Blocking Effect of Tween 20 and Bovine Serum Albumin in ELISA Microwells

ELISA provides a highly sensitive procedure for quantitating antigens and antibodies. In that assay, microwells are coated initially with a specific ligand and then saturated with inert molecules to minimize nonspecific background. Coating can be improved by pretreating the microwells with poly-l-lysine (PLL). Proteins and Tween 20 are most often used to block vacant binding sites in enzyme-linked immunosorbent assay (ELISA). In the present study the blocking effects of Tween 20 and bovine serum albumin (BSA) were estimated using an original novel approach. In the assay the magnitude of saturation of the microwells was quantitated by measuring the enzymatic activity of alkaline phosphatase adsorbed to residual vacant sites in the microwell. Tween 20 completely saturated ELISA microwells at concentrations higher than 2 μg/ml. If the microwells were pretreated with PLL, even high concentrations of the detergent did not completely saturate the wells. In contrast, BSA completely saturated both PLL-treated and nontreated microwells at 5 μg/ml. Complementation of Tween 20-induced saturation of PLL-treated microwells was achieved only by addition of BSA at concentration required for BSA alone to reach complete saturation. This approach is applicable for assessing binding to ELISA microwells of any reagent of choice either as a ligand or as a blocking reagent.

(μ-Oxo)diiron Complexes of Gated Glyoximes. Structural Changes Accompanying Ligation Trans to the Oxo Bridge

Structures of new low-spin oxo-bridged complexes of bis[(diphenylboryl)dimethylglyoximato]iron, Fe((DMG)BPh2)2, are reported. The pentacoordinate complex, 1 = [Fe((DMG)BPh2)2]2O, contains a bent Fe−O−Fe bridge (166.0°) with the irons each displaced 0.3 Å out of the N4 planes toward the oxo ligand. The Fe−μ-O bond length (1.7097(10) Å) is the shortest yet reported. Both axial phenyl groups are directed to the trans face, creating a superstructure which is collapsed around the vacant binding site. In the butylamine derivative, 2 = [(BuNH2)Fe((DMG)BPh2)2)]2O, the Fe−O−Fe bridge is linear (178.6°) with iron in the N4 plane and Fe−O bond lengths of 1.766(6) Å. The BuNH2 ligand is sandwiched between the axial phenyl groups in intimate contact with them. Structural changes accompanying ligation and their consequences are compared with those in heme systems. Crystal data for 1: orthorhombic, Pnna, a = 17.440(3) Å, b = 21.381(4) Å, c = 16.896(3) Å, V = 6300(2) Å3, Z = 4, R = 0.068. Crystal data for 2 (CHCl3 disolvate): triclinic, P1̄, a = 13.445(3) Å, b = 14.525(4) Å, c = 23.877(6) Å, α = 96.01°, β = 92.61°, γ = 117.40(1)°, V = 4094(2) Å3, Z = 2, R = 0.086.

Binding of phosphonate chelating agents and pyrophosphate to apotransferrin.

Difference ultraviolet spectroscopy has been used to monitor the binding of a series of phosphonate ligands to human apotransferrin. The ligands consist of pyrophosphate as well as the phosphonic acids (aminomethyl)phosphonic acid (AMPA), (hydroxymethyl)phosphonic acid (HMP), (phosphonomethyl)-iminodiacetic acid (PIDA), N,N-bis(phosphonomethyl)glycine (DPG), and nitrilotris(methylenephosphonic acid) (NTP). Equilibrium constants have been measured for the sequential binding of two ligands per molecule of apotransferrin. In addition, site-specific equilibrium constants have been measured for the binding of AMPA, HMP, and PIDA to the vacant binding site of both forms of monoferric transferrin. Since titrations of diferric transferrin produce no difference UV spectrum, it is proposed that the primary binding site for phosphonic acids includes the protein groups that bind the synergistic bicarbonate anion that is required for formation of a stable ferric transferrin complex. It is further proposed that those ligands with two phosphonate groups can simultaneously bind to cationic amino acid side chains that extend into the cleft between the two domains of each lobe of transferrin. From an inspection of the ferric transferrin crystal structure, the most likely anion binding residues in the cleft are Arg-632 and Lys-534 in the C-terminal lobe and Lys-206 and Lys-296 in the N-terminal lobe.