Substrate affinities for membrane transport proteins determined by 13C cross-polarization magic-angle spinning nuclear magnetic resonance spectroscopy

We have devised methods in which cross-polarization magic-angle spinning (CP-MAS) solid-state NMR is exploited to measure rigorous parameters for binding of (13)C-labeled substrates to membrane transport proteins. The methods were applied to two proteins from Escherichia coli: a nucleoside transporter, NupC, and a glucuronide transporter, GusB. A substantial signal for the binding of methyl [1-(13)C]-beta-d-glucuronide to GusB overexpressed in native membranes was achieved with a sample that contained as little as 20 nmol of GusB protein. The data were fitted to yield a K(D) value of 4.17 mM for the labeled ligand and 0.42 mM for an unlabeled ligand, p-nitrophenyl beta-d-glucuronide, which displaced the labeled compound. CP-MAS was also used to measure binding of [1'-(13)C]uridine to overexpressed NupC. The spectrum of NupC-enriched membranes containing [1'-(13)C]uridine exhibited a large peak from substrate bound to undefined sites other than the transport site, which obscured the signal from substrate bound to NupC. In a novel application of a cross-polarization/polarization-inversion (CPPI) NMR experiment, the signal from undefined binding was eliminated by use of appropriate inversion pulse lengths. By use of CPPI in a titration experiment, a K(D) value of 2.6 mM was determined for uridine bound to NupC. These approaches are broadly applicable to quantifying binding of substrates, inhibitors, drugs, and antibiotics to numerous membrane proteins.     

Derivatives of Some 2-Chloroethylphosphonic Acid Esters,with Plant Growth Regulating Activity

Abstract

A method for the synthesis of esters of 2-(dimethylsulfonium)ethylphos-phonic acid and the results of some trials showing the plant growth regulating activity of these compounds are presented. For the synthesis of the mentioned compounds, dimethyl sulphide is reacted with 2-chloroethylphosphonic acid esters; these esters are obtained through the complex[1] of AlCl₃, PCl₃ and 1,2-dichloroethan (1). Using the optimum reaction conditions, very good yields were obtained (96–99%). This complex is reacted with different alcohols to give 2-chloroethylphosphonic acid esters (2) (R?Me, Et, Pr, i-Pr, Bu, i-Bu, Pe). Using the optimum reaction conditions, in the case of methanol the maximum obtained yield was 50%. In the case of the other alcohols, the obtained yields were between 76 and 83%. An exception is i-propanol. whose ester was obtained with low yields and the reaction parameters modification have little influence on the yield. The reaction of the esters with dimethyl sulphide gives, in good yields (between 69 and 79%). esters of 2-(dimethylsulfonium)ethylphosphonic acid (3). substances with plant growth regulating activity.     

Determination of phenols and phenates in disinfectant formulations by liquid chromatography with UV detection: collaborative study

Fourteen collaborating laboratories assayed o-phenylphenol (OPP), p-t-amylphenol (PTAP), and o-benzyl-p-chlorophenol (OBPCP) in formulated products, both ready-to-use and concentrates, by RP-HPLC. The actives in the samples ranged from 0.03 to 11% OPP, 0.06 to 4% PTAP, and 0.07 to 10% OBPCP either in free forms or as salts. Seven blind duplicates were analyzed. The samples were diluted/extracted with acidified methanol, filtered, and analyzed by LC on a C18 column using gradient elution and UV detection at 285 nm. The concentration of the active ingredients was calculated from a standard curve. Each laboratory weighed each test sample twice within a single analytical run. The data were analyzed using all 14 laboratory results, with appropriate statistical tests to detect outliers. The repeatability RSDs ranged from 0.98 to 3.40% for the free phenols, and 1.26 to 2.51% for the salts. The reproducibility RSDs ranged from 5.31 to 7.80% for the free phenols, and 5.50 to 8.67% for the salts. The HorRat ranged from 0.86 to 2.17 for the free phenols, and 1.54 to 2.72 for the salts.     

Stöber silica particles as basis for redox modifications: particle shape, size, polydispersity, and porosity

The synthesis of Stöber silica particles as basis for redox modifications is optimized for desired properties, in particular diameter in a wide sub-micrometer range, spherical shape, monodispersity, the absence of porosity, and aggregation free isolability for characterization and later covalent modification. The materials are characterized by SEM, DLS, nitrogen sorption isotherms, helium as well as Gay-Lussac (water) pycnometry, and DRIFT spectroscopy. Particles with diameters between approximately 50 and 800 nm are obtained by varying the concentrations of the reagents and reactants, the type of solvent as well as the temperature. The use of high water concentrations and post-synthetic calcination at 600 °C results in silica particles that can be considered as nonporous with respect to the size of the active molecules to be immobilized. The effect of reaction temperature on size distribution is identified. Low polydispersity is achieved by performing the reaction in a temperature range in which a change in temperature has only a weak or no effect on the final particle diameter. Upon optimization of the sol–gel process, the shape of the particles is still spherical. The agreement between experimental and geometric data is within the expected precision of the characterization techniques.     

Kinetics and thermodynamics of chlorpromazine interaction with lipid bilayers: effect of charge and cholesterol

Passive transport across cell membranes is the major route for the permeation of xenobiotics through tight endothelia such as the blood–brain barrier. The rate of passive permeation through lipid bilayers for a given drug is therefore a critical step in the prediction of its pharmacodynamics. We describe a detailed study on the kinetics and thermodynamics for the interaction of chlorpromazine (CPZ), an antipsychotic drug used in the treatment of schizophrenia, with neutral and negatively charged lipid bilayers. Isothermal titration calorimetry was used to study the partition and translocation of CPZ in lipid membranes composed of pure POPC, POPC:POPS (9:1), and POPC:Chol:POPS (6:3:1). The membrane charge due to the presence of POPS as well as the additional charge resulting from the introduction of CPZ in the membrane were taken into account, allowing the calculation of the intrinsic partition coefficients (K(P)) and the enthalpy change (ΔH) associated with the process. The enthalpy change upon partition to all lipid bilayers studied is negative, but a significant entropy contribution was also observed for partition to the neutral membrane. Because of the positive charge of CPZ, the presence of negatively charged lipids in the bilayer increases both the observed amount of CPZ that partitions to the membrane (KP(obs)) and the magnitude of ΔH. However, when the electrostatic effects are discounted, the intrinsic partition coefficient was smaller, indicating that the hydrophobic contribution was less significant for the negatively charged membrane. The presence of cholesterol strongly decreases the affinity of CPZ for the bilayer in terms of both the amount of CPZ that associates with the membrane and the interaction enthalpy. A quantitative characterization of the rate of CPZ translocation through membranes composed of pure POPC and POPC:POPS (9:1) was also performed using an innovative methodology developed in this work based on the kinetics of the heat evolved due to the interaction of CPZ with the membranes.     

Selective photochemistry at stereogenic metal and ligand centers of cis-[Ru(diphosphine)2(H)2]: preparative, NMR, solid state, and laser flash studies

Three ruthenium complexes Λ-[cis-Ru((R,R)-Me-BPE)(2)(H)(2)] Λ-R,R-Ru1H(2), Δ-[cis-Ru((S,S)-Me-DuPHOS)(2)(H)(2)] Δ-S,S-Ru2H(2), and Λ-[cis-Ru((R,R)-Me-DuPHOS)(2)(H)(2)] Λ-R,R-Ru2H(2) (1 = (Me-BPE)(2), 2 = (Me-DuPHOS)(2)) were characterized by multinuclear NMR and CD spectroscopy in solution and by X-ray crystallography. The chiral ligands allow the full control of stereochemistry and enable mechanistic studies not otherwise available. Oxidative addition of E-H bonds (E = H, B, Si, C) was studied by steady state and laser flash photolysis in the presence of substrates. Steady state photolysis shows formation of single products with one stereoisomer. Solid state structures and circular dichroism spectra reveal a change in configuration at ruthenium for some Δ-S,S-Ru2H(2)/Λ-R,R-Ru2H(2) photoproducts from Λ to Δ (or vice versa) while the configuration for Λ-R,R-Ru1H(2) products remains unchanged as Λ. The X-ray structure of silyl hydride photoproducts suggests a residual H(1)···Si(1) interaction for Δ-[cis-Ru((R,R)-Me-DuPHOS)(2)(Et(2)SiH)(H)] and Δ-[cis-Ru((R,R)-Me-DuPHOS)(2)(PhSiH(2))(H)] but not for their Ru(R,R-BPE)(2) analogues. Molecular structures were also determined for Λ-[cis-Ru((R,R)-Me-BPE)(2)(Bpin)(H)], Λ-[Ru((S,S)-Me-DuPHOS)(2)(η(2)-C(2)H(4))], Δ-[Ru((R,R)-Me-DuPHOS)(2)(η(2)-C(2)H(4))], and trans-[Ru((R,R)-Me-DuPHOS)(2)(C(6)F(5))(H)]. In situ laser photolysis in the presence of p-H(2) generates hyperpolarized NMR spectra because of magnetically inequivalent hydrides; these experiments and low temperature photolysis with D(2) reveal that the loss of hydride ligands is concerted. The reaction intermediates [Ru(DuPHOS)(2)] and [Ru(BPE)(2)] were detected by laser flash photolysis and have spectra consistent with approximate square-planar Ru(0) structures. The rates of their reactions with H(2), D(2), HBpin, and PhSiH(3) were measured by transient kinetics. Rate constants are significantly faster for [Ru(BPE)(2)] than for [Ru(DuPHOS)(2)] and follow the substrate order H(2) > D(2) > PhSiH(3) > HBpin.     

[6,6]‐Open and [6,6]‐Closed Isomers of C70(CF2): Synthesis,Electrochemical and Quantum Chemical Investigation

Novel difluoromethylenated [70]fullerene derivatives, C₇₀(CF₂)_n (n=1–3), were obtained by the reaction of C₇₀ with sodium difluorochloroacetate. Two major products, isomeric C₇₀(CF₂) mono‐adducts with [6,6]‐open and [6,6]‐closed configurations, were isolated and their homofullerene and methanofullerene structures were reliably determined by a variety of methods that included X‐ray analysis and high‐level spectroscopic techniques. The [6,6]‐open isomer of C₇₀(CF₂) constitutes the first homofullerene example of a non‐hetero [70]fullerene derivative in which functionalisation involves the most reactive bond in the polar region of the cage. Voltammetric estimation of the electron affinity of the C₇₀(CF₂) isomers showed that it is substantially higher for the [6,6]‐open isomer (the 70‐electron π‐conjugated system is retained) than the [6,6]‐closed form, the latter being similar to the electron affinity of pristine C₇₀. In situ ESR spectroelectrochemical investigation of the C₇₀(CF₂) radical anions and DFT calculations of the hyperfine coupling constants provide evidence for the first example of an inter‐conversion between the [6,6]‐closed and [6,6]‐open forms of a cage‐modified fullerene driven by an electrochemical one‐electron transfer. Thus, [6,6]‐closed C₇₀(CF₂) constitutes an interesting example of a redox‐switchable fullerene derivative.     

Measurement of production date (age) of nanogram amount of uranium

JRC-Karlsruhe obtained a swipe sample from a highly enriched uranium seizure, which had taken place in 2011. Due to the very low amount of uranium (nanograms) a new method needed to be developed to determine the U production date (age). The particles on the swipe were collected on a pyrolytic graphite planchet using a vacuum impactor and they were subsequently leached with ccHNO₃. The “bulk” U isotopic composition (²³⁵U: 72.51?±?0.03 wt%) and the production date (December 1992?±?1 year) determined by MC-ICP-MS indicated that the material showed similarity with two other HEU cases seized earlier in Europe.