Bulky 4-phosphacyclohexanones: diastereoselective complexations, orthometallations and unprecedented [3.1.1]metallabicycles

The 4-phosphacyclohexanones, 2,2,6,6-tetramethyl-1-phenyl-4-phosphorinanone (La), 1,2,6-triphenyl-4-phosphorinanone ((Ph)Lb), 1-cyclohexyl-2,6-diphenyl-4-phosphorinanone ((Cy)Lb) and 1-tert-butyl-2,6-diphenyl-4-phosphorinanone ((Bu)Lb) have been made by modifications of literature methods. Phosphines (R)Lb are each formed as mixtures of meso- and rac-diastereoisomers. Isomerically pure rac-(Ph)Lb, rac-(Cy)Lb and meso-(Bu)Lb can be isolated by recrystallisation from MeCN. Heating mixtures of isomers of (R)Lb with TsOH leads to isomerisations to give predominantly the meso-(R)Lb. The complex trans-[PdCl2(La)2] (1) is readily made from [PdCl2(NCPh)2] but the analogous platinum complex 2 has not been detected and instead, cyclometallation at the 3-position (alpha to the ketone) in the phosphacycle occurs to give trans-[PtCl(La)(La-3H)] (3) (where La-3H = La deprotonated at the 3-position) featuring a [3.1.1]metallabicycle as confirmed by X-ray crystallography. The analogous palladabicycle 4 has been detected upon treatment of 1 with Et3N in refluxing toluene. The type of complex formed by (R)Lb depends on which diastereoisomer (meso or rac) is involved. rac-(Ph)Lb (a mixture of R,R- and S,S-enantiomers, labelled alpha and beta) forms trans-[MCl2(rac-(Ph)Lb)2], M = Pd (5) or Pt (6), as mixtures of diastereoisomers (alphaalpha/betabeta and alphabeta forms). The structure of alphaalpha-6 has been determined by X-ray crystallography. Ligand competition experiments monitored by 31P NMR showed that Pd(II) and Pt(II) have a significant preference to bind rac-(Ph)Lb over meso-(Ph)Lb. meso-(Bu)Lb reacts with [PtCl2(NCBu(t))2] under ambient conditions to give the binuclear complex [Pt2Cl2(meso-(Bu)Lb-2'H)2] (7) where orthometallation has occurred on one of the exocyclic phenyl substituents as confirmed by X-ray crystallography. rac-(Bu)Lb reacts with [PtCl2(NCBu(t))2] to give a mononuclear cyclometallated species assigned the structure trans-[PtCl(rac-(Bu)Lb-2'H)((Bu)Lb)] (8) on the basis of its 31P NMR spectrum. rac-(Cy)Lb reacts with [PtCl2(NCBu(t))2] in refluxing toluene to give trans-[PtCl2(rac-(Cy)Lb)2] (9) and the crystal structure of alphabeta-9 has been determined.     

Active sampling followed by solid-phase microextraction for the determination of pyrethroids in indoor air

A method based on solid-phase enrichment followed by headspace (HS)-solid-phase microextraction (SPME) is optimized to determine pyrethroids in air. By active sampling, pyrethroids present in air are retained in 25 mg of activated florisil and then transferred from the solid sorbent to an SPME fiber in the HS mode. A small volume of solvent is added to the adsorbent to favor this process. The selection of the adsorbent, as well as the optimization of certain parameters affecting the SPME, is performed using an experimental design strategy. Linearity is studied by external calibration in a wide range of concentrations using gas chromatography coupled to three different detection systems: electron capture detection, micro-electron capture detection, and mass spectrometry. An analysis of variance with a lack-of-fit test is run to validate the calibration data. Breakthrough of the adsorbent was studied sampling from 0.5 to 10 m(3) air, demonstrating that 1 m(3) air could be sampled without losses of pyrethroids. Quantitative recoveries are obtained at three concentration levels, with adequate repeatability. Limits of detection of the method are estimated at the sub-ng/m(3) level in most cases, well below the regulatory limits. Finally, several real indoor samples are collected and analyzed by the proposed method. Identification and quantitation of all target analytes present in the room air are possible.     

Redox non-innocence of thioether crowns: spectroelectrochemistry and electronic structure of formal nickel(III) complexes of aza-thioether macrocycles

The Ni^II complexes [Ni([9]aneNS₂‐CH₃)₂]²⁺ ([9]aneNS₂‐CH₃=N‐methyl‐1‐aza‐4,7‐dithiacyclononane), [Ni(bis[9]aneNS₂‐C₂H₄)]²⁺ (bis[9]aneNS₂‐C₂H₄=1,2‐bis‐(1‐aza‐4,7‐dithiacyclononylethane) and [Ni([9]aneS₃)₂]²⁺ ([9]aneS₃=1,4,7‐trithiacyclononane) have been prepared and can be electrochemically and chemically oxidized to give the formal Ni^III products, which have been characterized by X‐ray crystallography, UV/Vis and multi‐frequency EPR spectroscopy. The single‐crystal X‐ray structure of [Ni^III([9]aneNS₂‐CH₃)₂](ClO₄)₆?(H₅O₂)₃ reveals an octahedral co‐ordination at the Ni centre, while the crystal structure of [Ni^III(bis[9]aneNS₂‐C₂H₄)](ClO₄)₆?(H₃O)₃? 3H₂O exhibits a more distorted co‐ordination. In the homoleptic analogue, [Ni^III([9]aneS₃)₂](ClO₄)₃, structurally characterized at 30 K, the Ni? S distances [2.249(6), 2.251(5) and 2.437(2) Å] are consistent with a Jahn–Teller distorted octahedral stereochemistry. [Ni([9]aneNS₂‐CH₃)₂](PF₆)₂ shows a one‐electron oxidation process in MeCN (0.2 M NBu₄PF₆, 293 K) at E_1/2=+1.10 V versus Fc⁺/Fc assigned to a formal Ni^III/Ni^II couple. [Ni(bis[9]aneNS₂‐C₂H₄)](PF₆)₂ exhibits a one‐electron oxidation process at E_1/2=+0.98 V and a reduction process at E_1/2=?1.25 V assigned to Ni^II/Ni^III and Ni^II/Ni^I couples, respectively. The multi‐frequency X‐, L‐, S‐, K‐band EPR spectra of the 3+ cations and their 86.2 % ⁶¹Ni‐enriched analogues were simulated. Treatment of the spin Hamiltonian parameters by perturbation theory reveals that the SOMO has 50.6 %, 42.8 % and 37.2 % Ni character in [Ni([9]aneNS₂‐CH₃)₂]³⁺, [Ni(bis[9]aneNS₂‐C₂H₄)]³⁺ and [Ni([9]aneS₃)₂]³⁺, respectively, consistent with DFT calculations, and reflecting delocalisation of charge onto the S‐thioether centres. EPR spectra for [⁶¹Ni([9]aneS₃)₂]³⁺ are consistent with a dynamic Jahn–Teller distortion in this compound.     

Catalytic and Thermodynamic Properties of a Tannase Produced by Aspergillus niger GH1 Grown on Polyurethane Foam

Tannase is an inducible enzyme with important applications in the food and pharmaceutical industries. This enzyme was produced by the fungus Aspergillus niger GH1 under solid-state fermentation using polyurethane foam as solid support and tannic acid as sole carbon source and tannase inducer. Physicochemical properties of A. niger tannase were characterized, and the kinetic and thermodynamics parameters on methyl gallate hydrolysis were evaluated. The enzyme was stable in a pH range of 2-8 and a functional temperature range of 25-65 °C. The highest k(cat) value was 2,611.10 s(-1) at 65 °C. Tannase had more affinity for methyl gallate at 45 °C with a K(M) value of 1.82 mM and an efficiency of hydrolysis (k(cat)/K(M)) of 330.01 s(-1) mM(-1). The lowest E(a) value was found to be 21.38 kJ/mol at 4.4 mM of methyl gallate. The lowest free energy of Gibbs (ΔG) and enthalpy (ΔH) were found to be 64.86 and 18.56 kJ/mol, respectively. Entropy (ΔS) was -0.22 kJ/mol K. Results suggest that the A. niger GH1 tannase is an attractive enzyme for industrial applications due its catalytic and thermodynamical properties.     

Material properties of matrix lipids determine the conformation and intermolecular reactivity of diacetylenic phosphatidylcholine in the lipid bilayer

Photopolymerizable phospholipid DC(8,9)PC (1,2-bis-(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine) exhibits unique assembly characteristics in the lipid bilayer. Because of the presence of the diacetylene groups, DC(8,9)PC undergoes polymerization upon UV (254 nm) exposure and assumes chromogenic properties. DC(8,9)PC photopolymerization in gel-phase matrix lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monitored by UV-vis absorption spectroscopy occurred within 2 min after UV treatment, whereas no spectral shifts were observed when DC(8,9)PC was incorporated into liquid-phase matrix 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Liquid chromatography-tandem mass spectrometry analysis showed a decrease in the amount of DC(8,9)PC monomer in both DPPC and POPC environments without any change in the matrix lipids in UV-treated samples. Molecular dynamics (MD) simulations of DPPC/DC(8,9)PC and POPC/DC(8,9)PC bilayers indicate that the DC(8,9)PC molecules adjust to the thickness of the matrix lipid bilayer. Furthermore, the motions of DC(8,9)PC in the gel-phase bilayer are more restricted than in the fluid bilayer. The restricted motional flexibility of DC(8,9)PC (in the gel phase) enables the reactive diacetylenes in individual molecules to align and undergo polymerization, whereas the unrestricted motions in the fluid bilayer restrict polymerization because of the lack of appropriate alignment of the DC(8,9)PC fatty acyl chains. Fluorescence microscopy data indicates the homogeneous distribution of lipid probe 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine rhodamine B sulfonyl ammonium salt (N-Rh-PE) in POPC/DC(8,9)PC monolayers but domain formation in DPPC/DC(8,9)PC monolayers. These results show that the DC(8,9)PC molecules cluster and assume the preferred conformation in the gel-phase matrix for the UV-triggered polymerization reaction.     

Automated flow-based anion-exchange method for high-throughput isolation and real-time monitoring of RuBisCO in plant extracts

In this work, a miniaturized, completely enclosed multisyringe-flow system is proposed for high-throughput purification of RuBisCO from Triticum aestivum extracts. The automated method capitalizes on the uptake of the target protein at 4 °C onto Q-Sepharose Fast Flow strong anion-exchanger packed in a cylindrical microcolumn (105 × 4 mm) followed by a stepwise ionic-strength gradient elution (0-0.8 mol/L NaCl) to eliminate concomitant extract components and retrieve highly purified RuBisCO. The manifold is furnished downstream with a flow-through diode-array UV/vis spectrophotometer for real-time monitoring of the column effluent at the protein-specific wavelength of 280 nm to detect the elution of RuBisCO. Quantitation of RuBisCO and total soluble proteins in the eluate fractions were undertaken using polyacrylamide gel electrophoresis (PAGE) and the spectrophotometric Bradford assay, respectively. A comprehensive investigation of the effect of distinct concentration gradients on the isolation of RuBisCO and experimental conditions (namely, type of resin, column dimensions and mobile-phase flow rate) upon column capacity and analyte breakthrough was effected. The assembled set-up was aimed to critically ascertain the efficiency of preliminary batchwise pre-treatments of crude plant extracts (viz., polyethylenglycol (PEG) precipitation, ammonium sulphate precipitation and sucrose gradient centrifugation) in terms of RuBisCO purification and absolute recovery prior to automated anion-exchange column separation. Under the optimum physical and chemical conditions, the flow-through column system is able to admit crude plant extracts and gives rise to RuBisCO purification yields better than 75%, which might be increased up to 96 ± 9% with a prior PEG fractionation followed by sucrose gradient step.     

Comparison of luminescence ADP production assay and radiometric scintillation proximity assay for Cdc7 kinase

Several assay technologies have been successfully adapted and used in HTS to screen for protein kinase inhibitors; however, emerging comparative analysis studies report very low hit overlap between the different technologies, which challenges the working assumption that hit identification is not dependent on the assay method of choice. To help address this issue, we performed two screens on the cancer target, Cdc7-Dbf4 heterodimeric protein kinase, using a direct assay detection method measuring [(33)P]-phosphate incorporation into the substrate and an indirect method measuring residual ADP production using luminescence. We conducted the two screens under similar conditions, where in one, we measured [(33)P]-phosphate incorporation using scintillation proximity assay (SPA), and in the other, we detected luminescence signal of the ATP-dependent luciferase after regenerating ATP from residual ADP (LUM). Surprisingly, little or no correlation were observed between the positives identified by the two methods; at a threshold of 30% inhibition, 25 positives were identified in the LUM screen whereas the SPA screen only identified two positives, Tannic acid and Gentian violet, with Tannic acid being common to both. We tested 20 out of the 25 positive compounds in secondary confirmatory study and confirmed 12 compounds including Tannic acid as Cdc7-Dbf4 kinase inhibitors. Gentian violet, which was only positive in the SPA screen, inhibited luminescence detection and categorized as a false positive. This report demonstrates the strong impact in detection format on the success of a screening campaign and the importance of carefully designed confirmatory assays to eliminate those compounds that target the detection part of the assay.     

Formation and stability of prolinol and prolinol ether enamines by NMR: delicate selectivity and reactivity balances and parasitic equilibria

Enamine key intermediates in organocatalysis, derived from aldehydes and prolinol or J?rgensen-Hayashi-type prolinol ether catalysts, were generated in different solvents and investigated by NMR spectroscopy. Depending on the catalyst structure, trends for their formation and amounts are elucidated. For prolinol catalysts, the first enamine detection in situ is presented and the rapid cyclization of the enamine to the oxazolidine ("parasitic equilibrium") is monitored. In the case of diphenylprolinol, this equilibrium is fully shifted to the endo-oxazolidine ("dead end") by the two geminal phenyl rings, most probably because of the Thorpe-Ingold effect. With bulkier and electron-withdrawing aryl rings, however, the enamine is stabilized relative to the oxazolidine, allowing for the parallel detection of the enamine and the oxazolidine. In the case of prolinol ethers, the enamine amounts decrease with increasing sizes of the aryl meta-substituents and the O-protecting group. In addition, for small aldehyde alkyl chains, Z-configured enamines are observed for the first time in solution. Prolinol silyl ether enamines are evidenced to undergo slow desilylation and subsequent rapid oxazolidine formation in DMSO. For unfortunate combinations of aldehydes, catalysts, solvents, and additives, the enamine formation is drastically decelerated but can be screened for by a rapid and facile NMR approach. Altogether, especially by clarifying the delicate balances of catalyst selectivity and reactivity, our NMR spectroscopic findings can be expected to substantially aid synthetically working organic chemists in the optimization of organocatalytic reaction conditions and of prolinol (ether) substitution patterns for enamine catalysis.     

NMR investigations on the proline-catalyzed aldehyde self-condensation: Mannich mechanism, dienamine detection, and erosion of the aldol addition selectivity

The proline-catalyzed self-condensation of aliphatic aldehydes in DMSO with varying amounts of catalyst was studied by in situ NMR spectroscopy. The reaction profiles and intermediates observed as well as deuteration studies reveal that the proline-catalyzed aldol addition and condensation are competing, but not consecutive, reaction pathways. In addition, the rate-determining step of the condensation is suggested to be the C-C bond formation. Our findings indicate the involvement of two catalyst molecules in the C-C bond formation of the aldol condensation, presumably by the activation of both the aldol acceptor and donor in a Mannich-type pathway. This mechanism is shown to be operative also in the oligomerization of acetaldehyde with high proline amounts, for which the first in situ detection of a proline-derived dienamine was accomplished. In addition, the diastereoselectivity of the aldol addition is evidenced to be time-dependent since it is undermined by the retro-aldolization and the competing irreversible aldol condensation; here NMR reaction profiles can be used as a tool for reaction optimization.     

Far‐ and Mid‐Infrared Spectroscopic Analysis of the Substrate‐Induced Structural Dynamics of Respiratory Complex I

The catalytic activity of the respiratory NADH:ubiquinone oxidoreductase (complex I) is based on conformational reorganizations. Herein we probe the effect of substrates on the conformational flexibility of complex I by means of ¹H/²H exchange kinetics at the level of the amide proton in the mid‐infrared spectral range (1700–1500 cm^?1). Slow, medium, and fast exchanging domains are distinguished that reveal different accessibilities to the solvent. Whereas amide hydrogens undergo rapid exchange with the solvent in an open structure, hydrogens experience much slower exchange when they are involved in H‐bonded structures or when they are sterically inaccessible for the solvent. The results indicate a structure that is more open in the presence of both NADH and quinon. Complementary information on the overall internal hydrogen bonding of the protein was probed in the far infrared (300–30 cm^?1), a spectral range that includes a continuum mode of the hydrogen bonding signature.