Detection and identification of 4‐hydroxy‐2‐nonenal Schiff‐base adducts along with products of Michael addition using data‐dependent neutral loss‐driven MS3 acquisition: Method evaluation through an in vitro study on cytochrome c oxidase modifications

We report a data‐dependent neutral‐loss‐driven MS³ acquisition to enhance, in addition to abundant Michael adducts, the detection of Schiff‐base adducts of proteins and 4‐hydroxy‐2‐nonenal, a reactive end product of lipid peroxidation. In vitro modification of cytochrome c oxidase, a mitochondrial protein complex, was used as a model to evaluate the method. The technique allowed for a confident validation of modification sites and also identified a Schiff‐base adduct in subunit Vb of the protein complex.     

Kinetic simulation studies on the transient formation of the oxo‐iron(IV) porphyrin radical cation during the reaction of iron(III) tetrakis‐5,10,15,20‐(N‐methyl‐4‐pyridyl)‐porphyrin with hydrogen peroxide in aqueous solution

High‐valent oxo‐iron(IV) species are commonly proposed as the key intermediates in the catalytic mechanisms of iron enzymes. Water‐soluble iron(III) tetrakis‐5,10,15,20‐(N‐methyl‐4‐pyridyl)porphyrin (Fe(III)TMPyP) has been used as a model of heme‐enzyme to catalyse the hydrogen peroxide (H₂O₂) oxidation of various organic compounds. However, the mechanism of the reaction of Fe(III)TMPyP with H₂O₂ has not been fully established. In this study, we have explored the kinetic simulation of the reaction of Fe(III)TMPyP with H₂O₂ and of the catalytic reactivity of FeTMPyP in the luminescent peroxidation of luminol. According to the mechanism that has been established in this work, Fe(III)TMPyP is oxidized by H₂O₂ to produce (TMPyP)^·+Fe(IV)=O (k1 = 4.5 × 10⁴/mol/L/s) as a precursor of TMPyPFe(IV)=O. The intermediate, (TMPyP)^·+Fe(IV)=O, represented nearly 2% of Fe(III)TMPyP but it does not accumulate in suf?cient concentration to be detected because its decay rate is too fast. Kinetic simulations showed that the proposed scheme is capable of reproducing the observed time courses of FeTMPyP in various oxidation states and the decay pro?les of the luminol chemiluminescence. It also shows that (TMPyP)^·+Fe(IV)=O is 100 times more reactive than TMPyPFe(IV)=O in most of the reactions. These two species are responsible for the initial sharp and the sustained luminol emissions, respectively. Copyright © 2003 John Wiley & Sons, Ltd.     

Enantiopure 4‐oxazolin‐2‐ones and 4‐methylene‐2‐oxazolidinones as chiral building blocks in a divergent asymmetric synthesis of heterocycles

Enantiopure 3‐((R)‐ and 3‐((S)‐1‐phenylethyl)‐4‐oxazoline‐2‐ones were evaluated as chiral building blocks for the divergent construction of heterocycles with stereogenic quaternary centers. The N‐(R)‐ or N‐(S)‐1‐phenylethyl group of these compounds proved to be an efficient chiral auxiliary for the asymmetric induction of the 4‐ and 5‐positions of the 4‐oxazolin‐2‐one ring through thermal and MW‐promoted nucleophilic conjugated addition to Michael acceptors and alkyl halides. The resulting adducts were transformed via a cascade process into fused six‐membered carbo‐ and heterocycles. The structure of the reaction products depended on the electrophiles and reaction conditions used. Alternative isomeric 4‐methylene‐2‐oxazolidinones served as chiral precursors for a versatile and divergent approach to highly substituted cyclic carbamates. DFT quantum calculations showed that the formation of bicyclic pyranyl compounds was generated by a diastereoselective concerted hetero‐Diels‐Alder cycloaddition.     

Fluorescence enhancement of europium (III) perchlorate by 1,10‐phenanthroline on the 1‐(naphthalen‐2‐yl)‐2‐(phenylsulthio)ethanone complex and luminescence mechanism

A novel ligand, 1‐(naphthalen‐2‐yl)‐2‐(phenylsulthio)ethanone was synthesized using a new method and its two europium (Eu) (III) complexes were synthesized. The compounds were characterized by elemental analysis, coordination titration analysis, molar conductivity, infrared, thermo gravimetric analyzer‐differential scanning calorimetry (TGA‐DSC), ¹H NMR and UV spectra. The composition was suggested as EuL₅ · (ClO₄)₃ · 2H₂O and EuL₄ · phen(ClO₄)₃ · 2H₂O (L = C₁₀H₇COCH₂SOC₆H₅). The fluorescence spectra showed that the Eu(III) displayed strong characteristic metal‐centered fluorescence in the solid state. The ternary rare earth complex showed stronger fluorescence intensity than the binary rare earth complex in such material. The strongest characteristic fluorescence emission intensity of the ternary system was 1.49 times as strong as that of the binary system. The phosphorescence spectra were also discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Molecular characterization of the β‐amyloid(4‐10) epitope of plaque specific Aβ antibodies by affinity‐mass spectrometry using alanine site mutation

Alzheimer disease is a neurodegenerative disease affecting an increasing number of patients worldwide. Current therapeutic strategies are directed to molecules capable to block the aggregation of the β‐amyloid(1‐42) (Aβ) peptide and its shorter naturally occurring peptide fragments into toxic oligomers and amyloid fibrils. Aβ‐specific antibodies have been recently developed as powerful antiaggregation tools. The identification and functional characterization of the epitope structures of Aβ antibodies contributes to the elucidation of their mechanism of action in the human organism. In previous studies, the Aβ(4‐10) peptide has been identified as an epitope for the polyclonal anti‐Aβ(1‐42) antibody that has been shown capable to reduce amyloid deposition in a transgenic Alzheimer disease mouse model. To determine the functional significance of the amino acid residues involved in binding to the antibody, we report here the effects of alanine single‐site mutations within the Aβ‐epitope sequence on the antigen‐antibody interaction. Specific identification of the essential affinity preserving mutant peptides was obtained by exposing a Sepharose‐immobilized antibody column to an equimolar mixture of mutant peptides, followed by analysis of bound peptides using high‐resolution MALDI‐Fourier transform‐Ion Cyclotron Resonance mass spectrometry. For the polyclonal antibody, affinity was preserved in the H6A, D7A, S8A, and G9A mutants but was lost in the F4, R5, and Y10 mutants, indicating these residues as essential amino acids for binding. Enzyme‐linked immunosorbent assays confirmed the binding differences of the mutant peptides to the polyclonal antibody. In contrast, the mass spectrometric analysis of the mutant Aβ(4‐10) peptides upon affinity binding to a monoclonal anti‐Aβ(1‐17) antibody showed complete loss of binding by Ala‐site mutation of any residue of the Aβ(4‐10) epitope. Surface plasmon resonance affinity determination of wild‐type Aβ(1‐17) to the monoclonal Aβ antibody provided a binding constant K_D in the low nanomolar range. These results provide valuable information in the elucidation of the binding mechanism and the development of Aβ‐specific antibodies with improved therapeutic efficacy.     

An improved synthesis of (2S, 4S)‐ and (2S, 4R)‐2‐amino‐4‐methyldecanoic acids: assignment of the stereochemistry of culicinins

An improved synthesis of (2S, 4S)‐ and (2S, 4R)‐2‐amino‐4‐methyldecanoic acids was accomplished using a glutamate derivative as starting material and Evans' asymmetric alkylation as the decisive step. The NMR data of the two diastereomers were measured and compared with those of the natural product. As a result, the stereochemistry of this novel amino acid unit in culicinins was assigned as (2S, 4R). Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

One‐pot synthesis of (R)‐1‐(pyridin‐4‐yl)ethyl acetate using tandem catalyst prepared by co‐immobilization of palladium and lipase on mesoporous foam: Optimization and kinetic modeling

The synthesis of (R )‐1‐(pyridin‐4‐yl)ethyl acetate was achieved over tandem palladium‐lipase catalyst with 100% selectivity using 4‐acetyl pyridine as a reactant. The 2% w /w palladium and lipase catalyst was successfully co‐immobilized in the microenvironment of the mesocellular foam and characterized by various techniques. The palladium metal from catalyst hydrogenated 4‐acetyl pyridine to form 1‐(pyridin‐4‐yl)ethanol. The generated intermediate product then underwent kinetic resolution over lipase and selectively gave (R )‐1‐(pyridin‐4‐ yl)ethyl acetate. The catalytic conditions were then studied for optimal performance of both steps. The reaction conditions were optimized to 50 °C and toluene as a solvent. Both chemical and enzymatic kinetic models of the reaction were developed for a given set of reaction conditions and kinetic parameters were predicted. At optimal conditions, the obtained selectivity of intermediate (1‐(pyridin‐4‐yl)ethanol) was 51.38%. The final product yield of ((R )‐1‐(pyridin‐4‐yl)ethyl acetate) was 48.62%.     

Selective recognition of thymidine homopolymer (poly T) oligonucleotide with cobalt(II)–4‐[(5‐chloro‐2‐pyridyl)azo]‐1,3‐diaminobenzene complex

The interactions of cobalt(II)–4‐[(5‐chloro‐2‐pyridyl)azo]‐1,3‐diaminobenzene (5‐Cl‐PADAB) complex with different kinds of homopolymer oligonucleotides in basic medium were investigated based on the measurements of resonance light scattering, UV–vis, circular dichroism spectra and dark field light‐scattering imaging. Experiments showed that only thymidine homopolymer (poly T) oligonucleotides with the length in the range of poly T₆ to poly T₁₈ could interact with the Co(II)–5‐Cl‐PADAB complex in alkaline conditions and cause evident color and spectral change. Thus, the binary complex of Co(II)–5‐Cl‐PADAB could be employed as a visual probe for selectively recognizing the poly T oligonucleotides. Copyright © 2009 John Wiley & Sons, Ltd.     

Validated spectrofluorimetric method for the determination of carbamazepine in pharmaceutical dosage forms after reaction with 4‐chloro‐7‐‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl)

A sensitive and simple spectrofluorimetric method has been developed and validated for the determination of the anti‐epileptic drug carbamazepine (CBZ) in its dosage forms. The method was based on a nucleophilic substitution reaction of CBZ with 4‐chloro‐7‐nitrobenzo‐2‐ oxa‐1,3‐diazole (NBD‐Cl) in borate buffer (pH 9) to form a highly fluorescent derivative that was measured at 530 nm after excitation at 460 nm. Factors affecting the formation of the reaction product were studied and optimized, and the reaction mechanism was postulated. The fluorescence–concentration plot is rectilinear over the range of 0.6–8 µg/mL with limit of detection of 0.06 µg/mL and limit of quantitation of 0.19 µg/mL. The method was applied to the analysis of commercial tablets and the results were in good agreement with those obtained using the reference method. Validation of the analytical procedures was evaluated according to ICH guidelines. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of carbamazepine in human urine and serum samples by high‐performance liquid chromatography with post‐column Ru(bipy)‐Ce(SO4)2 chemiluminescence detection

A novel, sensitive and rapid CL method coupled with high‐performance liquid chromatography separation for the determination of carbamazepine is described. The method was based on the fact that carbamazepine could significantly enhance the chemiluminescence of the reaction of cerium sulfate and tris(2,2‐bipyridyl) ruthenium(II) in the presence of acid. The chromatographic separation was performed on a Kromasil® (Sigma‐Aldrich) TM RP‐C18 column (id: 150 mm × 4.6 mm, particle size: 5 µm, pore size: 100 Å) with a mobile phase consisting of methanol–water‐glacial acetic acid (70:29:1, v/v/v) at a flowrate of 1.0 mL/min, the total analysis time was within 650 s. Under optimal conditions, CL intensity was linear for carbamazepine in the range 2.0 × 10^?8 ~ 4.0 × 10^?5 g/mL, with a detection limit of 6.0 × 10^?9 g/mL (S/N = 3) and the relative standard detection was 2.5% for 2.0 × 10^?6 g/mL (n = 11). This method was successfully applied to the analysis of carbamazepine in human urine and serum samples. The possible mechanism of the CL reaction is also discussed briefly. Copyright © 2012 John Wiley & Sons, Ltd.     

α4 phosphoprotein interacts with EDD E3 ubiquitin ligase and poly(A)‐binding protein

Mammalian α4 phosphoprotein, the homolog of yeast Tap42, is a component of the mammalian target‐of‐rapamycin (mTOR) pathway that regulates ribogenesis, the initiation of translation, and cell‐cycle progression. α4 is known to interact with the catalytic subunit of protein phosphatase 2A (PP2Ac) and to regulate PP2A activity. Using α4 as bait in yeast two‐hybrid screening of a human K562 erythroleukemia cDNA library, EDD (E3 isolated by differential display) E3 ubiquitin ligase was identified as a new protein partner of α4. EDD is the mammalian ortholog of Drosophila hyperplastic discs gene (hyd) that controls cell proliferation during development. The EDD protein contains a PABC domain that is present in poly(A)‐binding protein (PABP), suggesting that PABP may also interact with α4. PABP recruits translation factors to the poly(A)‐tails of mRNAs. In the present study, immunoprecipitation/immunoblotting (IP/IB) analyses showed a physical interaction between α4 and EDD in rat Nb2 T‐lymphoma and human MCF‐7 breast cancer cell lines. α4 also interacted with PABP in Nb2, MCF‐7 and the human Jurkat T‐leukemic and K562 myeloma cell lines. COS‐1 cells, transfected with Flag‐tagged‐pSG5‐EDD, gave a (Flag)‐EDD–α4 immunocomplex. Furthermore, deletion mutants of α4 were constructed to determine the binding site for EDD. IP/IB analysis showed that EDD bound to the C‐terminal region of α4, independent of the α4‐PP2Ac binding site. Therefore, in addition to PP2Ac, α4 interacts with EDD and PABP, suggesting its involvement in multiple steps in the mTOR pathway that leads to translation initiation and cell‐cycle progression. J. Cell. Biochem. 110: 1123–1129, 2010. Published 2010 Wiley‐Liss, Inc.     

The role of the C(2) configuration and methyl substitution on the catalytic activity of novel 2,3,3‐ and 2,7,7‐trimethyl‐substituted γ‐aminonorbornan‐2‐ols

A new set of optically active 2,3,3‐ and 2,7,7‐trimethyl‐substituted γ‐aminonorbornan‐2‐ols have been obtained from 2‐methylenenorbornane‐1‐carbonitriles derived from (+)‐camphor and (?)‐fenchone and probed as chiral ligands for the enantioselective addition of diethylzinc to benzaldehyde. This has allowed the study of the structural factors influencing the chirality transfer, such as variation of the relative configuration at C(2) and steric hindrance at C(2), C(3), and C(7) positions of norbornane, which result in the observance of the important role played by the gem‐dimethyl position in γ‐aminonorbornan‐2‐exo‐ols. An empirical rationalization of the obtained experimental results has been realized on the basis of energetically‐favored diastereomeric Noyori‐like transition states. Chirality 2010. © 2010 Wiley‐Liss, Inc.     

Preparation of (2S,4R)‐2,4‐thiazolidinedicarboxylic acid by separation of diastereoisomeric salts with organic amines

L ‐Cysteine was condensed with glyoxylic acid monohydrate in acetic acid at 30°C to give (4R)‐2,4‐thiazolidinedicarboxylic acid [(4R)‐TDA] as a mixture of two diastereoisomers, (2R,4R)‐ and (2S,4R)‐TDA. An attempt was made to separate (2S,4R)‐TDA from the diastereoisomeric salts of (4R)‐TDA with 1‐propylamine, 2‐methyl‐2‐propylamine, benzylamine, and (R)‐ and (S)‐1‐phenylethylamines [(R)‐ and (S)‐PEA]. The salts of (2S,4R)‐TDA were preferentially crystallized as less soluble diastereoisomeric salts. When the salt with (R)‐PEA was employed, the separation was successfully achieved to afford optically pure (2S,4R)‐TDA in a yield of 41%, based on the starting amount of the diastereoisomeric mixture of (4R)‐TDA. Chirality 11:326–329, 1999. © 1999 Wiley‐Liss, Inc.     

Scale‐up and intensification of (S)‐1‐(2‐chlorophenyl)ethanol bioproduction: Economic evaluation of whole cell‐catalyzed reduction of o‐Chloroacetophenone

Escherichia coli cells co‐expressing genes coding for Candida tenuis xylose reductase and Candida boidinii formate dehydrogenase were used for the bioreduction of o‐chloroacetophenone with in situ coenzyme recycling. The product, (S)‐1‐(2‐chlorophenyl)ethanol, is a key chiral intermediate in the synthesis of polo‐like kinase 1 inhibitors, a new class of chemotherapeutic drugs. Production of the alcohol in multi‐gram scale requires intensification and scale‐up of the biocatalyst production, biotransformation, and downstream processing. Cell cultivation in a 6.9‐L bioreactor led to a more than tenfold increase in cell concentration compared to shaken flask cultivation. The resultant cells were used in conversions of 300 mM substrate to (S)‐1‐(2‐chlorophenyl)ethanol (e.e. >99.9%) in high yield (96%). Results obtained in a reaction volume of 500 mL were identical to biotransformations carried out in 1 mL (analytical) and 15 mL (preparative) scale. Optimization of product isolation based on hexane extraction yielded 86% isolated product. Biotransformation and extraction were accomplished in a stirred tank reactor equipped with pH and temperature control. The developed process lowered production costs by 80% and enabled (S)‐1‐(2‐chlorophenyl)ethanol production within previously defined economic boundaries. A simple and efficient way to synthesize (S)‐1‐(2‐chlorophenyl)ethanol in an isolated amount of 20 g product per reaction batch was demonstrated. Biotechnol. Bioeng. 2013; 110: 2311–2315. © 2013 Wiley Periodicals, Inc.     

Binding Pockets and Permeation Channels for Dioxygen through Cofactorless 3‐Hydroxy‐2‐methylquinolin‐4‐one 2,4‐Dioxygenase in Association with Its Natural Substrate, 3‐Hydroxy‐2‐methylquinolin‐4(1H)‐one. A Perspective from Molecular Dynamics Simulations

This work describes an investigation of pathways and binging pockets (BPs) for dioxygen (O₂) through the cofactorless oxygenase 3‐hydroxy‐2‐methylquinolin‐4‐one 2,4‐dioxygenase in complex with its natural substrate, 3‐hydroxy‐2‐methylquinolin‐4(1H)‐one, in aqueous solution. The investigation tool was random‐acceleration molecular dynamics (RAMD), whereby a tiny, randomly oriented external force is applied to O₂ in order to accelerate its movements. In doing that, care was taken that the external force only continues, if O₂ moves along a direction for a given period of time, otherwise the force changed direction randomly. Gates for expulsion of O₂ from the protein, which can also be taken as gates for O₂ uptake, were found throughout almost the whole external surface of the protein, alongside a variety of BPs for O₂. The most exploited gates and BPs were not found to correspond to the single gate and BP proposed previously from the examination of the static model from X‐ray diffraction analysis of this system. Therefore, experimental investigations of this system that go beyond the static model are urgently needed.     

Expressing an (E)-β-farnesene synthase in the chloroplast of tobacco affects the preference of green peach aphid and its parasitoid

(E)-β-Famesene(EβF) synthase catalyses the production of EβF,which for many aphids is the main or only component of the alarm pheromone causing the repellence of aphids and also functions as a kairomone for aphids' natural enemies.Many plants possess EβF synthase genes and can release EβF to repel aphids.In order to effectively recruit the plant-derived EβF synthase genes for aphid control,by using chloroplast transit peptide(CTP) of the small subunit of Rubisco(rbcS) from wheat(Triticum aestivum L.),we targeted AαβFS1,an EβF synthase gene from sweet wormwood(Artemisia annua L),to the chloroplast of tobacco to generate CTP + AαβFS1 transgenic lines.The CTP +AαβFS1 transgenic tobacco plants could emit EβF at a level up to 19.25 ng/day per g fresh tissues,4-12 fold higher than the AαβFS1 transgenic lines without chloroplast targeting.Furthermore,aphid/parasitoid behavioral bioassays demonstrated that the CTP + AαβFS1 transgenic tobacco showed enhanced repellence to green peach aphid(Myzus persicae) and attracted response of its parasitoid Diaeretiella rapae,thus affecting aphid infestation at two trophic levels.These data suggest that the chloroplast is an ideal subcellular compartment for metabolic engineering of plant-derived EβF synthase genes to generate a novel type of transgenic plant emitting an alarm pheromone for aphid control.     

The luminescent properties and crystal structure of Sr(1.5‐x)‐(1.5y)Mg0.5SiO4:xEu2+,yCe3+ blue phosphor synthesized by co‐precipitation method

In order to improve the luminescent performance of silicate blue phosphors, Sr_{(1.5‐x)‐(1.5y)}Mg_0.5SiO₄:xEu²⁺,yCe³⁺ phosphors were synthesized using one‐step calcination of a precursor prepared by chemical co‐precipitation. The crystal structure and luminescent properties of the phosphors were analyzed using X‐ray diffraction and fluorescence spectrophotometry, respectively. Because the activated ions (Eu²⁺) can occupy two different types of sites (Sr₁ and Sr₂), the emission spectrum of Eu²⁺ excited at 350 nm contains two single bands (EM₁ and EM₂) in the wavelength range 400–550 nm, centered at 463 nm, and the emission intensity first increases and then decreases with increasing concentrations of Eu²⁺ ions. Co‐doping of Ce³⁺ ions can greatly enhance the emission intensity of Eu²⁺ by transferring its excitation energy to Eu²⁺. Because of concentration quenching, a higher substitution concentration of Ce³⁺ can lead to a decrease in the intensity. Meanwhile, the quantum efficiency of the phosphor is improved after doping with Ce³⁺, and a blue shift phenomenon is observed in the CIE chromaticity diagram. The results indicate that Sr_{(1.5‐x)‐(1.5y)}Mg_0.5SiO₄:xEu²⁺,yCe³⁺ can be used as a potential new blue phosphor for white light‐emitting diodes.