Bromo dimethyl sulfoxide osmium(II) complexes. Molecular structure of cis,fac-[OsBr2(dmso-S)3(dmso-O)]

Bromo dimethyl sulfoxide osmium(II) complexes were synthesized: trans-[OsBr₂(dmso-S)₄] (1) was obtained by the reaction of K₂[OsBr₆] with DMSO in the presence of SnBr₂ at 100°C and cis,fac-[OsBr₂(dmso-S)₃(dmso-O)] (2) was prepared by thermal isomerization of 1 in a DMSO solution at 150°C. The coordination mode of DMSO molecules was determined by IR and ¹H and ¹³C NMR spectroscopy. X-ray diffraction analysis showed that compound 2 crystallizes in the monoclinic system, space group P2₁/n; a = 8.4711(5) Å, b = 27.7876(15) Å, c = 8.5569(5) Å, β = 115.7110(10)°; Z = 4. The coordination polyhedron of osmium is a distorted octahedron; the osmium environment is formed by two cis-arranged bromine atoms and three fac-S-coordinated and one O-coordinated DMSO molecules. The interconversion of complexes in solutions was studied by UV/Vis and ¹H and ¹³C NMR spectroscopy. In chloroform and DMSO, complex 2 isomerizes to cis-[OsBr₂(dmso-S)₄] and (in the light) to 1. The complexes trans-[OsX₂(dmso-d₆)₄], where X = Cl, Br, were isolated from DMSO-d ₆ and characterized by the IR spectra.     

Analysis of drug interactions with very low density lipoprotein by high-performance affinity chromatography

High-performance affinity chromatography (HPAC) was utilized to examine the binding of very low density lipoprotein (VLDL) with drugs, using R/S-propranolol as a model. These studies indicated that two mechanisms existed for the binding of R- and S-propranolol with VLDL. The first mechanism involved non-saturable partitioning of these drugs with VLDL, which probably occurred with the lipoprotein’s non-polar core. This partitioning was described by overall affinity constants of 1.2 (±0.3)?×?10⁶ M^?1 for R-propranolol and 2.4 (±0.6)?×?10⁶ M^?1 for S-propranolol at pH 7.4 and 37 °C. The second mechanism occurred through saturable binding by these drugs at fixed sites on VLDL, such as represented by apolipoproteins on the surface of the lipoprotein. The association equilibrium constants for this saturable binding at 37 °C were 7.0 (±2.3)?×?10⁴ M^?1 for R-propranolol and 9.6 (±2.2)?×?10⁴ M^?1 for S-propranolol. Comparable results were obtained at 20 and 27 °C for the propranolol enantiomers. This work provided fundamental information on the processes involved in the binding of R- and S-propranolol to VLDL, while also illustrating how HPAC can be used to evaluate relatively complex interactions between agents such as VLDL and drugs or other solutes.     

Isolation and Immobilization of Influenza Virus-Specific N-SA-α-2,3-Gal Receptors Using Magnetic Nanoparticles Coated with Chitosan and Maackia amurensis Lectin

Avian influenza viruses preferentially bind to sialic acid alpha-2,3-galactose (N-SA-α-2,3-Gal) receptors on epithelial cells. Herein, we describe a procedure we have developed for isolation of N-SA-α-2,3-Gal receptors from porcine trachea using magnetic nanoparticles (NPs) coated with chitosan (NP-Ch) and functionalized with Maackia amurensis lectin (NP-lectin). Magnetic nanoparticles were coated with chitosan in a one-step co-precipitation, and then M. amurensis lectin was immobilized covalently using glutaraldehyde. Lectin coated nanoparticles were incubated with sialic acid enriched fraction of tracheal homogenate, and N-SA-α-2,3-Gal receptor was extracted under magnetic field in two cycles. The presence of 66.4 kDa protein was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The interaction of immobilized receptor (NP-Ch-R) with M. amurensis lectin (NP-Ch-R-L) was demonstrated by Fourier transform infrared spectrometry (FTIR) and thermogravimetric analysis (TGA).     

Miscibility and crystallization behaviors of stereocomplex-type poly(l- and d-lactide)/poly(methyl methacrylate) blends

Stereocomplex-poly(l- and d-lactide) (sc-PLA) and poly(methyl methacrylate) (PMMA) blends were prepared by solution blending at PMMA loadings from 20 to 80 mass%. The miscibility and crystallization behaviors of the blends have been studied in detail by differential scanning calorimeter. The single-glass transition temperatures (T _g) of the blends demonstrated that the obtained system was miscible in the amorphous state. It was observed that the crystallization peak temperature of sc-PLA/PMMA blends was marginally lower than that of neat sc-PLA at various cooling rates, indicating the dilution effect of PMMA on the sc-PLA component to restrain the overall crystallization process. In the study of isothermal crystallization kinetics, the reciprocal value of crystallization peak time ( \( t_{\text{p}}^{ - 1} \) ) decreased with increasing PMMA content, indicating that the addition of non-crystalline PMMA inhibited the isothermal crystallization of sc-PLA at an identical crystallization temperature (T _c). Moreover, the negative value of Flory–Huggins interaction parameter (χ ₁₂ = ?0.16) of the blend further indicated that sc-PLA and PMMA formed miscible blends.     

Penning ionization electron spectrometry of hydrogen chloride

An electron spectrometric study has been performed on HCl using metastable helium and neon atoms as well as neon resonance photons. High resolution electron spectra were obtained with two different beam apparatuses for a mixed He(2¹ S, 2³ S) beam, a pure He(2³ S) beam, and, for the first time, state-selected pure Ne(3s ³ P ₂) and pure Ne(3s ³ P ₀) beams, and for NeI resonance photons. For the system He(2³ S)+HCl the vibrational populationsP(υ′) of the formed HCl⁺ (X ²∏ _i , υ′) and HCl⁺ (A ²Ω⁺, υ′) ions are found to differ from the Franck-Condon factors for unperturbed potentials, indicating slight bond stretching in HCl upon He(2³ S) approach. For He(2¹ S)+HCl the vibrational peak shapes and vibrational populations are substantially different from the He(2³ S) case, pointing to an additional, charge exchanged interaction (He⁺+HCl^?) in the entrance channel of the former system. For the first time, we have detected the electrons in both the He(2¹ S)+HCl and He(2³ S)+HCl spectra associated with the major mechanism for the formation of Cl⁺ ions: energy transfer to repulsive HCl** Rydberg states, dissociating toH(1s) and autoionizing Cl**(¹ D ₂ nl) atoms. For both Ne(³ P ₂)+HCl and Ne(³ P ₀)+HCl, the populationsP(υ′) of both final molecular states HCl⁺ (X, A) agree closely with the Franck-Condon factors at the average relative collision energyē _coll=55 meV and, for HCl⁺ (A ²Ω⁺), also atē _coll=130 meV.     

Kinetics of Water-Mediated Fluctuations in Room Temperature Ionic Liquid: N,N-Diethyl-N-methyl-N-(2-methoxyethyl) Ammonium Tetrafluoroborate

The kinetics of small-angle X-ray scattering (SAXS) was assessed in the room temperature ionic liquid (RTIL)–x mol% H₂O system, where the RTIL is N,N-diethyl-N-methyl-N-(2-methoxyethyl) ammonium tetrafluoroborate, [DEME][BF₄]. During equilibration of a non-equilibrium state, the largest time evolution of SAXS was observed at approximately 90 mol% H₂O. Above 85 mol% (x _c), the SAXS intensity increased gradually for 24 h. For the larger q region, the prepeak and principal peak in X-ray diffraction patterns have no time dependence in the water-rich region (70–91 mol%). The long time relaxation process observed in SAXS was related to the outstanding pH oscillations at 90–95 mol% over several days in the [DEME][BF₄]–water system. The x _c for nonequilibrium anomalies is related to the equilibrated crossover concentration from 65–85 mol% (from SAXS) to 85–95 mol% (for the prepeak in X-ray diffraction) in the [DEME][BF₄]–H₂O system. Inside [DEME][BF₄], the dynamic and static properties of hydrogen bonding of water changed drastically at x _c.     

On the determination of HCl+ (A,v′) vibrational populations from HCl+ (A→X) fluorescence spectra: direct comparison between photoionization and penning ionization

Using a beam apparatus, we have measured the HCl⁺ (A,v′→X,v″) fluorescence spectra of HCl⁺ (A,v′) ions formed in HeI (58.4 nm), and NeI (73.6 nm) photoionization and, for the first time, in He (2³ S) Penning ionization under single collision conditions with a wavelength bandwidth around 1 nm. In addition, we have studied Ne (3s ³ P _{2, 0}) Penning ionization of HCl at three different collision energies. The procedure and the problems in extracting HCl⁺ (A,v′) vibrational populations from the data are discussed in some detail. Thedirect comparison of photoionization and Penning ionization data allows definitive conclusions to be drawn on the question whether final state interactions in the Penning reaction change the “nascent” vibrational population (determined by electron spectrometry); for He (2³ S)+HCl, such changes are shown to be absent within the experimental uncertainty (<±10%). For Ne (3s ³ P _{2, 0})+HCl, the HCl⁺ (A,v′=0, 1) populations are also found to be close to those measured by electron spectrometry and essentially independent of collision energy in the range 34–96 meV. From measurements of the fluorescence intensity as a function of HCl density, we have evidence for a fast loss of HCl⁺ (A,v′) ions in collisions with HCl (rate constant around 5·10^?9 cm³s^?1).     

Lariat ethers in the chiral recognition of amino acid esters:electrospray ionization mass spectrometry investigation

The ability of the crown ethers (1–4), containing the ortho- or para- methoxyphenoxy-methyl substituents in their structure, to chiral recognition in reference to amino acid esters has been investigated by electrospray ionization mass spectrometry (ESI-MS). The method allows registering the diastereomeric complexes between the studied crowns as hosts and the protonated alanine, phenylglycine and phenylalanine methyl esters as guests in the gas phase. ESI-MS experiments using isotopically labeled guests provide robust and reproducible results, indicating a moderate degree of chiral discrimination in the series of the studied crown ethers. ESI-MS experiments using achiral amine as a reference yielded the results comparable with the previous method. It has been found that (S)-enantiomers of the crowns bind predominately (S)-enantiomers of the amino acid esters, and vice-versa. It has been shown that the chiral ortho-substituted crown (S)-1 demonstrates the more pronounced values for chiral discrimination as compared with the para-substituted crown (S)-2. This fact indicates the interrelationship between the chiral recognition and the lariat nature of crown 1. Increasing the size of the cavity and the presence of a flat aromatic moiety in crowns 3 and 4 strengthens their complexing ability, simultaneously weakening the enantioselectivity of the complexation.     

Graft copolymers via combination of cationic polymerization and atom transfer radical polymerization and their phase separation into spherical/worm-like nanostructures

Poly(p-chloromethyl styrene)-graft-poly(methyl methacrylate) (PCMS-g-PMMA) and poly(p-chloromethyl styrene)-graft-poly(benzyl methacrylate) (PCMS-g-PBzMA) graft copolymers with asymmetric branches are synthesized via the combination of cationic polymerization and atom transfer radical polymerization (ATRP). The process involves first, the preparation of poly(p-chloromethyl styrene) (PCMS-CH₂Cl) macroinitiator without any cross-linking or side reactions through pendant benzyl chloride (?CH₂Cl) functionality by cationic polymerization using a simple FeCl₃-based initiating system at 25 °C. The as-synthesized PCMS-CH₂Cl, without any transformation, is then used as the macroinitiator to graft PMMA and PBzMA branches by ATRP to produce PCMS-g-PMMA and PCMS-g-PBzMA graft copolymers of varying compositions with controlled molecular weight and moderately narrow polydispersities (M _w/M _n?≤?1.32). The resulting PCMS₂₁ -g-PMMA₂₃₂ graft copolymer in thin film form phase separates into spherical morphology with an average diameter of 170?±?72 nm. Whereas the PCMS₂₁ -g-PBzMA₁₅₆ graft copolymer gives worm-like nanostructures with an average length of 94 nm and width of 31 nm due to phase separation as visualized through atomic force microscopy. On the other hand, the phase-separated morphology is not very well-defined for other graft copolymers (PCMS₁₁₃ -g-PMMA₂₂₇ and PCMS₁₁₃ -g-PBzMA₁₅₄) thin films containing longer PCMS chains. This approach represents a rapid and convenient route to prepare unique spherical/worm-like polymer nanostructures. Figure

Well-defined poly(p-chloromethyl styrene)-graft-poly(methyl methacrylate) (PCMS-g-PMMA) and poly(p-chloromethyl styrene)-graft-poly(benzyl methacrylate) (PCMS-g-PBzMA) graft copolymers with asymmetric branches are synthesized by the combination of living cationic polymerization and atom transfer radical polymerization (ATRP). The resulting PCMS₂₁ -g-PMMA₂₃₂ and PCMS₂₁ -g-PBzMA₁₅₆ graft copolymers phase separate into nanostructured spherical and worm-like morphologies, respectively, in thin film form. The phase-separated morphology is not very well-defined for graft copolymers (PCMS₁₁₃ -g-PMMA₂₂₇ and PCMS₁₁₃ -g-PBzMA₁₅₄) thin films containing longer PCMS chains.     

Quantitative characterization by asymmetrical flow field-flow fractionation of IgG thermal aggregation with and without polymer protective agents

Complexes formed between poly(acrylates) and polyclonal immunoglobulin G (IgG) in its native conformation and after heat stress were characterized using asymmetric flow field-flow fractionation (AF4) coupled with on-line UV-Vis spectroscopy and multi-angle light-scattering detection (MALS). Mixtures of IgG and poly(acrylates) of increasing structural complexity, sodium poly(acrylate) (PAA), a sodium poly(acrylate) bearing at random 3 mol % n-octadecyl groups, and a random copolymer of sodium acrylate (35 mol %), N-n-octylacrylamide (25 mol %) and N-isopropylacrylamide (40 mol %), were fractionated in a sodium phosphate buffer (0.02 M, pH 6.8) in the presence, or not, of 0.1 M NaCl. The AF4 protocol developed allowed the fractionation of solutions containing free poly(acrylates), native IgG monomer and dimer, poly(acrylates)/IgG complexes made up of one IgG molecule and a few polymer chains, and/or larger poly(acrylates)/IgG aggregates. The molar mass and recovery of the soluble analytes were obtained for mixed solutions of poly(acrylates) and native IgG and for the same solutions incubated at 65 °C for 10 min. From the combined AF4 results, we concluded that in solutions of low ionic strength, the presence of PAA increased the recovery ratio of IgG after thermal stress because of the formation of electrostatically-driven PAA/IgG complexes, but PAA had no protective effect in the presence of 0.1 M NaCl. Poly(acrylates) bearing hydrophobic groups significantly increased IgG recovery after stress, independently of NaCl concentration, because of the synergistic effect of hydrophobic and electrostatic interactions. The AF4 results corroborate conclusions drawn from a previous study combining four analytical techniques. This study demonstrates that AF4 is an efficient tool for the analysis of protein formulations subjected to stress, an important achievement given the anticipated important role of proteins in near-future human therapies. ?      

Research on unsaturated azole derivatives

3-Formylindazoles are converted to 1-methyl- and 2-methyl-substitutedβ-(3-indazolyl)acrylic andα-bromoacrylic acids by the Wittig reaction. Dehydrohalogenation of theβ-(3-indazolyl)-α-bromoacrylic acid esters gaveβ-(3-indazolyl)propiolic acids, the decarboxylation of which gives isomeric 1-methyl- and 2-methyl-substituted 3-ethynylindazoles.     

Molar heats of transport of dilute aqueous HCl solutions: A potentiometric study

Heats of transport for dilute aqueous HCl solutions at 25°C have been determined from the measurements of thermoelectric powers of the thermocell $$(T){\text{ }}Ag{\text{ - }}AgCl/HCl(ag.)/Ag - AgCl{\text{ (T + }}\Delta {\text{T)}}$$ The variation of the heat of transport with concentration has been examined up to 0.04M and the molar heat of transport at infinite dilution obtained by extrapolation. Present experimental results may be summarized by the equation $${\text{Q}}^ * = {\text{ }}3397 - 3734I^{1/2} {\text{ + }}33610{\text{I}}^{{\text{3/2}}}$$ whereQ ^* is the heat of transport in cal-mole^?1 andI is the ionic strength.     

Preparation of immobilized sepharose-micrococcal nuclease derivatives: activity and stability

Micrococcal nuclease has been covalently attached to CNBr-activated Sepharose 4B by coupling through three different enzyme functions: (a) amino groups; (b) carboxyl groups; and (c) tyrosyl or histidyl residues. On the basis of coupling yield and catalytic efficiency, Sepharose-(NH₂) nuclease derivatives were chosen for further activity andstability studies. The activity of the insoluble enzyme has been evaluated with macromolecular (DNA) and small (synthetic nucleotide) substrates; with the latter the enzyme retains 70% of native enzyme activity. Good enhancement of enzyme stability in the 4–40°C range has been observed.     

Development of ssDNA Aptamers for the Sensitive Detection of Salmonella typhimurium and Salmonella enteritidis

Salmonella enterica subsp. enterica ser. enteritidis and Salmonella enterica subsp. enterica ser. typhimurium are the most common and severe food-borne pathogens responsible for causing salmonellosis in humans and animals. The development of an early and ultra-sensitive detection system is the first critical step in controlling this disease. To accomplish this, we used the cell systematic evolution of ligands by exponential enrichment (Cell-SELEX) technique to identify single-stranded DNA (ssDNA) aptamers to be used as detection probes that can specifically bind to S. enteritidis and S. typhimurium. A total of 12 target-specific ssDNA aptamers were obtained through ten rounds of Cell-SELEX under stringent selection conditions, and negative selection further enhanced the selectivity among these aptamers. Aptamer specificity was investigated using the gram-negative bacteria E. coli and P. aeruginosa and was found to be much higher towards S. enteritidis and S. typhimurium. Importantly, three candidate aptamers demonstrated higher binding affinities and the dissociation constants (Kd) were found to be in the range of nanomolar to submicromolar levels. Furthermore, individual aptamers were conjugated onto polyvalent directed aptamer polymer, which led to 100-fold increase in binding affinity compared to the individual aptamers alone. Taken together, this study reports the identification of higher affinity and specificity ssDNA aptamers (30mer), which may be useful as capture and detection probes in biosensor-based detection systems for salmonellosis.     

Polyfunctional imidazoles: VIII. 1-Aryl-4-chloro-5-[R-sulfanyl(sulfonyl)methyl]-1H-imidazoles

Alkylation of (1-aryl-4-chloro-1H-imidazol-5-yl)methanethiols with alkyl halides, propargyl bromide, or chloroacetic acid gave 1-aryl-5-(R-sulfanylmethyl)-4-chloro-1H-imidazoles. 1-Aryl-4-chloro-5-(methylsulfanylmethyl)-1H-imidazoles and [(1-aryl-4-chloro-1H-imidazol-5-yl)methylsulfanyl]acetic acids were oxidized to the corresponding sulfones with potassium permanganate.     

Ultrasensitive detection and identification of BRAF V600 mutations in fresh frozen,FFPE, and plasma samples of melanoma patients by E-ice-COLD-PCR

A number of molecular diagnostic methods have been developed for the detection and identification of mutations in tumor samples, which are important for the choice of treatment in the context of personalized medicine. For the treatment of metastatic melanoma, Vemurafenib is recommended for patients with BRAF V600 activating mutations. However, the different assays developed to date for the detection of these mutations lack sensitivity or specificity or do not allow a sequencing-based identification or validation of the mutation. Recently, enhanced improved and complete enrichment co-amplification at lower denaturation temperature-polymerase chain reaction (E-ice-COLD-PCR) has been developed as a sensitive method for the detection and identification of mutations in KRAS codons 12/13. Here, we present the first E-ice-COLD-PCR assay for the detection and identification of BRAF codon 600 mutations, which has a large dynamic range, as 25 pg to 25 ng can be used as DNA input without any reduction in mutation enrichment efficiency, and which can detect down to 0.01 % of mutated alleles in a wild-type background. The assay has been validated on fresh frozen, formalin-fixed paraffin-embedded (FFPE), and plasma samples of melanoma patients and has allowed the detection and identification of BRAF mutations present in samples appearing as wild type using standard pyrosequencing, endpoint genotyping, or Sanger sequencing. Thus, the BRAF V600 E-ice-COLD-PCR assay is currently one of the most powerful molecular diagnostic tools for the ultrasensitive detection and identification of BRAF codon 600 mutations.     

A new synthesis of bicyclic N,O- and N,S-enaminals by the anionic cyclization of alk-4-ynals with amino alcohols and amino thiols

A reaction of alk-4-ynals with aliphatic amino alcohols or 2-aminoethanethiol in the system DMSO—KOH gives bicyclic N,O- and N,S-enaminals: 6-methylidenehexahydro-2H-pyrrolo[2,1-b][1,3]oxazines, 5-methylidenehexahydropyrrolo[2,1-b]oxazoles, or 5-methyl-idenehexahydropyrrolo[2,1-b]thiazoles. The reaction proceeds through the formation of equilibrium mixtures of the corresponding imines and monocyclic aminals with subsequent 5-exodig-cyclization catalyzed by the superbasic system DMSO-KOH.     

A core-shell surface magnetic molecularly imprinted polymers with fluorescence for λ-cyhalothrin selective recognition

In this study, we report here a general protocol for making core-shell magnetic Fe₃O₄/SiO₂-MPS/MIPs (MPS = 3-(methacryloxyl) propyl trimethoxysilane, MIPs = molecularly imprinted polymers, Fe₃O₄/SiO₂-MPS as core, MIPs as shell) via a surface molecular imprinting technique for optical detection of trace λ-cyhalothrin. The fluorescent molecularly imprinted polymer shell was first prepared by copolymerization of acrylamide with a small quantity of allyl fluorescein in the presence of λ-cyhalothrin to form recognition sites without doping. The magnetic Fe₃O₄/SiO₂-MPS/MIPs exhibited paramagnetism, high fluorescence intensity, and highly selective recognition. Using fluorescence quenching as a detecting tool, Fe₃O₄/SiO₂-MPS/MIPs were successfully applied to selectively and sensitively detect λ-cyhalothrin, and a linear relationship could be obtained covering a wide concentration range of 0–50 nM with a correlation coefficient of 0.9962 described by the Stern-Volmer equation. The experimental results of practical detection revealed that magnetic Fe₃O₄/SiO₂-MPS/MIPs as an attractive recognition element was satisfactory for determination of trace λ-cyhalothrin in honey samples. This study, therefore, demonstrated the potential of MIPs for detection of λ-cyhalothrin in food.     

Multiplex genotyping of KRAS point mutations in tumor cell DNA by allele-specific real-time PCR on a centrifugal microfluidic disk segment

Point Mutations on the Kirsten rat sarcoma viral oncogene homolog (KRAS) have been identified as an important predictive biomarker for response to cancer therapy targeting the epidermal growth factor receptor. KRAS mutations are prevalent in up to 40 % of all colorectal carcinomas, and routinely conducted KRAS genotyping is becoming mandatory to predict therapy success and to reduce therapy costs. We report a low-cost, disposable and ready-to-use centrifugal microfluidic cartridge (termed GeneSlice) containing preloaded primers and probes. The GeneSlice cartridge enables the parallel detection of the seven most relevant KRAS point mutations by allele-specific real-time PCR. It represents a cost effective alternative to dideoxy-sequencing with a faster time-to-result (~ 2 h versus up to 20 h in case of dd-sequencing). Microfluidic processing of the GeneSlice along with allele-specific amplification and real-time detection are conducted in a slightly modified, commercially available PCR thermocycler. Intra-chip standard deviation of C_q values on the GeneSlices is negligible (GeneSlice 1: C_q,std.dev. = 0.13; GeneSlice 2: C_q,std.dev?=?0.26). In 23 of 24 experiments, the data for genotyping 6 cancer cell lines (n?=?4 per cell line) agreed with dd-sequencing. Additionally, DNA derived from microdissected formalin-fixed and paraffin embedded colorectal carcinomas of two cases was genotyped correctly and reproducibly (n?=?3 per patient; one GeneSlice excluded from evaluation). The GeneSlice therefore clearly demonstrated the potential to become a valuable tool for routine diagnostics of KRAS mutations by reducing costs and hands-on time. Figure

Photograph of a centrifugal microfluidic cartridge “GeneSlice” for multiplex genotyping of KRAS point mutations from tumor cell DNA by allele-specific real-time PCR. Information about the mutation status is required to predict success of state-of-the-art cancer therapy with antibodies