Coupling of a sheep anti-T3 IgG to a solid phase for a triiodothyronine radioimmunoassay

Optimal conditions for coupling sheep anti-T₃ IgG to a solid phase are presented. We found that the optimal activation of microcrystalline cellulose was achieved with 0.15M 1,1-carbonyldiimidazol (CDI) in acetone. We also found that using a 25 mg/cm³ anti-T₃ IgG solution, in barbitone buffer 0.05M, pH 8.0, we could get a reasonable yield of coupling and a remaining solution of anti-T₃ IgG (first supernatant) with a suitable concentration (10 mg/cm³) for another coupling. The solid phase anti-T₃ obtained in these two couplings present similar characteristics which make possible their use in a total T₃ RIA.     

On the High Viscosity of Aqueous Solution of Lysozyme Induced by Some Organic Solvents

A remarkably high viscosity has been induced in protein aqueous solutions by the addition of certain structurally related organic solvents. The effect has been observed for lysozyme aqueous solutions containing tetramethylurea (TMU), dimethylsulfoxide (DMSO), dimethylformamide, and hexamethylphosphortriamide. The effect has also been induced in ferrocytochrome c aqueous solutions by TMU. Critical concentrations for both the protein and organic solvent were verified for the onset of the viscosity increase. A common feature of the solvents which were able to induce the effect is a dipolar moiety (C=O, S → O and P → O) and a nonpolar region represented by the methyl groups. The resulting fluids show an extremely restricted flow and a typical non-Newtonian, pseudoplastic behavior. Use was made of¹H nuclear magnetic resonance (NMR) and Raman spectroscopy to characterize protein structural modifications and of¹³C NMR to investigate changes in relaxation times and chemical shifts in the solvent/water solutions. A systematic rheological characterization of the systems was undertaken for some of the solvents, and unusual patterns of viscous effects were identified for the solvent/water systems both with and without protein. The process was found to be at least partially reversible, as concluded from the recovered original solution rheological characteristics and the original protein¹H NMR spectrum, after eliminating the organic solvent by ultrafiltration. The whole process was characterized as consisting of two mutually independent stages. The first involves an extensive conformational transition of the polypeptide backbone, from a predominantly α-helical to increased random coiled and β-sheet structures, with the occurrence of nonorthodox protein secondary structures at regions above the solvent critical point. The second stage consists of short-lived interchain contacts leading to an entanglement of the macromolecular system as a whole. A microphase reversion in the organic solvent/water mixture, supported by¹³C NMR and rheological results, is proposed as the driving force causing the observed behavior.     

An Efficient and Simple Entry to N-Substituted beta-Enamino Acid Derivatives from 2-Alkyl-2-oxazolines and 2-Alkyl-2-thiazolines

Reaction of azaenolates of 2-alkyl-oxa(thia)zolines 6 with imidoyl chlorides 7 as electrophiles to furnish masked N-substituted beta-enamino acid derivatives 1-2 in 70-90% yield is described. Alternative routes are discussed. Compounds 1-2 generally appear in one tautomeric form, imino or enamino, depending on the nature of the imidoyl chloride. The configuration of the enamino moiety (Z) and the conformation (s-cis) of compounds 1-2 obtained were established by an NMR study and unequivocally set by nuclear Overhauser effect difference experiments. An X-ray structure of compound 1e is also reported, showing a strong intramolecular NH.N hydrogen bond. Ab initio calculations (HF/3-21G and HF/3-21+G) have been carried out on several representative examples (1e, 1p, and 1l) in an attempt to support and provide the correct geometry of these derivatives. Structural considerations among the possible isomers of compounds 1 are discussed. From these studies it was concluded that the theoretical calculations agree with the experimental results. In addition, a very simple one-pot procedure for the preparation of masked N-substituted alpha-alkylated beta-enamino acid derivatives 2 from 6, 7, and different alkyl halides (R(3)Y) is described.     

Effects of ethylene polymerization conditions on the activity of SiO2-supported zirconocene and on polymer properties

The effects of polymerization conditions were evaluated on the production of polyethylene by silica-supported (n-BuCp)₂ZrCl₂ grafted under optimized conditions and cocatalyzed by methylaluminoxane (MAO). The Al : Zr molar ratio, reaction temperature, monomer pressure, and the age and concentration of the catalyst were systematically varied. Most reactions were performed in toluene. Hexane, with the addition of triisobutilaluminum (TIBA) to MAO, was also tested as a polymerization solvent for both homogeneous and heterogeneous catalyst systems. Polymerization reactions in hexane showed their highest activities with MAO : TIBA ratios of 3 : 1 and 1 : 1 for the homogeneous and supported systems, respectively. Catalyst activity increased continuously as Al : Zr molar ratios increased from 0 to 2000, and remained constant up to 5000. The highest activity was observed at 333 K. High monomer pressures (≈ 4 atm) appeared to stabilize active species during polymerization, producing polyethylenes with high molecular weight (≈ 3 × 10⁵ g mol⁻¹). Catalyst concentration had no significant effect on polymerization activity or polymer properties. Catalyst aging under inert atmosphere was evaluated over 6 months; a pronounced reduction in catalyst activity [from 20 to 13 × 10⁵ g PE (mol Zr h)⁻¹] was observed only after the first two days following preparation. © 1999 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 37: 1987–1996, 1999     

Development and validation of a RP-HPLC method for Roflumilast and its degradation products

Roflumilast is a phosphodiesterase type 4 inhibitor that is administered orally as a long-term, in the clinical treatment of chronic obstructive pulmonary disease and asthma. Launched in 2010 for the European market, it currently does not have an official monograph. Here, a reproducible gradient RP-HPLC method was developed and validated for the separation and determination of Roflumilast in the presence of its six major degradation products. Separation was performed on a C₁₈ analytical column (250?×?4.6?mm, 5?µm) with a mobile phase-A of ACN and a phase-B of ammonium acetate buffer (5?mM, pH 4.2) containing triethylamine (0.5% v/v). The most effective RP-HPLC gradient program was determined to be 0/80, 35/10, 36/80, 40/80 (time in minutes/% mobile phase-B). The flow rate was 1.0?ml/min and the column temperature was 25°C. The success of separation of the degradation products with different chemical characteristics was obtained by extending the time of the gradient, changing the proportion of the mobile phases and increasing the velocity of the flow. Two detectors were evaluated for the identification of degradation products and Roflumilast: a diode-arrary detector and a charged aerosol detector. The inability of the charged aerosol detector to dectect one of the six degradation products indicated that the method developed with RP-HPLC and the diode-array detector was more suitable for Roflumilast analysis. The method was validated according to specificity, linearity, LOD, LOQ, accuracy, precision and robustness.     

Interrupted Baeyer–Villiger Rearrangement: Building A Stereoelectronic Trap for the Criegee Intermediate

The instability of hydroxy peroxyesters, the elusive Criegee intermediates of the Baeyer–Villiger rearrangement, can be alleviated by selective deactivation of the stereoelectronic effects that promote the 1,2‐alkyl shift. Stable cyclic Criegee intermediates constrained within a five‐membered ring can be prepared by mild reduction of the respective hydroperoxy peroxyesters (β‐hydroperoxy‐β‐peroxylactones) which were formed in high yields in reaction of β‐ketoesters with BF₃?Et₂O/H₂O₂.     

Preliminary investigation of the application of on-line membrane extraction of trifluoroacetic acid as an aid to improvement of negative ion electrospray mass spectrometry data

We have recently investigated the biodegradation of a number of acidic aromatic compounds that give excellent chromatography using trifluoroacetic acid (TFA) based HPLC methods. Unfortunately HPLC methods using TFA are not usually compatible with detection by negative ion mass spectrometry as TFA suppresses ionisation of the analyte during the electrospray process. We present a preliminary investigation of the use of an anion-exchange micro-membrane suppressor to remove TFA on-line post column with the aim of improvement of mass spectral data using an aromatic acid as an example, Thus LC-MS using a TFA based HPLC method with negative ion mass spectral detection is shown to be possible with good sensitivity.