Experimental parameters affecting sensitivity and specificity of a yeast assay for estrogenic compounds: results of an interlaboratory validation exercise

In vitro assays are considered as the first step in a tiered approach to compound screening for hormonal activity. Although many new assays have been developed in recent years, little attention has been paid towards assay validation. Our objective was to identify critical experimental parameters in a yeast estrogen screen (YES) that affect its sensitivity and specificity. We investigated the role of incubation time, solvent type, yeast inoculum growth stage and concentration on the outcome of the YES. Compounds tested included new and established agonists, antagonists and negative controls, and results were evaluated according to prefixed statistical criteria. In addition, we assessed the assay’s performance in a blind interlaboratory validation exercise (IVE). An incubation time of five days was necessary to positively identify the estrogenic properties of all agonists tested, when dissolved in DMSO. Longer incubation times were required when using an ethanol protocol. Similar estrogenic activity was reported for benzyl butyl phthalate, bisphenol-A, methoxychlor, permethrin and genistein in the IVE. One out of the three laboratories did not classify α,β-endosulfan, dissolved in DMSO, as an estrogen. The same was true for 4,4′-DDE and lindane, dissolved in ethanol, a result that might be attributable to an inappropriate yeast start concentration and/or growth stage. These validation experiments show that under appropriate experimental conditions the YES yields sensitive, specific and reliable results. Therefore it fulfills the requirements as a first step screening assay to evaluate the capacity of chemicals to interact with the estrogen receptor.     

End‐group modified poly(methyl vinyl ether): Characterization and LCST demixing behavior in water

A range of hydrophilic poly(methyl vinyl ether) (PMVE) polymers was synthesized by living cationic polymerization of methyl vinyl ether (MVE), having different hydrophilic or hydrophobic chain‐end functionalities. The dissimilar end‐groups were either introduced by end‐capping of the growing polymer chain with LiBH₄, methanol, and water or by functional initiation with 2‐bromo‐(3,3‐diethoxy‐propyl)‐2‐methylpropanoate. The synthesized PMVEs were characterized by ¹H NMR, size exclusion chromatography, and matrix‐assisted laser desorption ionization time of flight, displaying a narrow polydispersity. Modulated temperature DSC was applied to study the influence of the nature of the end‐groups on the solubility behavior of PMVE in water. Terminal‐modification with a hydroxyl function improves the solubility, whereas a Br‐containing end‐group causes the polymer to be insoluble in water at room temperature; however, the special type III lower critical solution temperature demixing behavior being maintained. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 461–469, 2006     

Matrix-isolation FT-IR study and theoretical calculations of the vibrational, tautomeric and H-bonding properties of hypoxanthine

The neutral compound hypoxanthine is investigated using the technique of matrix-isolation FT-IR spectroscopy combined with density functional theory (DFT) and ab initio methods. Two theoretical methods (RHF and DFT/B3-LYP) are compared for vibrational frequency prediction, and four methods (RHF//RHF, MP2//RHF, DFT//DFT and MP2//DFT) for prediction of the relative energies of the tautomers and the interaction energies of the complexes. All the possible tautomeric forms have been considered theoretically, and the results indicate that two oxo forms (O17 and O19) and one hydroxy form (H9-r1) are the three most stable forms. The experimental FT-IR spectra are consistent with this prediction, and nearly all the characteristic spectral features of these forms have been identified in the spectrum. A theoretical study of the H-bonded complexes of these three tautomers with water is also performed. Several structures have been found for each form and the results demonstrate that the closed complexes with two H-bonds are the most stable systems due to the H-bond cooperative effect.     

Asymmetric Ion‐Pairing Catalysis

Charged intermediates and reagents are ubiquitous in organic transformations. The interaction of these ionic species with chiral neutral, anionic, or cationic small molecules has emerged as a powerful strategy for catalytic, enantioselective synthesis. This review describes developments in the burgeoning field of asymmetric ion‐pairing catalysis with an emphasis on the insights that have been gleaned into the structural and mechanistic features that contribute to high asymmetric induction.     

Synthesis of poly(tetrahydrofuran)‐b‐polystyrene block copolymers from dual initiators for cationic ring‐opening polymerization and atom transfer radical polymerization

A dual initiator (4‐hydroxy‐butyl‐2‐bromoisobutyrate), that is, a molecule containing two functional groups capable of initiating two polymerizations occurring by different mechanisms, has been prepared. It has been used for the sequential two‐step synthesis of well‐defined block copolymers of polystyrene (PS) and poly(tetrahydrofuran) (PTHF) by atom transfer radical polymerization (ATRP) and cationic ring‐opening polymerization (CROP). This dual initiator contains a bromoisobutyrate group, which is an efficient initiator for the ATRP of styrene in combination with the Cu(0)/Cu(II)/N,N,N′,N″,N″‐pentamethyldiethylenetriamine catalyst system. In this way, PS with hydroxyl groups (PS‐OH) is formed. The in situ reaction of the hydroxyl groups originating from the dual initiator with trifluoromethane sulfonic anhydride gives a triflate ester initiating group for the CROP of tetrahydrofuran (THF), leading to PTHF with a tertiary bromide end group (PTHF‐Br). PS‐OH and PTHF‐Br homopolymers have been applied as macroinitiators for the CROP of THF and the ATRP of styrene, respectively. PS‐OH, used as a macroinitiator, results in a mixture of the block copolymer and remaining macroinitiator. With PTHF‐Br as a macroinitiator for the ATRP of styrene, well‐defined PTHF‐b‐PS block copolymers can be prepared. The efficiency of PS‐OH or PTHF‐Br as a macroinitiator has been investigated with matrix‐assisted laser desorption/ionization time‐of‐flight spectroscopy, gel permeation chromatography, and NMR. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 3206–3217, 2003     

Tautomery and H-bonding characteristics of 2-aminopurine: a combined experimental and theoretical study

An experimental and theoretical RHF, MP2 and DFT/6-31++G^** study is described of the matrix FT-IR spectra of monomer 2-aminopurine and H-bonded complexes of 2-aminopurine with water. 2-aminopurine occurs in Ar predominantly as the amino-N9H tautomer, but small amounts of the amino-N7H tautomer are also present. An approximate K_T value for this tautomeric equilibrium is found to be 0.016 (RHF) and 0.015 (DFT) using the infrared intensity measurement. Four H-bonded complexes of the abundant amino-N9H form with water are detected in the experimental FT-IR spectrum by their characteristic predicted absorptions, i.e. the three closed complexes N3 ^... H-O ^... H-N9, N1 ^... H-O ^... H-NH, N3 ^... H-O ^... H-NH and the open complex N7 ^... H-OH. From the experimental results, the proton affinity of the N7 atom in 2-aminopurine can be estimated. The dependence of the H-bond strength on the H-bond linearity is demonstrated by a correlation between the N ^... H distance and the N ^... H-O angle in closed N ^... H-O ^... H-N complexes. Received 10 December 2001 Published online 13 September 2002     

Cross-linked polyimide membranes for solvent resistant nanofiltration in aprotic solvents

Solvent stable nanofiltration membranes were prepared through the chemical cross-linking of asymmetric Matrimid^®-based polyimide membranes with p-xylylenediamine. The influence of this straightforward post-treatment on membrane stability, morphology and performance in dimethylformamide (DMF), N-methylpyrrolidinone (NMP), dimethylacetamide (DMAc) and dimethylsulfoxide (DMSO) was thoroughly investigated. With permeabilities up to 5.4 l/m² bar h and rejections up to 98% for low molecular weight dyes in these demanding solvents, optimally performing, truly solvent resistant nanofiltration membranes were obtained. Nanozeolite-filled membranes were prepared in parallel to study the effect of an inorganic filler on the cross-linking reaction and performance in aprotic solvents. The outstanding stability and performance of these membranes and their easy preparation clearly offer vast potential for applications in harsh solvent environments.