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High-content screening, typically defined as automated fluorescence microscopy combined with image analysis, is now well established as a means to study test compound effects in cellular disease-modeling systems. In this work, the authors establish several high-content screening assays in the 384-well format to measure the activation of the CC-type chemokine receptors 2B and 3 (CCR2B, CCR3). As a cellular model system, the authors use Chinese hamster ovary cells, stably transfected with 1 of the respective receptors. They characterize receptor stimulation by human monocyte chemoattractant protein-1 for CCR2B and by human eotaxin-1 for CCR3: Receptor internalization and receptor-induced phosphorylation of ERK1/2 (pERK) were quantified using fluorescence imaging and image analysis. The 4 assay formats were robust, displayed little day-to-day variability, and delivered good Z′ statistics for both CCRs. For each of the 2 receptors, the authors evaluated the potency of inhibitory compounds in the internalization format and the pERK assay and compared the results with those from other assays (ligand displacement binding, Ca 2+ mobilization, guanosine triphosphate exchange, chemotaxis). Both physiological agonists and test compounds differed significantly with respect to potencies and efficacies in the various profiling assays. The diverse assay formats delivered partially overlapping and partially complementary information, enabling the authors to reduce the probability of test compound-related technology artifacts and to specify the mode of action for individual test compounds. Transfer of the high-content screening format to a fully automated medium-throughput screening platform for CCR3 enabled the profiling of large compound numbers with respect to G protein signaling and possible tolerance-inducing liabilities. (Journal of Biomolecular Screening 2008:40-53)

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AbstractsEnantioselective synthesis of cyclic and functionalised a-amino acids has been achieved via asymmetric alkylation of a benzophenone-derived glycine-imine 3 mediated by catalysts 1,2(a) 2 or 1,2(b). 5 Tertiary butyl ester 4 of (S)-pipecolic acid has been prepared in 99% ee using this methodology. 5 It has more recently been shown that phosphazene bases allow the reaction to be carried out in homogeneous media. 4 Enantioselective epoxidation of a,b-unsaturated ketone 5 has been achieved with catalyst 1(c) and sodium hypochlorite as the stoichiometric oxidant to give 6 in 95% yield and 89% ee. 6 Alkylation of other enolates allows a variety of chiral functionalised molecules to be prepared. The chiral tetrahydropyran 9 has been synthesised via asymmetric alkylation with 1-chloro-3-iodopropane of the a,b-unsaturated ester 7 to give 8 in 95% ee. 7 Since the first reported use of asymmetric phase transfer catalysts (PTC) derived from the cinchona alkaloids in 1989, 1 the present third generation N-9-anthracenylmethyl quaternary salts 1 and 2 have been shown to catalyse a number of reactions, affording optically active products with high ee's. 2,3 The high enantioselectivity of these salts is due to their well-defined geometries, where three of the four tetrahedron faces around the quaternary nitrogen cation are effectively blocked by the other components of the structure, leaving one relatively open face allowing intimate contact with the counterion of the substrate. This complex can then be attacked enantioselectively. Effective mixing of phases during reaction is necessary, but more recently, catalysts 1(b) and 2(b) have been successfully employed with Schwesinger bases, in homogeneous solution, yielding similar ee's. 4Preparation: The pseudo-enantiomers 1 and 2 are easily prepared from the corresponding commercially available enantiomers of cinchonine via quaternisation with 9-halomethylanthracene. 2,3 The 2-hydroxymethylquinuclidine core has also been synthesised asymmetrically from pyridine-4-ethanol. 8 1437-20961999,0,09,1508,1508V00999ST.pdf SYNLETT Spotlight 9This feature focuses on a reagent chosen by a postgraduate, highlighting the uses and preparation of the reagent in current research