A one-bead, one-stock solution approach to chemical genetics: part 2

BACKGROUND: Chemical genetics provides a systematic means to study biology using small molecules to effect spatial and temporal control over protein function. As complementary approaches, phenotypic and proteomic screens of structurally diverse and complex small molecules may yield not only interesting individual probes of biological function, but also global information about small molecule collections and the interactions of their members with biological systems. RESULTS: We report a general high-throughput method for converting high-capacity beads into arrayed stock solutions amenable to both phenotypic and proteomic assays. Polystyrene beads from diversity-oriented syntheses were arrayed individually into wells. Bound compounds were cleaved, eluted, and resuspended to generate 'mother plates' of stock solutions. The second phase of development of our technology platform includes optimized cleavage and elution conditions, a novel bead arraying method, and robotic distribution of stock solutions of small molecules into 'daughter plates' for direct use in chemical genetic assays. This library formatting strategy enables what we refer to as annotation screening, in which every member of a library is annotated with biological assay data. This phase was validated by arraying and screening 708 members of an encoded 4320-member library of structurally diverse and complex dihydropyrancarboxamides. CONCLUSIONS: Our 'one-bead, multiple-stock solution' library formatting strategy is a central element of a technology platform aimed at advancing chemical genetics. Annotation screening provides a means for biology to inform chemistry, complementary to the way that chemistry can inform biology in conventional ('investigator-initiated') small molecule screens.     

A matrix isolation study of the photochemically induced reactions of nitrogen dioxide with 1,2-dibromoethene and 1,2-dichloroethene using Fourier transform infrared spectroscopy

Photolyses of matrices of either BrCHCHBr/NO2/Ar or ClCHCHCl/NO2/Ar using quartz-filtered radiation (lambda>240 nm) led to the appearance of infrared bands attributable to carbonyl, carbon monoxide, and ketene species; no bands belonging to a precursor complex NO2cdots, three dots, centeredXCHCHX (where X=Br or Cl) were observed upon matrix deposition. The possible reaction pathway is discussed.     

Primary ion beam energy effects on secondary ion emission from Ni(001)c(2 × 2)-CO: Classical dynamics calculations and sims

The variation of the SIMS spectra of Ni(001)c(2 × 2)-CO induced by bombardment of Ar⁺ primary ions with kinetic energy between 300 and 1200 eV has been examined in detail. The purpose of the study is to examine the influence of primary ion energy on a number of experimental observables and to test the accuracy of classical dynamics calculations for neutral particles in predicting the experimental results for ejected ions. The calculations were performed using two extreme forms of the Ar⁺ -substrate interaction potential to examine the sensitivity of the results to the parameters that need to be included in the model. We find excellent agreement between the NiCO⁺/Ni⁺ ion yield ratio measured between 300–1200 eV Ar⁺ ion energy and the computational results if the Molière form of the ion-substrate potential is used and if the calculated results are corrected by including an image force. The calculated angular distributions of the ejected particles also agree well with those observed experimentally. From the calculations we see that the extent of CO fragmentation relative to the amount of molecular CO ejection is roughly constant in the 300–1200 eV beam energy range with a slight increase seen at lower (~ 300 eV) energies. The implications of these results are discussed in terms of our ability to study the chemistry and structure of surfaces with SIMS.     

Room temperature electrical conductivity of a highly two dimensional synthetic metal: AsF5-graphite

We report here studies at room temperature of the electrical conductivity of AsF₅-graphite, a lamellar intercalation compound. Compounds with composition C_8nAsF₅ have been synthesized where n is the stage. Preliminary measurements of basal plane electrical conductivities indicating values comparable with OFHC copper have been confirmed. Associated anisotropy ratios $\text{α ≡}\text{σ}{}_{\mathrm{<mtext>a</mtext>}}\text{σ}{}_{\mathrm{<mtext>c</mtext>}}\text{> 10}{}^6$ are observed for n ≤ 3. Data for both the a-axis and c-axis conductivities as a function of stage for low stage compounds is reported.     

Synthesis of Ln(III) chloride tetraphenylporphyrin complexes

The isolation and identification of the first examples of anhydrous lanthanide chloride tetraphenylporphyrin complexes have been described. The purple complexes were generated by the reaction of dilithiotetraphenylporphyrin bis(dimethoxyethane) with lanthanide trichloride tris(tetrahydrofuran) salts to yield the products in up to 85% yield. The crystal structures for the holmium and ytterbium complexes are also presented.     

Comparative qualitative analysis of different classes of compounds in selected Australian and Indian Eucalyptus and Corymbia species: a convenient de-replication method for the eucalypts

JPC – Journal of Planar Chromatography – Modern TLC - Eucalypts are a large group of woody trees with about 900 taxa in 3 genera: Eucalyptus, Corymbia and Angophora. The secondary...     

Phenotyping Reveals Targets of a Pseudo-Natural-Product Autophagy Inhibitor

Pseudo-natural-product (NP) design combines natural product fragments to provide unprecedented NP-inspired compounds not accessible by biosynthesis, but endowed with biological relevance. Since the bioactivity of pseudo-NPs may be unprecedented or unexpected, they are best evaluated in target agnostic cell-based assays monitoring entire cellular programs or complex phenotypes. Here, the Cinchona alkaloid scaffold was merged with the indole ring system to synthesize indocinchona alkaloids by Pd-catalyzed annulation. Exploration of indocinchona alkaloid bioactivities in phenotypic assays revealed a novel class of azaindole-containing autophagy inhibitors, the azaquindoles. Subsequent characterization of the most potent compound, azaquindole-1, in the morphological cell painting assay, guided target identification efforts. In contrast to the parent Cinchona alkaloids, azaquindoles selectively inhibit starvation- and rapamycin-induced autophagy by targeting the lipid kinase VPS34.     

Process variables that control natural fiber welding: time, temperature, and amount of ionic liquid

A systematic study of variables that affect the fiber welding process is presented. Cotton cloth samples are treated with controlled amounts of 1-ethyl-3-methylimidazolium acetate for a series of times and temperatures. Diluting the ionic liquid with a volatile molecular co-solvent allows temporal and spatial control of the welding process not possible with neat ionic liquids. Materials are characterized by scanning electron microscopy, infrared spectroscopy, X-ray diffraction, and mechanical (tensile) testing. Results suggest careful management of process variables permits controlled, reproducible manipulation of chemical and physical properties.     

Determination of eleven coccidiostats in animal feed by liquid chromatography-tandem mass spectrometry at cross contamination levels

A confirmatory multi-residue method has been developed to allow for the detection, confirmation and quantification of eleven coccidiostats in animal feed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method can be used to determine halofuginone, robenidine, nicarbazin, diclazuril, decoquinate, semduramicin, lasalocid, monensin, salinomycin, narasin, maduramicin at levels relating to unavoidable carry over as stated in Regulation 2009/8/EC. Feed samples are extracted with water and acetonitrile with the addition of anhydrous magnesium sulphate and sodium chloride. The extract then undergoes a freezing out step before being diluted and injected onto the LC-MS/MS system. The LC-MS/MS system is run in MRM mode with both positive and negative electrospray ionisation and can confirm all eleven analytes in a run time of 19 min. The sensitivity of the method allows quantification and confirmation for all coccidiostats at a 0.5% carry over level. The method was validated over three days in accordance with of European legislation; Commission Decision 2002/657/EC. Validation criteria of accuracy, precision, decision limit (CCα), and detection capability (CCβ) along with measurement uncertainty are calculated for all analytes. The method was then successfully used to analyse a number of feed samples that contained various coccidiostat substances.