Small molecule-dependent genetic selection in stochastic nanodroplets as a means of detecting protein-ligand interactions on a large scale

Background: Understanding the cellular role of a protein often requires a means of altering its function, most commonly by mutating the gene encoding the protein. Alternatively, protein function can be altered directly using a small molecule that binds to the protein, but no general method exists for the systematic discovery of small molecule ligands. Split-pool synthesis provides a means of synthesizing vast numbers of small molecules. Synthetic chemists will soon be able to synthesize natural product-like substances by this method, so compatible screening methods that detect the activity of minute quantities of molecules among many inactive ones will be in demand.Results: We describe two advances towards achieving the above goals. First, a technique is described that uses a simple spray gun to create 5000–8000 droplets randomly, each having a volume of 50–200 nanoliters. The individual ‘nanodroplets’ contain a controlled number of cells and many also contain individual synthesis beads. As small molecules can be photochemically released from the beads in a time-dependent manner, the concentration of ligands that the cells are exposed to can be controlled. The spatial segregation of nanodroplets prevents the mixing of compounds from other beads so the effects of each molecule can be assayed individually. Second, a small molecule-dependent genetic selection involving engineered budding yeast cells was used to detect intracellular protein-ligand interactions in nanodroplets.Conclusions: The technique described here should facilitate the discovery of new cell-permeable ligands, especially when combined with a positive selection assay that detects intracellular binding of small molecules to proteins. Using ‘anchored combinatorial libraries’, it may be possible to screen entire libraries of natural product-like molecules against the entire collection of proteins encoded within cDNA libraries in a single experiment.     

Determination of the standard Gibbs free energies of formation of the silicates of cobalt,magnesium, and strontium by e.m.f. measurements

Standard Gibbs free energies of formation of the silicates in the quasi-binaries: CoO and SiO₂, MgO and SiO₂, and SrO and SiO₂ were determined with the help of solid-state galvanic cells in the temperature ranges 1273 to 1673 K, 1373 to 1573 K, and 1323 to 1473 K, respectively. Stabilized ZrO₂ was used as an electrolyte for the CoO and SiO₂ system. For the MgO and SiO₂ system MgF₂, and for the SrO and SiO₂ system SrF₂, was used as an electrolyte embedded in a silicate-matrix phase.     

SIMS analysis of poorly conducting surfaces

Summary Charge effects perturb the analysis of poorly conducting materials (glass, ceramics, polymers etc.) via modern surface physics methods which use charged primary and/or secondary particles. This is also true for secondary ion mass spectrometry (SIMS). The use of neutral primary particles (Neutral Primary Beam-SIMS = NPB-SIMS) reduces such perturbations appreciably: positive as well as negative secondary ions may be analyzed. The original distribution of very mobile species in the matrix to be analyzed will not be changed if certain conditions are observed. With this in mind the analysis of chemical and isotopic concentration profiles is possible as they are encountered in corrosion processes, tracer diffusion experiments, and surface engineering or semiconductor technology.

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SIMS-Analyse von Isolatoroberflächen

     

Thermodynamics of nickel orthosilicate

The standard Gibbs free energy, enthalpy, and entropy of formation of nickel orthosilicate have been determined with the help of a solid-electrolyte galvanic cell involving Ni²⁺ exchanged β-alumina electrolyte. The results have been compared with those obtained by other authors.     

A facilitated cyclic ether formation and its potential application in solid-phase peptide and organic synthesis

A "trimethyl lock" system has been known to facilitate lactonization reactions through what has been termed a stereopopulation control mechanism. We have found that a similar trimethyl lock system can also facilitate cyclic ether formation with the concomitant release of a carboxylic acid in the presence of anhydrous tetrabutylammonium fluoride. To study this base-mediated trimethyl lock-facilitated cyclic ether formation, we synthesized fifteen model compounds. All model compounds underwent base-mediated cyclic ether formation in high yields at 0 degree C to room temperature (r.t.) with the concomitant release of the attached carboxylate. Such a system potentially could be used for the development of a two-dimensional linker for solid phase peptide and organic synthesis.     

Racemization of an asparagine residue during peptide deamidation

The Asn residue in the pentapeptide Asn-Gln-Asn-Glu-Gly undergoes racemization at the Calpha center in the course of deamidation of this residue through a succinimide intermediate. The succinimide intermediate is known to racemize at the corresponding center, leading to racemized products of deamidation. Return of this intermediate to reactant Asn is very unlikely in dilute solution where attack of product ammonia on the succinimide is precluded, and Asn racemization has not been previously observed. We give evidence that the observed racemization occurs at the tetrahedral-intermediate stage preceding the succinimide intermediate. The observation is significant for protein stability in vivo and in vitro and has importance in medicine, food chemistry, and archaeological/palaeological dating.     

Mechanism of autoxidation of 5,7-dihydroxytryptamine: effect of fluorine substitution at positions 4 and/or 6

Analogs of 5,7-dihydroxytryptamine (5,7-DHT), namely, 4-fluoro-, 6-fluoro-, and 4,6-difluoro-5,7-DHT's (30a-c) were synthesized starting from 4-fluorophenol (7a), 4-fluorobenzyl alcohol (12) and 2,4-difluorophenol (7b), respectively. Regiospecific hydroxylation and formylation ortho to fluoro groups, both via aryllithium intermediates, were made possible by the blocking effect of tert-butyldimethylsilyloxy functions and allowed the conversion of the starting materials to the key intermediates, namely, 3,5-bis(tert-butyldimethylsilyloxy)-2-fluoro-, 4-fluoro- and 2,4-difluorobenzaldehydes (11a, b and 19, respectively). The latter were converted in one step to the corresponding benzyloxybenzaldehydes, from which indole-2-carboxylates 22a-c were synthesized via azidostyrenes 21a-c, respectively. Decarbonylation of the indole-2-carboxaldehydes (24a-c) produced from 22a-c in two steps gave 2,3-unsubstituted indoles 25a-c, respectively. Introduction of the aminoethyl side chains on C-3 of 25a-c via the corresponding indole-3-acetonitriles, and subsequent debenzylation generated the hydroxytryptamines, which were isolated as their creatinine sulfate salts 30a-c, respectively. Cyclic voltammetric studies indicated that like 5,7-DHT, 30a-c undergo electrochemical oxidation in 1 M H2SO4 via the corresponding p-quinoneimine derivatives 31a-c by an electrochemical-chemical-electrochemical (ECE) process. The voltammetrically detectable products of the ECE process appear to be the corresponding 5-hydroxytryptamine-4,7-dione (6) derivatives 33a-c. The nature of the interaction of dissolved O2 with 30a-c at pH 7.4 appears to be strikingly different from that of 5,7-DHT, which undergoes autoxidation at pH 7.4 via the 4-hydroperoxy derivative 4 to the quinone 6.(ABSTRACT TRUNCATED AT 250 WORDS)