A fluorescence lifetime-based binding assay for acetylpolyamine amidohydrolases from Pseudomonas aeruginosa using a [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) ligand probe

High-throughput assays for drug screening applications have to fulfill particular specifications. Besides the capability to identify even compounds with low potency, one of the major issues is to minimize the number of false-positive hits in a screening campaign in order to reduce the logistic effort for the subsequent cherry picking and confirmation procedure. In this respect, fluorescence lifetime (FLT) appears as an ideal readout parameter that is supposed to be robust against autofluorescent and light-absorbing compounds, the most common source of systematic false positives. The extraordinary fluorescence features of the recently discovered [1,3]dioxolo[4,5-f][1,3] benzodioxole dyes were exploited to develop an FLT-based binding assay with exceptionally robust readout. The assay setup was comprehensively validated and shown to comply not only with all requirements for a powerful high-throughput screening assay but also to be suitable to determine accurate binding constants for inhibitors against enzymes of the histone deacetylase family. Using the described binding assay, the first inhibitors against three members of this enzyme family from Pseudomonas aeruginosa were identified. The compounds were characterized in terms of potency and selectivity profile. The novel ligand probe should also be applicable to other homologues of the histone deacetylase family that are inhibited by N-hydroxy-N′-phenyloctandiamide.

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Interfacial rheology of mixed layers of food proteins and surfactants

Mixed protein–surfactant adsorption layers at liquid interfaces are described including the thermodynamic basis, the adsorption kinetics and the shear and dilational interfacial rheology. It is shown that due to the protrusion of hydrophobic protein parts into the oil phase the adsorption layers at the water–hexane interface are stronger anchored as compared to the water-air surface. Based on the different adsorption protocols, a sequential and a simultaneous scheme, the peculiarities of complexes between proteins and added surfactants are shown when formed in the solution bulk or at a liquid interface. The picture drawn from adsorption studies is supported by the findings of interfacial rheology.     

Two-dimensional electrophoretic analysis of Corynebacterium glutamicum membrane fraction and surface proteins

An improved protocol for the two-dimensional analysis of proteins of the Corynebacterium glutamicum cytoplasmic membrane fraction is described. By use of increased 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) concentrations (2-4%) and an optimized electrophoresis protocol, horizontal streaking of proteins of the cytoplasmic membrane fraction was almost completely avoided. More important, in contrast to a previously published method, both a sample tray and IPG-phor isoelectric focusing unit can be used for the in-gel application of proteins. The described protocol was also found to be suitable for hydrophilic cytoplasmic proteins. Additionally, the preparation and analysis of C. glutamicum cell surface proteins is described. Proteins were extracted with lauroyl sarcosinate and 100-120 spots were separated on two-dimensional (2-D) gels in comparison to 18-20 spots observed previously by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). C. glutamicum proteins can now be separated into three distinct fractions resembling different functional units of the bacterial cell.     

Sigma/pi Bonding Preferences of Solvated Alkali-Metal Cations to Ditopic Arylmethyl Anions

Arylmethyl anions allow alkali-metals to bind in a σ-fashion to the lateral carbanionic centre or a π-fashion to the aryl ring or in between these extremities, with the trend towards π bonding increasing on descending group 1. Here we review known alkali metal structures of diphenylmethane, fluorene, 2-benzylpyridine and 4-benzylpyridine. Next, we synthesise Li, Na, K monomers of these diarylmethyls using polydentate donors PMDETA or Me₆TREN to remove competing oligomerizing interactions, studying the effect that two aromatic rings has on negative charge (de)localisation via NMR, X-ray crystallographic and DFT studies. Diphenylmethyl and fluorenyl anions maintain C(H)−M interactions regardless of alkali-metal, although the adjacent arene carbons engage in interactions with larger alkali-metals. Introducing a nitrogen atom into the ring (at the 2- or 4-position) encourages relocalisation of negative charge away from the deprotonated carbon and onto nitrogen. Phenyl(2-pyridyl)methyl moves from an enamide formation at one extremity (lithium) to an aza-allyl formation at the other extremity (potassium), while C- or N-coordination modes become energetically viable for Na and K phenyl(4-pyridyl)methyl complexes.     

Activation autoradiography for a study of the selective and microheterogeneous distribution of Cu(II) in NH4Al(SO4)2·12H2O monocrystals

The possibility of the activation autoradiographic investigation of Cu(II) distribution in NH₄Al(SO₄)₂·12H₂O-monocrystals has been studied. The influence of irradiation and cooling times and the effect of application of filters made of neutron moderating materials on the sensitivity of the method is theoretically and experimentally evaluated.     

The GROMOS software for biomolecular simulation: GROMOS05

We present the latest version of the Groningen Molecular Simulation program package, GROMOS05. It has been developed for the dynamical modelling of (bio)molecules using the methods of molecular dynamics, stochastic dynamics, and energy minimization. An overview of GROMOS05 is given, highlighting features not present in the last major release, GROMOS96. The organization of the program package is outlined and the included analysis package GROMOS++ is described. Finally, some applications illustrating the various available functionalities are presented.     

Allosteric control of oligonucleotide hybridization by metal-induced cyclization

A bis(terpyridine) modified single-stranded DNA smoothly forms a stable cycle by Fe2+-assisted ring closure. A novel, complex type of allosteric behavior is observed with Zn2+, which reversibly off-regulates binding of a complementary oligonucleotide only in a narrow concentration range.     

Sequence selective hydrolysis of linear DNA using conjugates of Zr(IV) complexes and peptide nucleic acids

A series of conjugates of peptide nucleic acids (PNA) and Zr(IV) complexes was prepared. Their ability to hydrolyze DNA was tested using MALDI-TOF mass spectrometry and HPLC. The most efficient artificial restriction enzyme found, PNA conjugate with Zr(IV) complex of tris(hydroxymethyl)-aminomethane, cleaves DNA targets sequence selectively in close proximity to the Zr(IV) complex. It was demonstrated that cleavage products are substrates of terminal transferase.     

A catalytic asymmetric total synthesis of (–)-perophoramidine

We report a catalytic asymmetric total synthesis of the ascidian natural product perophoramidine. The synthesis employs a molybdenum-catalyzed asymmetric allylic alkylation of an oxindole nucleophile and a monosubstituted allylic electrophile as a key asymmetric step. The enantioenriched oxindole product from this transformation contains vicinal quaternary and tertiary stereocenters, and is obtained in high yield along with high levels of regio-, diastereo-, and enantioselectivity. To install the second quaternary stereocenter in the target, the route utilizes a novel regio- and diastereoselective allylation of a cyclic imino ether to deliver an allylated imino ether product in near quantitative yield and with complete regio- and diastereocontrol. Oxidative cleavage and reductive amination are used as final steps to access the natural product.