Conserved Tyr223(5.58) plays different roles in the activation and G-protein interaction of rhodopsin

Rhodopsin, a seven transmembrane helix (TM) receptor, binds its ligand 11-cis-retinal via a protonated Schiff base. Coupling to the G-protein transducin (G(t)) occurs after light-induced cis/trans-retinal isomerization, which leads through photoproducts into a sequence of metarhodopsin (Meta) states: Meta I ? Meta IIa ? Meta IIb ? Meta IIbH(+). The structural changes behind this three-step activation scheme are mediated by microswitch domains consisting of conserved amino acids. Here we focus on Tyr223(5.58) as part of the Y(5.58)X(7)K(R)(5.66) motif. Mutation to Ala, Phe, or Glu results in specific impairments of G(t)-activation measured by intrinsic G(t) fluorescence. UV-vis/FTIR spectroscopy of rhodopsin and its complex with a C-terminal G(t)α peptide allows the assignment of these deficiencies to specific steps in the activation path. Effects of mutation occur already in Meta I but do not directly influence deprotonation of the Schiff base during formation of Meta IIa. Absence of the whole phenol ring (Y223A) allows the activating motion of TM6 in Meta IIb but impairs the coupling to G(t). When only the hydroxyl group is lacking (Y223F), Meta IIb does not accumulate, but the activity toward G(t) remains substantial. From the FTIR features of Meta IIbH(+) we conclude that proton uptake to Glu134(3.49) is mandatory for Tyr223(5.58) to engage in the interaction with the key player Arg135(3.50) predicted by X-ray analysis. This polar interaction is partially recovered in Y223E, explaining its relatively high activity. Only the phenol side chain of tyrosine provides all characteristics for accumulation of the active state and G-protein activation.     

A fluorescent, shape-persistent dendritic host with photoswitchable guest encapsulation and intramolecular energy transfer

A fluorescent and photoresponsive host based on rigid polyphenylene dendrimers (PPDs) has been synthesized. The key building block for the divergent dendrimer buildup is a complex tetracyclone 12 containing azobenzenyl, pyridyl, and ethynyl entities. The rigidity of polyphenylenes is of crucial importance for a site-specific placement of different functions: eight azobenzene (AB) moieties into the rigid scaffold, a fluorescent perylenetetracarboxdiimide (PDI) into the core, and eight pyridin functions into the interior cavities. AB moieties of host-1 undergo reversible cis-trans photoisomerization and are photostable, as confirmed by various techniques: UV-vis, (1)H NMR, size exclusion chromatography, and fluorescence correlation (FCS). In this system, AB moieties act as photoswitchable hinges and enable control over (i) molecular size, (ii) intramolecular energy transfer between AB and PDI, and (iii) encapsulation and release of guest molecules. The presence of PDI allows not only following the effect of cis-trans photoisomerization on molecular size with highly sensitive FCS but also monitoring the efficiency of the intramolecular energy transfer process (from AB to PDI) by time-resolved optical spectroscopy. Pyridyl functions were incorporated to facilitate guest uptake via hydrogen bonds between the host and guests. Also, we have demonstrated that the photoswitchability of the host can be utilized to actively encapsulate guest molecules into its interior cavities. This novel, light-driven encapsulation mechanism could enable the design of new drug delivery systems.     

Evaluation of automated sample preparation, retention time locked gas chromatography-mass spectrometry and data analysis methods for the metabolomic study of Arabidopsis species

In this paper, automated sample preparation, retention time locked gas chromatography-mass spectrometry (GC-MS) and data analysis methods for the metabolomics study were evaluated. A miniaturized and automated derivatisation method using sequential oximation and silylation was applied to a polar extract of 4 types (2 types×2 ages) of Arabidopsis thaliana, a popular model organism often used in plant sciences and genetics. Automation of the derivatisation process offers excellent repeatability, and the time between sample preparation and analysis was short and constant, reducing artifact formation. Retention time locked (RTL) gas chromatography-mass spectrometry was used, resulting in reproducible retention times and GC-MS profiles. Two approaches were used for data analysis. XCMS followed by principal component analysis (approach 1) and AMDIS deconvolution combined with a commercially available program (Mass Profiler Professional) followed by principal component analysis (approach 2) were compared. Several features that were up- or down-regulated in the different types were detected.     

A Novel APPI-MS Setup for In Situ Degradation Product Studies of Atmospherically Relevant Compounds: Capillary Atmospheric Pressure Photo Ionization (cAPPI)

We report on the development of a novel atmospheric pressure photoionization setup and its applicability for in situ degradation product studies of atmospherically relevant compounds. A custom miniature spark discharge lamp was embedded into an ion transfer capillary, which separates the atmospheric pressure from the low pressure region in the first differential pumping stage of a conventional atmospheric pressure ionization mass spectrometer. The lamp operates with a continuous argon flow and produces intense light emissions in the VUV. The custom lamp is operated windowless and efficiently illuminates the sample flow through the transfer capillary on an area smaller than 1 mm². Limits of detection in the lower ppbV range, a temporal resolution of milliseconds in the positive as well as the quasi simultaneously operating negative ion mode, and a significant reduction of ion transformation processes render this system applicable to real time studies of rapidly changing chemical systems. The method termed capillary atmospheric pressure photo ionization (cAPPI) is characterized with respect to the lamp emission properties as a function of the operating conditions, temporal response, and its applicability for in situ degradation product studies of atmospherically relevant compounds, respectively.     

Enantioselective radical cyclisation reactions of 4-substituted quinolones mediated by a chiral template

Six 4-substituted quinolones 6-8, which bear an ω-iodoalkyl chain, were prepared and subjected to reductive radical cyclisation conditions employing BEt(3)/O(2) as the initiator and either Bu(3)SnH or TMS(3)SiH as hydride source. 4-(4-Iodobutyl)-quinolone (6a) and 4-(3-iodopropylthio)-quinolone (8a) gave the respective 6-endo-cyclisation products in good yields. 4-(3,3-Dimethyl-4-iodobutyl)-quinolone (6b) cyclised in a 5-exo-fashion, while the other substrates delivered only reduction products. The cyclisation reactions could be conducted in the presence of a chiral template (1) with high enantiomeric excess (94-99% ee). The association behaviour of substrate 6a to 1 was studied by NMR titration experiments. In the enantioselective cyclisation of 6b a significant nonlinearity was observed when comparing the product ee with the ee of the template.     

Influence of deuteration on lithium acetate dihydrate studied by inelastic X-ray scattering, density functional theory, thermal expansion, elastic and thermodynamic measurements

The influence of deuteration on the properties of lithium acetate dihydrate has been investigated by thermal expansion measurements, ultrasound spectroscopy and calorimetry. Inelastic X-ray scattering has been employed to investigate if the low temperature structural phase transition can be detected by a change in the vibrational spectrum. Density functional theory, DFT, calculations have been employed to complement the experimental investigations. The thermal expansion coefficients and the specific heat of the deuterated compound differ significantly from the protonated form. The differences in the elastic stiffness coefficients are just above the detection limit of the technique employed here. Temperature dependent inelastic X-ray spectroscopic measurements show no significant change of the vibrational spectrum when crossing the transition temperature. The DFT calculations show that the methyl group dynamics are best described in the framework of coupled rotators of opposing methyl groups. One of the coupled rotational modes corresponds to a hindered rotator with a barrier of 15 meV, while the other is a free rotator.     

New iron(II) α-iminopyridine complexes and their catalytic activity in the oxidation of activated methylene groups and secondary alcohols to ketones

A set of iron(II) complexes of the general formula [Fe(OTf)(2)L(2)] was synthesized in 32 to 78% isolated yields, where L represents a bidentate α-iminopyridine ligand. Four of the iron complexes were characterized structurally, revealing a rich coordination chemistry, because the coordination geometry of the iron complexes strongly depends on the substitution pattern exhibited by the ligands L. The catalytic activity of the new complexes was demonstrated in the oxidation of cyclohexane, activated methylene groups and secondary alcohols to the corresponding ketones utilizing H(2)O(2) and t-BuOOH as the oxidants. The oxidation of activated methylene groups and secondary alcohols to the corresponding ketones with t-BuOOH gave isolated yields between 22 and 91% (4 h, room temperature, 3% catalyst load). The influence of the structure of the ligand on the activity of the corresponding metal complex is also reported. Furthermore, UV-vis experiments were performed which provided evidence for the formation of an [Fe-O-O-t-Bu] intermediate.     

2,5,8,11-Tetraboronic ester perylenediimides: a next generation building block for dye-stuff synthesis

Via an unprecedentedly reported ruthenium catalyzed reaction, an efficient and straightforward method was developed for the synthesis of 2,5,8,11-tetraboronate perylenediimide derivatives. A possible reaction mechanism is proposed. The synthesis of 2,5,8,11-tetra-iodo and tetra-amino perylenediimides derivatives is also reported.     

DEKOIS: demanding evaluation kits for objective in silico screening--a versatile tool for benchmarking docking programs and scoring functions

For widely applied in silico screening techniques success depends on the rational selection of an appropriate method. We herein present a fast, versatile, and robust method to construct demanding evaluation kits for objective in silico screening (DEKOIS). This automated process enables creating tailor-made decoy sets for any given sets of bioactives. It facilitates a target-dependent validation of docking algorithms and scoring functions helping to save time and resources. We have developed metrics for assessing and improving decoy set quality and employ them to investigate how decoy embedding affects docking. We demonstrate that screening performance is target-dependent and can be impaired by latent actives in the decoy set (LADS) or enhanced by poor decoy embedding. The presented method allows extending and complementing the collection of publicly available high quality decoy sets toward new target space. All present and future DEKOIS data sets will be made accessible at www.dekois.com.