CD‐sensitive Zn‐porphyrin tweezer host‐guest complexes,part 2: Cis‐ and trans‐3‐hydroxy‐4‐aryl/alkyl‐β‐lactams. A case study

This article describes an application of the host‐guest chiral recognition approach called tweezer methodology for the determination of the absolute configuration of 3‐hydroxy‐β‐lactams. These substrates represent challenging cases due to their chemical reactivity, the presence of multiple stereogenic centers and several functional groups which offer various possibilities of binding to the Zn‐porphyrin host. OPLS‐2005, the force field used in this work to predict the interporphyrin twist, modeled correctly the host‐guest complexation mechanism and revealed conformational details of the bound substrates. The computational study also suggested that in cases where an increase in the magnitude of the stereodifferentiation and an intense experimental CD are observed, the bound conformation of the conjugates are hydrogen bonded. The present investigation provides evidence that when the tweezer method is assisted by the OPLS‐2005 based computational approach, it can be successfully applied to the configurational and conformational elucidation of multi‐functional compounds with multiple stereogenic centers. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Enantioseparation of ketoconazole and miconazole by capillary electrophoresis and a study on their inclusion interactions with β‐cyclodextrin and derivatives

A chiral separation method coupled with capillary electrophoresis (CE) analysis for ketoconazole and miconazole enantiomers using chiral selectors such as β‐cyclodextrin (β‐CD) and hydroxypropyl‐β‐CD (HP‐β‐CD) was developed in this study, which included the optimisation, validation and application of the method on the antifungal cream samples. The formation of inclusion complex between the hosts (β‐CD and HP‐β‐CD) and guests (ketoconazole and miconazole) were compared and analysed using ultraviolet–visible spectrophotometry, nuclear magnetic resonance (NMR) spectroscopy and molecular docking methods. Results from the study showed that in a concentration that ranged between 0.25 and 50 mg L^?1, the linear calibration curves of each enantiomer had a high coefficient of regression (R² > 0.999), low limit of detection (0.075 mg L^?1) and low limit of quantification (0.25 mg L^?1). The relative standard deviation (RSD) of the intraday and interday analyses ranged from 0.79% to 8.01% and 3.30% to 11.43%, respectively, while the recoveries ranged from 82.0% to 105.7% (RSD < 7%, n = 3). The most probable structure of the inclusion complexes was proposed based on the findings from the molecular docking studies conducted using the PatchDock server.     

Interaction of β3/β2‐Peptides,Consisting of Val‐Ala‐Leu Segments,with POPC Giant Unilamellar Vesicles (GUVs) and White Blood Cancer Cells (U937) – A New Type of Cell‐Penetrating Peptides,and a Surprising Chain‐Length Dependence of Their Vesicle‐ and Cell‐Lysing Activity

Many years ago, β²/β³‐peptides, consisting of alternatively arranged β²‐ and β³h‐amino‐acid residues, have been found to undergo folding to a unique type of helix, the 10/12‐helix, and to exhibit non‐polar, lipophilic properties (Helv. Chim. Acta 1997 , 80, 2033). We have now synthesized such ‘mixed’ hexa‐, nona‐, dodeca‐, and octadecapeptides, consisting of Val‐Ala‐Leu triads, with N‐terminal fluorescein (FAM) labels, i.e., 1 – 4 , and studied their interactions with POPC (=1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine) giant unilamellar vesicles (GUVs) and with human white blood cancer cells U937. The methods used were microfluidic technology, fluorescence correlation spectroscopy (FCS), a flow‐cytometry assay, a membrane‐toxicity assay with the dehydrogenase G6PDH as enzymatic reporter, and visual microscopy observations. All β³/β²‐peptide derivatives penetrate the GUVs and/or the cells. As shown with the isomeric β³/β²‐, β³‐, and β²‐nonamers, 2, 5 , and 6 , respectively, the derivatives 5 and 6 consisting exclusively of β³‐ or β²‐amino‐acid residues, respectively, interact neither with the vesicles nor with the cells. Depending on the method of investigation and on the pretreatment of the cells, the β³/β²‐nonamer and/or the β³/β²‐dodecamer derivative, 2 and/or 3 , respectively, cause a surprising disintegration or lysis of the GUVs and cells, comparable with the action of tensides, viral fusion peptides, and host‐defense antimicrobial peptides. Possible sources of the chain‐length‐dependent destructive potential of the β³/β²‐nona‐ and β³/β²‐dodecapeptide derivatives, and a possible relationship with the phosphate‐to‐phosphate and hydrocarbon thicknesses of GUVs, and eukaryotic cells are discussed. Further investigations with other types of GUVs and of eukaryotic or prokaryotic cells will be necessary to elucidate the mechanism(s) of interaction of ‘mixed’ β³/β²‐peptides with membranes and to evaluate possible biomedical applications.     

PMC,a potent hydrophilic α‐tocopherol derivative,inhibits NF‐κB activation via PP2A but not IκBα‐dependent signals in vascular smooth muscle cells

The hydrophilic α‐tocopherol derivative, 2,2,5,7,8‐pentamethyl‐6‐hydroxychromane (PMC), is a promising alternative to vitamin E in clinical applications. Critical vascular inflammation leads to vascular dysfunction and vascular diseases, including atherosclerosis, hypertension and abdominal aortic aneurysms. In this study, we investigated the mechanisms of the inhibitory effects of PMC in vascular smooth muscle cells (VSMCs) exposed to pro‐inflammatory stimuli, lipopolysaccharide (LPS) combined with interferon (IFN)‐γ. Treatment of LPS/IFN‐γ‐stimulated VSMCs with PMC suppressed the expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase‐9 in a concentration‐dependent manner. A reduction in LPS/IFN‐γ‐induced nuclear factor (NF)‐κB activation was also observed in PMC‐treated VSMCs. The translocation and phosphorylation of p65, protein phosphatase 2A (PP2A) inactivation and the formation of reactive oxygen species (ROS) were significantly inhibited by PMC in LPS/IFN‐γ‐activated VSMCs. However, neither IκBα degradation nor IκB kinase (IKK) or ribosomal s6 kinase‐1 phosphorylation was affected by PMC under these conditions. Both treatments with okadaic acid, a PP2A‐selective inhibitor, and transfection with PP2A siRNA markedly reversed the PMC‐mediated inhibition of iNOS expression, NF‐κB‐promoter activity and p65 phosphorylation. Immunoprecipitation analysis of the cellular extracts of LPS/IFN‐γ‐stimulated VSMCs revealed that p65 colocalizes with PP2A. In addition, p65 phosphorylation and PP2A inactivation were induced in VSMCs by treatment with H₂O₂, but neither IκBα degradation nor IKK phosphorylation was observed. These results collectively indicate that the PMC‐mediated inhibition of NF‐κB activity in LPS/IFN‐γ‐stimulated VSMCs occurs through the ROS‐PP2A‐p65 signalling cascade, an IKK‐IκBα‐independent mechanism. Therapeutic interventions using PMC may therefore be beneficial for the treatment of vascular inflammatory diseases.     

Arabinoxylan, β‐glucan and pectin in barley and malt endosperm cell walls: a microstructure study using CLSM and cryo‐SEM

The architecture of endosperm cell walls in Hordeum vulgare (barley) differs remarkably from that of other grass species and is affected by germination or malting. Here, the cell wall microstructure is investigated using (bio)chemical analyses, cryogenic scanning electron microscopy (cryo‐SEM) and confocal laser scanning microscopy (CLSM) as the main techniques. The relative proportions of β‐glucan, arabinoxylan and pectin in cell walls were 61, 34 and 5%, respectively. The average thickness of a single endosperm cell wall was 0.30 µm, as estimated by the cryo‐SEM analysis of barley seeds, which was reduced to 0.16 µm after malting. After fluorescent staining, 3D confocal multiphoton microscopy (multiphoton CLSM) imaging revealed the complex cell wall architecture. The endosperm cell wall is composed of a structure in which arabinoxylan and pectin are colocalized on the outside, with β‐glucan depositions on the inside. During germination, arabinoxylan and β‐glucan are hydrolysed, but unlike β‐glucan, arabinoxylan remains present in defined cell walls in malt. Integrating the results, an enhanced model for the endosperm cell walls in barley is proposed.     

Parasitoid wasp sting: A cocktail of GABA,taurine, and β‐alanine opens chloride channels for central synaptic block and transient paralysis of a cockroach host

The wasp Ampulex compressa injects venom directly into the prothoracic ganglion of its cockroach host to induce a transient paralysis of the front legs. To identify the biochemical basis for this paralysis, we separated venom components according to molecular size and tested fractions for inhibition of synaptic transmission at the cockroach cercal‐giant synapse. Only fractions in the low molecular weight range (<2 kDa) caused synaptic block. Dabsylation of venom components and analysis by HPLC and MALDI‐TOF‐MS revealed high levels of GABA (25 mM), and its receptor agonists β‐alanine (18 mM), and taurine (9 mM) in the active fractions. Each component produces transient block of synaptic transmission at the cercal‐giant synapse and block of efferent motor output from the prothoracic ganglion, which mimics effects produced by injection of whole venom. Whole venom evokes picrotoxin‐sensitive chloride currents in cockroach central neurons, consistent with a GABAergic action. Together these data demonstrate that Ampulex utilizes GABAergic chloride channel activation as a strategy for central synaptic block to induce transient and focal leg paralysis in its host. © 2006 Wiley Periodicals, Inc. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006