Synthesis of the AB ring system of clifednamide utilizing Claisen rearrangement and Diels-Alder reaction as key steps.

In order to construct the functionalized AB ring system of clifednamide, member of the class of macrocyclic tetramic acid lactams, a synthesis was developed which utilized an Ireland-Claisen rearrangement and an intramolecular Diels-Alder reaction. Starting from di-O-isopropylidene-d-mannitol the allyl carboxylate precursor for the sigmatropic rearrangement was prepared. This rearrangement proceeded diastereoselectively only in the presence of an allyl silyl ether instead of the parent enone in the side chain, as suggested by deuteration experiments. A subsequent Diels-Alder reaction yielded the target ethyl hexahydro-1H-indene-carboxylate with high diastereoselectivity. Quantum-chemical investigations of this intramolecular Diels-Alder reaction support the proposed configuration of the final product.

The synthesis and biological evaluation of desepoxyisotedanolide and a comparison with desepoxytedanolide.

The tedanolides are biologically active polyketides that exhibit a macrolactone constructed from a primary alcohol. Since polyketidal transformations only generate secondary alcohols, it has been hypothesized by Taylor that this unique lactone could arise from a postketidal transesterification. In order to probe this hypothesis and to investigate the biological profile of the putative precursor of all members of the tedanolide family, we embarked on the synthesis of desepoxyisotedanolide and its biological evaluation in comparison to desepoxytedanolide. The biological experiments unraveled a second target for desepoxytedanolide and provided evidence that the proposed transesterification indeed provides a survival advantage for the producing microorganism.

SAR studies on hydropentalene derivatives--Important core units of biologically active tetramic acid macrolactams and ptychanolides.

Structurally diverse bicyclo[3.3.0]octanes were prepared and tested for their biological activity. Both the antiproliferative activity and the results of phenotypic characterization varied with the substitution patterns. Two derivatives displayed high inhibitory (IC50 ≤3µM) activity against the L-929 cell line, but differed in their mode of action. A cluster analysis with impedance profiling data showed the two compounds in relationship to microtubule interfering compounds. In PtK2 cells treated with both derivatives a perturbing effect on the microtubular network was observed, whereas the actin cytoskeleton in incubated PtK2 cells was disturbed only by one compound. The effects on tubulin and actin polymerization could be confirmed by in vitro polymerization experiments.

The marine polyketide myriaporone 3/4 stalls translation by targeting the elongation phase.

Myriaporone 3/4, a cytotoxic polyketide, has been reported as an inhibitor of eukaryotic protein synthesis. However, the mechanism by which it inhibits translation was unknown. Here we show that myriaporone 3/4 stalls protein synthesis in the elongation phase by inducing phosphorylation of eukaryotic elongation factor 2. The phosphorylation results from direct binding of myriaporone 3/4 to eukaryotic elongation factor 2 kinase. Our study also shows that myriaporone 3/4 in the nanomolar range inhibits in vitro tube formation by endothelial cells without being cytotoxic. In general, myriaporone 3/4 was at least 300 times less toxic to primary cells than to tumor cells.

Synthesis of penicillenol C1 and of a bis-azide analogue for photoaffinity labeling.

Two diasteroisomers of the Penicillium metabolite penicillenol C1 were synthesized for the first time by 3-acylation of an L-threonine-derived tetramic acid with enantiopure 2-methyloct-(6E)-enoic acids. The 5S,6R,9S isomer has NMR spectra and optical rotation identical with those of the natural compound. A bis-azide-tagged penicillenol analogue was also synthesized for photoaffinity labeling of target proteins. The photolysis of the bis-azide in the presence of methanol as a protein-mimicking nucleophile led to reaction only of the aryl azide, while leaving the benzyl azide available for pull-downs or the attachment of fluorescent tracers. As a proof of concept, the distribution of this bis-azide-tagged tetramic acid in living cells was visualized via a Staudinger ligation between the azide tag and a phosphane fluorophore.

Modular synthesis of polyene side chain analogues of the potent macrolide antibiotic etnangien by a flexible coupling strategy based on hetero-bis-metallated alkenes.

An efficient procedure for the concise synthesis of hetero-bis-metallated alkenes as useful building blocks for the modular access to highly elaborate polyenes and stabilized analogues is reported. By applying these bifunctional olefins in convergent Stille/Suzuki-Miyaura couplings, novel, carefully selected side chain analogues of the potent RNA polymerase inhibitor etnangien were synthesized by a modular late stage coupling strategy and evaluated for antibacterial and antiproliferative activities.

Synthesis and biological evaluation of paleo-soraphens.

Synthesis can provide molecules such as paleo-soraphens A and B that are genetically encoded but not obtained from the natural source. Although it is unclear whether this is part of an evolutionary process or the consequence of the chemical synthesis, the biological evaluation of these genetically encoded natural products can shed light on how natural products are structurally optimized with respect to their biological profile.

Understanding the Inhibitory and Antioxidant Effects of Pyrroloquinoline Quinone (PQQ) on Copper(II)-Induced α-Synuclein-119 Aggregation.

Parkinson's disease (PD) is associated with the aggregation and misfolding of a-synuclein (a-syn) protein in dopaminergic neurons. The misfolding process is heavily linked to copper dysregulation in PD. Experimental evidence supports the hypothesis that the co-presence of Cu(II) and α-syn facilitates the aggregation of α-syn, affecting the pathological development of PD. Recent literature has shown that pyrroloquinoline quinone (PQQ) contains strong neuroprotective activity by reducing the reactive oxygen species (ROS) production by α-syn. Despite these known facts, minimal studies have been done on the antioxidant effect of PQQ against ROS formation in the presence of Cu(II) and α-syn-119. Thus, it is of great significance to study the interaction between all three components, PQQ, Cu(II), and α-syn-119. In this proof-of-concept study, a variety of chemical techniques were employed to examine the antioxidant effect of PQQ on ROS that α-syn-119 produced in the presence of Cu(II). Our results showed that PQQ effectively prevented ROS formation in SH-SY5Y human differentiated neuronal cells. Thioflavin T (ThT) fluorescence assay, circular dichroism (CD) spectroscopy, and transmission electron microscopy (TEM) were applied, where PQQ was able to actively prevent fibrillation of α-syn-119 in the presence of Cu(II). This finding was further confirmed using electrochemical impedance spectroscopy (EIS), where the binding of PQQ to the α-syn-119 suppressed the aggregation process on the electrode surface. With these encouraging results, we envisage that PQQ and its derivatives can be a promising candidate for further studies as a multitarget therapeutic agent toward PD therapy.

Attenuation of Oxidative Stress by Cannabinoids and Cannabis Extracts in Differentiated Neuronal Cells.

In this proof-of-concept study, the antioxidant activity of phytocannabinoids, namely cannabidiol (CBD) and Δ9- tetrahydrocannabinol (THC), were investigated using an in vitro system of differentiated human neuronal SY-SH5Y cells. The oxidative stress was induced by hydrogen peroxide, as reactive oxygen species (ROS). Alzheimer's disease (AD)-like pathological conditions were mimicked in vitro by treating the differentiated neuronal cells with amyloid-ß1-42 (Aß1-42) in the presence of Cu(II). We showed that THC had a high potency to combat oxidative stress in both in vitro models, while CBD did not show a remarkable antioxidant activity. The cannabis extracts also exhibited a significant antioxidant activity, which depended on the ratio of the THC and CBD. However, our results did not suggest any antagonist effect of the CBD on the antioxidant activity of THC. The effect of cannabis extracts on the cell viability of differentiated human neuronal SY-SH5Y cells was also investigated, which emphasized the differences between the bioactivity of cannabis extracts due to their composition. Our preliminary results demonstrated that cannabis extracts and phytocannabinoids have a promising potential as antioxidants, which can be further investigated to develop novel pharmaceuticals targeting oxidative stress therapy.

Laser microdissection and pressure catapulting of single human motor neurons for RNA editing analysis.

Glutamate is the major excitatory neurotransmitter in the mammalian nervous system. The properties of their ionotropic glutamate receptors largely determine how different neurons respond to glutamate. RNA editing in pre-mRNAs encoding subunits of glutamate receptors, particularly the GluR 2 subunit of AMPA receptors, controls calcium permeability, response time, and total ion flow in individual receptors as well as the density of AMPA receptors at synapses through effects on ER assembly, sorting, and plasma membrane insertion. When RNA editing fails in a neuron, calcium influx through AMPA receptors may cause neuron death by glutamate excitotoxicity, as in the case of vulnerable hippocampal CA1 pyramidal neurons that die after transient forebrain ischemia. Elevated cerebrospinal glutamate is common in ALS and loss of GluR 2 Q/R site RNA editing has been reported to occur selectively in lower motor neurons in a majority of Japanese sporadic ALS patients. We describe our methods for laser microdissection followed by RT-PCR analysis to study RNA editing in single motor neurons.