Total synthesis of 8,9-Dehydro-Epothilone C

Magdeburg, Univ., Fak. für Verfahrens- und Systemtechnik, Diss., 2010

Progress towards the total synthesis of sorangicin

Magdeburg, Univ., Fak. für Verfahrens- und Systemtechnik, Diss., 2012

Synthesis of medium-chain-length polyhydroxyalkanoates in arabidopsis thaliana using intermediates of peroxisomal fatty acid beta-oxidation.

Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids. These polymers have properties of biodegradable thermoplastics and elastomers. Medium-chain-length PHAs (MCL-PHAs) are synthesized in bacteria by using intermediates of the beta-oxidation of alkanoic acids. To assess the feasibility of producing MCL-PHAs in plants, Arabidopsis thaliana was transformed with the PhaC1 synthase from Pseudomonas aeruginosa modified for peroxisome targeting by addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. Immunocytochemistry demonstrated that the modified PHA synthase was appropriately targeted to leaf-type peroxisomes in light-grown plants and glyoxysomes in dark-grown plants. Plants expressing the PHA synthase accumulated electron-lucent inclusions in the glyoxysomes and leaf-type peroxisomes, as well as in the vacuole. These inclusions were similar to bacterial PHA inclusions. Analysis of plant extracts by GC and mass spectrometry demonstrated the presence of MCL-PHA in transgenic plants to approximately 4 mg per g of dry weight. The plant PHA contained saturated and unsaturated 3-hydroxyalkanoic acids ranging from six to 16 carbons with 41% of the monomers being 3-hydroxyoctanoic acid and 3-hydroxyoctenoic acid. These results indicate that the beta-oxidation of plant fatty acids can generate a broad range of R-3-hydroxyacyl-CoA intermediates that can be used to synthesize MCL-PHAs.

Stereoselective total synthesis of some insect pheromones: (±)-serricornine and (±)-invictolide

An efficient (12 steps, 12% overallyield) and stereoselective total synthesis of (±)-serricornine (1) the sex pheromone of the cigarette beetle (Lasioderma serricornine F) is described. The preparation of intermediate 5, which encompasses the proper relative configuration of three contiguous chiral centers of (±)-invictolide, (3), is discussed.

Methodology in structural determination and synthesis of insect pheromone

By means of ethereal washing of insect pheromone glands of female moths, GC-MS detection along with microchemical reactions and electroantennogram (EAG) survey, six economically important insect species were targeted for pheromone identification. The discovery of a natural pheromone inhibitor, chemo-selectivity and species isolation by pheromone will be described. The modified triple bond migration and triethylamine liganded vinyl cuprate were applied for achiral pheromone synthesis in double bond formation. Some optically active pheromones and their stereoisomers were synthesized through chiral pool or asymmetric synthesis. Some examples of chiral recognition of insects towards their chiral pheromones will be discussed. A CaH2 and silica gel catalyzed Sharpless Expoxidation Reaction was found in shortening the reaction time.

Induction of synthesis of the rat cystatin S protein by the submandibular gland during the acute phase of experimental Chagas disease

Rats experimentally infected with Trypanosoma cruzi Y strain exhibited hypertrophy of the submandibular gland at 18 days after infection.SDS-PAGE of infected rats saliva revealed the presence of an additional band with an apparent molecular weight of about 13KDa. Electrophoresis of protein salivaand immunochemical analysis with antibody against rat cystatin S confirmed that the protein was identical to that induced by beta adrenergic stimulation.

Highly inducible synthesis of heterologous proteins in epithelial cells carrying a glucocorticoid-responsive vector.

A glucocorticoid-responsive vector is described which allows for the highly inducible expression of complementary DNAs (cDNAs) in stably transfected mammalian cell lines. This vector, pLK-neo, composed of a variant mouse mammary tumor virus long terminal repeat promoter, containing a hormone regulatory element, a Geneticin resistance-encoding gene in a simian virus 40 transcription unit, and a polylinker insertion site for heterologous cDNAs, was used to express the polymeric immunoglobulin (poly-Ig) receptor and the thymocyte marker, Thy-1, in Madin-Darby canine kidney (MDCK) cells and in murine fibroblast L cells. A high level of poly-Ig receptor or Thy-1 mRNA accumulation was observed in MDCK cells in response to dexamethasone with a parallel ten- to 200-fold increase in protein synthesis depending on the recombinant protein and the transfected cell clone.

Synthesis of novel biomaterials in plants.

Metabolic engineering of plants allows the possibility of using crops for the synthesis of novel polymers having useful material properties. Strong and flexible protein-based polymers, which are based on the structure of silk and elastin have been synthesized in transgenic plants. A wide range of polyhydroxyalkanoates having properties ranging from stiff plastics to soft elastomers and glues have been synthesized in various compartments of plants, such as the cytoplasm, plastid and peroxisome. These plant biomaterials could replace, in part, the synthetic plastics, fibers and elastomers produced from petroleum, thus offering the advantage of renewability, sustainability and biodegradability.

Target-guided synthesis of metal-based phosphatase inhibitors

Report for the scientific sojourn at the Imperial College of London, United Kingdom, from 2007 to 2009. PTEN is a tumour suppressor enzyme that plays important roles in the PI3K pathway which regulates growth, proliferation and survival and is thus related to many human disorders such as diabetes, neurodegenerative diseases, cardiovascular complications and cancer. It is hence of great interest to understand in detail its molecular behaviour and to find small molecules that can switch on/off its activity. For this purpose, metal complexes have been synthesized and preliminary studies in vivo show that all are capable of inhibiting PTEN.

tagO is involved in the synthesis of all anionic cell-wall polymers in Bacillus subtilis 168.

Sequence homologies suggest that the Bacillus subtilis 168 tagO gene encodes UDP-N-acetylglucosamine:undecaprenyl-P N-acetylglucosaminyl 1-P transferase, the enzyme responsible for catalysing the first step in the synthesis of the teichoic acid linkage unit, i.e. the formation of undecaprenyl-PP-N-acetylglucosamine. Inhibition of tagO expression mediated by an IPTG-inducible P(spac) promoter led to the development of a coccoid cell morphology, a feature characteristic of mutants blocked in teichoic acid synthesis. Indeed, analyses of the cell-wall phosphate content, as well as the incorporation of radioactively labelled precursors, revealed that the synthesis of poly(glycerol phosphate) and poly(glucosyl N-acetylgalactosamine 1-phosphate), the two strain 168 teichoic acids known to share the same linkage unit, was affected. Surprisingly, under phosphate limitation, deficiency of TagO precludes the synthesis of teichuronic acid, which is normally induced under these conditions. The regulatory region of tagO, containing two partly overlapping sigma(A)-controlled promoters, is similar to that of sigA, the gene encoding the major sigma factor responsible for growth. Here, the authors discuss the possibility that TagO may represent a pivotal element in the multi-enzyme complexes responsible for the synthesis of anionic cell-wall polymers, and that it may play one of the key roles in balanced cell growth.

Selection of peptides and synthesis of pentameric peptabody molecules reacting specifically with ErbB-2 receptor.

The HER-2/ErbB-2 oncoprotein is overexpressed in human breast and ovarian adenocarcinomas and is clearly associated with the malignant phenotype. Although no specific ligand for this receptor has been positively identified, ErbB-2 was shown to play a central role in a network of interactions with the related ErbB-1, ErbB-3 and ErbB-4 receptors. We have selected new peptides binding to ErbB-2 extracellular domain protein (ECD) by screening 2 newly developed constrained and unconstrained random hexapeptide phage libraries. Out of 37 phage clones, which bound specifically to ErbB-2 ECD, we found 6 constrained and 10 linear different hexapeptide sequences. Among the latter, 5 consensus motifs, all with a common methionine and a positively charged residue at positions 1 and 3, respectively, were identified. Furthermore, 3 representative hexapeptides were fused to a coiled-coil pentameric recombinant protein to form the so-called peptabodies recently developed in our laboratory. The 3 peptabodies bound specifically to the ErbB-2 ECD, as determined by enzyme-linked immunosorbent assay and BIAcore analysis and to tumor cells overexpressing ErbB-2, as shown by flow cytometry. Interestingly, one of the free selected linear peptides and all 3 peptabodies inhibited the proliferation of tumor cells overexpressing ErbB-2. In conclusion, a novel type of ErbB-2-specific ligand is described that might complement presently available monoclonal antibodies.

Synthesis of limonene β-amino alcohol derivatives in support of new antileishmanial therapies

A series of seven limonene β-amino alcohol derivatives has been regioselectively synthesised in moderate to good yields. Two of these compounds were found to be significantly effective against in vitro cultures of the Leishmania (Viannia) braziliensis promastigote form in the micromolar range. The activities found for 3b and 3f were about 100-fold more potent than the standard drug, Pentamidine, in the same test, while limonene did not display any activity. This is the first report of antileishmanial activity by limonene β-amino alcohol derivatives.

Uptake of macromolecules by cercariae during skin penetration and transformation to schistosomula (Schistosoma mansoni)

Here, we observed the uptake of membrane-impermeant molecules by cercariae as they penetrate the skin and are transformed into schistosomula. We propose that membrane-impermeant molecules, Lucifer Yellow, Propidium iodide and Hoechst 33258 enter the parasite through both thenephridiopore and the surface membrane and then diffuse throughout the body of the parasite. We present a hypothesis that the internal cells of the body of the schistosomulum represent a new host-parasite interface, at which skin-derived growth factors may stimulate receptors on internal membranes during transformation of the cercariae into the schistosomulum.

Convenient synthesis of heterobifunctional poly(ethylene glycol) suitable for the functionalization of iron oxide nanoparticles for biomedical applications.

A straightforward route is proposed for the multi-gram scale synthesis of heterobifunctional poly(ethylene glycol) (PEG) oligomers containing combination of triethyloxysilane extremity for surface modification of metal oxides and amino or azido active end groups for further functionalization. The suitability of these PEG derivatives to be conjugated to nanomaterials was shown by pegylation of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles (NPs), followed by functionalization with small peptide ligands for biomedical applications.

Saccharomyces cerevisiae, a tool to study the β-oxidation through the synthesis of polyhydroxyalkanoates

Summary Polyhydroxyalkanoates (PHAs) represent a family of polyesters naturally synthesized by a wide variety of bacteria. Through their thermoplastic and elastomeric qualities, together with their biodegradable and renewable properties, they are predicted to be a good alternative to the petroleum- derived plastics. Nevertheless, as PHA production costs using bacteria fermentation are still too high, PHA synthesis within eukaryotic systems, such as plants, has been elaborated. Although the costs were then efficiently lowered, the yield of PHAs produced remained low. In this study, Saccharomyces cerevisae has been used as another eukaryotic model in order to reveal the steps which limit PHA production. These cells express the PHA synthase of Pseudomonas aeruginosa and the PHAs obtained were analyzed to understand the flux of fatty acids towards and through the peroxisomal β-oxidation core cycle, generating the main substrate of the PHA synthase. When S. cerevisiae wild-type cells are grown in a media containing glucose as carbon source as well as fatty acids, the PHA monomer composition is largely influenced by the nature of the external fatty acid used. Thus, even-chain PHA monomers are generated from oleic acid (18:1Δ9cis) and odd- chain PHA monomers are generated from heptadecenoic acid (17:1Δ. 10 cis). Moreover, PHA synthesis is dependent on the first two enzymes of the 0-oxidation core cycle, the acyl-CoA oxidase and the multifunctional enzyme enoyl-CoA hydratase II / R-3-hydroxyacyl-CoA dehydrogenase. S. cerevisiae mutant cells growing on oleic or heptadecenoic acid and deficient in either the R-3- hydroxyacyl-CoA dehydrogenase or in the 3-ketothiolase activity, the last β-oxidation cycle steps, surprisingly contained PHAs of predominantly even-chain monomers. This is also noticed in wild- type and mutants grown on glucose or raffinose, indicating that the substrate used for PHA synthesis is generated from the degradation of intracellular short- and medium-chain fatty acids by the 3- oxidation cycle. Inhibition of fatty acid biosynthesis by cerulenin blocks the synthesis of PHAs from intracellular fatty acids but still enables the use of extracellular fatty acids for polymer production. Together, these results uncovered the existence of a substantial futile cycle whereby short- and medium-chain intermediates of the cytoplasmic fatty acid biosynthetic pathway are directed towards the peroxisomal β-oxidation pathway. In this thesis, no increase of the yield of PHA produced could be obtained. But the PHA synthesis confirmed the carbon flux into and through the β-oxidation core cycle and unveiled the existence of novel mechanisms. It is thus a good tool to study in vivo the flux of carbons in S. cerevisiae cells. Résumé Les polyhydroxyalkanoates (PHAs) sont une famille de polyesters naturellement synthétisés par un grand nombre de bactéries. Ayant des propriétés de thermoplastiques, d'élastomères et étant des ressources biodégradables et renouvelables, les PHAs représentent une bonne alternative aux plastiques dérivés du pétrole. Pour pallier aux coûts considérables de la production de PHAs par fermentation bactérienne, la synthèse de PHAs par des systèmes eucaryotes telles les plantes a été élaborée. Les coûts ont ainsi efficacement été diminués, mais le rendement de PHAs produits reste faible. Dans cette étude, Saccharomyces cerevisiae a été utilisé comme autre modèle eucaryote pour révéler les étapes limitantes de la production de PHAs. Les PHAs obtenus dans les cellules exprimant la F'HA synthase de Pseudomonas aeruginosa ont été analysés afin de comprendre le flux d'acides gras vers et à travers le cycle péroxisomal de la β-oxidation, principal producteur du substrat de la PHA synthase. Lorsque la souche S. cerevisiae de type sauvage se développe dans un milieu contenant du glucose et des acides gras, la composition des monomères de PHAs est influencée par la nature des acides gras extracellulaires. Ainsi, les monomères pairs sont générés par l'acide oléique (18:1Δ9cis), tandis que les impairs le sont par l'acide heptadécénoïque (17:1Δ10cis). La synthèse de PHAs est dépendante des deux premières enzymes de la β-oxidation; l'acyl-CoA oxidase et l'enzyme multifonctionnelle enoyl-CoA hydratase II / R-3-hydroxyacyl-CoA déshydrogénase. Les souches mutantes ne possédant pas les activités de la R-3-hydroxyacyl-CoA déshydrogénase ou de la 3- ketothiolase contiennent, en présence d'acide oléique ou heptadécénoïque, des PHAs composés essentiellement de monomères pairs. Cela a également été observé en présence de glucose ou de raffinose uniquement. Le substrat utilisé pour la synthèse de PHAs a ainsi été généré par la dégradation d'acides gras intracellulaires à chaîne courte et moyenne via le cycle de la β-oxidation. L'inhibition de la synthèse d'acides gras par la cérulénine a bloqué la synthèse de PHAs par les acides gras internes. Ces résultats ont révélés l'existence d'un cycle futile par lequel des intermédiaires à chaîne courte et moyenne de la synthèse cytoplasmique d'acides gras sont dirigés vers le cycle péroxisomal de la β-oxidation. Dans cette étude, le rendement de PHAs produits reste inchangé, mais l'analyse des PHAs permet de confirmer le flux de carbones vers et à travers le cycle péroxisomal de la β-oxidation et l'existence de nouveaux méchanismes a été dévoilée. Cette synthèse s'avère être un bon outil pour étudier in vivo le flux de carbones dans les cellules de S. cerevisiae.