Berlin, Techn. Univ., Diss., 2001. / Computerdatei im Fernzugriff.

Thesis (Ph. D.)--University of Oregon, 2006. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 115-129). Also available for download via the World Wide Web; free to University of Oregon users.

Cyanobacteria are rich in biologically active secondary metabolites, many of which have potential application as anticancer or antimicrobial drugs or as useful probes in cell biology studies. A Jamaican isolate of the marine cyanobacterium, Lyngbya majuscula was the source of a novel antifungal and cytotoxic secondary metabolite, hectochlorin. The structure of hectochlorin suggested that it was derived from a hybid PKS/NRPS system. Unique features of hectochlorin such as the presence of a gem dichloro functionality and two 2,3-dihydroxy isovaleric acid prompted efforts to clone and characterize the gene cluster involved in hectochlorin biosynthesis. Initial attempts to isolate the hectochlorin biosynthetic gene cluster led to the identification of a mixed PKS/NRPS gene cluster, LMcryl, whose genetic architecture did not substantiate its involvement in the biosynthesis of hectochlorin. This gene cluster was designated as a cryptic gene cluster because a corresponding metabolite remains as yet unidentified. The expression of this gene cluster was successfully demonstrated using RT-PCR and these results form the basis for characterizing the metabolite using a novel interdisciplinary approach. A 38 kb region putatively involved in the biosynthesis of hectochlorin has also been isolated and characterized. The hct gene cluster consists of eight open reading frames (ORFs) and appears to be colinear with regard to hectochlorin biosynthesis. An unusual feature of this gene cluster includes the presence of a ketoreductase domain in an NRPS module and appears to be the first report of such an occurrence in a cyanobacterial secondary metabolite gene cluster. Other tailoring enzymes present in the gene cluster are two cytochrome P450 monooxygenases and a putative halogenase. The juxtaposition of two ORF's with identical modular organization suggests that this gene cluster may have resulted from a gene duplication event. Furthermore, biochemical characterization of two adenylation domains from this cluster strengthens its involvement in the biosynthesis of hectochlorin. / Graduation date: 2006

Marine cyanobacteria have been shown to produce a variety of biologically active and stucturally diverse secondary metabolites. These compounds are of interest to natural products researchers mainly because of their potential application as biomedicinals, biochemical probes, and agrichemicals. The metabolic pathways utilized by the cyanobacterium Lyngbya majuscula to generate curacin A, a potent antimitotic and its cometabolite, the molluscicidal barbamide, have been studied. Application of methods including radioisotope and stable isotope labeling have revealed the role of acetate and the amino acids methionine, valine, cysteine, and leucine as potential precursors in the biosynthesis of curacin and barbamide. An analytical technique based upon GC-EIMS methodology has also been developed to monitor the production levels of curacin A from cultures of Lyngbya majuscula with respect to growth. This method which makes use of the thiazole analog of curacin A, curazole as an internal standard, has also been preliminarily applied to the curacin A production in response to environmental factors associated with changes in geographical locations at or near sites where the original collections of the cyanophyte were made in Curacao, Netherlands Antillies. This was performed by a series of transplantation experiments envolving high and trace curacin A producing strains of L. majuscula. Interest in the bioactive profile of curacin A prompted a pilot scale up of the cultured tissue for isolation of the metabolite. These efforts provided a framework of methods that can be used industrially to obtain large quantites of the compound to meet possible future pharmaceutical or dignostics demands. / Graduation date: 1997

This thesis details my investigations of marine cyanobacterial natural products that resulted in the discovery of eighteen new secondary metabolites. Isolation and characterization of these unique molecules were carried out using different chromatographic techniques and by careful analyses of 1D and 2D NMR data, respectively. Preliminary bioassays of the crude organic extract of the marine cyanobacterium Lyngbya semiplena collected from Papua New Guinea in 1999, showed good activity in the brine shrimp toxicity model at 10 ppm. Guided by this assay, seven new anandamide-like fatty acid amides, semiplenamides A to G, together with four cyclic depsipeptides, wewakpeptins A to D, were identified. Due to the structural resemblance of the novel ethanolamide derivatives (semiplenamide A-G) with anandamide, (N-arachidonoyl-ethanolamine), an endogenous agonist of cannabinoid receptors compounds, semiplenamide A-G were tested on the well characterized proteins of the endocannabinoid system: 1) the "central" cannabinoid CB₁ receptors; 2) the anandamide membrane transporter (AMT), which is responsible for anandamide cellular uptake; and 3) the fatty acid amide hydrolase (FAAH), which catalyses anandamide hydrolysis. Three showed modest potency in displacing radiolabeled anandamide from the cannabinoid receptor (CB₁), and one was a modest inhibitor of the anandamide membrane transporter (AMT). The wewakpeptins were tested for cytotoxicity to NCI-H460 human lung tumor and neuro-2a mouse neuroblastoma cells. Intriguingly, wewakpeptin A and B were approximately 10-fold more toxic than C and D to these cell lines. Lyngbya majuscula has been recognized as a chemically and biologically rich strain. Five new lyngbyabellin analogs, lyngbyabellins E-I, along with the known compound dolabellin, originally isolated from sea hare Dolabella auricularia, were identified from a 2002 Papua New Guinea collection of the marine cyanobacterium Lyngbya majuscula. The lyngbyabellins were tested for cytotoxicity to NCI-H460 human lung tumor and neuro-2a mouse neuroblastoma cells and had LC₅₀ values between 0.2 and 4.8 μM. Intriguingly, lyngbyabellin E and H appeared to be more active against the H460 cell line with LC₅₀ values of 0.4 μM and 0.2 μM, respectively, compared to LC₅₀ values of 1.2 and 1.4 μM in the neuro-2a cell line. Lynbyabellin I was the most toxic to neuro-2a cells (LC₅₀ 0.7 μM), whereas lyngbyabellin G, was the least cytotoxic of all compounds to either cell line. On the basis of this limited screening, it appears that lung tumor cell toxicity is enhanced in the cyclic representatives, and overall potency is increased in those containing an elaborated side chain. Additionally, two new cytotoxins, aurilides B and C, which are closely related to aurilide, originally isolated from the sea hare Dolabella auricularia, have been identified from the same extract where the lyngbyabellins E-I were isolated. Aurilides B and C were tested for cytotoxicity to NCI-H460 human lung tumor and neuro-2a mouse neuroblastoma cells. Interestingly, aurilide B was approximately 4-fold more toxic than C to these cell lines. The LC₅₀ for Aurilide B was 0.01 μM and 0.04 μM for neuro-2a and H460 cells, respectively, and 0.05 μM and 0.13 μM, respectively, for aurilide C. Aurilide B was evaluated in the NCI 60 cell line panel and found to exhibit a high level of growth inhibition in leukemia, renal, and prostate cancer cell lines with a GI₅₀ less than 10 nM. Aurilide B showed net tumor cell killing activity in the NCI's hollow fiber assay, an in vivo model for assessing a chemical's anticancer activity. / Graduation date: 2006

Marine algae have been recognized as a rich resource of new and unusual organic molecules with diverse biological properties. The current need to develop new antifungal, anticancer, antibiotic and antiviral drugs has led to an intense research effort into the discovery, isolation and structure determination of potential medicinal agents from marine algae. In the past two years, I have participated in a drug discovery program designed for antitumor, antifungal and other agents of potential pharmaceutical utility from the marine cyanobacterium Lyngbya majuscula. This research utilized modern chromatographic and spectrochemical techniques including 2D NMR spectroscopy. Brine shrimp toxicity guided the fractionation that led to the discovery of the biologically active compound kalkitoxin from a Curacao Lyngbya majuscula extract. The structure of this new thiazoline ring-containing lipid was determined spectroscopically by interpretation of 2D-NMR experiments, including heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple-bond coherence spectroscopy (HMBC) and ��H-��H COSY at room temperature and elevated temperature. Kalkitoxin shows modest molluscicidal toxicity, good brine shrimp toxicity and extremely potent ichthyotoxicity. From the same extract of Lyngbya majuscula, I also isolated two other secondary metabolites, malyngamide J and malyngamide L. The structures of these new compounds, including stereochemistry, were determined by spectroscopic techniques including 2D-NMR experiments and by comparison with other known malyngamides. / Graduation date: 1997

Graduation date: 2003

Thesis (M.Sc.). / Written for the Dept. of Plant Science. Title from title page of PDF (viewed 2008/07/29). Includes bibliographical references.

Techn. Universiẗat, Diss., 2001--Berlin.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.