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 is an account of my explorations into the field of natural products chemistry. These investigations led to the discovery of several novel secondary metabolites isolated from the marine cyanobacterium Lyngbya majuscula. In addition, biosynthetic investigations were undertaken using stable isotope-labeled precursors. The dominant role that NMR spectroscopy plays in the field of natural products chemistry has led to the development of several novel pulse sequences. Hectochlorin was discovered during a phytochemical investigation of a cultured L. majuscula originally collected off the coast of the Caribbean Island, Jamaica. The absolute stereochemistry was determined by X-ray crystallography. Through a series of biological evaluations, this compound was found to stimulate actin polymerization. The jamaicamide class of compound was isolated from the same organism that yielded hectochlorin. The structures were elucidated utilizing a variety of NMR methods, including a newly developed pulse sequence. Because the producing organism was in culture, a biosynthetic pathway investigation ensued to elucidate the carbon framework in jamaicamide A. The marine natural product barbamide is intriguing due to the incorporation of a trichloromethyl group into its molecular constitution. Further investigation into the timing of the chlorination reaction has been pursued. In addition, the isolation of dechlorobarbamide and the determination of the absolute stereochemistry assignment of barbamide was accomplished. A reevaluation of the stereochemistry of antillatoxin necessitated a correction in the original assignment. Four antillatoxin stereoisomers were obtained from a collaborator and found to possess differing levels of biological activity. The three dimensional solution structures of these isomers were evaluated in an effort to understand the role these stereochemical features play in the observed bioactivity. The structures were determined utilizing NMR-derived constraints applied to molecular modeling calculations. The development of two new pulse sequences for the determination of long-range heteronuclear coupling constants was also accomplished. The 1,1 ADEQUATE experiment was modified to yield an ACCORDIAN experiment which can be optimized to observe of a wide range of ��J[subscript cc] couplings. This new experiment is demonstrated for a model compound as well as for the new marine natural product jamaicamide A. / Graduation date: 2002

Graduation date: 1993