Kerr, Russell G.

Ecteinascidin is a tetrahydroisoquinoline alkaloid isolated from the colonial ascidian Ecteinascidia turbinata. Ecteinascidin exhibits tremendous activity against P388 murine leukemia, however its yields from the tunicate are as low as 10^-4%. A biosynthetic investigation, using in vivo and in vitro methods, has resulted in the identification of the metabolic precursors of ecteinascidin as tyrosine, DOPA and cysteine. Reactive intermediates such as tyrosine and DOPA diketopiperazines have also been identified. These preliminary experiments set the stage for subsequent protein isolation.

9,11-secogorgosterol is a secondary metabolite from the gorgonian Pseudopterogorgia americana which acts as a chemical defense. The soft coral lives in a symbiotic association with unicellular algae known as zooxanthellae. A biosynthetic investigation, using in vivo and in vitro methods, has resulted in the identification of the metabolic precursor of 9,11-secogorgosterol as gorgosterol. This finding is significant as it indicates that the conversion of gorgosterol to 9,11-secogorgosterol is due to gorgonian metabolism. Since gorgosterol is known to be a product of zooxanthellae metabolism, this would be the first example of a defensive secondary metabolite being produced by two organisms living in symbiosis. A viable acetone powder has been generated from the crude cell-free extract and has demonstrated the efficient transformation of gorgosterol to 9, 11-secogorgosterol. This indicates possible future value as a synthetic tool for secosterol production.

Chemical analysis of large number of specimens of the sponge Xestospongia muta have revealed the existence of three distinct sterol chemotypes. Since sponges are well known to contain a diverse array of all classes of lipids, the utility of fatty acid composition as a chemotaxonomic tool for Xestospongia muta has been addressed. Further the biosynthetic capabilities of the three chemotypes are being evaluated for possible taxonomic use. Further the utility of TLC and NMR as additional taxonomic tools has also been evaluated.

Parkeol, Lanosta-9(11),24-dien-3beta-ol, was identified as the triterpene precursor of saponins which contain Delta9(11) unsaturation from sea cucumbers and shown not to be the precursor in starfish. Further, the site of saponin biosynthesis in sea cucumber was shown to be the cuvier gland. These results were obtained by "feeding" radiolabeled precursors to live specimens of sea cucumbers and starfish and by conducting incubation experiments with cell-free extracts.

Bryostatin 1 is a macrocyclic lactone isolated from the marine bryozoan Bugula neritina. It is currently in clinical trials at the National Cancer Institute as a promising new antileukemic agent. Bryostatin 1 is found in Bugula neritina in poor yields (10^-4% dry weight). The focus of this research was to develop the biochemical and biosynthetic techniques that could be utilized to optimize the production of bryostatin 1 in Bugula neritina through aquaculture. To that end, the basic biosynthetic pathway to bryostatin 1 in the marine bryozoan was evaluated by using radiolabeled precursors. Techniques for the incorporation of these precursors were also developed. Other experiments involved an analysis of the composition of a specific population of Bugula neritina in terms of its bryostatin and lipid content. A determination of which lipids are produced de novo and which are exogenous was also undertaken.

The marine environment has proven to be an extremely rich source of novel natural products with activities in a variety of biological assays. The field of marine natural products chemistry has enjoyed an explosion of growth over the past 40 years and currently there are marine-derived metabolites in clinical trials for cancer, analgesia, allergy and cognitive diseases. Even with this tremendous development, it is clear that chemists and pharmacologists are only beginning to scratch the surface of the biomedical potential of marine organisms. One problem with this source of new pharmaceuticals is that the supply of such compounds can be problematic. The thesis describes projects directed at fundamental biosynthetic questions regarding terpenes from marine corals. In a general sense, these projects are directed as addressing the supply issue identified above. One project examined the origin of terpene building blocks in the coral Pseudopterogorgia elisabethae. A second project focused on the characterization of terpenes from Leptogorgia minimata. Here, a new cembranoid diterpene was isolated. Experiments were also conducted to identify a microbial source of terpene biosynthesis in this system. Data strongly supported a bacterial origin of these compounds.

Erythropodium caribaeorum is an octocoral known for its production of novel anticancer agents, such as eleutherobin and its analogs as well as for its complex association with symbiotic dinoflagellates (Zooxanthellae; genus Symbiodimum). With this in mind, two sets of experiments were conducted using the extracts of the symbiotic algal cells from Erythropodium caribaeorum: an isolation and characterization of a novel tri-hydroxy sterol and a bioassay-guided isolation of three additional compounds. All compounds exhibit significant bioactivity against the following three cell lines: the human melanoma cell line M14P, the colon cancer cell line RKO and the breast cancer cell line MDA-MB-231. Importantly, this study reports the first isolation of a bioactive polyhydroxy sterol from a gorgonian's symbiont and further suggests that these algae represent a promising sustainable resource for drug discovery.

A-nor sterols have been isolated and characterized from the marine sponge Teichaxinella morchella using High Performance Liquid Chromatography, Nuclear Magnetic Resonance, and Mass Spectrometry. A cell free extract of T. morchella was used to determine the biosynthetic origin of the sterols. Incubation experiments with radiolabled Farnesyl Pyrophosphate and cholesterol showed that the sterols are coming from a dietary source. Subcellular localization studies showed that the sterols are part of the cell membrane.

The pseudopterosins are a class of diterpene glycosides isolated from the Caribbean sea whip, Pseudopterogorgia elisabethae. Pseudopterosins A-D possess potent anti-inflammatory and analgesic properties (ED50 ca. 3.0 mg/kg). There is a large demand in the pharmaceutical and cosmetic industries for pseudopterosin derivatives and presently, the only source of these chemicals is from wild-collected gorgonians. Preliminary experiments were performed to develop a reliable, cost-effective production method of the pseudopterosins in order to avoid large-scale harvesting. We exposed the gorgonians to various forms of "stress" in an attempt to increase the amount of pseudopterosins produced. We have also evaluated the feasibility of raising Pseudopterogorgia elisabethae in an aquaculture setting by studying their growth rates in different environments. Our studies indicate that aquaculture used with some simple induction methods can allow for the commercialization of this important biochemical resource.

Gymnodinium breve is responsible for toxic blooms causing epizootics and, in humans, neurotoxic shellfish poisoning. Toxicity is from a family of polyketides known as brevetoxins. Proposed biosynthetic mechanisms of brevetoxin production are analogous to fatty acid biosynthesis. As part of an investigation of brevetoxin production, the fatty acid synthase from Gymnodinium breve was characterized. Cell free extracts of G. breve incorporated [3H]-acetate and [3H]-acetyl-CoA into palmitate with lesser incorporation into stearate, myristate, and laurate. Fatty acid synthase activity was associated with the chloroplast, appeared to be composed of membrane-associated and soluble components, and was not completely inhibited by cerulenin. These results suggests that G. breve possesses a Type II fatty acid synthase similar in organization to those found in other photosynthetic organisms. Isolation of the enzymes, on both an enzymatic and genomic level, were hindered by low amounts G. breve cells and bacteria present within the cultures.