Kerr, Russell G.

Research into the chemical constituents of marine organisms is a relatively new
and growing field, given that much of the marine environment has only in the last half
century become accessible. Despite the strides that have been made in ocean exploration,
the marine biome has still been only barely explored. Much of the impetus for the
isolation and identification of chemical entities from marine organisms has been related
to their potential as medicines. For a number of reasons, including synthetic efficiency,
this has meant that many marine compounds isolated and studied have been small
molecules. In recent years, large strides have been made in techniques for the isolation
and identification of biological macromolecules, especially proteins. The understanding
of these molecules, and their relationship to the biochemical processes of the marine
organisms from which they have been isolated, is important not only because of the
potential for this information to help in the synthesis of medicines, but because it may help in the recognition of processes that affect the very viability of marine organisms
increasingly exposed to anthropogenic threats to their environment. This work consists of
four related studies involving the development of methods for the separation and
identification of proteins from a number or gorgon ian species. Chromatographic and gel
based methods were used to isolate an elisabethatriene synthase (ELS) which shows
promise as a biotechnological tool for the production or pseudopterosins. A number of
gorgonians were screened for the presence of proteins that might have antimicrobial
properties, and several organisms were identified that might be of interest in this context.
Two-dimensional gel electrophoresis methods were then developed to allow the
construction of gel maps for the azooxanthellate gorgonian Leptogorgia minimata and the
xoozanthellae gorgonian Pseudopterogorgia e/isahethae, the separated proteins were
digested and analyzed by LC/MSMS. and the information generated was used to examine
the proteome of the organism for functional and phylogenetic relationships.
Finally, the gorgonian Euniceafi<sca was exposed to several environmental stressors in
the laboratory. and the two-dimensional proteomic methods developed were used to
examine the effect of the stressors on the organism .

Nature has served human kind m many ways, one of which is a source of
medicines. Natural products from marine sources represent a relatively new area
of research and have shown tremendous potential as a source of new chemical
entities in drug discovery. Caribbean gorgomans corals of the genus
Pseudopterogorgia have been shown to produce a variety of chemically
interesting and biologically significant secondary metabolites. ln this dissertation,
the Caribbean coral Pseudopterogorgia acerosa has been investigated for the
presence of novel diterpenes and these compounds were found to belong to three
different classes: pseudopteranoids, bis-pseudopteranoids and lipidyl
pseudopteranes. Nine of these were new compounds. The structural elucidation of these compounds was performed using spectroscopic means such as l D and 20
NMR, and mass spectroscopy.
There is growing evidence that secondary metabolites isolated from manne
invertebrates may actually be produced by a bacterial symbiont. The research
studies in our laboratory regarding the source of diterpenes in the selected
gorgonian corals suggested a bacterial origin. The hypothesis that coral associated
bacteria are the source of diterpenes in the coral P. acerosa, was evaluated using
the series of experiments and evidence supported this biosynthetic origin.
A study comparing the "gall" tissue and healthy coral tissue in terms of diterpene
content and culturable bacterial communities showed that different groups of
diterpenes were concentrated in different coral tissue types. It was also observed
that the bacterial populations associated with the "gall" and healthy tissues were
considerably different. Furthermore, observed specificity in antimicrobial activity
of certain groups of compounds against bacteria isolated from the same coral
suggested the ecological role of these compounds. This work with "gall" tissue
supports the hypothesis that diseased coral tissue represents an excellent source of
bioactive natural products for drug discovery.
Additionaly, a simple LC-MS method was developed for the analysis of
anticancer drug carmustine in plasma.

The overall goal of this research was to isolate key genes involved in the
diterpene biosynthesis from Euniceafusca and Erythropodium caribaeorum using
molecular biology techniques. The initial goal was to use fuscol induced cell
cultures of Symbiodinium sp. isolated from E. fusca and to develop an approach
based on differential display of mRNA-reverse transcription-PeR. Together with
inverse PCR, these techniques ultimately provided a full-length farnesyl
diphosphate synthase sequence. Functional expression of this enzyme was
demonstrated with the addition of appropriate substrates and confirmed by
chromatography. From this data, degenerate primer based PCR was used to
isolate putative geranylgeranyl diphosphate biosynthetic genes from E.
caribaeorum. Both chemical and genetic examinations of Pseudopterogorgia
elisabethae eggs and their associated Symbiodinium sp. were employed to identify
the biosynthetic origin of their diterpenes. Terpene content and biosynthetic
capabilities of azooxanthellae eggs demonstrated the presence of pseudopterosins
but also indicated that the eggs were not capable of producing these compounds.
Likewise, no correlation could be observed for the phylogenetic relationships
inferred for the Symbiodinium sp., with that of the terpene chemistry present in P.
elisabethae. This finding leads us to speculate about an additional source of
terpene production within this coral.
Based on these and other recent findings suggesting symbiotic bacteria as
the source of secondary metabolites from marine invertebrates, bacterial
assemblages from E. caribaeorum were examined. This study revealed
considerable phylogenetic bacterial diversity within this coral and the
identification of several bacteria known to produce terpenes in other organisms.

Pseudopterogorgia elisabethae is a known source of structurally interesting bioactive
metabolites. A detailed search for new, related compounds was undertaken in this study
which resulted in the isolation and characterization of more than ten new diterpenes with
serrulatane and ileabethane skeletons. Some of the new compounds isolated are closely
related terpenes with significant biological activity and others are likely to be key
biosynthetic intermediates. As a component of the development of a production method
of anti-inflammatory compounds such as seco-pseudopterosin and elisabethadione, a
synthesis of a seco-pseudoperosin aglycone from elisabethatriene was developed.

The marine environment is a prolific source of novel compounds for therapeutic
use due to the complex biological and chemical diversity. Throughout the past 30-40
years, over 15,000 natural products have been discovered from the oceans, many of
which display a broad range of potential clinical and commercial applications. Many
marine invertebrates are sessile organisms that lack physical protection, and which
chemical defense may be a possible explanation for these secondary metabolites. Despite
the promise marine natural products have as potent pharmaceutical agents, one of the
major factors delaying clinical use is the supply issue. These bioactive compounds are
often found in trace amounts in the host organism, which makes harvesting from the reefs
unfeasible. A general goal in our lab was to investigate the biosynthesis of secondary
metabolite terpenes to ultimately provide a production method of these potent marine
derived compounds. Eleutherobin and desmethyleleutherobin are diterpenes isolated from the
Caribbean soft coral Erythropodium caribaeorum. These extremely valuable anticancer
agents disrupt cell division by polymerizing and stabilizing microtubules, and have
demonstrated tumor tissue selectivity toward selected breast, renal, ovarian and lung
cancer cell lines. Determining the first intermediate in terpene biosynthesis is the initial
step in developing a biotechnological production method of these cytotoxic agents. We
investigated the complex chemistry of this coral using a radioactivity-guided isolation
procedure, and isolated and partially characterized a diterpene hydrocarbon from E.
caribaeorum.
The close association between marine invertebrates, zooxanthellae and numerous
bacteria gives rise to the question of the identity of the producer of secondary metabolites
in marine organisms. If the symbiont produces these therapeutic agents, cell culture
methods could be employed to supply the compounds rather than obtaining them from
coral reefs. Sesquiterpenes have been isolated from the gorgonian Plexaurella spp.,
however, no investigations concerning host/symbiont contribution of the sesquiterpenes
have been reported. We investigated the biosynthetic source of terpenes in this coral, and
experimental evidence indicates that bacteria are responsible for sesquiterpene
production. We also examined sesquiterpene variation of Plexaurella spp. from various
locations, and found sesquiterpene content to vary within and between species,
identifying Plexaurella as a chemically indistinguishable genus.

The bryostatins are a family of macrolide lactones isolated from the
marine bryozoan Bugu/a neritina. Since its detection in 1968, bryostatin 1
has demonstrated remarkable anticancer, immunopotentiating,
biomodulatory and radioprotective effects which result mainly from its
ability to activate protein kinase C, a family of isozymes involved in cellular
signal transduction. It is currently being tested in several phase I and phase
II clinical trials as a potential anticancer drug for leukemia, melanoma and
nephrotoma. A series of experiments was undertaken to elucidate the
biosynthetic origins of bryostatin, using a fortified crude cell-free enzyme
preparation and radiolabelled precursors. A regional characterization of
Bugula neritina from Sicily, Italy and Daytona Beach, Florida is also
described.

The octocoral Eunicea fusca is the source of important anti-inflammatory compounds
such as the diterpenes fuscol and the fuscosides. Evidence suggests that these
compounds are being produce by bacterial sources that live in symbiosis with the coral.
As part of an investigation to better understand the role of bacteria associated with E.
fusca , the characterization of the bacterial community using two different techniques
(culture dependent technique and culture independent technique) and sea water samples
was done. Sea water samples were used as a control to determine how closely associated
bacteria are to this octocoral. The polymerase chain reaction (PCR) with universal
bacterial primers FC27 and RC 1492 specific to prokaryotic 16S rDNA gene sequences
was used to characterize the total bacterial population when using both the culturable and
the non-culturable approaches. The results indicate a diverse group of bacteria associated to E. fusca composed of 10 different groups. The pro teo bacteria group was the most
predominant group when both techniques were used. The a-proteobacteria represented
the highest percentage of bacteria associated to E. fusca.