Thesis (Ph.D.)--Victoria University of Wellington, 2008. / Includes bibliographical references.

I investigated the ecological functions of halogenated secondary metabolites from the red alga Asparagopsis armata, their localisation in specialised cells and also their cost of production. A. armata produces large amounts of halogenated metabolites ( &lt 20 ??g / mg dry weight) that are sequestered in gland cells, as was demonstrated with light, epifluorescence and transmission electron microscopy. Cellular structures were identified that likely assist the release of metabolites from the gland cells to the algal surface. The halogenated metabolites of A. armata have multiple ecological roles, functioning as both inhibitors of bacterial fouling and as herbivore deterrents. Their activity against bacteria and herbivores was measured by a novel test in which the metabolites were manipulated in A. armata by omitting bromide ions from the culture media. This technique prevented the production of halogenated metabolites, but did not impact on other aspects of algal biology. Algae lacking halogenated metabolites (bromide [-] algae) had higher densities of epiphytic bacteria than those that continued to produce metabolites (bromide [+] algae). Bioassays with pure compounds against individual bacterial isolates further supported an inhibitory role for the halogenated metabolites against epiphytic bacteria, and also indicated an affect on bacterial community structure as well as abundance. Bromide (+) A. armata produced halogenated metabolites that also deterred feeding by two herbivores (an amphipod and an abalone), but not a third (an opisthobranch mollusc). A novel outcome from these feeding assays was the demonstration of a relationship between herbivore size and consumption of the chemically defended A. armata by the abalone Haliotis rubra. In addition to the fitness benefits gained from chemical defence, there were also costs for allocating resources to secondary metabolites. These costs were only detected under limiting light resources, consistent with predictions of the plant defence models. The integration of chemical analyses and cellular measures of chemical defence proved essential in elucidating resource allocation to chemical defence in the filamentous stage of A. armata. This thesis highlights that the simple relationships between growth and defence in filamentous algae can provide an excellent model for studies of the ecology and evolution of chemical defences in marine algae.

Molecular genetic and enzymological techniques have been employed to study secondary metabolite biosynthesis. These investigations have focused on two projects: the cloning and heterologous expression of biosynthetic gene clusters from unculturable marine organisms and the characterization of individual enzymes involved in the biosynthesis of the antifungal agent blasticidin S. The marine environment is proving to be a valuable source of biologically active compounds, but problems associated with sustainable harvest, laboratory culture, and organic synthesis make obtaining sufficient quantities of compounds for drug development both difficult and expensive. A method has been developed for the isolation of biosynthetic gene clusters from complex marine microbe/invertebrate associations. Using this method a mixed polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene cluster has been cloned from the marine sponge Jaspis splendens. The cloned gene cluster was found to code for a PKS with three extension modules and an NRPS with three extension modules. In addition, several open reading frames (ORFs) were identified that may be involved in the biosynthesis of the PKS starter molecule. Partial characterization of catalytic domains from the NRPS was also completed. The second project centers on the characterization of enzymes involved in blasticidin S (BS) biosynthesis. Two ORFs were identified in the BS gene cluster encoding gene products predicted to be involved in the early steps of BS biosynthesis. The blsG gene product has sequence similarity to lysine 2,3-aminomutase and is believed to be involved in the formation of the β-arginine moiety of BS. A series of heterologous expression studies were undertaken to determine the function of B1sG. The product of blsM exhibits sequence homology with several nucleosidetransferases. blsM was cloned from the BS gene cluster, heterologously expressed in E. coli, and shown to catalyze the formation of cytosine using cytidine 5'- monophosphate as the preferred substrate. Point mutations were introduced in blsM to generate three B1sM mutant enzymes: S92D, E98A, and E98D. All three mutants lost cytidine 5'-monophosphate hydrolysis activity. Surprisingly, the B1sM S92D mutant exhibits cytidine deaminase activity when incubated with cytidine or deoxycytidine, resulting in the formation of uridine and deoxyuridine, respectively. / Graduation date: 2005

Graduation date: 2003

Graduation date: 1993

I investigated the ecological functions of halogenated secondary metabolites from the red alga Asparagopsis armata, their localisation in specialised cells and also their cost of production. A. armata produces large amounts of halogenated metabolites ( &lt 20 ??g / mg dry weight) that are sequestered in gland cells, as was demonstrated with light, epifluorescence and transmission electron microscopy. Cellular structures were identified that likely assist the release of metabolites from the gland cells to the algal surface. The halogenated metabolites of A. armata have multiple ecological roles, functioning as both inhibitors of bacterial fouling and as herbivore deterrents. Their activity against bacteria and herbivores was measured by a novel test in which the metabolites were manipulated in A. armata by omitting bromide ions from the culture media. This technique prevented the production of halogenated metabolites, but did not impact on other aspects of algal biology. Algae lacking halogenated metabolites (bromide [-] algae) had higher densities of epiphytic bacteria than those that continued to produce metabolites (bromide [+] algae). Bioassays with pure compounds against individual bacterial isolates further supported an inhibitory role for the halogenated metabolites against epiphytic bacteria, and also indicated an affect on bacterial community structure as well as abundance. Bromide (+) A. armata produced halogenated metabolites that also deterred feeding by two herbivores (an amphipod and an abalone), but not a third (an opisthobranch mollusc). A novel outcome from these feeding assays was the demonstration of a relationship between herbivore size and consumption of the chemically defended A. armata by the abalone Haliotis rubra. In addition to the fitness benefits gained from chemical defence, there were also costs for allocating resources to secondary metabolites. These costs were only detected under limiting light resources, consistent with predictions of the plant defence models. The integration of chemical analyses and cellular measures of chemical defence proved essential in elucidating resource allocation to chemical defence in the filamentous stage of A. armata. This thesis highlights that the simple relationships between growth and defence in filamentous algae can provide an excellent model for studies of the ecology and evolution of chemical defences in marine algae.

Thesis (Ph.D. (Chemistry)) - Rhodes University, 2008.

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

This thesis comprises both a natural product investigation and a synthetic component. The natural product investigations are presented in Chapters Two and Three. In Chapter Two the isolation and spectroscopic identification of the new isocopalane diterpene 12S,13R,14Sisocopalan- 13-ol-12,14-diacetate (2.1) and two known 3-(14S)-isocopal-12-ene-15-oyl-1- acetyl-sn-glycerol (2.2) and 3-(14S)-isocopal-12-ene-15-oyl-2-acetyl-sn-glycerol (2.3) from a single, large, unidentified sub-Antarctic nudibranch, collected near Marion Island, approximately 2000 km south of Cape Town are described. Chapter Three discusses the isolation, spectroscopic structure elucidation and anti-oesophageal cancer activity (3.1-3.4 only) of two known labdane diterpenes 6β,7α-diacetoxylabda-8,13E-dien-15-ol (3.1) and 2α,6β,7α-triacetoxylabda-8,13E-dien-15-ol (3.2) and one new 6β,7α,15-triacetoxylabda 8,13E-diene (3.3), as well as new 3α,11-dihydroxy-9,11-seco-cholest-4,7-dien-6,9-dione (3.4) and cholest 7-en-3,5,7-triol (3.5) from the endemic pulmonate mollusc, Trimusculus costatus. The absolute configuration of 3.2, and hence 3.1 and 3.3 (from biogenetic arguments) was determined through X-ray diffraction of a single crystal of the camphanate ester of 3.2. The absolute configuration of the secondary hydroxyl at C-3 of 3.4 was established using the Modified Mosher’s method. The synthetic component of the thesis commences in Chapter Four with the semi-synthesis of labdane diterpene nitriles 9α-cyano-15,16-epoxy-7β-hydroxylabda-13(16),14-dien-6-one (4.1), 9α-cyano-15,16-epoxy-7-hydroxylabda-7,13(16),14-trien-6-one (4.2) and 9α-cyano-15,16- epoxy-6β,7β dihydroxylabda-13(16),14-diene (4.3) from the terrestrial labdane diterpene, hispanolone (4.4). This work is an extension of previous synthetic studies directed towards the synthesis of T. costatus metabolites. Diterpenes 4.1-4.3 exhibited in planta activity against the economically important crop pathogens, Magnaporthea grisea and Puccinia recondita. Chapter Five describes the successful semi-synthesis of two isomeric marine molluscan labdane diterpene aldehyde metabolites, labd-13E-ene-8β-ol-15-al (5.1) and labd-13Z-ene- 8β-ol-15-al (5.2) from the commercially available, terrestrial plant derived, labdane diterpene manool (5.3). Diterpenes 5.1 and 5.2, originally isolated from the Mediterranean nudibranch,Pleurobranchaea meckelii and selected diterpenes arising from this synthesis were evaluated for their activity against an oesophageal cancer cell line (WHCO1). Chapter Six further develops the research discussed in Chapter Five, where ethyl 17-norabiet-13(15)-E-en-8β-ol- 16-oate (5.49) and ethyl 17-norabiet-13(15)-Z-en-8β-ol-16-oate (5.50) were first semisynthesized serendipitously. Based on their structural relationship to naturally occurring tricyclic diterpenes with anti-plasmodial activity, tricyclic diterpenes, 17-norpimaran-13α- ethoxy-8,16-olactone (6.6), 17-norisopimar-15-ene-8β,13β-diol (6.7), 17-norisopimarane- 8β,16-diol (6.8) and 17-norabiet-13(15)-ene-8β,16-diol (6.9) were semi-synthesized from the terrestrial labdane diterpene, 5.3, and critically evaluated for their antimalarial potential from parasite inhibition and haemolytic studies.

An in depth chemical investigation of the major and minor pyrroloiminoquinone metabolites produced by four species of endemic South African Latrunculid sponges, collected from Algoa Bay and the Tsitsikamma Marine Reserve off the south eastern coast of South Africa, yielded eleven new and twelve known pyrroloiminoquinone metabolites. The structures of the new metabolites were determined using standard spectroscopic techniques. Tsitsikamma pedunculata was shown to contain 7,8-dehydro-3-dihydro-discorhabdin C (2.1), 14-bromo-7,8-dehydro-3-dihydro-discorhabdin C (2.2), discorhabdin S (2.3), 14-bromo-1-hydroxy-discorhabdin S (2.4), 1-bromo-2-hydroxy-4-debromo-discorhabdin S (2.5), and 2,4-debromo-3-dihydro-discorhabdin C (2.6), together with the known compounds 14-bromo-discorhabdin C (1.51), 14-bromo-3-dihydro-discorhabdin C (1.52) and 3-dihydro-discorhabdin C. The metabolites from T. pedunculata were characterised by the presence of a reduced C-3 carbonyl and bromination at C-14. Compounds isolated from a second Latrunculid sponge, Latrunculia lorii, ranged from a substituted bicyclic pyrrolecarboxylic acid, makaluvic acid A (1.47), to the simple tricyclic known pyrroloiminoquinones makaluvamine C (1.33) and damirone B (1.20) and the more complex discorhabdin D type metabolites, discorhabdin M (3.2), 1-amino discorhabdin D (3.3), 1-methoxy discorhabdin D (3.4) and 1-alanyl discorhabdin D (3.5). Discorhabdin G* (3.1) was also isolated and characterised. This is the first reported occurrence of the known compounds 1.20, 1.33 and 1.47 in a Latrunculia sponge. Discorhabdin and bis-pyrroloiminoquinone type compounds predominated in Tsitsikamma favus. Three known, tsitsikammamines A (1.71) and B (1.72), 1.52, and five new pyrroloiminoquinones, tsitsikammamine N-oxime (4.1), tsitsikammamine B N-oxime (4.2), 2.1, 2.4 and 2.6, were isolated from this sponge. A fourth Latrunculid sponge (Strongylodesma sp.) yielded three known compounds, discorhabdins A (1.57), D (1.61) and 1.53, and one new pyrroloiminoquinone 3.3. The dual role of these metabolites as cytotoxic agents and pigments resulted in an attempt to relate the photochemical properties of these metabolites to their cytotoxicity. The pyrroloiminoquinone metabolites studied exhibited moderate singlet oxygen quantum yields, while three compounds (1.57, 4.1 and 4.2) were shown to be capable of producing radicals at a wavelength of 532 nm. The possibility of a correlation between the electrochemical properties and anti-cancer (HCT-116) activity of selected pyrroloiminoquinones was explored. A study of the oesophageal and ovarian cytotoxicities of two pyrroloiminoquinones (1.57 and 1.72), together with an investigation into the intercalation and topoisomerase I inhibitory activity of the bis-pyrroloiminoquinones (1.71, 1.72, 4.1 and 4.2), are presented.