This thesis described the chemical investigations of natural products from Australian marine sponge-derived fungi. Sponge samples were collected from the Great Barrier Reef, Queensland, Australia, by Queensland Museum. The thesis is divided into eight chapters and can be devided into two major parts. The first three chapters comprised the first part of the thesis: Chapter 1 outlined the research background, literature review of marine fungal secondary metabolites; Chapter 2 introduced fungal culture and storage background knowledge, and the list of isolated marine fungal strains. Chapter 3 introduced the background of the thrombin inhibition assay and assay results. The second part (Chapter 4 to 7) of this thesis is focused on chemical isolation and structure elucidation of secondary metabolites from isolated fungal strains, mostly active strains against thrombin. An unidentified fungal strain, FS-G315858 (T)-Y, isolated from the frozen sponge sample Dysidea sp.1400 produced five peptide compounds (chapter 4, 16-20). Compound 16 is a polypeptide which features the same relative configuration with a known compound unguisine A, and compounds 17-20 are diketopiperazines. Active fungal strains FS-G315695 (T)-Y and FDPS-61732-YB were isolated from different sponge samples. However, they were identified to be the identical fungal strain Eurotium rubrum; the chemical isolation of FS-G315695 (T)-Y from its mycelia EtOAc extract resulted in three compounds (chapter 5, 17-19). Compounds 18 and 19 were identified to be flavoglaucin and iso-dihydroauroglaucin. Compound 17 was identified to have the same relative configuration with a known compound neo-echinulin A. The chemical isolation of FDPS-61732-YB from its broth EtOAc extract resulted in several diketopiperazines (chapter 5, 27-29). Another active fungal strain FS-G315695 (T)-WY was identified as Aspergillus ochraceous, the chemical isolation of its mycelia EtOAc extract resulted in one benzodiazepine compound (chapter 6, 18), together with two fatty acids (chapter 6, 16-17). The structure of compound 18 was elucidated and identified to have same relative configuration with the known compound circumdatin E. Media comparison for active fungal strain FS-G315695 (T)-Y was conducted and this work resulted in producing several neo-echinulin analogues (chapter 7, 1-3). The isolation and structure elucidation of these compounds were reported in chapter 7.

Isolation and structure elucidation of natural products from marine sponges and an invertebrate were performed. The marine sponges and invertebrate were obtained from three locations including South East Queensland, in Australia, Pontianak in West Kalimantan, and Tulamben, in Bali, Indonesia. The natural products were purified using chromatographic techniques, the structures were elucidated by means of extensive 1D and 2D NMR spectroscopy and some were confirmed by X-ray crystallography. Five known compounds and potentially a new metabolite were identified from three different sponges and a species of nudibranch obtained in Mooloolaba, South East Queensland in Australia. Furospinosulin-1 (4.5), a linear sesterterpenoid, was identified from a sponge coded 20-1-07-1-7 while imidazole alkaloids, preclathridine A (4.9) and clathridine (4.10), were characterized from sponge 22-4-07-2-1. The ethyl acetate extract of sponge 14-7-07-1-1 yielded a polyacetylene fulvinol-like compound (4.15) and potentially a new metabolite compound 5 (4.21). Additionally, a specimen of Chromodoris kuiteri furnished a cyclic macrolide latrunculin A (4.27). There were six secondary metabolites identified from Aaptos aaptos, two of which to the best of our knowledge were new compounds. Aaptamine (5.1), a chemotaxonomic marker of the sponge A. aaptos, 9-demethylaaptamine (5.3) and a biosynthetically unrelated compound (-)-jaspamide (5.31) were found as major constituents of the dichloromethane extract of the sponge. On the other hand, a known indole-3-carbaldehyde (5.10), and the two new natural products methyl 3-(8,9-dimethoxy-4H-benzo[de][1,6]naphthyridin-4-yl)propanoate (5.11) and 8,9-dimethoxy-4H-benzo[de][1,6]naphthyridine-5,6-dione (5.30) were isolated as minor components. During a two-month fieldwork trip to Tulamben, Bali, six different sponges were obtained. Investigation of a blue colored sponge Petrosia sp. afforded two isoquinolinequinone metabolites, namely mimosamycin (5.35) and O-demethylrenierone (5.36). A new 3-alkylpiperidine metabolite, tetradehydrohaliclonacyclamine A (6.28), was isolated from a sponge Halichondria sp. The structure and relative stereochemistry of 6.28 were determined from analysis of 2D NMR data and interpretation of coupling constants. Suitable crystals that were grown from hexane: ethyl acetate (1:3) allowed the determination of the absolute configuration of 6.28 and it was established to be 2S, 3S, 7S, and 9S on the basis of X-ray crystallographic data. The isolated compound (6.28) appeared to be as a single enantiomer according to chiral HPLC. The parent compounds, haliclonacyclamine A (6.19) and B (6.20), were re-isolated from a sample (coded BK-Hal-12-AIK) obtained from a previous project on Haliclona in our group. Their absolute configurations were determined for the first time by X-ray crystallography and they were established to be 2R, 3R, 7R, and 9R.

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: 2001

Recent developments in NMR hardware have extended the reach of natural products chemists to structure elucidation of compounds isolated on a nanomolar scale. In parallel with advances in hardware new NMR techniques for structure elucidation have evolved, e.g. quantitative NOEs, residual dipolar couplings (RDCs)and diffusion-ordered spectroscopy (DOSY). The application of recent NMR techniques was utilized in the screening and structural assignment of natural products isolated from a 2004 collection of South African tunicates. Assignment of the planar structures of mandelalides A-D from a new Lissoclinum sp. was feasible only after data acquisition on a 700 MHz magnet equipped with 5 mm ����C cryogenic probe. While relative configuration of the polyketide mandelalides was established after extensive J-coupling and NOE analysis, quantitative ROESY and RDC measurements were also explored before the absolute configuration was accomplished by chemical degradation and comparison with standards. The application of DOSY, a greatly under-appreciated technique in natural products research, led to the identification of new rubrolide analogues, compounds with moderate antibacterial properties from the new species Synoicum globosum. Finally, relative and absolute configuration of new and known unstable amino alcohols from two Pseudodistoma species was based on J-coupling analysis and application of the Mosher method. / Graduation date: 2013

Graduation date: 1997

Vinigrol was isolated in 1987 from the fungal strain Virgaria nigra F-5408 by Hashimoto and co-workers. This compound was identified as having antihypertensive and platelet aggregation properties as well as being recognized as a tumor necrosis factor inhibitor. Aside from its interesting biological activities, vinigrol also possesses a unique structural motif consisting in a decahydro-1,5-butanonaphthalene core decorated with 8 contiguous stereocenters. Despite synthetic efforts by many research groups since its isolation, it wasn’t until 2009 that the first total synthesis of vinigrol was reported by Baran and co-workers. Herein is presented a formal synthesis of this highly compact molecule which relies upon a highly diastereoselective ketal Claisen rearrangement as the stereodefining step and an intramolecular Diels-Alder reaction to access the tricyclic structure of the molecule. (+)-Digitoxigenin is a cardiac glycoside used in the treatment of many ailments such as congestive heart failure. It is a member of the cardenolides, a sub-type of steroid containing certain structural differences such as cis A/B and C/D ring junctions, a tertiary hydroxyl group at C14 and a butenolide substituent at C17. Although a few syntheses of this class of compounds have been reported, general strategies to access their framework is scarce. Herein we report our studies towards the total synthesis of digitoxigenin which rely upon a cascading gold-catalyzed cycloisomerization (or enyne metathesis)/Diels-Alder reaction.

This thesis is an account of investigation on the natural products deriving from various marine algae and has resulted in the discovery of eleven novel bioactive metabolites. Isolation and characterization of these new molecules were carried out using different chromatographic techniques and by analyses of different spectroscopic data, respectively. Using bioassay guided fractionation (brine shrimp toxicity assay), I isolated and identified five new, biologically active compounds [2β,3α-epitaondiol, flabellinol, flabellinone, stypoaldehyde and stypohydroperoxide], together with five known compounds [2-geranylgeranyl-6-methyl-1, 4-benzoquinone, (-) epistypodiol, (-) stypoldione, fucoxanthin and iditol] from the marine brown alga Stypopodium flabelliforme, collected from Papua New Guinea. All of the new compounds were found to have cytotoxic activity (EC₅₀ ranges from 0.8-10 μg/ml) in human lung cancer (NCI-H460). 2β,3α-epitaondiol and flabellinol exhibited strong sodium channel blocking activity (EC₅₀=0.3 and 0.9 μg/ml, respectively). As a result of efforts to identify bioactive agents from marine algae, I have isolated and identified one new halogenated monoterpene [(-)-(5E,7Z)-3,4,8- trichloro-7-dichloromethyl-3-methyl-1,5,7-octatriene] in addition to another three known halogenated monoterpene compounds from the red alga Plocamium cartilagineum collected from the eastern coast of South Africa. [(-)-(5E,7Z)-3,4,8- trichloro-7-dichloromethyl-3-methyl-1,5,7-octatriene] was found to be active as a cytotoxic agent in human lung cancer (NCI-H460) and mouse neuro-2a cell lines (EC₅₀ 4 μg/ml). As part of continued search for bioactive secondary metabolites from marine sources using a bioassay guided fractionation approach (anti-trypanosome activity), I examined the organic extract of a Papua New Guinean collection of the green alga Udotea orientalis growing on a coral wall and collected in September 1998. Successive HPLC separations resulted in the isolation of three new compounds; (+) curcuepoxide A, (+) curcuepoxide B and (+)-l0α-hydroxycurcudiol. In addition I isolated four known compounds; (+)-10β- hydroxycurcudiol, (+) curcuphenol, (+) curcudiol and (+) curcudiol-10-one. A bioassay guided investigation approach (anti-Sirt2) of a Lyngbya majuscula collection from Key West Florida, led to the discovery of two novel bioactive natural products [(+)-malyngamide X and one cyclic depsipeptide, (+)-floridamide]. The new cyclic depsipeptide, (+)-floridamide contains four amino acids units beside the unique unit, 2,2-dimethyl-3-hydroxyoctanoic acid (Dhoaa). / Graduation date: 2004

The antioxidant and redox properties of L-ascorbic acid are closely associated with the electron rich 2, 3-enediol moiety of the molecule and therefore selective functionalization of the 2- and 3-OH groups is essential for the detailed structure-activity studies. Reactions of 5- and 6-OH protected ascorbic acid with electrophilic reagents exclusively produce the corresponding 3-O-alkylated products under mild basic conditions due to the high nucleophilicity of the C-3-OH. Based on the density functional theory (B3LYP) electron density calculations, a novel and general method was devised for the direct alkylation of the 2-OH group of ascorbic acid with complete regio- and chemo-selectivity. A complete spectroscopic analysis of two complementary series of 2- O -acetyl-3- O -alkyl and 2- O -alkyl-3- O -acetyl ascorbic acid derivatives was carried out to define their spectroscopic characteristics and to resolve common inconsistencies in the literature. The asymmetric approach to the synthesis of natural products or other biologically active compounds is impeded by low abundance of natural sources as well as a limited number of efficient synthetic methods. Nevertheless, carbohydrate-based systems such as the aldono-1,4-lactones (also known as aldono-γ-lactones) which generate a host of chiral compounds have been particularly rewarding in this respect. This study shows a practical approach using 5,6- O -isopropylidene-L-ascorbic acid (ketal of L-ascorbic acid) as a single common starting material for facile asymmetric synthesis of protected, optically pure and functionalized aldono-1,4-lactones derivatives, valuable in the synthesis of derivatives of various pharmacologically active agents for structure-activity studies. The practicality of this new approach is demonstrated by the convenient synthesis of a series of thermal Claisen-rearranged products of 5,6- O -isopropylidene-3- O -allyl-L-ascorbic acid and 5,6- O -isopropylidene-2- O -allyl-L-ascorbic acid as the corresponding 5,6- O -isopropylidene-2-allyl-3-keto-L-galactono-γ-lactone and 5,6- O -isopropylidene-3-allyl-2-keto-L-galactono-γ-lactone respectively. The synthetic routes are economical, efficient, diastereospecific, and proceed in high yields. / "August 2005." / Thesis (Ph.D.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Chemistry.

Curcumin is the active component of tumeric, and this polyphenolic compound has been extensively investigated as an anticancer drug that modulates multiple pathways and genes. We demonstrated that curcumin inhibited 253JB-V and KU7 bladder cancer cell growth, and this was accompanied by induction of apoptosis and decreased expression of the proapoptotic protein survivin and the angiogenic proteins vascular endothelial growth factor (VEGF) and VEGF receptor 1 (VEGFR1). Since expression of survivin, VEGF and VEGFR1 are dependent on specificity protein (Sp) transcription factors, we also investigated the effects of curcumin on downregulation of Sp protein expression as an underlying mechanism for the apoptotic and antiangiogenic activity of this compound. Curcumin decreases expression of Sp1, Sp3 and Sp4 in blader cancer cells indicating that the cancer chemotherapeutic activity of curcumin is due, in part, to decreased expression of Sp transcription factors and Sp-dependent genes. Betulinic acid (BA) and curcumin are phytochemical anticancer agents, and we hypothesized that both compounds decrease EGFR expression in bladder cancer through downregulation of specificity protein (Sp) transcription factors. BA and curcumin decreased expression of EGFR, Sp1, Sp3, Sp4 and Sp-dependent proteins in 253JB-V and KU7 cells; EGFR was also decreased in cells transfected with a cocktail (iSp) containing small inhibitory RNAs for Sp1, Sp3 and Sp4 showing that EGFR is an Sp-regulated gene. Methyl 2-cyano-3,11-dioxo-18?-olean-1,12- dien-30-oate (CDODA-Me) is a synthetic triterpenoid derived from glycyrrhetinic acid which inhibits proliferation of KU7 and 253JB-V bladder cancer cells. CDODA-Me also decreased expression of specificity protein-1 (Sp1), Sp3 and Sp4 transcription factors. Similar results were observed for a structurally-related triterpenoid, methyl 2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me), which is currently in clinical trials for treatment of leukemia. Celastrol, a naturally occurring triterpenoid acid from an ivy-like vine exhibits anticancer activity against bladder cancer cells. Celastrol decreased cell proliferation, induced apoptosis and decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and several Sp-dependent genes like Fibroblast growth factor receptor 3 (FGFR3). In vivo studies using KU7 cells as xenografts showed that celastrol represents novel class of anticancer drugs that acts, in part, through targeting downregulation of Sp transcription factors.