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Polyketide secondary metabolites of the Dominican sponge Plakortis angulospiculatus Porter, Justin M.J. 2004

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POLYKETIDE SECONDARY METABOLITES OF T H E D O M I N I C A N S P O N G E PLAKORTIS ANGULOSP/CULATUS  by JUSTIN M.J. PORTER H.B.Sc. University of Toronto, 2000 B.Ed. OISE/University of Toronto, 2001 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES Department of Chemistry We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September 2004 © Justin M.J. Porter, 2004  Library Authorization  In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  Justin Michael James Porter  21/09/2004  Name of Author (please print)  Date (dd/mm/yyyy)  Title of Thesis:  Polyketide Secondary Metabolites of the Dominican Sponge Plakortis  Degree:  Master of Science  Department of  Chemistry  The University of British Columbia Vancouver, B C Canada  Year:  angulospiculatus  2004  ABSTRACT  As part of a search for new antifungal agents, the polyketide cyclic peroxides, compounds 32 and 33, were isolatedfromthe Dominican sponge Plakortis angulospiculatus. Both compounds showed potent in vitro activity against Candida albicans. Chemical genetic profiling of the crude sponge extract and compound 33 indicated a non-specific mode of action against the yeast, Saccharomyces cerevisiae. Further exploration of the sponge extract yielded two furano polyketides, the newly discovered a,P-unsaturated ester, 37, and the previously isolated compound, glanvillic acid B (38). The structures were elucidated by one- and two-dimensional spectroscopic analysis of 32,33,37, and 38.  ii  TABLE O F CONTENTS  Page ii  Abstract Table of Contents  iii  List of Figures  v  List of Schemes  vii  List of Tables  vii  List of Abbreviations  viii  Dedication  ix  Acknow ledgements  x  CHAPTER 1  General Introduction  1.1  Natural Product Chemistry  1  1.2  Marine Invertebrates as a Source of Drugs  2  1.3  Chemical Genetics and Drug Discovery  6  1.4  Polyketide Biosynthesis: The Plakortis Polyketide  1.5  Current Study's Aim  CHAPTER 2  13 23  Antifungal Cyclic Peroxide Polyketides from Plakortis angulospiculatus  2.1  Introduction  25  2.2  Isolation  25  2.3  Structure Elucidation of 3,6-epidioxy-8-memyl-4,6,10-triethyltrideca-4-enoic acid (32)  2.4  26  Structure Elucidation of 3,6-epidioxy-8-methyl-4,6,lO-triethyltetradeca-4,11-dienoic acid (33)  36  2.5  Proposed Stereochemistry and Biosynthesis of Structures 32 and 33  45  2.6  Biological Activity  53  2.7  Conclusion  54  iii  CHAPTER 3  Furano Polyketides from Plakortis angulospiculatus  3.1  Introduction  55  3.2  Isolation  55  3.3  Structure Elucidation of methyl-6,10-diethyl-4,8-dimethyl-3,6-epoxytrideca-2,4-dienoate (37)  56  3.4  Structure Elucidation of Glanvillic Acid B (38)  69  3.5  Proposed Stereochemistry and Biosynthesis of Structures 37 and 38  3.6  Conclusion  CHAPTER 4  78 80  General Conclusions  4.1  Comparison of the Polyketide Biosynthesis for Compounds 32,33, 37, and 38....  81  4.2  Chemical Genetic Profiling of Marine Invertebrate Extracts  84  EXPERIMENTAL SECTION  General Experimental Procedures  90  Animal Material  91  Extraction of P.angulospiculatus and Isolation of Compounds 32, 33, 37, and 38 Bibliography  92 93  iv  LIST O F FIGURES  Page 7  Figure 1.3.1  Classical versus Chemical Genetics  Figure 1.3.2  Reverse-versus Forward-Chemical Genetics  8  Figure 1.3.3  Drug-Induced Haploinsufficiency Assay  10  Figure 1.3.4  Chemical Genetic Profile of Twelve Inhibitory Compounds of S. cerevisiae  12  Figure 1.4.1  Plakortis Cyclic Peroxide Polyketides  16  Figure 1.4.2  Plakortis Furan Polyketides  20  Figure 1.4.3  Plakortis Lactone, Cyclic Ether, Spiculane-Type Polyketides  22  Figure 2.3.1  n  C Spectrum (100 MHz, CDCI3) of 32  28  Figure 2.3.2  *H Spectrum (500MHz, CDC1 ) of 32  29  Figure 2.3.3  HSQC Spectrum (400 MHz, CDC1 ) of 32  30  Figure 2.3.4  COSY Spectrum (500 MHz, CDC1 ) of 32  31  Figure 2.3.5  HMBC Spectrum (500 MHz, CDC1 ) of 32  32  Figure 2.3.6  Methine HMBC Correlations for Compound 32: Fragment A  33  Figure 2.3.7  Methyl HMBC Correlations for Compound 32: Fragment A  34  Figure 2.3.8  Methyl HMBC Correlations for Compound 32: Fragment B  35  Figure 2.4.1  13  Figure 2.4.2  !  3  3  3  3  C Spectrum (100 MHz, CDC1 ) of 33  38  3  H Spectrum (500MHz, CDC1 ) of 33  39  3  Figure 2.4.3  HSQC Spectrum (400 MHz, CDC1 ) of 33  40  Figure 2.4.4  COSY Spectrum (500 MHz, CDC1 ) of 33  41  Figure 2.4.5  HMBC Spectrum (500 MHz, CDC1 ) of 33  42  Figure 2.4.6  Expanded HSQC Spectrum (400 MHz, CDC1 ) of 33  44  Figure 2.4.7  Selected COSY and HMBC Correlations of 33  45  Figure 2.5.1  Selected Coupling Constants for 32 and 33  46  Figure 2.5.2  Selected NOE Correlations of 32  47  Figure 2.5.3  Selected NOE Correlations of 33  47  Figure 2.5.4  ROESY Expanded Spectrum (400 MHz, CDC1 ) of 32  48  Figure 2.5.5  ROESY Expanded Spectrum (500 MHz, CDC1 ) of 33  49  Figure 3.3.1  3  3  3  3  3  3  13  C Spectrum (100 MHz, CDCI3) of 37  58  v 1  Figure 3.3.2  'H Spectrum (500MHz, CDC1 ) of 37  59  Figure 3.3.3  HSQC Spectrum (400 MHz, CDC1 ) of 37  60  Figure 3.3.4  COSY Spectrum (500 MHz, CDC1 ) of 37  61  Figure 3.3.5  HMBC Spectrum (500 MHz, CDC1 ) of 37  62  Figure 3.3.6  Selected HMBC Correlations for Compound 37: Fragment A  63  Figure 3.3.7  Selected HMBC Correlations for Compound 37: Fragment A  64  Figure 3.3.8  Selective Gradient 1 -D NOE (400 MHz,  65  Figure 3.3.9  Resonance structures for Fragment A (37)  3  3  3  3  CDCI3)  of 37  66  Figure 3.3.10 Methyl HMBC Correlations for Compound 37: Fragment B  67  Figure 3.3.11 Selected HMBC and COSY Correlations for Compound 37: Fragment B  68  Figure 3.3.12 Selected HMBC Correlations for Compound 37  68  Figure 3.4.1  71  13  C Spectrum (100 MHz,  CDCI3)  of 32  Figure 3.4.2  'H Spectrum (500MHz, CDC1 ) of 32  72  Figure 3.4.3  HSQC Spectrum (400 MHz,  of 32  73  Figure 3.4.4  COSY Spectrum (500 MHz, CDC1 ) of 32  74  Figure 3.4.5  HMBC Spectrum (500 MHz,  75  Figure 3.4.6  Selected HMBC Correlations for Compound 38: Fragment A  76  Figure 3.4.7  Selected HMBC Correlations for Compound 38: Fragment B  77  Figure 4.2.1  Haploid Deletion Mutant Fitness Test Profde  85  Figure 4.2.2  Scatterplot and Fitness Test Profile Expansion  3  CDCI3) 3  CDCI3)  of 32  for Clotrimazole and Fluconazole Figure 4.2.3  Scatterplot and Fitness Test Profile Expansion for Fluconazole and Camptothecin  Figure 4.2.4  86 87  Scatterplot and Fitness Test Profile Expansion for Extracts 00-132 and 00-192  89  vi  LIST OF SCHEMES  Scheme 1.4.1 Chain Extension in Polyketide Biosynthesis  Page 14  Scheme 1.4.2 Biosynthesis of Amphotericin B  15  Scheme 1.4.3 Biosynthesis of Triophamine: Butyrate Incorporation  18  Scheme 1.4.4 Proposed Biosynthesis for Monotriajaponide A: Cyclic Peroxide Precursor  19  Scheme 1.4.5 Proposed Formation of the Cyclic Peroxide Moiety  19  Scheme 1.4.6 Proposed Cyclic Peroxide to Furano Conversion  20  Scheme 1.4.7 Production of Glanvillic Acid A from a Hypothetical Precursor Scheme 2.5.1 Proposed Biosynthesis of Compounds 32 and 33  21 51  Scheme 2.5.2 Proposed Biosynthesis of the Cyclic Peroxide Moiety in Compounds 32 and 33  52  Scheme 3.5.1 Proposed Biosynthesis of Compounds 37 and 38 from Cyclic Peroxides  79  Scheme 4.1.1 Polyketide Linear Chain Representations for 32,33,37, and 38  82  Scheme 4.1.2 Proposed Biosynthetic Schemes for Compounds 32,33,37, and 38  83  LIST O F T A B L E S  Page Table 2.3.1  13  C and H NMR Data for 32  27  Table 2.4.1  13  C and'H NMR Data for 33  37  Table 2.4.2  13  C and H NMR Chemical Shift Comparison for 33  43  Table 2.6.1  Minimum Inhibitory Concentration of 32 and 33  Table 3.3.1  13  C and *H NMR Data for 37  57  Table 3.4.1  I3  C and 'H NMR Data for 38  70  ]  1  53  Table 4.2.1  Drugs in Fitness Test Profde  84  Table 4.2.2  Marine Invertebrate Extracts in Fitness Test Profde  86  vii  LIST O F ABBREVIATIONS  AcN amu br s [OC] D 23  COSY d dd 8 DNA EtOAc HEX HMBC HMQC HPLC HRESIMS HSQC m MeOH MTT NMR NOE ODS q ROESY PCR Rf s S SCUBA sp. t TLC TFA UV w  acetonitrile, CH CN atomic mass unit broad singlet specific optical rotation at wavelength of sodium D line (23°C) homonuclear correlation spectroscopy doublet double doublet chemical shift (ppm) deoxyribonucleic acid ethyl acetate, C H 3 C O O C H 2 C H 3 hexanes heteronuclear multiple bond correlation heteronuclear multiple quantum coherence high performance liquid chromatography high resolution electrospray ionization mass spectrometry heteronuclear single quantum coherence multiplet methanol, CH OH methylthiazolyldiphenyl tetrazolium bromide nuclear magnetic resonance nuclear Overhauser enhancement octadecyl silyl quartet rotating frame nuclear overhauser enhancement spectroscopy polymerase chain reaction retardation factor singlet solvent signal self-contained underwater breathing apparatus species (singular) triplet thin-layer chromatography trifluoroacetic acid ultraviolet water signal 3  3  DEDICATION  I must have gotten the chemistry bugfromthis fellow, my great-grandfather, William Alexander Brackley. He graduatedfromCentral Technical School (Toronto) as a chemist. I understand hefirstworked for a paint and varnish company before going to the T. Eaton Company. He is shown above in the quality testing labs of the T. Eaton Co. during the late 1920s. I would like to dedicate this thesis to his son, my grandfather, William James Brackley (1932-2004) and to my grandmother Margaret Porter (1919-2003). Both were very influential people in my life.  ix  ACKNOWLEDGEMENTS  I would like to acknowledge my supervisor, Dr. Raymond Andersen, for the opportunity to do this research. Also, I would like to thank him for his patience and many ideas from which I have learned a great deal. I would also like to acknowledge the following people: Helen Wright for the countless biological assays. Ainslie Parsons for her chemical genetic work out at the University of Toronto. Rob Van Soest for identifying the sponge at the core of this study. Mike LeBlanc for all the gear and stuff. Dave Williams for his invaluable insight into marine natural product chemistry. Xin-Hui Huang for his technical expertise and friendly nature. Dr. Burlinson and the NMR staff for helping to tame temperamental NMR machines. Starbucks coffee® for keeping me awake... too awake. Fellow grad students Alban, Kate, Kelsey and Roger for anytime dive buddies and for always being open to discussing new ideas. Also, Harry and Lu for their advice on a wide array of topics. Mom, Dad and, my lil'sis, Araby who have continued to support me throughout my degree and my life. And, of course, where would I be without Louise.  x  CHAPTER 1  1.1  General Introduction  Natural Product Chemistry One of the primary goals of natural product chemistry is to isolate and characterize  secondary metabolites. Secondary metabolites are small molecular weight compounds (<1500 amu) that are not essential for the development, growth, or reproduction of the producing organism. Indeed, biosynthetic pathways yielding secondary metabolites are specific to certain cell types. By comparison, primary metabolites (e.g. nucleic acids, amino acids, polysaccharides, etc.) are common to all cells and are essential for the existence of the organism. Although one might consider any chemical of biological origin a natural product, the term "natural product" usually implies a secondary metabolite.  1  Three penchants spur on the discovery and isolation of a natural product: 1) The secondary metabolite is of novel origin or structure, possibly indicating a unique biogenesis leading to a previously undescribed carbon skeleton or functional group array. 2) The secondary metabolite plays an important role in the producing organism, such as protecting the organism against predation (i.e. the study of Chemical Ecology.) 3) The secondary metabolite displays in vitro biological activity (i.e. the source of lead compounds for drug discovery.) The third penchant provides one of the strongest motivations to discover new moleculesfromnature. Roughly half of all papers published for new marine natural products contain biological activity data. Furthermore, the majority of natural product chemists use a 2  1  "bioassay-guided fractionation" scheme, in conjunction with a purification regiment and spectroscopic data, to discover new, relatively complex chemical entities that, in some cases, become drugs in human therapeutics.  1.2  Marine Invertebrates as a Source of Drugs  Approximately half of the best selling drugs in clinical use are natural products or their synthetic derivatives. Given the great chemical diversity of natural products and the 3  likelihood that many natural products have evolved to target specific proteins, they remain a rich source of lead compounds for drug development. One of the best known examples of a 4  natural product turned drug is Taxol (1, paclitaxel), a tricyclic diterpene, originally isolated from the bark of the Pacific Yew tree, Taxus brevifolia. By stabilizing microtubules during 5  cell division, paclitaxel has a mode of action against cancer cell lines that was previously unknown. Today, Taxol has become one of the best-selling drugs in the treatment of breast and ovarian cancers, reaching sales of 1.5 billion USD in 2000. From this example we can 6  see how a natural product not only has the potential to add to the scientific pool of knowledge, but how it may also bear great therapeutic and commercial rewards.  2  The marine environment, covering over 70% of the earth's surface and presenting a much larger volume of inhabitable space than terrestrial surfaces, provides a wealth of biodiversity. There are roughly 100,000 species of invertebrates in the world's oceans, with possibly more yet to be discovered. Representative invertebrate phyla including Bryozoa, Coelenterata, Porifera, and Echinodermata only exist in aquatic regions and have adapted their metabolism to survive the uniqueness of their saline environments. A great number of the organisms in these phyla lack any form of physical protection, such as shells or spines, but they do make use of biologically active secondary metabolites to defend against predation. These are the chemicals that marine natural product chemists have sought after 7  for the past three and a half decades. Such compounds isolatedfromorganisms in areas of high biodiversity represent an ideal source of new secondary metabolites with diverse chemical structures and potent biological activity. Many novel bioactive molecules have already been discoveredfrommarine invertebrates, some of which are promising drug leads. One compound, the diterpene glycoside, eleutherobin (2),fromthe Australian soft-coral Eleutherobia sp., works by a similar mode of action as Taxol, in that it is microtubule stabilizing. Eleutherobin is 100 times more potent than Taxol against a number of different cancer cell lines in vitro. Other 8  exciting new natural products with strong potential as drug leads have been discovered from the extracts of marine invertebrates. Some examples include bryostatin 1 (3), a macrolide isolated from extracts of the bryozoan, Bugula neritina, now in phase II clinical trials , and 9  discodermolide (4), an anticancer polyketidefromCaribbean sponge Discodermia dissoluta. These marine natural products, or one of their synthetic derivatives, may one day 10  represent viable candidates for medicinal use.  3  4, discodermolide  Although the majority of compounds isolated are directed towards finding anticancer agents, in part due to societal demand and funding concerns, there is a pressing need to find lead compounds for other therapeutics. With the rise in immunocompromised individuals (e.g. AIDS, cancer chemotherapy, transplant recipients, etc.) there exists a great necessity to add to the limited arsenal of current antifungal drugs. Over the past two decades there has been a 400% increase in the cases of systemic mycoses, echoed by a 10-fold increase in the frequency of candidiasis from the infection of Candida albicans. The two major classes of n  antifungal drugs, polyenes (e.g. amphotericin B) and azoles (e.g. fluconazole), are problematic in that the polyenes can be toxic and the azoles aren't always very effective.  4  Part of the difficulty in finding effective antifungal agents is that fungi are eukaryotic and possess a metabolism with similarities to mammals, thus limiting the number of viable cellular targets. Targets for antifungal agents are usually focused on the cell envelope and the fungal membrane sterol ergosterol and its biosynthesis. The search for antifungal lead 13  compounds must seek out agents which are selective to fungal targets and yet remain broadspectrum in the treatment of different fungal pathogens. Once again we see examples of marine natural products as potential lead compounds for drug discovery, specifically in the treatment of fungal infections. One antifungal compound is 15G256y (5), a cyclic lipopeptide isolatedfroma mangrove-derived fungus, Hypoxylon oceanicum. This compound appears to inhibit cell wall biosynthesis and is 14  currently being evaluated in preclinical trials for its usefulness as an antifungal drug. Another compound, spongistatin 1 (6), purifiedfromthe Eastern Indian Ocean sponge Spongia sp. was originally isolated for its strong growth inhibition of a subset of the National Cancer Institute's (NCI) 60 human cancer cell lines. Spongistatin 1, a macrocyclic lactone 15  polyether, has also displayed broad-spectrum antifungal activitiy. It is proposed that 16  spongistatin 1 works by a similar mode of action against cancer cells and fungi, in that it is an antimicrotubule agent.' Although there are many other examples of other invertebrate7  derived antifungal agents, 5 and 6 are interesting as the respective activity studies seem to indicate they act in specific ways against fungi. However, to become lead compounds for drug development, these compounds must also be selective in their cellular targeting, avoiding the toxic effects that would limit their therapeutic benefits for humans.  5  1.3  Chemical Genetics and Drug Discovery Chemical genetics employs small molecules to probe the function of proteins, thereby  allowing scientists to understand and govern the physiological and cellular function of such gene products. By binding to protein targets, small molecules either activate (e.g. nuclear hormone receptors are steroid hormone-activated transcription factors) or inactivate the proteins (e.g. colchicines inactivate tubulin) giving information about the function of the gene product. This chemical genetic approach mimics classical genetics in which 18  biochemists activate genes by mutations or inactivate genes (e.g. a deletion or "knock-out" mutant) to explore the function of a particular gene product (Figure 1.3.1). Chemical 19  genetics has the advantage over the classical genetic approach in that the small molecules act as conditional mutations, either inducing or suppressing the formation of a specific phenotype. Since the small molecules act quickly and reversibly, they act as provisional mutations that can be turned on and off, thus imparting the ability to study dynamic cellular  6  processes. By linking the phenotype, caused by a small compound, to a specific target (or not), chemical genetics purports to be of invaluable utility in the drug discovery process.  CLASSICAL GENETICS  CHEMICAL GENETICS  Figure 1.3.1 Classical versus Chemical Genetics  There are two different types of chemical genetic screens: forward and reverse (Figure 1.3.2). Reverse-chemical genetic screens test a large number of small molecules against a protein of interest until an appropriate bioactive ligand is found. The forwardchemical genetic approach takes place in three steps. In analogy to creating random genetic mutations, the first step of forward-chemical genetics involves treating the organism or cell type of interest with a varied collection of small molecules. In turn, the next step involves identifying the phenotype, or observable effect, within the collection of compound-treated cells and finally identifying the actual drug target in the screening organism. Given that 21  fewer than 5% of employable human proteins have been utilized as drug targets, forward22  chemical genetics is an advantageous means of finding new protein targets for drug discovery.  7  REVERSE  Figure 1.3.2 Reverse- versus Forward-Chemical Genetics Sharing a high degree of gene conservation with the human genome, the yeast, Saccharomyces cerevisiae, is a model organism for drug discovery using chemical genetic screens. Amongst the -6000 genes found from sequencing of the S. cerevisiae genome, there are several hundred gene products with similar sequences to proteins implicated in many human diseases. In addition, a number of drugs used against human targets, such as lovastatin, will specifically inhibit the related yeast protein. In the search for antifungal 24  8  drugs, researchers seek viable protein targetsfromgenes conserved in S. cerevisiae and other fungi, but absentfromhumans. Inherent within the ~1100 essential genes of S. cerevisiae are -350 genes that do not match sequences found in the human genome. A portion of these -350 genes are thought to be highly conserved with other fungi and are important for immediate viability (i.e. inhibiting the gene product would prove fungicidal). Hence, small 25  molecules that would target the products of this subset of the -350 genes would have the potential to be ideal antifungal compounds. Recently, a yeast haploinsufficiency profiling assay has been gaining widespread acceptance as an invaluable tool to determine the exact cellular target of a molecular inhibitor. This phenotype-based assay lowers the amount of a gene encoding a drug target by transforming a wild-type diploid cell into a heterozygous diploid cell. This effectively leaves one copy of the gene encoding the cellular target (we began with two) often leaving the heterozygote mutant hypersensitive to a normally sublethal concentration of the drug - this is called "drug-induced haploinsufficiency" (Figure 1.3.3). Molecular barcodes, unique oglionucleotides surrounded by common PCR primers, are integrated at the site of each gene deletion allowing for detection and quantification of each strain. The abundance of each strain is quantified with respect to time and the heterozygotes showing hypersensitivity to the drug are detected by a DNA microarray analysis of the PCR-amplified barcode pool. By this methodology, haploinsufficiency profiling is advantageous as it may indicate the cellular target of the drug as sensitive strains drop out of the pool. Furthermore, many of the targets for drugs are the products of essential genes, such that, by assaying strains that retain one copy of a given gene, haploinsufficiency profiling can examine all -6000 potential genes in the S. cerevisiae genome.  26  9  PHENOTYPE ^> Alive  A complementary assay to haploinsufficiency profiling uses chemical genetic and genetic interaction data to connect bioactive compounds to cellular pathways. The main difference between the two assays is the gene copy number. Whereas the haploinsufficiency assay makes use of heterozygote deletion mutants, missing one of two copies of a particular gene, the other assay uses haploid deletion mutants that are missing the one and only copy of a particular gene. This chemical genetic, haploid deletion assay can only produce strains based upon the -5000 non-essential genes of S. cerevisiae. Furthermore, this screen reveals information about the general pathways and cellular processes affected by the drug, instead of finding a specific target. To make sense of chemical genetic profiles generated from haploid deletion mutants, genetic interaction profiles are generated for the targets of known compounds by synthetic genetic array (SGA) analysis. Basically, if two genes are deleted and  10  cell death occurs, when neither deletion by itself is lethal to the organism, the cell death is referred to as a "synthetic lethal" interaction. These relationships can occur for genes within a single biochemical pathway or for genes acting in two distinct pathways. Similarly, when a drug inhibits a gene product, combined with the deletion of a different gene, a "synthetic lethal" interaction may occur. This allows for comparison between the chemical genetic profiles of compounds and genetic interaction data for the haploid deletions of 27  S. cerevisiae.  Inhibitory compounds with characterized targets are used to construct chemical genetic profiles to test if a set of haploid deletion mutants will not only show hypersensitivity to the drug, but also indicate the correct pathway in the chemical-genetic interaction. Figure 1.3.4 shows the chemical-genetic profiles for twelve inhibitory compounds of S. cerevisiae: cyclosporine A, FK506, fluconazole, suffometuron methyl, wortmannin, tunicamycin, caffeine, rapamycin, camptothecin, hydroxyurea, cycloheximide, and benomyl. The rows of the cluster plot display the individual profiles for each compound. Red bars within the rows indicate the sensitive genes, 647 different genes in total for all 12 compounds. Of interest, the compound benomyl (bottom row, Figure 1.3.4) is the only compound shown enriched for mitosis, chromosome structure, and cell structure. Benomyl showed chemical-genetic interactions with genes involved in the structure of tubulin (TUB3), the prefoldin actin/tubulin chaperone complex (GIM3, GIM4, GIM5, PAC2, PAC10, PFD1, YKE2),  tubulin folding (CIN1, CIN2, CIN4), and the mitotic spindle checkpoint (MAD1, MAD2, BUB3). These interactions are consistent with the fact that benomyl is a microtubule depolymerizing agent. Two compounds with similar profiles are cyclosporin A and FK506 (top two rows, Figure 1.3.4). Their chemical genetic interaction profiles are enriched for  11  vacuolar structure and function as well as genes involved in cell wall organization. Indeed, both compounds are calcineurin inhibitors and calcineurin plays an established role in vacuolar function and cell wall homeostasis. Based upon these drug signatures it is possible to characterize the cellular targets for varying compounds. Most importantly, such chemicalgenetic profiles give researchers an efficacious tool to find the target of new compounds, by comparing profiles or by directly characterizing the genes and pathways inhibited by the 2X  compound.  J  i"j  P  =  :  :  :  ^ E i r ^ r t f l T D Cyclosporin A FK506  I I! UJ  Fluconazole Surfometuron methyl Wortmannin Tunicamycin Caffeine Rapamyan Camptothecin Hydroxyurea  CycSoheximide  I!  Genes associated with vacuolar structure and function, and cell wall organization  I  | Benomyl  Genes associated with mitosis, chromosome structure, and cell structure  Figure 1.3.4 Chemical-Genetic Profile of Twelve Inhibitory Compounds of S. cerevisiae  12  Natural products make ideal small molecules to be used in such chemical genetic profiling. By prescreening crude extractsfrommarine based organisms in this manner, one is in effect screening hundreds of compounds not only for a desired phenotypic response, but for biological activity that acts in a specific way. This focuses the search for lead compounds in drug discovery.  29  1.4  Polyketide Biosynthesis: The Plakortis Polyketide  Polyketides are a class of secondary metabolites with a wide range of functional, structural, and biological diversity. Biological activities found for this class include antibacterial, anticancer, antifungal, antiparasitic and immunosuppressive, making for a medicinally important group of compounds. The main building block of a polyketide is acetate in the form of acetyl Coenzyme A. A collection of enzymes known as the Polyketide Synthases (PKS) act in an assembly-line fashion (Scheme 1.4.1) to construct polyketide chains of varying length in a manner similar to fatty acid biosyntheis. First, a starter unit, usually acetyl CoA, is loaded onto the keto synthase (KS) by acyltransferase (AT). Malonyl Co A, formedfromthe carboxylation of acetyl CoA, is loaded onto an acyl carrier protein (ACP), again by acyltransferase (AT), where it undergoes a carbon-carbon bond forming Claisen ester condensation with the acyl residue bound to the KS. This results in an elongated chain in the form of a f3-keto ester bound to the ACP. The chain extension is followed by several steps including reduction by keto reductase (KR), dehydration by dehydratase (DH), and reduction by enoyl reductase (ER). The cycle then repeats after transfer of the chain from the ACP to the KS. Finally the polyketide chain is transferred to a thioesterase (TE) and is cleaved off the enzyme as afreeacid or an acyl ester. Post-PKS modifications, including the  13  formation of rings and the addition of various functional groups, produces polyketides with the diverse set of structures found in nature.  30  LOAD CHAIN  Scheme  j  1.4.1 Chain Extension in Polyketide Biosynthesis  Originally isolated in 1955, the polyketide amphotericin B (8) is today the most commonly used antifungal drug in human therapeutics. An isolate of the soil bacterium Streptomyces nodosus,fromthe bed of the Orinoco river in South America, produced amphotericin B (8). Elucidation of its full structure in the 1970s by X-ray crystallography hinted at its polyketide origin. In 1997 the polyketide biogenesis of amphotericin B was supported by a Double Quantum Filtered (DQF)-HSQC experiment. Labeled precursors  14  sodium [ C2]acetate, sodium [l- C]proprionate and sodium [3- C]proprionate were fed to 13  13  13  S. nodosus . Sixteen acetates and three propionate units were incorporated into amphotericin B (8) (Scheme 1.4.2) by the PKS at the expected positions. The chain was releasedfromthe enzyme complex to give the polyene intermediate 7. Further oxidations and glycosylations result in thefinalproduct of amphotericin B (8). Of note in the biosynthesis of 31  amphotericin B is the inclusion of propionate units, leading to methyl branches in the gross structure. As will be seen momentarily, the polyketide building blocks include, not only acetate and propionate residues, but also butyrate.  8  § H  Scheme 1.4.2 Biosynthesis of Amphotericin B  15  Sponges of the family Plakinidae, specifically within the genus Plakortis, produce many novel polyketides with potent biological activities. Representative polyketide structural classes include cyclic peroxides, furans, lactones, cyclic ethers, and spiculane-type compounds. Many of these compounds are biosynthetically related and, at first glance, there appears to be a great deal of similarities between the polyketide synthases that make them. However, one must use caution in using these compounds as chemotaxonomic markers. Structures previously isolated from Plakortis sponges have been found in sponges of different genera. Time may tell whether or not the original producer of these polyketides is a sponge or a bacterial symbiont, an occurrence that does not go without precedence. Cyclic peroxide polyketides are the most prevalent structures isolatedfromPlakortis sponges. A few representative polyketides are shown in Figure 1.4.1. Plakortide M (9) and 32  haterumadioxin A (10), isolatedfromPlakortis halichondrioides and Plakortis lita, 33  respectively, represent two of the most common types of cyclic peroxide polyketides.  12  "COOH  13 Figure 1.4.1 Plakortis Cyclic Peroxide Polyketides  16  Both compounds were shown to possess potent cytotoxic activity against numerous cancer cell lines, with the authors of the plakortide M paper noting that this compound did not appear to be selective against the different cell lines. Structurally, 9 and 10 are typical of Plakortis polyketides with either a saturated or unsaturated six-membered peroxide ring and ethyl group substitutions on the ring and chain. Usually methyl or ethyl esters are serendipitously discovered with the free acid as artifacts of the extraction process. In terms of structure activity relationships, the esters appear to show weaker levels of biological activity than do thefreeacids. Compound 11fromthe sponge Plakortis simplex, contains two 1,2dioxene rings and its methyl ester had an IC50 of <0. lug/ml against P388 murine leukemia cells (the authors of this paper speculated that the unstable free acid would have had a lower IC50 had it been available for testing). Twofive-memberedcyclic peroxides plakinic acid D 34  (12) and plakortide E (13) are bothfromPlakortis sp.. Plakinic acid D was cytotoxic 35  36  against certain cancer cell lines, while no activity data was listed for plakortide E. The structures do bear similarities to the six-membered cyclic peroxides, however, one striking difference is the phenyl acetate starter unit of 13. Biosynthetic studies of Triopha catalinae were the first to show de novo polyketide biosynthesis in a dorid nudibranch and the first demonstration of biosynthesis involving butyrate units in a marine invertebrate. Skin extracts of T. catalinae produced triophamine (14), a symmetrical diacylguanidine. The existence of ethyl branches in triophamine hinted at the possibility of butyrate incorporation in a processive polyketide biosynthetic scheme. A biosynthetic feeding experiment of T. catalinae used [2,3- C2]butyrate and [2,3- C2]ethyl 13  13  malonate (Scheme 1.4.3). It was found that butyrate was incorporated via the commonly  17  accepted intermediate 2-ethylmalonate. This study was important as it showed that butyrate units are used to form ethyl branches in the polyketide biosynthesis of marine invertebrates.  Scheme 1.4.3  Biosynthesis of Triophamine: Butyrate Incorporation  Although feeding experiments, involving labeled precursors, have not been performed on sponges to demonstrate polyketide biosynthesis, one can envision a biosynthetic proposal for the cyclic peroxide polyketidesfromsponges. For example, Monotriajaponide A (15) was discovered from the sponge, Monotria japonica, along with other Plakortis-type cyclic peroxides. Monotriajaponide A (15), althoughfroma sponge of 38  a different genus, could be the precursor to a compound such as haterumadioxin A (10). Scheme 1.4.4 shows the possible polyketide biogenesis that would lead to 15.  18  Scheme 1.4.4 Proposed Biosynthesis for Monotriajaponide A: Cyclic Peroxide Precursor  Monotriajaponide A (15) could then be converted to a radical by abstraction of a hydrogen by triplet oxygen as shown in Scheme 1.4.5. This would form a resonance stabilized radical intermediate. An enzymatic intramolecular Michael type addition would form the six-membered peroxide similar to the mechanism described for norterpene cyclic peroxides, thus yielding haterumadioxin A (10). 39  H  COOH  Scheme 1.4.5 Proposed Formation of the Cyclic Peroxide Moiety  19  A smaller, but related class of compounds are the furano polyketides from Plakortis sponges (Figure 1.4.2). These compounds are most likely biosynthetically derived from the cyclic peroxides. Two representative examples are glanvillic acid A (16) and the methyl 40  ester (17). Both were isolatedfromPlakortis halichondrioides and no in vitro biological 41  activity was reported for either compound.  H3000C  Figure 1.4.2 Plakortis Furan Polyketides As just mentioned, it is believed that furano-type polyketides are derivedfromthe cyclic peroxides (Scheme 1.4.6). Abstraction of an a-protonfromthe cyclic peroxide 18 opens the ring and forms an epoxide intermediate. Intramolecular attack on the epoxide would form the furano type ring system shown for 19. Loss of H2O would produce the final conjugated structure of the furano polyketide, 20.  42  Scheme 1.4.6 Proposed Cyclic Peroxide to Furano Conversion 20  Furthermore, the true furans, the glanvillic acids are thought to be created via a protoninitiated reaction (Scheme 1.4.7). However, to this point intime,there have been no reports in the literature of such a precursor as 21. In most cases, analogs of the hypothetical precursor 21 would be a methyl ester and have ethyl or methyl substitution at carbon six, instead of an abstractable proton.  .COOH  Scheme 1.4.7 Production of Glanvillic Acid A from a Hypothetical Precursor  In brief, other polyketides from Plakortis sponges include lactones, cyclic ethers and spiculane-type structures (Figure 1.4.3). Plakortone D (22) was discoveredfromthe Carribean sponge, Plakortis halichondrioides. Compound 22 was shown to be an activator of cardiac SR-Ca -pumping ATPase. The lactone (23), y-lactone (24), 8-lactone (25) were 2+  43  41  44  also discoveredfromPlakortis halichondrioides. The 1,2-dioxane peroxylactone, plakortolide H (26), was isolatedfromPlakortis simplex. Only one article reports finding 4S  cyclic ethers, as shown by plakortethers C, E, F (27,28,29)fromPlakortis simplex. These compounds are, once again, derivedfromcyclic peroxides and plakortether E (28) exhibits selective cytotoxicity against the RAW 264-7 cell line (murine macrophage). A specimen 46  of Plakortis lita yielded homo-plakotenin (30) which was found to reduce the proliferation of rheumatoid synovial fibroblasts. Spiculoic Acid A (31) was discoveredfroma sample of 47  the Caribbean sponge Plakortis angulospiculatus. Its central ring system is proposed to be  21  formed by a Diels Alderase catalyzed intramolecular [4+2] cycloaddition reaction. Furthermore, 31 displayed in vitro cytotoxicity against human breast cancer MCF-7 cells.  30  Figure 1.4.3  31  Plakortis Lactone, Cyclic Ether, Spiculane-Type Polyketides  22  As can be seen from the preceding examples, sponges of the genus Plakortis have evolved to produce a varied selection of polyketide structures. Possibly Plakortis sponges make these compounds as chemical defenses to discourage predation in the wild. As a result, Plakortis polyketides not only display a wealth of structural diversity, but, also, demonstrate a wide-range of potent in vitro bioactivity that has prompted many scientists to study them.  1.5  Current Study's Aim The main goal of this study was to isolate novel antifungal agents. To accomplish  this, a total of 64 marine invertebrate and 24 bacterial extracts were screened against Candida albicans for antifungal activity. The extract with the most potent antifungal activity, a Dominican sponge (collection number 00-303) was chosen as an isolation project.  23  In collaboration with Dr. Charlie Boone, a sample of the crude extract (00-303) was sent to his lab in the Banting and Best Department of Medical Research, University of Toronto. Our intent was to send other fractions throughout the purification process for Dr. Boone's labtotest against their S. cerevisiae haploid and heterozygote deletion mutant arrays. It was hoped that by using this chemical genetic screen the biological target of the active metabolite would be revealed. In addition, by sending samples of increasing purity throughout the isolation, we wished to assess the viability of screening crude extracts by the S. cerevisiae deletion mutant arrays. Such screens would allow our group to focus future research on discovering antifungal agents that target specific proteins of interest. The results of this study are discussed in the following chapters.  24  CHAPTER 2  Antifungal Cyclic Peroxide Polyketides from Plakortis angulospiculatus  2.1 Introduction  The most prolific class of biologically active compoundsfromPlakortis sp. is the cyclic peroxides. Herein, the isolation and structural elucidation is described for two cyclic peroxides, 32 and 33. Compound 32 has not been previously isolated, however, the related cyclic peroxide 33, differing by one degree of unsaturation and an extra carbon in the chain, was previously reported. The absolute configuration of both compounds is suggested as it has been deduced via spectroscopic data and a survey of the literature. Both compounds were pursued for their potent activity against the yeast, Candida albicans.  2.2 Isolation  A portion of the collected Plakortis angulospiculatus (50 g, wet weight) was immersed in and extracted twice with MeOH. The methanol extract was concentrated and reduced to dryness in vacuo, leaving a brown gum (1.76 g) that was partitioned between EtOAc (5 x 100 mL) and H 0 (200 mL.) The EtOAc layer was dried over MgS0 , filtered, 2  4  and reduced to dryness in vacuo (yellow-brown oil, 366 mg). Alamar Blue® assays against Candida albicans placed the activity in the organic layer residue (6.3 (Xg/mL) and no activity at 100u\g/mL in the aqueous layer residue. Bioautographic assay of the organic layer residue  25  displayed a zone of inhibition corresponding to an Rf of 0.21 (10 pg, 1:1 EtOAc/HEX). Consequently, the EtOAc residue was chromatographed on Sephadex LH-20®(4:1 MeOH/CbbCbJ. Active fractions displaying the characteristic Rf were pooled (yellow oil, 156 mg, 3.3 p-g/mL against C.albicans) and further separated by normal phase silica gradient flash column chromatography (7:93 EtOAC/CH Cl2 -» EtOAc). Final purification of the 2  pooled active fractions (31 mg) by reversed-phase HPLC (3:2 AcN/H 0, 0.1% TFA) yielded 2  two pure compounds 32 (6 mg) and 33 (6 mg) as clear oils.  2.3 Structure Elucidation of 3,6-epidioxy-8-methyl-4,6,10-triethyltrideca-4-enoic acid (32)  Compound 32 was isolated as an optically active, clear oil, that gave a positive mode HRES1MS [M+Na] peak at m/z 363.2505 appropriate for a molecular formula of C20H36O4 +  (calc'd 363.2511 for C2oH 6C>4Na). Accordingly, 32 had three degrees of unsaturation. The 3  13  C NMR (Figure 2.3.1), 'H NMR (Figure 2.3.2), and HSQC (Figure 2.3.3) spectra  revealedfivemethyl carbons, eight methylene carbons, three methine carbons (8c 26.0, 36.1, 76.2), one quarternary carbon (8c 83.8), two olefinic carbons (8c 125.3,137.5) and one carbonyl carbon (8c 175.2). Two fragments, a peroxide ring and a carbon chain, were constructed and connected based upon COSY (Figure 2.3.4) and HMBC (Figure 2.3.5) spectra to provide the gross structure of 32. A detailed summary of the 1- and 2-D NMR spectroscopic data is listed in Table 2.3.1.  26  Table 2.3.1  c#  13  13  C and *H NMR Data for 32  C 5, ppm  a  *H 5, ppm (Int., m, J(Hz))  COSY  HMBC  b  H2, H2\ H3 175.2 1 H3 2 36.9 2.93 (1, dd, 16.2, 9.6) H2\ H3 2' 2.61 (l,dd, 16.3, 2.8) H2, H3 H2, H2', H5, H14 4.57 (l,br d,8.3) H2, H2', H5 76.2 3 H2, H3,H5,H7,H14,H15 4 137.5 H3, H7, H14, H16,H16' H3, H14 125.3 5.49(l,br,s) 5 H5,H7, H8,H16, H16', H17 6 83.8 H5,H8,H9',H16,H16',H18 1.40 (2,m) H8 7 42.9 1.52 (l,m) H7, H9',H18 H7,H18 26.0 8 H8, H9\ H10 H7,H18 1.08 (l,m) 9 43.3 1.00 (l,m) H8, H9 9' 1.23 (l,m) H9 H8, H9, H9', H20 10 36.1 35.4 1.15 (2, m) H9,H9',H13 11 1.23 (2, m) HI 3 H10, H11,H13 12 19.5 H12 H11,H12 0.86 (3, t, 7.3) 13 14.6 2.01 (2, m) H5,H15 H5,H15 14 24.9 H14 H14 1.07 (3, t, 7.4) 15 11.6 H7,H17 31.0 1.75 (l,dq, 14.1,7.5) H16',H17 16 1.63 (l,dq, 14.1,7.4) H16, H17 16' 17 8.2 0.88 (3, t, 7.5) HI 6, H16' H16, H16' H8 H7, H8, H9 22.4 0.87 (3, d, 6.7) 18 H9, H9',H10, H11,H20 26.4 1.23 (2, m) H20 19 H19 H19 10.8 0.81 (3, t, 7.0) 20 "Recorded at 100 MHz, calibrated to the CDC1 C 8 of 77.0 ppm Recorded at 500 MHz, calibrated to the CDC1 *H 5 of 7.24 ppm 13  3  3  27  28  29  17 15 20 13 —  10  12 1819 8 14 =  20 30  16  2 10  11  40  97  50 1—60 70 1—80 90 100 110 120 130 — 140 — 150 — 160 170 tppm (tl) I |  I  I II |  I  5.50 5.00 ppm (t2)  I I I | I I  4.50  Figure 2.3.3  I  I | I I I I | I  4.00  3.50  I  I I | I I I I | I I I I |  3.00  2.50  I  2.00  I I I | I I I I | I II  1.50  1.00  HSQC Spectrum (400 MHz, CDC1 ) of 32 3  30  17 18 1.00  19 12 10 1.50  -2.00  h-2.50  h-3.00  -3.50  -4.00  -4.50  [-5.00  — 5.50 _ppm (tl) innrpnn^r|nT^rxjT"rn r"nrn p _  5.50  5.00  4.50  4.00  _  3.50  rrrpm 3.00  pnnn~p~rrT~]  2.50  2.00  i i i r y n r r  1.50  1.00  ppm (t2)  Figure 2.3.5 COSY Spectrum (500 MHz, CDC1 ) of 32 3  31  10 20  30  !—40 50  h-  60  -70  -80  -90  100  -110  M20  —130 r  140  — 150  -160  ^ 170  -PPm (tl) T T ' n " y T r n ^ n ~ n - p r n n ~ i ~ ] ~ r r n _  |  5.50 5.00 ppm (t2)  4.50  4.00  3.50  | i i i i | i M  3.00  2.50  i j i i M  2.00  prrrTyi i i i  1.50  1.00  Figure 2.3.5 HMBC Spectrum (500 MHz, CDC1 ) of 32 3  32  One carbonyl carbon and two olefmic carbons accounted for 2 out of 3 degrees of unsaturation. Based upon the molecular formula, the lack of any other olefmic or carbonyl carbons, and the unaccounted for degree of unsaturation, compound 32 had to contain a ring. Two characteristic hydrogen signals, the olefmic proton at SH 5.49 (H5) and the methinic proton at 5 4.57 (H3), gave HMBC correlations to Cl through C7 allowing for the H  assignment of the unsaturated peroxide ring, Fragment A (Figure 2.3.6).  HMBC Correlation  Figure 2.3.6 Methine HMBC Correlations for Compound 32: Fragment A  A pair of two-bond correlations from 5H 5.49 (H5) and 5H 4.57 (H3) to the olefmic carbon at 8c 137.5 (C4), established the C3/C4/C5 connectivity and positioned the double bond between C4 and C5 (8 125.3.) Further HMBC correlationsfrom8H 4.57 (H3) to the C  carbon at 8c 36.9 (C2) and the carboxylic carbon, 8c 175.2 (Cl), established the connectivity C1/C2/C3. The C3/C2 bond was confirmed by COSY correlations between H3 and H2/2'. As one would expect, the geminally coupled a-methylene protons, H2/2', were deshielded and appear downfield at 8 2.61 and SH 2.93. The signal at 8 5.49 (H5) also showed HMBC H  H  correlations to 8c 83.8 (C6) and 8c 42.9 (C7). The more deshielded quarternary carbon, C6 (8 83.8), was appropriate for an oxygen bearing carbon directly attached to the olefmic C  carbon at 8c 125.3 (C5). The lack of an HMBC correlation between H3 and C6 is consistent  33  with a peroxide linkage forming a six-membered ring. Both C3 (8 76.2) and C6 (8 83.8) C  C  possess chemical shifts appropriate for oxygen bearing carbons. Based upon the molecular formula and the chemical shift data, it is suggested that Fragment A is an unsaturated 6membered peroxide ring. Two triplet *H NMR signals at 8 0.88 (HI7) and SH 1.07 (HI 5) were evident in the H  one-dimensional spectrum. HMBC correlationsfromthe two methyls established the connectivity of C17/C16 and C15/C14, allowing for the placement of two ethyl branch substituents on theringat C6 (8 83.8) and C4 (8 137.5 ) (Figure 2.3.7), respectively. C  C  17  COOH  •or  Figure 2.3.7 Methyl HMBC Correlations for Compound 32: Fragment A  The key to constructing Fragment B resided in the strong HMBC correlations between the three remaining methyl group resonances and the chain carbons (Figure 2.3.8). Two triplet signals (8 0.81,0.86) and one doublet (8 0.87) in the *H NMR spectrum H  H  indicated chain branching as two ethyls and one methyl, respectively. Two of the branching points were located at the remaining methine carbons 8 26.0 (C8) and 8 36.1 (C10). HMBC C  C  correlations showed the connectivity to be C8/C18, for the methyl branch, and C10/C19/C20, for the ethyl branch.  34  20  HMBC Correlation  Figure 2.3.8 Methyl HMBC Correlations for Compound 32: Fragment B  The final ethyl group (8H0.86, HI3) correlated into a methylene carbon (8c 35.4, Cl 1) to form the fragment Cl 1/C12/C13. This fragment was found to be the terminus of the main carbon chain backbone. As the protons of the C8/C18 fragment had three-bond HMBC correlations into C7 (8 42.9) and C9 (8 43.3), C8/C18 was connected through C7 directly C  C  to the ring moiety. Thus, the C10/C19/C20 fragment was central to the chain and attached to the Cl 1/C12/C13 terminus. COSY correlations supported the assignment of Fragment B. The signal at 8H 1.52 (H8) was found to have three key COSY signals to 8 1.40 (H7), 8 1.00 (H9') and 8H 0.87 H  H  (HI8.) Also, S 1.08 (H9) correlated into the methinic proton at 8 1.23 (H10), further H  H  extending the chain. The placement of the two methylene carbons, Cl 1 and C12, was confirmed by a COSY correlation between HI2 (8H 1.23) and HI 3 (8R 0.86), but not HI 1 (8H1.15) and HI 3 (8 0.86). H  All assignments were consistent with unsaturated cyclic peroxides possessing polyketide chains similar to those that have been reported in the literature.  35  2.4 Structure Elucidation of 3,6-epidioxy-8-methyl-4,6,10-triethyltetradeca-4,l 1-dienoic acid (33)  Compound 33 was isolated as an optically active, clear oil, that gave an HRESIMS [M+Na] peak at m/z 375.2502 appropriate for a molecular formula of C 2 1 H 3 6 O 4 (calc'd +  375.2511 for C^iH^C^Na). Accordingly, 33 had four degrees of unsaturation. The C NMR 13  (Figure 2.4.1), 'H NMR (Figure 2.4.2), and HSQC (Figure 2.4.3) spectra revealed five  methyl carbons, seven methylene carbons, three methine carbons (8c 25.9,42.3, 76.3), one quarternary carbon (8c 83.7), four olefinic carbons (8c 125.3, 132.2, 133.2,137.2) and one carbonyl carbon (8 175.4). The C NMR and 'H NMR spectra for 32 and 33 were similar, 13  C  the major difference being two extra olefinic signals in both the C NMR (8c 132.2,133.2) 13  and the *H NMR (8H 4.95, 5.36) spectra of 33. This accounted for the extra degree of unsaturation of 33 in comparison to the related structure, 32. A detailed analysis of COSY (Figure 2.4.4) and HMBC (Figure 2.4.5) spectroscopic data (Table 2.4.1) afforded the  assignment of the gross structure, 33. Comparison of NMR chemical shift values reported in the literature (Table 2.4.2) supported this structure with the exception of two inappropriately assigned carbons (C9 and C20).  36  Table 2.4.1  c# 1 2 2' 3 4 5 6 7  13  13  C and H NMR Data for 33 !  C 8, ppm  a  H 8, ppm (Int., m, J(Hz)) r  COSY  HMBC  b  175.4 36.8 76.3 137.2 125.3 83.7 43.3  2.92 (l,dd, 16.3, 9.6) H2\ H3 2.58 (l,dd, 16.3, 2.8) H2, H3 H2, H2' 4.55(1, brd, 8.5) 5.49(1, brs) 1.40 (2,m)  H8  H2, H2' H3 H2, H5,H15 H2, H5,H15,H16 H7,H15,H17 H5,H7, H8, HI7, HI 7', HI8 H5,H8, H9,H9',H17,H17\ H19 H7, H9, H9'H10,H19 H7, H8,H10,H19, H20  H7, H9',H19 1.52 (l,m) 25.9 8 H9',H10 1.19 (l,m) 9 44.5 1.05 (l,m) H8, H9 9' H11,H9 H9, H9',H11,H12,H20, H21 1.81 (l,m) 10 42.3 H9, H9', H10, HI3, H20, H20' 11 133.2 4.95(1, dd, 15.3, 9.2) H10, H12 H10,H13,H14 5.36(1, dt, 15.2, 6.4) H11,H13 12 132.2 1.97 (2,m) H12,H14 H11,H12,H14 25.7 13 H12,H13 0.94 (3, t, 7.5) H13 14 14.3 H5,H16 1.99 (2,m) H16 24.9 15 1.07 (3,t, 7.4) HI 5 16 11.6 HI 5 1.73 (l,dq, 14.1,7.5) H17',H18 H7,H18 30.9 17 1.62 (l,dq, 14.1,7.5) H17,H18 17' 0.87 (3, t, 7.5) H17,H17' H17,H17' 18 8.1 H8 H7, H8, H9, H9' 0.83 (3, d, 6.5) 19 21.3 H9, H9',H10, H11,H21 1.28 (l,m) H20', H21 28.9 20 H20, H21 1.14 (l,m) 20' H10,H20,H20' H20, H20' 21 11.7 0.80 (3, t, 7.4) Recorded at 100 MHz, calibrated to the CDC1 C 8 of 77.0 ppm 13  3  Recorded at 500 MHz, calibrated to the CDC1 'H 8 of 7.24 ppm 3  37  38  39  16 14 9* 18 19 21 13 15  7 20' 17  8  20  I  18  10  16 21  14 20  19 8 13  15. 17  97  20  30  40  10  h-50  -60  h - 70  80  90  — 100  ^-110  — 120  130 12  11 140  — 150  — 160  ' - 170 ippm (tl) I I | I I I I | I I I I j I I I I | I I I I | I I I I | I I I I | I I II  5.50 5.00 ppm (12)  4.50  Figure 2.4.3  4.00  3.50  3.00  2.50  | I I II  2.00  | I I I I | I I I I  1.50  1.00  HSQC Spectrum (400 MHz, CDC1 ) of 33 3  40  Figure 2.4.4 COSY Spectrum (500 MHz, CDC1 ) of 33 3  41  — 150  ^-160  ^-170  *  | fppm (tl)  *  I I | I I I I | I I I I | I I I I | I I I I | I I I I | I I I I | I I I I | I I I I | I II  5.50 ppm (t2)  5.00  4.50  4.00  3.50  3.00  2.50  2.00  1.50  I | I I I I | I  1.00  Figure 2.4.5 HMBC Spectrum (500 MHz, CDC1 ) of 33 3  42  13  1  The cyclic peroxide moieties of both 32 and 33 are identical. The C and HNMR ,J  chemical shifts for both rings were nearly identical. COSY and HMBC correlations confirmed the nature of the ring. However, the carbon chain connected to the ring of 33 was found to be extended by one carbon and contain a double bond. The double bond was placed between Cl 1 (5 133.2, 6H 4.95) and C12 (Sc 132.2, 5H 5.36). Coupling constants of 15 Hz C  for both protons indicated an E stereochemistry across the double bond. Structural assignments for 33 were consistent with, but not in complete concurrence with the values reported in the literature (Table 2.4.2).  Table 2.4.2  13  C and 'H NMR Chemical Shift Comparisons for 33  c# 1 2 2' 3 4 5 6 7 8  1111 Ell  ;  10 11 12 13 14 15 16 17 17' 18 19 20 " 20' 21  Literature Values  Found Values  C5, ppm 175.4 36.8 ,3  76.3 137.2 125.3 83.7 43.3 25.9 44 •> 42.3 133.2 132.3 25.7 14.3 24.9 11.6 30.9 8.1 21.3 2S «  11.7  a  1  H 8, ppm  2.92 2.58 4.55 5.49 1.40 1.52  C5, ppm 176.6 35.3 13  76.5 137.5 124.9 83.6 43.3 25.9 2S < > •  1.81  4.95 5.36 1.97 0.94 1.99 1.07 1.73 1.62 0.87 0.83  BflBflNHMI 0.80  42.3 133.2 132.1 25.7 14.2 24.8 11.7 30.9 8.1 21.3  HBB 11.6  a  'H S, ppm 2.86 2.53 4.55 5.46 1.38 1.50  HHHPBHHBNR 1.80 4.95 5.35 1.98 0.94 1.98 1.06 1.69 1.58 0.86 0.83  •HBHBHHHI 0.79  43  As found in the current study, Gunasekera et al. who were the first to isolate 33, have switched the values of C9 and C20. In addition, the chemical shift values of H9 (5H 1.19) 49  and H9' (5H 1.05) do not correspond to those chemical shift values reported in the literature (5H 1.39 and 1.15, respectively). Also, the chemical shift of H20 and H20' do match the chemical shift values found in the current study. In the expanded HSQC spectrum (Figure 2.4.6) it is evident that the protons with 8 1.19 (H9) and 5H 1.05 (H9') are attached to the H  carbon with a chemical shift of §c 44.5 (C9). Again, in the expanded HSQC the protons with  "i—i—|—i—i—r  2.00 ppm (t2)  1.75  ~\—i—r  i—i—r 1.50  1.25  Figure 2.4.6 Expanded HSQC Spectrum (400 MHz, CDCfe) of 33  44  8H 1.28 (HBO) and 5H 1.14 (H20') are clearly bonded to the carbon with a chemical shift of 28.9 ppm (C20). Furthermore, the assignments in the current study correspond to the integration of the peaks in the H NMR spectrum. It is impossible to have H9 with a chemical ]  shift of 8H 1.39 as there is an isolated multiplet at SH 1.40 which integrates to only two protons - not three. Inspection of the H NMR spectrum of 33 from Gunasekera et al. we see l  a similar integration pattern. Figure 2.4.7 displays the observed H20/H21, H8/H9 COSY 50  correlations, and the H19/C9, H7/C9 HMBC correlations that are impossible if C9 and C20 are switched. 21  HMBC Correlation ™™ COSY Correlation Figure 2.4.7  Selected COSY and HMBC Correlations of 33  Finally, the trend in the carbon chemical shifts found in 32 and the assignments of other Plakortis polyketides with similar chains reported in the literature are consistent with the current analysis of 33.  2.5 Proposed Stereochemistry and Biosynthesis of Structures 32 and 33  It is proposed here that the absolute configuration of both 32 and 33 is (3S,6R,8S,10S) and (3S,6R,8S,10R), respectively. This stereochemical assignment is largely based upon the study by Gang Yao and Kosta Steilou in which they synthesized two methyl ester diastereomers of 33 (34 and 35). Based upon similarities in the C and H NMR data 51  13  !  45  from the methyl ester natural product derivative (36), isolated by Gunasekera and coworkers, Yao and Steilou proposed that thefreeacid, identical to 33, would have the same absolute configuration as the methyl ester, 36.  35 (3S,6R,8R,10R)  34 (3R,6S,8R,10R)  36 (3S,6R,8S,10R)  Experimental data supports the suggestion that 32 and 33 have the same relative stereochemistry as 36. As shown in Figure 2.5.1, the coupling constants between H2/H2' and H3 are the same for both 32 and 33. Based upon these couplings there is a preferred staggered conformation across the C2/C3 bond. H2 couples to H3 with a constant of 9.6 Hz making these two protons anti to each other. H2' and H3 are gauche with a coupling constant of 2.8 Hz. This is consistent with the Karplus curve. COOH  ^ ^ Coupling Constant x  Figure 2.5.1  Selected Coupling Constants for 32 and 33 46  Given the preferred conformation of C2/C3, the relative stereochemistry at positions 3 and 6 of the ring is supported by nuclear Overhauser correlations for both compounds 32 and 33. Figure 2.5.2 shows the NOE correlations from the ROESY spectrum (Figure 2.5.4) of compound 32. Of note are the correlations of H2 to H8 and HI 8 on the top-side of the ring. Furthermore, H3 and HI 7 correlate indicating that this ethyl branch and the methine proton both exist on the bottom side of the ring. COOH  ^  Figure 2.5.2  N O E Correlation  Selected NOE Correlations of 32  A similar pattern is repeated in the ROESY spectrum of 33 (Figure 2.5.5). However, here there are only nuclear overhauser correlations on the top-side of the ring (Figure 2.5.3). These correlations are found between H2 and H7, H8, and HI9. COOH  N O E Correlation  Figure 2.5.3  Selected NOE Correlations of 33  47  —1.00  — 1.25  —1.50  — 1.75  — 2.00  — 2.25  — 2.50  — 2.75  — 3.00 _ppm (fl) Ii|I  I  I I|I I I I|I I  5.50 5.25 ppm (fi)  Figure 2.5.4  5.00  I I|1  4.75  I I I|I  I I I|I  4.50  4.25  I I I|II  4.00  I  I |  3.75  I I  I  3.50  I  | I  I  3.25  il | M  3.00  I I | I I  2.75  I  I | I I II  | I  2.50  ROESY Expanded Spectrum (400 MHz, CDC1 ) of 32 3  48  I I I  I  I I I I  I  5.50 5.25 ppm (t2)  I I I I  5.00  Figure 2.5.5  I  I I I I  4.75  II I  I I  4.50  I II 4.25  I I  I  I I I I  4.00  I II  3.75  II  I  3.50  I I I I  I  3.25  I I I I  I !  I I I  I  I I I I  I  I I I I  3.00 2.75  ROESY Expanded Spectrum (500 MHz, CDC1 ) of 33 3  49  I  Given the preferred conformation across C2/C3 and the NOE correlations of both 32 and 33, the relative stereochemistry of the ring moiety is (3S,6R)*. Furthermore both compounds had negative optical rotations (32: 33:  [OC] D 23  [OC] D 23  = -37.8° (c 0.26,  CHCI3);  = -30.7° (c 0.25, CHCI3)) and it has been noted for such compounds that the sign  of the optical rotation tends to be governed by the peroxide ring moiety. It is then quite possible that theringsin 32 and 33 have the same absolute configuration as that of 36 ([cx] = -89° (c 0.1, CHC1 )). That is, both 32 and 33 have an absolute configuration of D  3  (3S,6R) for theringmoiety. As it was already proposed, 33 has the same absolute configuration as that of the methyl ester 36. It is very likely that 32 has the same spatial orientation as 33 since they were isolatedfromthe same sponge and the chain branching is identical. One can envision a similar biosynthetic origin for 32 and 33, employing a related polyketide synthase to construct both compounds. The difference in both pathways would occur during the initial step, with the two pathways converging, thereafter. Biosynthetically, that is, 32 would most likely have a propionate starter unit and 33 would begin with either an intact butyrate starter unit or with the combination of two acetates (Scheme 2.5.1). The remainder of both compounds would be constructedfromthree butyrates, one propionate, and an acetate residue with the stereochemistry determined enzymatically. Furthermore, the methyl and ethyl substituents at positions 8* and 10*, respectively, would remain syn to one another in both compounds. This would result in 33 having an absolute configuration of (8S,10R) in the chain and compound 32 existing as (8S,10S), as the assignment of priority groups about C10 is reversed without the double bond. Thefinalstep in the biosynthesis of 32 and 33 would  50  Compound 33  COCH  Scheme 2.5.1  Proposed Biosynthesis of Compounds 32 and 33  involve the enzymatic addition of oxygen and cyclization, possibly via an intramolecular Michael addition of the peroxide (Scheme 2.5.2). r'P.—8*  Scheme 2.5.2  Proposed Biosynthesis of the Cyclic Peroxide Moiety in Compounds 32 and 33  The absolute stereochemistry of 33 is proposed to be (3S,6R,8S,10R) based upon the aforementioned synthetic study by Yao and Steilou. It is tentatively proposed here that the absolute stereochemistry of 32 is most likely (3S,6R,8S,10S) as it appears to share a similar biosynthetic origin with 33. Mosher's empirical method could be used to confirm the absolute configuration of 32 and 33, however, this would be dependent upon the relative configuration determined in the previously mentioned synthetic study. Therefore, the two cyclic peroxide polyketides are proposed as: (3S,6R,8S,10S)-3,6-epidioxy-8-methyl-4,6,10triethyltrideca-4-enoic acid (32) and  (3S,6R,8S,10R)-3,6-epidioxy-8-methyl-4,6,10-  triethyltetradeca-4,11 -dienoic acid (33).  52  2.6 Biological Activity  The minimum inhibitory concentrations (MIC) for compounds 32 and 33 (Table 2.6.1) were established by an Alamar Blue® microdilution assay. Both compounds showed potent activity against Candida albicans and, to a lesser extent, activity against various bacterial pathogens. Interestingly, compounds 32 and 33 have the greatest activity against yeast (C. albicans), gram-positive bacteria (MRSA, VRE), and gram-negative bacteria (E.coli, P.cepacia), in that order. In addition, compound 32 was found to be consistently more active than compound 33. Possibly, compound 32 is more soluble than 33 in aqueous environments, increasing the availability of 32 to the pathogens in the broth assays. Otherwise, the functionalized end of both molecules, the cyclic peroxide moiety, is identical and cannot account for the difference in activities.  Table 2.6.1  Minimum Inhibitory Concentration of 32 and 33  Pathogen  M I C of Compound 32  M I C of Compound 33  (lig/mL)  (ng/mL)  Candida albicans  0.8  Methicillin Resistant  25  5Q  1.6  Vancomycin Resistant  25  100  Enterococcus Pseudomonas cepacia Escherichia coli  100 > 100  100 > 100  Staphylococcus aureus  Yeast deletion mutant profiling against the crude extract of Plakortis angulospiculatus and compound 33 yielded no clear targets or affected pathways. Both the  53  profiles appeared similar and only a few strains were identified as sensitive. The strains that were identified as sensitive are strains that often show general, non-specific drug sensitivity.  2.7 Conclusion Cyclic peroxide polyketides from Plakortis sponges constitute the majority of compounds isolated from such organisms and are usually found to be saturated within the ring moiety. Indeed, cyclic peroxides, such as compounds 32 and 33, with an extra degree of unsaturation in the peroxide ring, form a smaller subset of the body of compounds discovered from Plakortis sponges. Compound 32 was new, in that, its carbon chain had one less carbon than a related compound cited in the literature. Both 32 and 33 showed potent activity against Candida albicans. However, upon further study, compound 33 did not appear to have a particularly unique or interesting fungal cellular target. It may be possible that most, if not all, Plakortis-type cyclic peroxides have general modes of action against fungi that would negate their usefulness as lead compounds for drug discovery.  54  CHAPTER 3  Furano Polyketides from Plakortis angulospiculatus  3.1 Introduction In addition to the search for the biologically active natural products, this research project was concerned with chemical prospecting for new secondary metabolites. By chemical prospecting one searches for new and novel molecular structures based upon interesting NMR signatures. Such an approach to isolating natural products has the potential to yield unprecedented carbon skeletons that may be overlooked in a particular bioassayguided fractionation scheme. In such a manner, by U NMR data, two furano polyketides l  have been isolated: the oc,(3-unsaturated furano ester, 37, and the previously discovered furan polyketide, glanvillic acid B (38). Compound 37 is thefirstexample of a Plakortis type furano ester with a methyl branch attached to the ring at position 4. Glanvillic acid B, 38, was isolated in itsfreeacid form for the first time and spectroscopic data is reported accordingly.  37  COOCH  3  38  3.2 Isolation A portion of the collected sponge Plakortis angulospiculatus (50 g, wet weight) was immersed in and extracted twice with MeOH. The methanol extract was concentrated and reduced to dryness in vacuo, leaving a brown gum (1.76 g) that was partitioned between  55  EtOAc (5 x 100 mL) and H 0 (200 mL). The EtOAc layer was dried over MgS0 , filtered, 2  4  and reduced to dryness in vacuo (yellow-brown oil, 366 mg). Subsequently, the EtOAc residue was chromatographed on Sephadex LH-20®(4:1 MeOH/CHhCb). One fraction (pale yellow oil, 33 mg) displaying a characteristic H NMR signal of 6.21 ppm (in CDCU) was !  chromatographed on a gradient normal phase silica flash column (95:5 HEX/EtOAc — > 100%EtOAc). Fractions containing the aforementioned NMR signal were pooled (9.5 mg) and purified by reversed-phase HPLC (78:22 MeOH/H 0, 0.1% TFA) yielding one pure 2  compound, 37 (colourless oil, 1.2 mg). A separate LH-20 fraction (pale yellow oil, 26 mg) was subjected to a normal phase silica flash column (95:5 HEX/EtOAc — > 50:50 HEX/EtOAc). One fraction (clear oil, 4.5 mg) containing a characteristic *H NMR signal of 5.85 ppm (in CDC1 ) was purified by reverse-phased HPLC (3AcN:2H 0, 0.1 % TFA) to 3  2  yield one pure compound, 38 (clear oil, 0.5 mg). 3.3 Structure Elucidation of methyl-6,10-diethyl-4,8-dimethyl-3,6-epoxytrideca-2,4-dienoate (37)  37  C00CH3  Compound 37 was isolated as an optically active, clear oil, that gave a positive mode HRESIMS [M+Na] peak at m/z 345.2415 appropriate for a molecular formula of CMH34O3 +  (calc'd 345.2406 for C2oH 40 Na). Accordingly, 37 had four degrees of unsaturation. The 3  13  3  C NMR (Figure 3.3.1), H NMR (Figure 3.3.2), and HSQC (Figure3.3.3) spectra revealed l  six methyl carbons, six methylene carbons, two methine carbons (5c 26.1, 36.0), one  56  quarternary carbon (8c 98.1), four olefinic carbons (8c 84.0,133.8,141.6, 172.3) and one carbonyl carbon (8c 166.9). Two fragments, a ring and a carbon chain, were constructed and connected based upon COSY (Figure 3.3.4) and HMBC (Figure 3.3.5) spectra to provide the gross structure of 37. A detailed summary of the 1- and 2-D NMR spectroscopic data is listed in Table 3.3.1.  Table 3.3.1  c#  13  13  C and 'H NMR Data for 37  C8,ppm  a  'H 8, ppm (Int., m, J(Hz))  COSY  HMBC  b  H20 1 166.9 4.79 (l,s) 2 84.0 H2,H14 3 172.3 H2, H5,H14 4 133.8 H14 H15,H15' 141.6 5 6.21 (l,s) H8, H15, H15',H16 98.1 6 H8, H9, H9',H17 1.68 (2, d,5.7) H8 7 45.1 1.36 (l,m) H7, H9',H17 H7,H17 8 26.1 H9' H7, H8,H17 1.07 (l,m) 9 43.0 0.97 (l,m) H8, H9 9' Hll H8, H9, H9',H11,H18,H19 10 36.0 1.19 (l,m) H13 1.11 (2,m) H10 11 35.4 1.21 (2, m) H13 H13 12 19.5 Hll 0.84 (3, t, 7.2) H12 13 14.6 H5 H5 14 11.0 1.83 (3, d, 1) H7,H16 1.82 (l,m) H15\H16 15 32.0 H15,H16 15' 1.70 (l,m) H15,H15' 0.78 (3, t, 7.4) H15,H15' 16 8.0 0.84 (3, d, 6.5) H8 H7, H8, H9 17 21.7 H9,H9',H10,H11,H19 1.19(2, m) H19 18 26.3 0.79 (3, t, 7.1) H18 19 10.7 3.67 (3, s) 20 50.5 Recorded at 100 MHz, calibrated to the CDC1 C 8 of 77.0 ppm 13  3  Recorded at 500 MHz, calibrated to the CDC1 *H 8 of 7.24 ppm 3  57  58  59  14 15  13 17 19 16 10 18 12 8 119  16  10  19 14 13 12  20  17 188  1—30 11 10  40 50  20  60 70 80  — I 90 100 110  — I 120 130 1—140 150 160 170 tppm (tl) I I I I | I I I I | I I I I | I II I | I I I I | I I I I | I I I I | I I I I | I I I I | I I I I | I I I I | I I I I  6.00 ppm (t2)  5.50  5.00  Figure 3.3.3  4.50  4.00  3.50  3.00  2.50  2.00  1.50  1.00  HSQC Spectrum (400 MHz, CDC1 ) of 37 3  60  61  I |=PPm (tl) I I I I | I I I I | I I I I | I ll  6.00 ppm (t2)  5.50  5.00  Figure 3.3.5  I | I I I I | I I I I | ll  4.50  4.00  3.50  I I | I I I I | I I I I | I I I I | II  3.00  2.50  2.00  1.50  i i | i i i i | i  1.00  0.50  HMBC Spectrum (500 MHz, CDC1 ) of 37 3  62  Two distinctive proton singlets at 8H 6.21 and 5H 4.79 were useful in solving the structure of the ring moiety, Fragment A. By HSQC data, the proton at 5H 6.21 was attached to the olefinic carbon 5c 141.6 (C5). Using this proton resonance as a starting point for assigning the gross ring structure, HMBC correlations (Figure 3.3.6) were seen for 8c 133.8 (C4) and the methyl group carbon at 8c 11.0 (C14). This suggested that the carbon at 8c 133.8 (C4) was the olefinic partner of the carbon bearing the distinctive proton (8H 6.21 (H5)). In addition, the methyl group protons at 8H 1.83 (HI 4) correlated into 8c 133.8 (C4), such that this olefinic carbon would be substituted by this methyl group. A three-bond correlation between 8 1.83 (H14) and 8 172.3 (C3) indicated that 8 133.8 (C4) was in all H  C  C  likelihood bonded to 8 172.3 (C3). C  HMBC Correlation  COOCH  3  Figure 3.3.6 Selected HMBC Correlations for Compound 37: Fragment A  Once again, by HSQC correlations, the other characteristic proton singlet at SH 4.79 was shown to be attached to a carbon with a chemical shift of 8c 84.0 (C2). As shown in Figure 3.3.7, this proton had HMBC correlations to the carbons bearing chemical shifts of 8c 172.3 (C3) and 8c 166.9 (Cl). Furthermore, the methyl ester was characterized by a strong HMBC correlation of the methyl protons at 8H 3.67 into the carbon at 8c 166.9 (Cl). Given the C5/C4/C3 order had already been ascertained, and the carbon at 8c 166.9 (Cl) is an ester, it was apparent that the carbon with 8c 84.0 (C2) would most likely be positioned between  the carbons at 5c 172.3 (C3) and 5c 166.9 (Cl). Looking back at the carbon with a chemical shift of 5c 141.6 (C5) it was indirectly shown to be connected to the quaternary carbon at 5c 98.1(C6) by HMBC correlationsfromthe methylene protons (5 1.70(H15), 1.82(H15')) of H  an ethyl group attached to 5c 98.1(C6). Thus, the core carbon connectivity of the ring, Cl through C6, was established.  HMBC Correlation  COOCH  3  Figure 3.3.7 Selected HMBC Correlations for Compound 37: Fragment A  The stereochemistry of the C2-C3 double bond was determined to be Z based upon a selective 1-D gradient NOESY spectrum (Figure 3.3.8). By pulsing the proton at 4.79 ppm (H2) a nuclear overhauser enhancement was seen for the methyl protons at 1.83 ppm (H14) indicating that they were on the same side of the double bond. The five-membered ring was formed by placing an ether-type linkage between the quarternary carbon, 5c 98.1(C6), and the olefmic carbon, 5c 172.3 (C3). The quarternary carbon at 5c 98.1(C6) would be consistent with an oxygen bearing quartnerary carbon bonded to an olefin. Interestingly, at 172.3 ppm, C3 was downfield of the carbonyl carbon, 5c 166.9 (Cl). As can be seen in Figure 3.3.9, there are three resonance contributors to the proposed ring system. Resonance structure I is thought to be the major resonance contributor as it would place a positive charge on C3, accounting for the deshielding of this carbon.  64  65  Furthermore, resonance structure I would be the most stable possessing a tertiary carbon bearing a positive charge stabilized by an adjacent anionic oxygen. By comparison, resonance structure III has greater charge separation and the positive charge is located on a secondary carbon, C5, which causes III to be less energetically favourable than I. Resonance structure II places a positive charge on the oxygen attached to C3. Theoretically this positively charged oxygen could be stabilized by the adjacent negatively charged oxygen attached to C2. This would result in an inductive effect that would destabilize the positive charge at C3. However, given the fact that the carbon chemical shift of C3 is well downfield at 172.3 ppm, the empirical data does not support resonance structure II as a major contributor. Thus, the rationale for the deshielding at C3 is based upon the electron withdrawing effects of the adjacent oxygen and the partial positive charge of C3, as shown by resonance structure I.  Figure 3.3.9 Resonance structures for Fragment A (37)  The existence of the two double bonds, one carbonyl, and the heterocycle in Fragment A satisfied the four degrees of unsaturation. From the mass spectrometry data it was obvious that ten carbons and twenty-one hydrogens remained to construct a saturated hydrocarbon chain (Fragment B). As with the cyclic peroxide polyketide chains from  66  Chapter 2, the key to elucidating the carbon chain for 37 lies in the strong HMBC correlations of the terminal methyls. Two triplets at 5 0.79 (8c 10.7 (C19)) and §H 0.84 (8c H  14.6 (C13)), and one doublet at 8 0.84 (8 21.7 (C17)) indicated the main chain must H  C  possess one methyl group and one ethyl group branch. HMBC correlations (Figure 3.3.10) from SH 0.79 (HI 9) to the methylene carbon at 8c 26.3 (C18) and the methinic carbon at 8c 36.0 (C10) established the ethyl branch carbons as 8 10.7 (C19), 26.3 (C18) extending from C  the main chain at 8 36.0 (C10). The methyl branch protons 8 0.84 (HI 7) showed HMBC C  H  correlations into the methine carbon at 8c 26.1 (C8) and two methylene carbons at 8c 43.0 (C8) and 8c 45.1 (C7). Furthermore, the remaining methyl carbon, 8c 14.6 (C13), corresponded to the terminus of the main carbon chain. These terminal methyl protons, 8H 0.84 (H13), had HMBC correlations into to methylene carbons 8 19.5 (C12) and 8 35.4 C  C  (C13). These correlations accounted for all the carbons and hydrogens remaining, giving three pieces to be put together.  HMBC Correlation  Figure 3.3.10 Methyl HMBC Correlations for Compound 37: Fragment B  The three sub-fragments were attached based upon HMBC correlations (Figure 3.3.11) into the central methine carbon at 8c 36.0 (C10). The geminally coupled methylene protons 8H 0.97,1.07 (H9, H9') showed two-bond correlations into the methine carbon at 8c 36.0 (C10). In addition, the methylene protons at 8H 1.11 (HI 1) had an HMBC correlation  67  into the methine carbon at 8c 36.0 (CIO) and they had a three-bond COSY correlation to the methine proton at 8H 1.19 (H10). These correlations were important as they clarified the ordering of the carbons of the main chain as 8 45.1 (C7), 8 26.1 (C8), 8 43.0 (C9), 8 36.0 C  C  C  C  (CIO), 8 35.4 (Cll), 8 19.5 (C12), and 8c 14.6 (C13). C  C  HMBC Correlation COSY Correlation H  i-r  H  Figure 3.3.11 Selected HMBC and COSY Correlations for Compound 37: Fragment B  Fragment A andfragmentB could only be joined by a covalent bond between the carbon at 8c 98.1 (C6) and 8c 45.1 (C7). In support of this connectivity are two HMBC correlations (Figure 3.3.12). The methine proton 8H 136 (H8) correlates to the carbon 8c 98.1 (C6). In addition, the methylene protons 8H 1-68 (H7) correlate to the carbon 8c 32.0 (C15). Hence, the gross structure of 37 was established.  HMBC Correlation H  H COOCH  3  Figure 3.3.12 Selected HMBC Correlations for Compound 37  68  3.4 Structure Elucidation of Glanvillic Acid B (38)  COOH  38 Compound 38 was isolated as an optically active, clear oil, that gave a negative mode HRESIMS [M-H]" peak at m/z 293.2121 appropriate for a molecular formula of C 1 8 H 3 0 O 3 (calc'd 293.2117 for C 1 8 H 2 9 O 3 ) . Accordingly, 38 had four degrees of unsaturation. The C 13  NMR (Figure 3.4.1), *H NMR (Figure 3.4.2), and HSQC (Figure 3.4.3) spectra revealed  four methyl carbons, six methylene carbons, two methine carbons (5c 30.0, 36.0) and one olefmic carbon (8 107.7). By HMBC data three additional olefmic (5 124.5,139.5,154.5) C  C  carbons and one carbonyl carbon (8c 172.2) were seen. Two fragments, a fiiran ring and a carbon chain, were constructed and connected based upon COSY (Figure 3.4.4) and HMBC (Figure 3.4.5) spectra to provide the gross structure of compound 38. A detailed summary of the 1- and 2-D NMR spectroscopic data is listed in Table 3.4.1.  69  Table 3.4.1  c# 1 2 3 4 5 6 7 7' 8 9 9' 10 11 12 13 14 15 16 17 18  13  13  C8,  C and H NMR Data for 38 ]  H 8, ppm (Int., m, J(Hz))  COSY  3.60 (2, br s)  H5  r  ppm  172.2 31.6 139.5 124.5 107.7 154.5 35.9  H2  C  C  C c  5.85 (1, br s)  H2  2.50 (l,dd, 14.8,5.7) 2.32 (1, m) 1.82 (l,m) 1.16 (l,m) 1.04 (l,m) 1.31 (l,m) 1.17(2, m). 1.22(2, m) 0.85 (3, t, 6.9) 2.32 (2, q, 7.5) 1.11 (3,t, 7.5) 0.85 (3, d, 6.6) 1.27 (2,m) 0.81 (3, t, 7.3)  H7' H7 H7, H7', H9, H9' H16 H8, H9' H8, H9  c  30.0 41.0 36.0 35.4 19.4 14.5 18.0 14.7 20.1 26.4 10.8  HMBC  b  a  HI 3 H12 H15 H14 H8 H18 HI 7  H2, H5,H14 H2, H5,H14,H15 H7, H7',H14 H7, H7' H9, H9',H16 H7, H7',H9, H9',H16 H7, H7',H16 H9,H9',H18 H13 H13 H15 H14 H7, H7' H9, H9',H18  Recorded at 100 MHz, calibrated to the CDC1 C 8 of 77.0 ppm Recorded at 500 MHz, calibrated to the CDC1 'H 8 of 7.24 ppm 3  3  c l 3  C 8 recorded from HSQC and HMBC data  70  71  72  'j I  I  I I  5.50 ppm (t2)  I  I  I  5.00  II  I  4.50  I I I I  4.00  I  3.50  I I  3.00  I  I  I  2.50  I I I I  2.00  I  1.50  I I I  I  I  I  1.00  Figure 3.4.3 HSQC Spectrum (400 MHz, CDC1 ) of 38 3  73  I  I I  I  pTTTTTT"in pnrTTTTTTTjT-m | i i i r|H~rrT^TTT"qnnrrrjT^rTT-n~rq _  5.50 ppm (t2)  Figure 3.4.5  5.00  4.50  4.00  3.50  3.00  2.50  2.00  1.50  1.00  HMBC Spectrum (500 MHz, CDC1 ) of 38 3  75  Due to the limited amount of material, not all of the carbons displayed signals in the 13  C spectrum, presumably as a result of low isotopic abundance and long relaxation times.  Consequently, five carbon chemical shifts (8c 31.6, 124.5,139.5,154.5, 172.2) could only be assigned based upon HMBC correlations. The ring moiety, Fragment A, was completely assigned based upon the HMBC data collected (Figure 3.4.6). Starting with the broad singlet resonance at 8H 5.85 (8c 107.7 (C5)), HMBC correlations were seen for olefmic carbons at 8c 124.5 (C4) and 139.5 (C3). By further inspection of the HMBC spectrum, an ethyl branch was found to be substituted at 8c 124.5 (C4) and an acyl group was attached to 8c 139.5 (C3). The question remained which of the carbons of 8c 124.5 (C4) and 139.5 (C3) was bonded directly to 8 107.7 (C5). An HMBC correlation between 8 2.32 (H14) and 8c 107.7 C  H  (C5) indicated that the carbon bearing the ethyl group, 8c 124.5 (C4), was directly bonded to 8 107.7 (C5). Additional correlations of the methylene protons 2.50 (H7), 8H 2.32 (H7') (8 C  C  35.9 (C7)) into 8 107.7 (C5) and 154.5 (C6) established the final carbons of the ring C3-C6. C  A 2,3,5-trisubstituted furan ring could then be formed.  HMBC Correlation  1COOH  Figure 3.4.6  Selected HMBC Correlations for Compound 38: Fragment A  76  Having satisfied all four degrees of unsaturation, a fully saturated chain (Fragment B) was constructed, beginning with the methylene carbon (8c 35.9 (C7)) attached directly to the ring moiety. As can be seen in Figure 3.4.7, two  H  M  B  C  correlations for each of the geminal  protons at 2 . 5 0 ( H 7 ) and S H 2 . 3 2 ( H 7 ' ) into the methinic carbon at 8 3 0 . 0 ( C 8 ) and the C  methylene carbon at 8c  4  1  .  0 (  C  9  )  .  Further  H  M  B  C  correlationsfromthe methyl protons at 8H  0 . 8 5 ( C 1 6 ) into the carbon at 8 3 5 . 9 ( C 7 ) indicated that the methine carbon, 8 3 0 . 0 ( C 8 ) C  C  must be bonded directly to the methyl and to the carbon with the chemical shift of 8c 35.9 (  C  7  )  .  This clarified the ordering as  C  7  /  C  8  /  C  9  ,  with a methyl branch as  C  1  from  6  C  8  .  18  HMBC Correlation  Figure 3.4.7 Selected HMBC Correlations for Compound 38: Fragment B  The methyl carbon at 8c 14.5 (Cl 3) showed HMBC correlations into two methylene carbons (8 19.4 (C12) and 8 35.4 (Cll)), whereas the methyl carbon at 8 10.8 (C18) C  C  C  showed HMBC correlations into a methylene carbon (8c 26.4 (C17)) and a methine carbon at 8 36.0 (C10). By a COSY correlation between 8H 0.81 (HI 8) and 5H 1.27 (HI 7) it was C  decided that the methyl at 8c 10.8 (C18) was part of an ethyl group which branchedfromthe main chain at the methine carbon 8c 36.0 (C10). This resulted in a carbon chain that possesses the same connectivity as the saturated hydrocarbon chains seen in compounds 32 and 37. As no HMBC or COSY correlations could  77  aid in assigning the order of the two methylene carbons, Cl 1 and C12, near the terminus of the main chain, it was necessary to compare the carbon chemical shifts. By comparison of the chemical shift data, the order of the methylene carbons was found to be 5c 35.4 at Cl 1 and 5c 19.4 at C12. This was consistent with the previous analysis of spectroscopic data for compounds 32 and 37, but inconsistent with the ordering of these two compounds for the previous isolation of methyl glanvillate B. In the characterization of methyl glanvillate B, Cl 1 was assigned a shift of 5c 19.5 and C12 was given a chemical shift of 5c 35.5, opposite to the current assignment. No mention of the rationale for the assignment of these carbons was given in the paper. Running a C NMR simulation for 38 with ACD/CNMR Predictor® ,3  software (v.7.05) gave chemical shifts of 5c 35.5 for Cl 1 and 5c 19.5 for Cl 2. Based upon this result and in keeping with the assignments of compounds 32 and 37, Cl 1 was assigned as 5c 35.4 and C12 as 5c 19.4. Thus, the full assignment of compound 38 was completed.  3.5 Proposed Stereochemistry and Biosynthesis of Structures 37 and 38  As previously mentioned in the General Introduction, Plakortis furano polyketides are thought to be derived from cyclic peroxide polyketides. Both Faulkner et al. and 42  Fattorusso et a/. have biosynthetically rationalized the stereochemistry of furan polyketides 53  based upon the stereochemistry of their parent cyclic peroxide polyketides. In a similar fashion, the current study proposes the stereochemistry of the furano polyketides, 37 and 38, based upon their hypothetical cyclic peroxides precursors. The cyclic peroxide precursors of 37 and 38, shown in Scheme 3.5.1, would follow the same biosynthetic pathway as cyclic peroxides 32 and 33 discussed in section 2.5. Hence, it is reasonable to assume the stereochemistry of the cyclic peroxide precursors for 37 and 38 would be the same as 32 and  78  33. In Scheme 3.5.1 two cyclic peroxides are shown, the methyl ester as precursory to 37 and the free acid as precursory to 38. Possibly the methyl ester is formed by the action S-adenosyl methionine upon thefreeacid. In both cases, abstraction of an a-protonfromthe cyclic peroxide opens the ring and forms an epoxide intermediate. Intramolecular attack on  38 Scheme 3.5.1 Proposed Biosynthesis of Compounds 37 and 38fromCyclic Peroxides  79  the epoxide by the anionic oxygen would form the furano type ring system. Loss of H2O would produce the final conjugated structure of the furano polyketide, 37. Addition of a proton at position two and abstraction of the proton at position six produces the furan ring in compound 38. In this manner, it is proposed that the two furan polyketides are methyl (2Z,6R,8S,10S)-6,10-diemyl-4,8-dimemyl-3,6-epoxytrideca-2,4-dienoate (37) and (8S,10S)4,10-diethyl-3,6-epoxy-8-methyltrideca-3,5-dienoic acid (38, glanvillic acid B).  3.6 Conclusion  At the time of this study, only five other compounds have been isolated with a similar oc,p-unsaturated furano ester ring system like compound 37. None of these related compounds possess a methyl group attached to the ring at position four nor do they bear the same carbon chain. Glanvillic acid B (38) was previously isolated in its methyl ester form via synthetic modification. This study represents the first time glanvillic acid B was isolated in itsfreeacid form. Only glanvillic acid A, which has one degree of unsaturation in its carbon chain, shares the same 2,3,5-trisubstituted furan structure. In addition, glanvillic acids A & B were originally isolatedfromthe Dominican sponge, Plakortis halichondrioides, which is of different species than Plakortis angulospiculatus, the spongefromwhich glanvillic acid B (38) was isolated in the current study. Interestingly, compounds 37 and 38 possess identical carbon chains, in terms of their connectivity, as compounds 32 and 33. It is very likely that the methyl and ethyl substituents on the carbon chains of compounds 37 and 38 are in a syn orientation, as was proposed for compounds 32 and 33. Further stereochemical investigations would need to be conducted to fully prove the absolute configuration of compounds 37 and 38.  80  CHAPTER 4  4.1  General Discussion  Comparison of the Polyketide Biosynthesis for Compounds 32,33,37, and 38  The rich structural diversity of polyketides stemsfromslight differences in the assembly of small carbon units by the polyketide synthases and further modifications to the resulting carbon chain. Different combinations of acetate, propionate, and butyrate units, along with variations in the starter unit, produce linear carbon chains that are released from the PKS complex of enzymes. Post-polyketide synthase modifications allow for the introduction of many structural features, including rings and functional groups. This structural diversity results in a wide range of potent biological activities that has led to important drugs, such as the previously mentioned antifungal drug, amphotericin B. As can be seen with the structures of 32,33, 37, and 38 (Scheme 4.1.1), differences in the ordering of acetate, propionate, and butyrate units leads to structural diversity. Compounds 32,37, and 38 share a similarly branched carbon chain. Accordingly, the first three units assembled by the PKS would be propionate-butyrate-propionate for 32,37, and 38. The extension by one extra carbon in the chain of 33 would be consistent with a butyrate starter unit, instead of a propionate residue. Structures 32 and 33 have an identical ordering of units after their respective starter units. The ethyl branch attached to the quaternary carbon of the ring in compounds 32,33, and 37 is derivedfroma butyrate placed as the fourth residue. By comparison, structure 38 has an acetate residue where the other three compounds have butyrates. The ethyl branch attached to the double bond of the rings in compounds 32, 33, and 38 is derivedfroma butyrate placed as thefifthresidue. By comparison, structure 37  81  has a propionate unit as the fifth residue, giving a methyl branch. All of the compounds terminate with an acetate residue. 1  2  Pr  33  Bu  3  4  Bu  Pr  Bu  Pr  5  Bu  6  Bu  Ac  Bu  Bu  Ac  Bu  Pr  Ac  Bu  Ac  "=> Pr  Bu  Pr  Pr  Bu  Pr  COOCHj  Ac  Ac - acetate Pr = propionate Bu = butyrate  Scheme 4.1.1 Polyketide Linear Chain Representations for 32,33,37, and 38 It is proposed that all the structures shown are converted into cyclic peroxides as either theirfinalstructure or as a transitory intermediate (Scheme 4.1.2). Compounds 32 and 33 would be converted directly into theirfinalcyclic peroxide forms via the enzymatic incorporation of oxygen, as shown previously in Scheme 2.5.2 (pg. 52). Compound 37 would go one step further, by conversion of its six-membered peroxide ring into a furano structure according to Scheme 3.5.1 (pg. 77). With formation of a methyl ester, possibly through the 82  action of S-adenosyl methionine, compound 38 would be converted from a furano to its fixran form by abstraction of the proton from the methinic carbon of the ring, as according to Scheme 3.5.1 (pg. 77). The comparison of these compounds highlights some of the structural differences that arise in polyketide biogenesis. It isfromthis structural variance of polyketides that results in a great diversity of biological activity.  83  4.2  Chemical Genetic Profiling of Marine Invertebrate Extracts  The cluster plot, shown in Figure 4.2.1, displays the haploid mutant, chemicalgenetic profiles for 17 compounds of known action, two compounds of unknown action, and the crude extracts of five marine invertebrates. Dr. Charlie Boone's lab (U of T) tested -4500 yeast haploid deletion mutants strains in parallel for sensitivity or resistance to particular compounds. Compounds and extracts are clustered on the horizontal axis of this fitness test profile based upon the similarity of their signatures. Yeast strains that are sensitive to the compound or extract are represented in red, those that are resistant are represented in green, and those with no significant change are represented in black.  Table 4.2.1  Drugs in Fitness Test Profile  Abbreviation  Full Name  Mode of Action  Clot FLC Gelda Nocod Tuni Thia  Clotrimazole Fluconazole Geldanamycin Nocodazole Tunicamycin Thialysine Sulfometuron methyl FK506 Cycloheximide Agelasine E Cytochalasin A Brefeldin A Camptothecin Mitomycin C Wortmannin Methylmethane sulfonate Caspofungin Papuamide B Rapamycin  ERG11 inhibitor (ergosterol biosynthesis) ERG11 inhibitor (ergosterol biosynthesis) Hsp90 inhibitor (chaperone) Microtubule depolymerizing agent Alg7 inhibitor (protein glycosylation)  SMM  FK506 Cyh Agel E Cyto BFA CPT MitoC Wort MMS CAS PapB Rapa  amino acid biosynthesis inhibitor calcineurin inhibitor inhibits protein synthesis unknown Actin depolymerizing agent inhibits secretion topoisomerase I inhibitor phosphatidyl inositol inhibitor DNA damaging agent inhibits cell wall biosynthesis unknown inhibits protein translation and more  84  Table 4.2.2  Marine Invertebrate Extracts in Fitness Test Profile Organism Sponge Sponge Sponge Sea Cucumber Sponge  Extract/Compound 00-303 (303C) 95-57 00-89 00-132 00-192  Collection Location Dominica Papua New Guinea Indonesia Dominica Indonesia  The profiles of fluconazole (FLC) and clotrimazole (Clot) cluster together (see tree dendrogram atop Figure 4.2.1). This is expected given that they target the same protein, in that both inhibit the product of ERG11, an essential gene in the yeast ergosterol biosynthesis pathway. Figure 4.2.2 displays a scatterplot and an expanded portion of the chemical genetic profile for fluconazole and clotrimazole. Each strain is represented by a blue box whose  Resistant  F L C Sensitive-*  Clot  FLC  Figure 4.2.2 Scatterplot and Fitness Test Profile Expansion for Clotrimazole and Fluconazole  86  coordinates on the scatterplot are correlated with the degree to which each strain is resistant or sensitive to the two drugs. The majority of strains that showed no sensitivity or resistance are found together around the coordinates (0,0) on the scatterplot. These strains would be shown in black on the on the profile shown to the right of Figure 4.2.2. The upper right quadrant of the scatterplot shows the strains that are significantly sensitive to both drugs. Although the correlations in the chemical genetic profile aren't as obvious at first sight, statistical analysis of the scatterplot shows a significant correlation between the profiles of the two drugs. Based upon this correlation of sensitive strains it is deduced that clotrimazole and fluconazole inhibit similar pathways and have the same cellular target. In the case of fluconazole and camptothecin (Figure 4.2.3), the lack of correlation between the profiles of fluconazole, an inhibitor of ergosterol biosynthesis, and the profile of  — 3  3  s  1  «— Resistant  Figure 4.2.3  3  3  ;  C P T Sensitive—*  3  FLC  CPT  Scatterplot and Fitness Test Profile Expansion for Fluconazole and Camptothecin  87  camptothecin, a topoisomerase inhibitor, can clearly be seen. Both the scatterplot and the chemical genetic profile expansion show a lack of correlation. The scatterplot shows the most sensitive strains for either compound are not the same. This is echoed in the profile where the most intensely red bands (the most sensitive strains) do not correspond to one another. The crude extract of from the sponge Plakortis angulospiculatus (00-303), albeit very potent against C. albicans and S. cerevisiae, gave a non-specific profile. Few yeast strains were shown to be sensitive in thefitnesstest profile (Figure 4.2.1) of the crude extract of 00303. Those strains that were sensitive to the extract often showed general, non-specific drug sensitivity. In addition, the extract of 00-303 did not show any specificity in a haploinsufficiency assay. It was hoped that by testing the purified compound 33, by excluding the other secondary metabolites in the extract, the profile might be cleared of "noise" and reveal a specific target or pathway. This had never been attempted before with the cyclic peroxide class of polyketide compounds. Unfortunately, the results for 33 showed a similar profile to that of the crude extract 00-303 in both the haploid and heterozygote deletion assays. Hence, the crude 00-303 and compound 33 seem to target generally drugsensitive strains of S. cerevisiae. This is consistent with the diverse biological activity listed in the literature for Plakortis cyclic peroxide polyketides. Due to timing considerations, the sponge extract 00-303 was selected as an isolation project and as an archetype for the chemical genetic analysis of marine invertebrate extracts. The crude 00-303 was selected based upon its potency against C. albicans and S. cerevisiae. Although the crude extract did not show a specificfitnesstest profile, the isolation part of the project had already begun. Providing the purification was underway and the biological activity data was very strong, the isolation of the active compounds 32 and 33 was  88  completed. Later, it was discovered that two marine invertebrate extracts 00-132, from an Indonesian sponge, and 00-192, from a Dominican Sea Cucumber, showed intriguing fitness test profiles. They both displayed profiles strongly sensitive for a number of yeast strains, indicative of some mode of specificity. Interestingly, the two crudes possessed similar profiles to one another and, in turn, both of their profiles were similar to rapamycin (Figure 4.2.4). Rapamycin is a polyketide that targets the TOR proteins in yeast which are involved in regulating protein translation, as well as many other cellular processes. The initial assays of the extracts of 00-132 and 00-192 showed comparable activity against C.albicans, but considerably less than the extract of 00-303. At the time of this thesis, both of these extracts are under study to isolate the compounds responsible for this activity.  Figure 4.2.4  Scatterplot and Fitness Test Profile Expansion for Extracts 00-132 and 00-192  89  EXPERIMENTAL SECTION  General Experimental Procedures One- and two-dimensional NMR spectra were recorded on either a 400 MHz Bruker Avance 400 spectrometer or a 500 MHz Bruker AMX-500, both housing a 5mm BBI-Z probe. One-dimensional C spectra were recorded on a Bruker AM-400, with a normal 5mm 13  probe, at 100 MHz. All samples were run in deuterated chloroform (99.96% isotopic purity) and NMR data was calibrated to the solvent signals of 8H 7.24 ppm and 8c 77.0 ppm. All NMR spectra were initially processed by XWIN-NMR® and, subsequently, processed on MestRe-C® (v.3.7.9) software for inclusion in this thesis. Electrospray ionization (ESI) mass spectrometry was run by staff in the UBC Mass Spectrometry Centre, Department of Chemistry, on a Bruker Esquire ion trap mass spectrometer. All chromatography was performed using HPLC grade solvents, including doublydistilled, 0.22 umfilteredH2O. Size exclusion chromatography used LH-20®, lipophilic sephadex, as a solid phase. Normal phase silica gel (230-400 mesh) was used in running flash columns. HPLC was performed using a Waters 2487 Dual X Absorbance Detector and a Waters 1525 Binary HPLC Pump with Breeze® software. A CSC-Inertsil, 150AAODS-2, 5um reverse phase HPLC column was employed in the final purification of all compounds. Thinlayer chromatography (normal and reverse phase) was performed on commercial plates. Staining with 1% /?-anisaldehyde, 2% H2SO4 in acetic acid solution and visualization under UV light (254 nm) facilitated the identification of unknown compounds. Biological testing against Candida albicans and bacterial pathogens was conducted in the department of Chemistry, UBC, by Helen Wright. Minimum inhibitory concentrations (MIC) were determined by an Alamar Blue® microdilution assay using Mueller-Hinton broth 90  with incubation at 37°C. Bioautographic assays were also done with the help of Helen Wright on 8 x 8 cm normal phase TLC plates, staining with MTT (thiazolyl blue). Haploinsufficiency profding with Saccharomyces cerevisiae was performed by Ainslie Parsons in Dr. Charlie Boone's laboratory, Department of Biochemistry, University of Toronto.  Animal Material  The sponge Plakortis angulospiculatus (Carter, Homosclerophorida/Plakinidae) was collected by SCUBA, September 3, 2000, near Pennville, Dominica, 15° 37.4'N, 61°25.1'W. The sponge was found on vertical walls at a depth of 10 to 15 m and was described to be medium brown outside, tan inside. A voucher specimen has been deposited (museum # ZMAPOR 17723) in the Zoologisch Museum, University of Amsterdam.  91  Extraction of P. angulospiculatus and Isolation of Compounds 32,33,37, and 38  See Section 2.2 and 3.2 for isolation method. 32: clear oil; [a] = -37.8 (c 0.26, CHC1 ); 'HNMR 0.81 (3, t), 0.86 (3, t), 0.87 (3, d), 0.88 (3, t), 1.00 (1, m), 1.07 (3, t), 1.08 (1, m), 1.15 (2, m), 1.23 (4, m), 1.40 (2, m), 1.52(1, m), 1.63 (1, dq), 1.75 (1, dq), 2.01 (2, m), 2.61 (1, dd), 2.93 (1, dd), 4.57 (1, br d), 5.49 (1, br s); C NMR 8.2, 10.8, 11.6, 14.6, 19.5, 22.4, 24.9, 26.0, 26.4, 31.0, 35.4, 36.1, 36.9, 42.9, 43.3, 76.2, 83.8, 125.3,137.5,175.2; positive ion HRESIMS [M+Naf m/z 363.2505 (calc'd 363.2511 forC2oH360Na) 23  D  3  13  4  33: clear oil; [<x] = -30.7 (c 0.25, CHC1 ); 'HNMR 0.80 (3, t), 0.83 (3, d), 0.87 (3, t), 0.94 (3,t), 1.05 (l,m), 1.07 (3,t), 1.14 (l,m), 1.19 (l,m), 1.28 (l,m), 1.40 (2,m), 1.52 (l,m), I. 62 (1, dq), 1.73 (1, dq), 1.81 (1, m), 1.97 (2, m), 1.99 (2, m), 2.58 (1, dd), 2.92 (1, dd), 4.55(1, brd), 4.95(1, dd), 5.36(1, dt), 5.49(1, brs); CNMR8.1,11.6, 11.7, 14.3,21.3,24.9, 25.7, 25.9, 28.9, 30.9, 36.8, 42.3, 43.3, 44.5, 76.3, 83.7, 125.3, 132.2, 133.2, 137.2, 175.4; positive ion HRESIMS [M+Na] m/z 375.2502 (calc'd for375.2511 for C iH 0 Na ) 23  D  3  13  +  2  36  4  37: clear oil; [a] = -104.2 (c 0.24, CHC1 ); *H NMR 0.78 (3, t), 0.79 (3, t), 0.84 (3, d), 0.84 (3, t), 0.97 (1, m), 1.07 (1, m), 1.11 (2, m), 1.19 (3, m), 1.21 (2, m), 1.36 (1, m), 1.68 (2, d), 1.70 (l,m), 1.82(1, m), 1.83 (3, d), 3.67 (3, s),4.79 (l,s), 6.21 (1, s); CNMR8.0, 10.7, II. 0,14.6, 19.5, 21.7, 26.1, 26.3, 32.0, 35.4, 36.0, 43.0,45.1, 50.5, 84.0, 98.1,133.8,141.6, 166.9,172.3; positive ion HRESIMS [M+Na] m/z 345.2415 (calc'd 345.2406 for C oH 0 Na) 24  D  3  13  +  2  34  3  38: clear oil; 'H NMR 0.81 (3, t), 0.85 (3, d), 0.85 (3, t), 1.04 (1, m), 1.11 (3, t), 1.16 (1, m), 1.17(2, m), 1.22 (2,m), 1.27 (2, m), 1.31 (l,m), 1.82 (l,m), 2.32 (l,m),2.32 (2, q), 2.50 (1, dd), 3.60 (2, br s), 5.85 (1, br s); C NMR 10.8,14.5, 14.7,18.0,19.4, 20.1, 26.4, 30.0, 31.6, 35.4, 35.9, 36.0,41.0,107.7,124.5,139.5,154.5,172.2; negative ion HRESIMS [M-H]"m/z 293.2121 (calc'd 293.2117 for Ci H 0 ) 13  8  29  3  92  Bibliography Cannell, RJ.P. in Methods in Biotechnology: Natural Products Isolation. Ed. Cannell, R.J.P.. Human Press. Totowa, New Jersey. 1998. ch. 1. p. 1-2. 1  Faulkner, D.J. Antonie van Leeuwenhoek 2000, 77,135-145.  2  a) Newmann, D.J.; Cragg, G.M.; Snader, K.M. J. Nat. Prod. 2003, 66, 1022-1037.; b) Newman, D.J.; Cragg, G.M.; Snader, K.M. Natural Products Reports 2000,17,215-234. 3  Tulp, M ; Bohlin, L. Trends in Pharmacological Sciences 2002, 23, 225-231.  4  Wani, M.C.; Taylor, H.L.; Wall, M.E.; Coggon, P.; McPhail, A.T. J. Am. Chem. Soc. 1971, 93, 2325-2327. 5  Mann, J. Nature Reviews: Cancer 2002, 2, 143-148.  6  a) Andersen, R.J.; Williams, D.E. Pharmaceuticalsfromthe Sea in Issues in Environmental Science and Technology, Vol. 13. Ed. Harrison, R.M.; Hester, R.E.. The Royal Society of Chemistry. Cambridge. 2000. p. 55-79.; b) Brusca, R.C.; Brusca, G.J. Invertebrates. Sinauer Associates. Sunderland. MA. 1990. p. 1-6. 7  Lindel, T.; Jensen, P.R.; Fenical, W.; Long, B.H.; Casazza, A.M.; Carboni, J.; Fairchild, C.R. J. Am. Chem. Soc. 1997,119, 8744-8745. 8  Pettit, G.R.; Herald, C.L.; Clardy, J.; Arnold, E.; Doubek, D.L.; Herald, D.L. J. Am. Chem. Soc. 1982,104, 6848. 9  ter Haar, E.; Kowalski, R.J.; Hamel, E.; Lin, CM.; Longley, R.E.; Gunasekera, S.P.; Rosenkranz, H.S.; Day, B.W. Biochemistry 1996, 35,243. 10  "a) Gupte, M.; Kulkarni, P.; Ganguli, B.N. Appl. Microbiol. Biotechnol. 2002,58,46-57. b) Dasgupta, F. Antifungal agents - past, present, future prospect. Ed. Varma, R.S.; Khan, I.K.; Singh, A.P.. National Academy of Chemistry and Biology. Lucknow. 1998. p. 29-54. Jiang, B.; Bussey, H.; Roemer, T. Current Opinion in Microbiology 2002, 5,466-471.  12  Odds, F.C.; Brown, A.J.P.; Gow, N.A.R. TRENDS in Microbiology 2003, 11,272-279.  13  a) Abbanat, D.; Leighton, M.; Maiese, W.; Jones, E.B.G.; Pearce, C ; Greenstein, M. Journal ofAntibiotics 1998, 51, 296-302.; b) Schlingmann, G.; Milne, Lisa; Williams, D.R.; Carter, G.T. Journal ofAntibiotics 1998, 51, 303-316.; c) Albaugh, D.; Albert, G.; Bradford, P.; Cotter, V.; Froyd, J.; Gaughran, J.; Kirsch, D.R.; Lai, M.; Rehnig, A.; Sieverding, E.; Silverman, S. Journal ofAntibiotics 1998, 51, 317-322. 14  93  Pettit, G.R.; Cichacz, Z.A.; Herald, C.L.; Boyd, M.R.; Schmidt, J.M.; Hooper, J.N.A.  15  J.Org. Chem. 1993, 58,1302-1304.  Pettit, R.K.; McAllister, S.C; Pettit, G.R.; Herald, C.L.; Johnson, J.M.; Cichacz, Z.A. Int.  16  J. Antimicrobial Agents 1998, 9, 147-152.  Ovechkina, Y.Y.; Pettit, R.K.; Cichacz, Z.A.; Pettit, G.R.; Oakley, B.R. Antimicrobial  17  Agents and Chemotherapy 1999,43, 1993-1999.  Shreiber, S.L. Bioorg.Med.Chem 1998, 6,1127-1152.  18  19  Tan, D.S.; Foley, M.A.; Stockwell, B.R.; Shair, M.D.; Schreiber, S.L. J.Am.Chem.Soc.  1999,121, 9073-9087. Stockwell, B.R. Trends in Biotechnology 2000,18,449-455.  20  Mayer, T.U. Trends in Cell Biology 2003,13, 270-277.  21  Drews, J. Nat.Biotech. 1996,14, 1516-1518.;  22  a)Bassett, D.E. Nature: Genetics 1997,15, 339-344.; b) Bostein, D.; Chervitz, S.A.; Cherry, J.M. Science 1997, 277,1259-1260.  23  Bach, T.J.; Lichtenhaler, H.K. Z. Naturforsch 1983, 38, 212-219.  24  Hughes, T.R. Fund. Integr. Genomics 2002, 2, 199-211.  25  a) Hughes, T.; Andrews, B.; Boone, C. Cell 2004,116, 5-7.; b) Lum, P.Y.; Armour, CD.; Stepaniants, S.B. et al. Cell 2004,116,121-137.; c) Giaever, G.; Flaherty, P.; Kumm, J.; Proctor, M.; Nislow, C ; Jaramillo, D.F.; Chu, A.M.; Jordan, M.I.; Arkin, A.P.; Davis, R.W.  26  Proc. Nat. Acad. Sci. 2004,101, 793-798.  a)Tong, A.H.Y.; Evangelista, M.; Parsons, A.B.; Xu, H.; Bader, G.D.; Page, N.; Robinson, M.; Raghibizadeh, S.; Hogue, C.W.V.; Bussey, H.; Andrews, B.; Tyers, M.; Boone, C. Science 2001,294, 2364-2368. b) Marton, M.J.; DeRisi, J.L.; Bennett, H.A.;Iyer, V.R.; Meyer, M.R.; Roberts, C.J.; Stoughton, R.; Burchard, J.; Slade, D.; Dai, H.; Bassett, D.E.; Hartwell, L.H.; Brown, P.O.; Friend, S.H. Nature Medicine 1998,4, 1293-1301. 27  Parsons, A.B.; Brost, R.L.; Ding, H.; Li, Z.; Zhang, C ; Sheikh, B.; Brown, G.W.; Kane, P.M.; Hughes, T.R.; Boone, C Nature Biotechnology 2004, 22, 62-69.  28  a) Lokey, RS. Curr.Op.Chem.Bio. 2003, 7, 91-96.; b) Salomon, C.E.; Magarvey, N.A.; Sherman, D.H. Nat. Prod. Rep. 2004, 21,105-121.  29  Staunton, J.; Weissman, K.J. Nat. Prod. Rep. 2001,18, 380-416.  30  94  McNamara, CM.; Box, S.; Crawforth, J.M.; Hickman, B.S.; Norwood, T.J.; Rawlings, B.J. J. Chem. Soc, Perkin Trans. 1 1998, 83-87. 31  Jimenez, M.S.; Garzon, S.P.; Rodriguez, A.D. J. Nat. Prod. 2003, 66. 655-661.  32  Takada, N.; Wantabe, M.; Yamada, A.; Suenaga, KI.; Yamada, K.; Ueda, K.; Uemura, D. J. Nat. Prod. 2001, 64. 356-359. 33  Rudi, A.; Talpir, R; Kashman, Y.; Benayahu, Y.; Schleyer, M. J. Nat. Prod. 1993, 56, 2178-2182.  34  Patil, A.D.; Freyer, A.J.; Bean, M.F.; Carte, B.K.; Westley, J.W.; Johnson, R.K.; Lahouratate, P. Tet. 1996, 52, 377-394.  35  Davidson, B.S. J. Org. Chem. 1991, 56,6722-6724.  36  a)Gustafson, K.; Andersen, RJ. J. Org. Chem. 1982,47, 2167-2169.; b)Graziani, E.I.; Andersen, R.J. Chem. Comm. 1996, 2377-2378.; c)Kubanek, J.; Andersen, RJ. Tet. Lett. 1997, 38, 6327-6330. 37  Yanai, M.; Ohta, S.; Ohta, E.; Hirata, T.; Ikegami, S. Bioorg. Med. Chem. 2003,11, 17151721.  3 8  Ovenden, S.P.B.; Capon, R.J. J. Nat. Prod. 1999, 62, 214-218.  39  Williams, D.E.; Allen, T.M.; Soest, R.V.; Behrisch, H.W.; Andersen, R.J. J. Nat. Prod. 2001,64, 281-285.  40  Stierle, D.B.; Faulkner, D.J. J. Org. Chem. 1980,45, 3396-3401.  41  Compagnone, R.S.; Pina, I.C; Rangel, H.R.; Dagger, F.; Suarez, A.I.; Reddy, M.V.R; Faulkner, D.J. Tet. 1998, 54, 3057-3068. 42  a)Patil, A.D.; Freyer, A.J.; Bean, M.F.; Carte, B.K.; Westley, J.W.; Johnson, R.K.; Lahouratate, P. Tet. 1996, 52, 377-394.; b) Hayes, P.Y.; Kitching, W. J. Am. Chem. Soc. 2002,124, 9718-9719.  43  44  Ravi, B.N.; Armstrong, R.W.; Faulkner, D.J. J. Org. Chem. 1979,44, 3109-3113.  Rudi, A.; Afanii, R.; Gravalos, L.G.; Aknin, M.; Gaydou, E.; Vacelet, J.; Kashman, Y J. Nat. Prod. 2003, 66, 682-285. 45  Campagnuolo, C ; Fattorusso, E.; Taglialatela-Scafati, O.; Ianaro, A.; Pisano, B. Eur. J. Org. Chem. 2002,1,61-69.  46  95  Qureshi, A.; Stevenson, C.S.; Albert, C.L.; Jacobs, R.S.; Faulkner, D.J. J. Nat. Prod. 1999, 62,1205-1207.  47  Huang, X.H.; van Soest, R.; Roberge, M.; Andersen, R.J. Org. Lett. 2004, 6, 75-78.  48  Gunasekera, S.P.; Gunasekera, MM.; Gunawardana, G.P.; McCarthy, P.; Burres, N. J. Nat. Prod. 1990, 53, 669-674. 49  Supporting Information for Reference 51 available via the Internet: http://pubs.acs.Org/subscribe/journals/orlef7/suppinfo/4/i04/ol016943y/ol016943y_sl .pdf 50  51  Yao, G.; Steliou, K. Org. Lett. 2002,4,485-488.  Capon, R.J.; Macleod, J.KL Tetrahedron 1985, 41, 3391-3404.  52  Cafieri, R; Fattorusso, E.; Taglialatela-Scafati, O.; Ianaro, A. Tet. 1999, 7045-7056.  53  96  


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