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No description available.

An examination of an organic extract of the cyanobacterium Lyngbya majuscula collected from Wasini Island off the southern Kenyan coast led to the isolation of the known cyclodepsipeptide antanapeptin A (7), recently isolated from a Madagascan collection of L. majuscula, and a new bioactive cyclodepsipeptide, homodolastatin 16 (42). Although L. majuscula is a common, pantropical cyanobacterium this study represents the first investigation of the natural product chemistry of a Kenyan population of L. majuscula. The structures of the two cyclodepsipeptides were determined from 2D NMR and mass spectrometry data. The L- stereochemistry of the proline, valine, and N-methylphenylalanine amino acids in 7 and the L – proline configuration in 42, was confirmed by Marfey’s HPLC method. Chiral GC was used to determine the absolute stereochemistry of the hydroxyisovaleric acid moiety in 7 and 42, the lactate residue in 42 and tentatively propose an L-stereochemistry for the Nmethylisoleucine amino acid in 42. Homodolastatin 16, a higher homologue of the potential anti-cancer agent, dolastatin 16, exhibited moderate activity against two oesophageal cancer cell lines.

Natural products play a historical role in the discovery of medicine but present unique challenges for chemical isolation, identification and production. In this work we describe the identification of twenty novel diterpenoids. These were isolated by use of chromatography, and the structures determined by spectroscopic methods, primarily 1D and 2D NMR. Six of these possess unprecedented diterpenoid skeletons and two of them show significant growth inhibitory effects on cancer cell lines in vitro (GI50 < 10 μM). The biomimetic semisynthesis of diterpendoids and analogues is also presented. Access to the bielschowskyane carbon skeleton by dearomatization of a furanocembranoid precursor is described. Highlights include a stereoselective alkene epoxidation, a novel kinetic furan dearomatization method, and an efficient [2+2] photochemical cycloaddition. The role of conformational steering was studied spectroscopically using VT 1H-NMR and NOESY as well as quantum chemical calculations at the DFT level of theory. We also disclose a biomimetic synthesis of providencin using a photochemical Norrish-Yang cyclization. This provided the absolute configuration by chemical correlation with the precursor bipinnatin E, the latter determined by x-ray diffraction. An unexpected, regioisomeric byproduct was observed and a possible mechanism is proposed. A biomimetic synthesis of the diterpene alkaloid aceropterine is also described, using an epoxidation-rearrangement cascade. This work led to a revised structure of aceropterine, formulated by spectroscopic methods. Finally, the isolation and structure elucidation of a novel, cyclic lipopeptide from Pseudomonas sp. is described. The compound was obtained using a unique antibiotic crowd sourcing approach and the structure determined by spectroscopic methods and advanced Marfey’s analysis. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

Bacterial type I modular polyketide synthases (PKS) are large multifunctional enzyme systems responsible for the biosynthesis of complex polyketide natural products such as erythromycin, pikromycin, and borrelidin. Type I systems are comprised of a loading module which generally selects an appropriate acyl group starter unit, and multiple discrete extension modules, responsible for each single round of acyl group incorporation into the final polyketide core structure. These modules can exist naturally as either single discrete polypeptides, such as modules 5 and 6 from the pikromycin PKS (PikA3 and PikA4 respectively), or as multimodular polypeptides fused together by short intrapolypeptide linkers such as the loading module and the first and second extension modules of the erythromycin and pikromycin PKSs (DEBS1 and PikAI respectively). While short peptide linkers between modules on the same polypeptide facilitate the transfer of polyketide intermediates from one module to the next via their close proximity to one another, docking domains found at the C-terminus of one module and the N-terminus of the next subsequent module facilitate the needed protein-protein interactions for the passage of biosynthetic intermediates between modules on separate polypeptides. The ability to utilize docking domains in place of intrapolypeptide linkers was explored in the pikromycin and erythromycin PKSs by dissecting the tri-modular PikAI and DEBS1 polypeptides with matched docking domains. It has been shown that PikAI can be separated into two proteins at either of these linkers, only when matched pairs of docking domains from a heterologous modular phoslactomycin PKS are used in place of the intrapolypeptide linker. In both cases the yields of pikromycin produced by the S. venezuelae host mutant, which is a PikAI deletion strain were 50% of that of an S. venezuelae strain expressing the native trimodular PikAI. Additionally, expression of module 2 as a monomodular protein fused to a heterologous N-terminal docking domain was also observed to give almost a 10-fold improvement in the in vivo generation of pikromycin from a synthetic diketide intermediate. The utilization of docking domains to separate linked modules was also demonstrated in the erythromycin PKS. Expression of the first protein involved in erythromycin biosynthesis (DEBS1) with the DEBS thioesterase fused to the C-terminal (DEBS1-TE) in S. venezuelae results in the production of triketide lactone products. Separation of DEBS1-TE resulted in 50% triketide lactone production, consistent with the observations in the pikromycin system. Published work has shown that the DEBS loading module has relaxed substrate specificity, and is capable of incorporating acetate, butyrate and isobutyrate in addition to the normally observed propionate starter unit, which typically predominates. However, in the current study when the DEBS loading module is separated from module 1 with matched docking domains, a dramatic shift in the starter unit, favoring the isobutyrate derived tri-ketide lactone is observed. This apparent shift in starter unit preference for a dissected PKS system has resulted in insights into the kinetics of acyl group loading, off loading, as well as the hydrolysis and transfer from the AT to ACP domains. In addition to the separation of multimodular PKS polypeptides with docking domains, it has also been shown that the individual catalytic domains of single discrete module, BorA5 from the borrelidin PKS can be expressed as stand alone proteins while retaining catalytic functionality in vitro. This work has provided a basis for future studies of this module, which has been proposed to function iteratively, catalyzing three rounds of chain elongation.

<p> The extensive;: use of antibiotics in the clinic, veterinary medicine, and agricultures has imposed an immense selective pressure for the emergence of antibiotic resistant bacteria In order to maintain the upper hand against pathogenic bacteria, we must constantly seek new antimicrobials. Most antibiotics used in the clinic were discovered as natural products or are derivatives thereof. Therefore, we must seek means of increasing the chemical diversity of natural products in our quest for new antibiotics. Herein, we investigate methyltransferases as agents to increase chemical diversity. More specificity, we have performed biochemical studies on a tetracycline and a glycopeptide methyltransferase. </p> <p> In our studies of the putative tetracycline N-methyltransferase OxyT, we determined the conditions required to overexpress the protein in an E. coli host. Subsequently, using purified protein we examined substrate specificity using commercially available compounds. However, we were unable to detect methylation of the compounds tested and therefore we made an effort to secure a biologically relevant substrate by insertionally inactivating the oxyT gene in S. rimosus but were unsuccessful. </p> <p> In our studies of the glycopeptide N-methyltransferase MtfA, we examined the biochemical activity of this enzyme on the glycopeptide desulfo-A47934. We purified desulfo-A47934 as a fermentation product of S. toyocaensis ΔstaL and determined an extinction co-efficient of 4200 Lmol^-1cm^-1. Furthermore, based on a crystal structure of MtfA we biochemically characterized the enzyme and its four mutants Y32F, E144A, H228A, and R230A to study residues involved in substrate binding and catalysis. We demonstrated that these mutations did not alter quaternary protein structure but did lead to a significant decrease in enzyme activity as compared to the wild-type enzyme. </p> / Thesis / Master of Science (MSc)

As part of an ongoing search for bioactive natural products from the endemic rainforests and surrounding ocean in Madagascar, a total of four extracts were comprehensively studied and were found to contain novel and/or bioactive compounds. The following dissertation discusses the isolation, structure elucidation, and bioactivity studies of these isolates. The following compounds from plants of Madagascar's rainforest are discussed in the order they were studied: flavonoids and long-chain compounds from Schizolaena hystrix, a cyclohexene derivative and butenolides from Artabotrys madagascariensis, and limonoids from Malleastrum sp. From the Malagasy marine ascidian Trididemnum sp. collected in the Indian Ocean, the identification as well as the potential biosynthetic origin of polyketide derived bistramides is reported. n an attempt to explore other facets of natural products chemistry, the second part of this dissertation discusses the process of designing potential anticancer agents based on the scaffold of a natural product. The biomolecular target of these studies is an enzyme that is overexpressed in tumor cells, namely Cdc25B, whose inhibition catalyzes cell cycle arrest at the G2/M transition of the cell cycle. Several analogs of a potent Cdc25B inhibitor were synthesized and tested in the enzyme-based assay. / Ph. D.

Bioassay directed fractionation of the methyl ethyl ketone extract of Chiloscyphus rivularis yielded eight sesquiterpenoids, and detailed spectroscopic interpretation led to the assignment of their structures as 12-hydroxychiloscyphone, chiloscypha-2,7-dione, 12-hydroxychiloscypha-2,7-dione, chiloscypha-2,7,9-trione, rivulalactone, 4-hydroxy oppositant-7-one, chiloscyphone, and intermedeol. The structure and stereochemistry of rivulalactone, a novel trinorsesquiterpenoid, was confirmed by its synthesis starting from chiloscyphone. 12-Hydroxychiloscyphone, chiloscypha-2,7-dione, 12-hydroxychiloscypha-2,7-dione, chiloscypha-2,7,9-trione, rivulalactone are new. 12-Hydroxychiloscyphone showed selective bioactivity towards DNA repair-deficient yeast mutants and cytotoxicity to human lung carcinoma cells. In order to improve the activity of cytotoxic furanonaphthoquinones by affixing a hydroxyamino side chain, 2-methyl-2-[2'-(4',9'-dihydronaphtho[2',3'-b]furan-4',9'-dionyl methyl)amino]-1,3-propanediol and its analogs have been synthesized. Bioassay data showed they act by a different mechanism of action than their parental furanonaphthoquinone derivatives. / Ph. D.

As part of an International Cooperative Biodiversity Groups (ICBG) program and a continuing search for anticancer natural products from the Madagascar rainforest, twenty extracts from Madagascar were selected for investigation based on their antiproliferative activity. Bioassay-guided fractionation of five of the extracts yielded sixteen new compounds, and their structures were determined using a combination of 1D and 2D NMR experiments, including COSY, HSQC/HMQC, HMBC, and ROESY/NOESY sequences, mass spectrometry, and chemical conversion. In addition, ten known compounds were obtained from five of the extracts. Studies on the remaining extracts were suspended due to various reasons. A multi-step synthesis of the sesquiterpenoid, (7R*)-opposite-4(15)-ene-1beta,7-diol, was also described. The first chapter of this dissertation reviews the new compounds isolated from Malagasy plants and marine organism in the last two decades. Chapters II to VI discuss the isolation, structure elucidation and bioactivities of new compounds from Scutia myrtina, Cordyla madagascariensis ssp. madagascariensis, Elaeodendron alluaudianum, Cassipourea lanceolata, and Sclerocarya birrea subsp. caffra. Chapter VII describes the synthesis and bioactivity of the sesquiterpenoid,(7R*)-opposite-4(15)-ene-1beta,7-diol. The isolation of known compounds is discussed briefly in the last chapter. / Ph. D.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Cancer is one of the leading causes of death in developed countries and rising fast as a cause of death in developing countries. The increase of cancer prevalence in developing countries can be attributed to westernisation trends, with lifestyle cancers such as colorectal and lung cancer being amongst the most commonly reported malignant neoplasms. This means that the development of novel methods of treatment is essential in combatting this disease in the developing world. Combinational chemotherapy is one of the best candidates for treatment, but it is reliant on effective compounds targeting different modes of action. It also means that these compounds should be easily and cheaply available. Makaluvamines have been identified as a class of compounds that may have a novel mode of action on top of being known as topoisomerase II inhibitors. This study attempted to devise a short and concise synthetic strategy, based on reported procedures, to construct makaluvamine C analogues. This involved the introduction of a methyl group to an indole intermediate (7,8-dimethoxy-1,3,4,5-tetrahydropyrrolo[4,3,2-de]quinoline), before oxidation to a quarternized pyrroloiminoquinone (7-methoxy-5-methyl-8-oxo-1,3,4,8- tetrahydropyrrolo[4,3,2-de]quinolin-5-ium chloride). The introduction of this methyl group proved problematic, as the indole substrate proved to be difficult to handle and tended to degrade under reaction conditions. The lack of initial success prompted the deviation from the initial route by quarternizing a quinoline intermediate to form a quinolinium iodide salt (4- (dimethoxymethyl)-6,7-dimethoxy-1-methyl-5-nitroquinolin-1-ium iodide). Upon reduction to give 4-(dimethoxymethyl)-6,7-dimethoxy-1-methyl-1,2,3,4-tetrahydroquinolin-5-amine, it was discovered that the subsequent ring-closing reaction to produce 7,8-dimethoxy-5-methyl- 1,3,4,5-tetrahydropyrrolo[4,3,2-de]quinoline was still problematic. The synthesis of the target compounds has not yet been successfully completed, but will still be pursued so these compounds can be evaluated for their anticancer activity and have their mode of action tested. / AFRIKAANSE OPSOMMING: Kanker lewer van die grootste bydrae tot mortaliteit in ontwikkelde lande en is vining aan die toeneem in ontwikkelende lande. Die toename van kanker voorvalle in ontwikkelende lande kan toegedra word aan die verwesteringstendens, met kankers soos kolo-rektale- en long kanker onder die mees algemene kwaadaardige neoplasmsas wat aangemeld word – kankers wat gekoppel word aan leefstyl keuses. Dit beteken dat daar ‘n dringende nood is aan nuwe metodes van behandeling van die siekte in ontwikkelende lande. Kombinasie chemoterapie is een van die beste kandidate vir behandel, sienende dat dit gebruik maak van middels was verskillende aspekte van die siekte uitbuit. Om effektief te wees, moet die antikanker middels goedkoop en maklik beskikbaar te wees. Makaluvamines is geïdentifiseer as ‘n klas van antikanker middele wat moontlik ‘n nuwe metode van inhibisie het, tesame met hul topoïsomerase II inhibisie. Hierdie study het daarom gepoog om ‘n korter en meer bondige sintetiese roete saam te stel, wat gebaseer is op literatuur prosedures, om analoeë van makaluvamine C te produseer. Dit het die aanhegging van ‘n metiel group aan ‘n indool tussenproduk (7,8-dimetoksie-1,3,4,5- tetrahidropirolo[4,3,2-de]kinolien) behels, gevolg deur die oksidasie tot die kwaternêre piroloiminokwinoon (7-metoksie-5-metiel-8-oxo-1,3,4,8-tetrahidropirolo[4,3,2-de]kinolin-5-ium chloried). Om hierdie metiel groep aan te voeg was, nietemin, problematies, aangesien die indool produk moeilik was om te hanteer sienende dat dit onder reaksie toestande gedegradeer het. Die aanvanklike onsuksesvolle pogings het daartoe gelei dat die sintetiese roete herdink was en is aangepas om eerder gebruik te maak van ‘n kinolinium jodied sout (4-(dimetoksiemetiel)-6,7-dimetoksie-1-metiel-5-nitrokinolin-1-ium jodied). Die reduksie van hierdie sout en agtereenvolgende annulasie reaksie om 7,8-dimetoksie-5-metiel-1,3,4,5- tetrahidropirolo[4,3,2-de]kinolien te vorm was egter steeds problematies. Die sintese van die beoogde produkte was tot dusver nog nie suksesvol nie, maar sal egter steeds aangedurf word om hulle ten einde suksesvol te sintetiseer en dan te stuur om hulle biologiese eienskappe te toets. Dit sluit hulle antikanker aktiwiteit in asook hul metode van inhibisie.