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This thesis presents results from investigations of secondary metabolites from marine organisms. The first part pursued the isolation of new compounds from soft bodied marine organisms, the study of aqueous extracts from the different algae and soft bodied marine organisms permitted the selection of one extract from the red alga <i>Laurencia </i>sp., from which six new sesquiterpenes cyclic ethers were isolated. The investigation of the sponge <i>Stylotella aurantium </i>collected from Fijian waters, resulted in isolation of seven previously reported pyrrole alkaloids. The second part focused on the exploration of three novel actinomycete microbes isolated from Mariana Trench sediment, collected at depth 10,898 m (Challenger Deep; 11°19’911’’ N; 142°12’372’’E) for the production of bioactive compounds. Seven novel phenazine alkaloids, dermacozines (A-I), were isolated from the fermentation broth of two Mariana Trench microbes <i>Dermacoccus abyssi</i> strain MT1.1 and <i>Dermacoccus </i>strain MT1.2. The investigated cytotoxic activity against leukaemia cell line (K562) showed that dermacozine F and G have moderate activity with IC_50s of 9 and 7 <i>μ</i>M, respectively, while the highest radical scavenger activity was observed with dermacozine C with an IC₅₀ value of 21 <i>μ</i>M. Investigation of <i>Micromonspora</i> sp. isolated from Mariana Trench sediment led to the isolation of six compounds including: one unusual depeptide and the known deferoxamine together with four known diketopiprazines. Finally, the last part of work focused on the investigation of molecular basis of cold temperature and high pressure adapted growth in extremophilic microbes such as <i>Photobacterium profundum sp.</i> The overall aim of this project was to understand more about the high pressure and cold-adapted growth of two novel <i>P. profundum</i> strains SS9R (Piezophile) and 3TCK (Piezosensitive) by studying the surface of polysaccharides which play an important role in the cold-adapted growth of <i>P. profundum</i> <i>SS9.</i>

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of biophysics and physical chemistry Candidate: Šárka Slezáková Supervisor: Ing. Vladimír Kubíček, CSc. Title of diploma thesis: HPLC Determination of Benzimidazoles In this thesis we have investigated the possibility of establishing benzimidazole with HILIC chromatography using a chromatographic column Ascentis Express HILIC 10.0 cm x 3.0 mm; 2.7 microns. Two groups of benzimidazoles were tested. The first one was focused on albendazole and its metabolites. Experiments with these substances did not produce satisfactory results, because the mobile phase composition which enables separation of the studied analytes was not found. The second group was formed by flubendazole and its reduced and hydrolyzed form. In this case, several mobile phase compositions were tested. Finally, distribution of individual analytes in a mixture, using a mobile phase ACN:HCOOH 0.03 mol/l (90:10), was successfully achieved. Ricobendazol was chosen as an internal standard. When separation conditions were found, calibration curve for the determination of reduced flubendazole in biological samples was subsequently constructed using ricobendazol as the internal standard with the use of fluorescence detection. Keywords: HILIC, HPLC, albendazole,...

Zuzana Jindřišková The Study of Secondary Metabolites in Explant Culture of Trifolium pratense L. The basic prerequisite for a successful elicitation that is used to increase the production of secondary metabolites is, among others, finding a suitable elicitor, its concentration and optimal duration of effect of the elicitor on the plant in vitro culture, which was the main subject of this diploma thesis. The focus of our observations was the influence of 6-, 24- , 48- and 168-hour effect of nickel chloride solution (in the concentrations of 0.1 mmol, 1 mmol, 10 mmol and 100 mmol) and zinc sulphate (in the concentrations of 0.1 µmol, 1 µmol, 10 µmol a 100 µmol) on the production of flavonoids and isoflavonoids in the suspension culture of Trifolium pratense L. variety Tempus. The culture was cultivated on the Gamborg nutrien medium with the addition of 2 mg.l-1 2,4- dichlorophenoxyacetic acid and 2 mg.l-1 6- benzylaminopurine at 25řC and the light period of 16 hours light/ 8 hours dark. The maximum content of flavonoids, which was found out by the photometric determination of the Czech Pharmacopoeia 2009, was proved in the suspension culture of Trifolium pratense L. variety Tempus (0.406%) after 48-hour elicitation of nickel chloride solution in the concentration of 0.1 mmol, when there was a...

A Dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the Degree of Master of Science. / An outline of reported synthetic routes to the Lupine alkaloids, epilamprolobine [2] and lamprolobine [3] and a review of the use of vinylogous amides and urethanes as precursors for the synthesis of alkaloids are presented in Chapter 1. This is followed by a presentation of our strategy for synthesis of the two Lupine alkaloids. Vinylogous cyanamide intermediate 1- (3-hydroxypropyl) -2- cyanomethylenepiperidine [68] plays a key role in this strategy, since exploitation of its ambident nucleophilicity forms the central theme of this project, The successful route to the intermediate [68] involved the preliminary preparation of the tertiary thiolactam, 1-(2- ethoxycarbonylethyl)piperidine-2-thione [83][ by thiation of the secondary lactam 2-piperidinone [72] and conjugate addition at nitrogen with ethyl acrylate in a Michael reaction. Sulphur extrusion of the salt made from [83] and bromoacetonitrile and subsequent reduction of the ester group provided the pivotal vinylogous cyanamide intermediate. A number; of alternative routes based on 5- bromopentanoic acid [80], 1-allyl-2-piperidinone [73] and thiolactams [84J and [105] were unsuccessful. Cyclisation of the intermediate [68] was achieved by an intramolecular c-alkylative ring closure via the corresponding tosylate [l16] to forln an unsaturated functionalised quinolizidine [69]. Stereoselective carboncarbon double bond reduction and nitrile reduction resulted in the synthesis of two quinolizidines. lupinamine [11] and epilupinamine [112]. Further transformations led to the formation of the derivatives, N-acetyllupinamine [113] and N-acetylepilupinamine [114], and also to the target alkaloids, epilamprolcbine [2] and lamprolobine [3]. / Andrew Chakane 2018

The projects described in this dissertation are focused on compounds derived from marine organisms collected from the western Atlantic marine environment. Chapter 1 provides an introduction to the study of natural products chemistry, marine natural products, and overview of the research undertaken from natural product chemists. Chapter 2 describes the isolation and structure elucidation of a series of rare diterpenoids from the gorgonian Briareum asbestinum, together with their conformational analysis and biosynthetic interconversions. These rare diterpenes from Briareum asbestinum are linked by an unusual transannular oxa-6π electrocyclization which is described in detail and this work demonstates the biomimetic hemisynthesis of briareolate esters L (19) to B (22) achieved via an intermediary, briareolate ester G (2), through a controlled set of photoinduced isomerizations and a unique photochromic transannular oxa-6π electrocyclization. This work focuses largely on the mechanistic understanding of the photochemical production of these briarane diterpenoids and illustrates a unique UVA/UVC, photochromic switch which induces a transannular oxa- 6π electrocyclization. Chapter 3 describes the assay-guided isolation of marine antioxidants. This chapter focuses on the screening of marine organism extracts using the Ferric Reducing Antioxidant Power (FRAP) assay for antioxidant activity guided isolation of marine natural products. The chapter concludes with the activity guided isolation and structural elucidation of 1-O-palmitoyl-2-O-myristoyl-3-O-(6-sulfo-α-D-quinovopyranosyl)- glycerol (40) to show direct antioxidant potential through FRAP analysis. Chapter 4 describes the isolation, structural elucidation and pharmacological evaluation of the novel secondary metabolites iso-PsA(45), Iso-PsC (46), iso-PsD (47) as well as known Pseudopterosins A(41), B(42), C(43), D(44), K(48), K2’OAc(49), K2’OAc(50). These secondary metabolites were evaluated for both cytotoxicity. The chapter concludes with the screening of these compounds as αβ-amyloid fibril modulators utilizing atomic force microcopy (AFM). / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

Unbranched heterocytous cyanobacteria exhibit complex filament and colony architectures and variable life strategies from symbionts to free living planktic and non-planktic species. They are counted among microbial groups showing an extensive production of secondary metabolites, resulting in both pharmaceutically important and toxic compounds. The main focus of this thesis is to broaden our knowledge on bioactive secondary metabolite potential in this widespread group of cyanobacteria. An effective combination of methods including whole genome sequencing, bioinformatic analysis, and analytical chemistry techniques are applied to accomplish this task. The discrepancies in distribution of various classes of compounds among ecological groups defined by different life strategies are discussed. Additionally, the thesis endeavours to test multidisciplinary approaches to tackle taxonomic assignments of unresolved unbranched heterocytous cyanobacteria using morphological, phylogenetic and ecophysiological methods, including a meta-analysis of morphological traits.

Many bacteria secrete secondary metabolites, whose production is decoupled from active growth in laboratory cultures. Historically, the advantages of secondary metabolite production have mostly been explored in the context of cellular interactions, such as antibiotic effects on competing organisms, damage caused to host tissues during infection, or cell density-dependent signaling. However, recent studies in the opportunistic pathogen Pseudomonas aeruginosa have brought into focus the physiological effects of secondary metabolites on their producer and their implications for multicellular behavior. P. aeruginosa produces antibiotics called phenazines, which can act as mediators to transfer reducing power to an extracellular oxidant and thereby support bacterial survival when oxygen is not accessible. In the crowded environments of biofilms, communities of bacteria surrounded by self-made matrices, this property of phenazines could support energy generation for cells in anoxic subzones. As biofilm formation is a hallmark of P. aeruginosa colonization at various infection sites within the body, I was motivated to investigate the regulation of phenazine production at the level of synthesis and transport, the distribution of phenazines in P. aeruginosa biofilms, and the effects of individual phenazines on P. aeruginosa gene expression and colony biofilm morphogenesis. As part of this work, a novel electrochemical device was developed that enables direct detection of phenazines released from intact colony biofilms. Application of this device and other electrochemical techniques enabled detection of the reactive phenazine intermediate 5-Me-PCA, which was found to be the primary phenazine affecting P. aeruginosa colony morphogenesis. The production of this phenazine was found to be sufficient for activation of the redox-active transcription factor SoxR and full induction of the RND efflux pump MexGHI-OpmD. Finally, results described in this thesis show that 5-Me-PCA is transported by MexGHI-OpmD, constituting a unique demonstration of the self-protective role of an efflux pump in a gram-negative antibiotic-producing bacterium. These findings raise broad questions about the effects of individual phenazines on biofilm cell physiology and have implications for the contributions of individual phenazines to virulence and survival during infection. The technology developed also has potential applications in novel diagnostic and therapeutic approaches. Chapters 1-3 introduce and highlight advances made in understanding secondary metabolite production, with a focus on P. aeruginosa. Chapter 1 provides an introduction to antibiotic production, the concept of self-resistance and other physiological effects of antibiotics in their producers, and infections caused by P. aeruginosa. Chapter 2 reviews recent studies that have brought into focus the physiological effects of secondary metabolites on their producers and their implications for multicellular behavior. Chapter 3 provides an overview of our current understanding of the regulation of phenazine production in pseudomonads and other bacterial species. Chapter 4 describes the development of an integrated circuit-based platform for detection of redox-active metabolites released from multicellular samples, and demonstrates its application to mapping phenazines released from P. aeruginosa biofilms. The study described in Chapter 5 investigates the role of the P. aeruginosa SoxR regulon, which is induced by phenazines, in phenazine transport and shows that the understudied reactive phenazine 5-methylphenazine-1-carboxylic acid (5-Me-PCA) is transported by the RND efflux pump MexGHI-OpmD and is required for wild-type biofilm formation. Chapter 6 describes the development of an assay for 5-Me-PCA production and studies exploring the role of the regulator PsrA in controlling phenazine biosynthesis. Chapter 7 provides an overview of the findings and open questions to be explored in future research. The P. aeruginosa genome contains two nearly identical operons that encode biosynthetic enzymes for the production of phenazine-1-carboxylic acid, the precursor to all of the other phenazines. The study described in Appendix A characterizes the respective contributions of these operons to phenazine production in shaken liquid cultures and biofilms. Appendix B presents evidence that electron acceptor availability influences, and is influenced by, the morphogenesis of P. aeruginosa colony biofilms. Finally, Appendix C describes a screen for commercially available compounds that inhibit production of the phenazine pyocyanin by P. aeruginosa. Together, these findings reveal the unique physiological roles of specific phenazine-related genetic loci and regulatory proteins and of 5-Me-PCA, a phenazine that was previously overlooked due to the technical challenges associated with its detection. They have also uncovered novel aspects of phenazine production in both shaken liquid cultures and biofilms relevant for the development of therapeutics.