O fato de esteróides de plantas apresentarem tanto funções fisiológicas quanto ecológicas, talvez tenha sido fator limitante de seu uso em quimiossistemática. Até hoje, os trabalhos do nosso grupo têm se restringido a marcadores com apenas funções ecológicas. Neste trabalho, utilizamos esteróides como marcadores quimiossistemáticos, com o intuito de descobrir se eles poderiam ser analisados com a metodologia usual desenvolvida por nosso grupo. Devido à ampla distribuição dessas moléculas, limitamos o trabalho a angiospermas. Inicialmente, realizamos um levantamento bibliográfico no Chemical Abstracts (até dezembro de 1986) e, em uma segunda etapa, codificamos e armazenamos os dados em um microcomputador IBM-PC. A presença de alguns esteróis (campesterol, sitosterol, colesterol e estigmasterol) em praticamente todos organismos, levou-nos a eliminar deste trabalho esse grupo de substâncias, pois características imutáveis não têm valor quimiossistemático. Baseados em semelhanças estruturais, separamos os esteródes em oito tipos, a saber: esteróides, brassinolídeos, ecdisonas, sapogeninas, witanolídeos, pregnanos, cardenolídeos e bufodienolídeos. Esta classificação foi legitimada por análises estatísticas (testes não-paramétricos). Os índices de oxidação e especialização de esqueleto (metodologia do nosso grupo) não revelaram muitas diferenças dentro de cada tipo esteroidal. De um modo geral, estes tipos esteroidais apresentam-se predominantemente distribuídos em certos grupos de angiospermas. Este é, por exemplo, o caso das ecdisonas em Caryophyllales e Lamiales, dos witanolídeos em Solanales e, dos cardenolídeos e pregnanos em Gentianales. Por outro lado, enquanto superordens, como Magnoliiflorae, apresentam apenas esteróis (tipo menos oxidado e menos especializado), outras superordens, como Ranunculiflorae, Solaniflorae e Liliiflorae, apresentam uma fantástica diversidade de tipos. As monocotiledôneas se caracterizam pela presença de saponinas, e os outros tipos (exceto esteróis) aparecem com distribuição esparsa. Comparando nossos dados com os de pteridófitas gimnospermas (apenas esteróis e ecdisonas), podemos concluir que a diversidade dessa classe de micromoléculas em angiospermas coloca os esteróides na posição de uma característica evoluída de plantas. / Plant steroids perform not only ecological, but also physiological functions. Hence it was thought a priori that their use as chemosystematic markers should be of doubtful value. The present work aimed to evaluate the correctness of this concept. In view of the vast natural distribution of steroids we restricted our efforts to the angiosperms. Initially we undertook a literature survey in Chemical Abstracts (up to 1986) and then we codified and stored the data in a microcomputer IBM-PC. Some of the steroids (campesterol, colesterol, sitosterol and stigmasterol) occur practically in alI organisms. Since constant characters are of little systematic value, such compounds were omitted from our study. According to structural analogies we separated plant steroids in eight types: simple steroids, brassinolides, ecdysons, sapogenins, withanolides, pregnanes, cardenolides and bufodienolides. This classification was authenticated by statistical analyses (non parametric tests).The skeletal speciaIizations and the oxidation levels (according to methodology developed by our group) did not differ greatly wthin each steroidal group. However the general distribution of steroids in angiosperms is quite selective. This is the case e.g. for ecdysons in Caryophyllales and Lamiales. On the other hand, while superordens such as Magnoliiflorea are characterized only by steroids (the least specialized and oxygenated type), other superordens such as Ranunculiflorae, Solaniflorae and Liliiflorae are characterized by a very considerable number of types. The monocotyledons contain chiefly saponins, other types (except steroids) appearing more sparsly distributed. In contrast to angiosperms pteridophytes and gymnosperms posses only few steroidal types (simple steroids and ecdysons). Hence diversity of this micromolecular class is an indication of phylogenetic advance in the plant kingdom.

Na última década, o estudo de espécies ativas de oxigênio e nitrogênio e o seus papeis em um grande número de patologias revelou que substâncias antioxidantes são capazes de prevenir os efeitos do estresse oxidativo. A reação quimiluminescente de luminol e peróxido de hidrogênio, na presença de hemina como catalisador, tem sido utilizada como método de avaliação de atividade antioxidante, uma vez que a emissão pode ser suprimida por este tipo de substância. Foi realizado um estudo cinético para estabelecer as condições experimentais do ensaio. As concentrações dos reagentes foram variadas em diferentes condições experimentais na ausência e na presença de antioxidantes, e os resultados obtidos levaram ao melhor entendimento acerca do sistema. Um novo método para o tratamento de dados foi também utilizado, permitindo a correlação entre o efeito antioxidante e o número de fótons suprimidos. Vários antioxidantes conhecidos (trolox, ácido ascórbico e ácido úrico) foram utilizados para estabelecer a metodologia. A atividade antioxidante foi calculada a partir da correlação entre a área de supressão obtida e a concentração de antioxidante, usando trolox como composto de referência. Empregando esta metodologia foi possível determinar a atividade antioxidante dos extratos e do produto majoritário isolado de Photomorphe umbellata, o 4-nerolidicatecol. / In the last decade the study of active oxygen and nitrogen species and their role in a large number of chronicle diseases, including cancer, heart disease and even aging itself, revealed that natural and synthetic antioxidants are able to prevent the effects caused by oxidative stress. Several methods can be used to evaluate the total antioxidant activity in body fluids, complex mixtures and isolated substances. Simple trapping assays can quantify the total antioxidant content in a sample, which is expressed as TRAP (total radical-trapping potential) or TEAC (trolox equivalent antioxidant capacity) and these indexes are well accepted due to its high sensitivity and operational facilities. The determination of the antioxidant potential of plant extracts and isolated natural products may constitute a simple tool to evaluate the potential biological activity of plant constituents. The chemiluminescence reaction of luminol and hydrogen peroxide in the presence of hemin as catalyst has been used as the method to evaluate the antioxidant activity, since the chemiluminescence emission can be suppressed by antioxidants, and a linear relationship between antioxidant concentration and the observed induction time is obtained. A kinetic study was performed to establish the ideal experimental conditions for the assay. The reactant concentrations were varied in the absence and the presence of antioxidants, and the results lead to a better understanding of the system. A new method for data treatment is also used, which allows the correlation of the antioxidant effect to the number of photons suppressed. Several well-known antioxidants (trolox, ascorbic and uric acid) were used to establish the methodology. TRAP values were calculated from the correlations between the number of photons suppressed and the antioxidant concentration, using trolox as reference compound. Using this methodology we were able to determine the antioxidant activity of Photomorphe umbellata extracts, and of its isolated major compound, 4-nerolidylcatechol.

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

Trypanosomiasis and Leishmaniasis are tropical diseases caused by the parasites Trypanosoma and Leishmania, that cause severe medical and economical problems for millions of people in the developing world. Trypanosomiasis can be divided into African and American trypanosomiasis, which are caused by Trypanosoma brucei and Trypanosoma cruzi respectively. There are more than 20 different species of Leishmania worldwide that cause Leishmaniasis, but the most severe infection, visceral leishmaniasis, is caused by Leishmania donovani. Both diseases are transmitted by blood sucking insects like the tsetse fly and the sand fly. The majority of existing drugs for trypanosomiasis and leishmaniasis are either too toxic or have low efficacy, and in some cases parasites have also developed resistance. There is therefore a pressing need to develop new chemotherapeutic agents, and in this context, the enzyme trypanothione reductase (TryR) has emerged as an attractive validated target for drug design. The natural product cadabicine, extracted from the plant Cadaba farinosa, is a diphenyl ether-containing macrocyclic spermidine alkaloid which has been identified as a potential inhibitor of TryR by virtual screening. In order to investigate the potential of cadabicine as a TryR inhibitor, an efficient synthetic route to the natural product was delivered. This work was focused on the preparation and combination of three key synthetic units, namely an orthogonally protected spermidine derivative and two functionalised cinnamic acid units. This approach lead to the formation of the macrocycle by an intramolecular nucleophilic aromatic substitution followed by a convenient conversion to the natural product. In the same manner cyclic and noncyclic analogues of cadabicine were prepared, in order to examine the structure-activity relationship of these alkaloids to TryR.

Na última década, o estudo de espécies ativas de oxigênio e nitrogênio e o seus papeis em um grande número de patologias revelou que substâncias antioxidantes são capazes de prevenir os efeitos do estresse oxidativo. A reação quimiluminescente de luminol e peróxido de hidrogênio, na presença de hemina como catalisador, tem sido utilizada como método de avaliação de atividade antioxidante, uma vez que a emissão pode ser suprimida por este tipo de substância. Foi realizado um estudo cinético para estabelecer as condições experimentais do ensaio. As concentrações dos reagentes foram variadas em diferentes condições experimentais na ausência e na presença de antioxidantes, e os resultados obtidos levaram ao melhor entendimento acerca do sistema. Um novo método para o tratamento de dados foi também utilizado, permitindo a correlação entre o efeito antioxidante e o número de fótons suprimidos. Vários antioxidantes conhecidos (trolox, ácido ascórbico e ácido úrico) foram utilizados para estabelecer a metodologia. A atividade antioxidante foi calculada a partir da correlação entre a área de supressão obtida e a concentração de antioxidante, usando trolox como composto de referência. Empregando esta metodologia foi possível determinar a atividade antioxidante dos extratos e do produto majoritário isolado de Photomorphe umbellata, o 4-nerolidicatecol. / In the last decade the study of active oxygen and nitrogen species and their role in a large number of chronicle diseases, including cancer, heart disease and even aging itself, revealed that natural and synthetic antioxidants are able to prevent the effects caused by oxidative stress. Several methods can be used to evaluate the total antioxidant activity in body fluids, complex mixtures and isolated substances. Simple trapping assays can quantify the total antioxidant content in a sample, which is expressed as TRAP (total radical-trapping potential) or TEAC (trolox equivalent antioxidant capacity) and these indexes are well accepted due to its high sensitivity and operational facilities. The determination of the antioxidant potential of plant extracts and isolated natural products may constitute a simple tool to evaluate the potential biological activity of plant constituents. The chemiluminescence reaction of luminol and hydrogen peroxide in the presence of hemin as catalyst has been used as the method to evaluate the antioxidant activity, since the chemiluminescence emission can be suppressed by antioxidants, and a linear relationship between antioxidant concentration and the observed induction time is obtained. A kinetic study was performed to establish the ideal experimental conditions for the assay. The reactant concentrations were varied in the absence and the presence of antioxidants, and the results lead to a better understanding of the system. A new method for data treatment is also used, which allows the correlation of the antioxidant effect to the number of photons suppressed. Several well-known antioxidants (trolox, ascorbic and uric acid) were used to establish the methodology. TRAP values were calculated from the correlations between the number of photons suppressed and the antioxidant concentration, using trolox as reference compound. Using this methodology we were able to determine the antioxidant activity of Photomorphe umbellata extracts, and of its isolated major compound, 4-nerolidylcatechol.

Natural products represent a large and diverse array of molecules. Natural products and their derivatives play important roles in the human sphere, serving as pharmaceuticals, biofuels, and more. However, the structural complexity of many promising natural products prohibits industrial production sufficient to make full use of their capabilities. The challenge posed by natural products has spurred many advances in multiple fields. Despite these achievements, ignorance of the native metabolic pathways and inefficiencies in manipulating the genes involved has slowed the ability of science to capitalize on the enormous potential of natural products. In Chapter 1, we begin by surveying the fields concerned with the production or variation of natural products. This begins with organic synthesis, continues with in vivo and in vitro biocatalytic methods, and concludes with the “combination” techniques that seek to unite the strengths of biocatalysis and organic chemistry: precursor-driven biosynthesis, mutasynthesis and semi-synthesis. After examining the advantages and disadvantages of the extant technologies, in Chapter 2 we describe a novel strategy to develop semi-synthetic routes to underexplored classes of natural products. While it employs features of existing techniques, our strategy originates from a fundamentally different conception of natural product production, which looks away from the native precursors of a single target, and towards versatile precursors amenable to multiple forms of chemical modification. We then carry out a demonstration of this strategy by first biosynthetically producing 2Z,7E-farnesol from heterologously expressed Mycobacterium tuberculosis synthetases, and subsequently derivatizing this unnatural precursor into a set of novel Ambrox© analogs. Complex biocatalytic applications rely on DNA manipulation technologies to rapidly construct and diversify metabolic pathways. When components of the targeted pathway are unknown or poorly understood, the creation of large libraries of variant pathways can be employed to circumvent these limitations and rapidly develop the desired phenotype. In Chapter 3, we harness our existing library building technology, Reiterative Recombination, to the yeast sexual reproduction cycle for the purpose of combining separately constructed library strains via simple mating and chromosome segregation into an exponentially larger combinatorial library. This chapter describes the design, construction, and initial validation of this system, termed Reiterative Segregation. Finally, in Chapter 4, we explore possible elaborations of the Reiterative Segregation design, and work towards combining libraries of alternative sugar metabolic pathways as an application relevant to biofuel production.

>Magister Scientiae - MSc / Antimicrobials are chemical compounds that destroy or inhibit the growth of microorganisms. The majority of these antimicrobials are actually natural products or natural product derived with key examples being the pioneer antibiotics penicillin and cephalosporin. Antimicrobials are an extremely important class of therapeutic agents; however, the development of drug resistance and slow pace of new antibiotic discovery is one of the major health issues facing the world today. There is therefore a crucial need to discover and develop new antibacterial agents. In this study, the potential of marine algae as a source of new antibiotics was explored. Crude organic extracts and chromatographic fractions obtained from small-scale extraction of 17 different marine algae were used to prepare a pre-fractionated library that would be tested against several disease causing microorganisms. The activity of the pre-fractionated library and purified compounds was determined against a panel of drug resistant microorganisms namely Acinetobacter baumannii ATCCBAA®-1605™, Enterococcus faecalis ATCC® 51299™, Escherichia coli ATCC® 25922™, Staphylococcus aureus subsp. aureus ATCC® 33591™ and Candida albicans ATCC® 24433™. Finally, cytotoxicity tests of 50 selected library extracts and isolated compounds were done against two cell lines namely MCF-7 (breast cancer) and HEK-293 (kidney embryonic). Based on their antimicrobial activity and interesting chemical profiles, the seaweeds Plocamium sp. and Stypopodium multipartitum were selected for further study. Three new and unusual halogenated monoterpenes (4.16, 4.17 and 4.18) were isolated from Plocamium sp., and an unusual meroditerpenoid (5.8) was isolated from Stypopodium multipartitum. The metabolites were purified using preparative (silica gel) chromatography as well as semipreparative normal phase HPLC. The structures of purified compounds were determined from spectroscopic data, including nuclear magnetic resonance (NMR) spectroscopy. A small library of 153 fractions was generated from collections of South African marine algae. Pre-fractionated crude extracts showed excellent antimicrobial activity against all microbes but particularly against Staphylococcus aureus. The compounds were generally active against the Gram positive bacteria and the yeast. In conclusion, three antimicrobial halogenated monoterpenes and an unusual monoterpene were isolated from a Plocamium sp. and Stypopodium multipartitum respectively. Antimicrobial activity of crude fractions was excellent but that of isolated compounds was not as great as anticipated.

Streptomyces are known to produce a large variety of antibiotics and other bioactive compounds with remarkable industrial importance. Streptomyces sp. COUK1 was found as a contaminant on a plate in which Rhodococcus erythropolis was used as a test strain in a disk diffusion assay and produced a zone of inhibition against the cultured R. erythropolis. The identity of the contaminant was confirmed as Streptomyces through 16S rRNA sequencing. This Streptomyces produces a strong inhibitory compound in different growth media. A culture extract from inorganic salts starch agar was found to be very active; producing a large zone of inhibition against several Gram positive and Gram negative test strains. The active molecules in this extract have been detected via TLC and bioautography. The difference in the antibacterial activity and chromatographic properties of extracts recovered from different growth media suggests that this Streptomyces strain could produce more than one type of inhibitory compound.

No description available.

In spite of significant shortcomings among existing clinical antifungal agents, the rate of development of new therapeutics has been sluggish, and the mortality rate caused by fungal diseases has remained almost unchanged. Natural products have provided useful templates for the development of several of the most important therapeutic antifungal agents. In particular, fungi have been rich sources of antifungal natural products, and many fungal species remain chemically underexplored. Our research program has focused on fungicolous and coprophilous fungal niche groups. These types of fungi often show antagonistic effects toward host or competitor fungi—a phenomenon hypothesized to be associated in part with the production of antifungal metabolites. Earlier results reported from our research program have shown that studies of such fungi can be effective approaches to the discovery of new bioactive natural products, including antifungal agents. During our continued efforts to discover new antifungal and antiinsectan natural products, diverse fungal metabolites were obtained from complex fermentation extracts by use of various chromatographic methods. In addition to previously known compounds and simple analogues thereof, structurally interesting new metabolites were encountered. Those isolated from fungicolous fungi include ten new caryophyllene-type sesquiterpenoids from a Pestalotiopsis sp., three of which contain previously undescribed ring systems or new skeletons. The remainder are oxidized analogues of punctaporonins. Seven new polyketide-derived metabolites were obtained from another Pestalotiopsis isolate, in this case, P. disseminata, and two unusual ring systems were identified. A distinctive biosynthetic pathway was proposed for these seven polyketides. Members of another class of polyketides (pyrenocines), which contain pyrone or thiopyran units, were encountered from Penicillium paxilli. One of the three new pyrenocine analogues obtained contains an adenine unit—a rare feature among fungal secondary metabolites. Chemical investigation of another Penicillium sp., P. lanosum, afforded a new fumiquinazoline analogue, as well as a compound previously described in a thesis of a member of our research group. In work described here, the original stereochemical assignment was revised, and the compound was renamed as lanosindole. Metabolites isolated from this fungicolous Penicillium isolate have amino acid origins in common, and two of them showed antiinsectan activity. Seven new polyketide alcohols were obtained from the coprophilous fungus Podospora appendiculata. Two of them contain a tetrahydropyran unit and three are acyclic, differentiating them from other known members of this class. Finally, funiculosin B, an antifungal metabolite of mixed biogenetic origin containing a rare tetrahydroxycyclopentanyl moiety, was isolated from a flower-colonizing isolate of Capnodium sp. The structures of the compounds described above were determined mainly by analysis of NMR and MS data. Synthetic modification, X-ray crystallographic analysis, and ECD data analysis in conjunction with molecular modeling were applied to their stereochemical assignments. The results described in this thesis indicate that fungicolous and coprophilous fungi are prolific producers of new natural products, some of which display activity in assays of medical and agricultural relevance. Although most of the new compounds described here were inactive against pathogenic fungi, the rich diversity of chemistry encountered suggests potential for this ecology-based approach in the discovery of new bioactive natural products.