Concise syntheses of the natural products brosimones A and B have been achieved using sequential dehydrogenative Diels-Alder (DHDA) cycloadditions. The syntheses employ either Pt/C-cyclopentene or DDQ to effect dehydrogenation of prenylchalcone substrates in combination with silver nanoparticles (AgNP’s) to promote subsequent Diels-Alder cycloadditions. A concise, biomimetic approach to sorbiterrin A has been developed employing consecutive Michael additions of a 4-hydroxypyrone to a sorbicillinol derivative and silver nanoparticle-mediated bridged aldol/dehydration to construct the [3.3.1] ring system. The relative stereochemistry of sorbiterrin A was unambiguously confirmed by X-ray crystallographic analysis. Metal-catalyzed, double Claisen rearrangement of a bis-allyloxyflavone has been utilized to enable a concise synthesis of the hydrobenzofuro[3,2-b]chromenone core structure of the natural products sanggenon A and sanggenol F. In addition, catalytic, enantioselective [4+2] cycloadditions of 2’-hydroxychalcones have been accomplished using B(OPh)3/BINOL complexes. Asymmetric syntheses of the flavonoid Diels-Alder natural products sanggenons C and O have been achieved employing a stereodivergent reaction of a racemic mixture (stereodivergent RRM) involving [4+2] cycloaddition. Diaporine is a natural product containing a novel epoxyquinol dimer framework. An efficient annulation involving pyrone addition to a quinone has been developed for rapid assembly of the γ-naphthopyrone core structure. Dimerization was achieved through a Pd(II)-mediated dehydrogenative coupling. A natural product and precursor to diaporine, aurofusarin, was synthesized in excellent yield through an oxidation and demethylation sequence. In addition, diastereoselective epoxidation of aurofusarin was achieved using a phase transfer catalytic system.

O estudo fitoquímico de Peperomia oreophila revelou a presençca de duas lignanas furofurânicas (7R, 8R, 7R, 8R)-3,4,5-trimetóxi-3,4-metilenodioxi-5-metóxi- 8.8,7.O.9,7.O.9-lignana (1), (7R, 8R, 7R, 8R)-3,4,5-trimetóxi-3,4,5-trimetóxi- 8.8,7.O.9,7.O.9-lignana (2); as duas amidas (2E)-N-isobutil-3-(5-metóxi-7,8- benzodioxol-1-il)acrilamida (3), (2E)-N-isobutil-3-(3,4,5-trimetóxifenil)acrilamida (4), três derivados de acido cinâmico (2E)-3-(3,4,5-trimetóxifenil)acrilato de metila (5), (2Z)-3- (3,4,5-trimetóxifenil)acrilato de metila (6), (2E)-3-(5-metóxi-7,8-benzodioxol-1-il)acrilato de metila (7); os dois policetídeos fenólicos [(2E)-3,7-dimetilocta-2,6-dien-1-il]-5- metil-2-(3-metilbut-2-en-1-il)benzeno-1,3-diol (8) (inédita) e [(2E)-3,7-dimetilocta- 2,6-dien-1-il]-2,2,7-trimetil-2H-cromen-5-ol (9); de P. arifolia: o policetídeo fenólico [(2E)-3,7-dimetilocta-2,6-dien-1-il]-5-metil-2-(3-metilbut-2-en-1-il) benzeno-1,3-diol, isolada também de P. oreophila (10) (inédita); de P. urocarpa: o policetídeo fenólico 5- metil-2-[(2E,6E)-3,7,11-trimetildodeca-2,6,10-trien-1-il] benzeno-1,3-diol (11) e o ácido 2,4-dihidróxi-6-metil-3-[(2E,6E)-3,7,11-trimetildodeca-2,6,10-trien-1-il] benzóico, (12); de P. nitida: o fenilpropanoide apiol (1-alil-3,6-dimetóxi-10,11- benzodioxol) (13), os cromenos 7-hidróxi-2,2,5-trimetil-2H-cromeno-carboxilato de metila (14) e o 7-metóxi-2,2,5-dimetil-2H-cromeno-6-carboxilato de metila (15). O policetídeo 2-hidróxi-4,6-dimetóxiacetofenona, principal metabólito das folhas de P. glabella, teve sua biossíntese investigada utilizando-se como precursores o acetil-CoA e o malonil-CoA. Foram realizados estudos de otimização da atividade de policetídeo sintase (PKS) em função do pH, tempo de reação, temperatura e saturação de substratos, além de estudos da variação circadiana. Estudos de genes de PKS resultaram em amplificações cujo seqüenciamento poderá determinar a identidade dessas regiões e homologia entre as seqüências dessas Peperomias e a região KS do gene AviM de Streptomyces viridochromogenes que expressa o ácido orselínico / The phytochemical investigation carried out on Peperomia oreophila revealed the accumulation of two furofuran lignans (7R,8R,7R,8R)-3,4,5-trimethoxy-3,4- methylenedioxy-8.8, 7.O.9, 9.O.7-lignan (1), (7, 8R, 7R, 8)-3,4,5-trimethoxy-3,4,5- trimethoxy-8.8-7.O.9, 9.O.7-lignan (2); two amides (2´E)-N-isobutyl-3´-(5-methoxy-7,8- benzodioxol-1-yl) acrylamide (3), (2E)-N-isobutyl-3-(3,4,5-trimethoxyphenyl)acrylamide (4), three derivate cinâmic acid methyl (2E)-3-(3,4,5-trimethoxyphenyl)acrylate (5), methyl (2Z)-3-(3,4,5-trimethoxyphenyl)acrylate (6), methyl (2E)-3-(5-methoxy-7,8- benzodioxol-1-yl)acrylate (7); two phenolic polyketides [(2E)-3,7-dimethylocta-2,6- dien-1-yl]-5-methyl-2-(3-methylbut-2-en-1-yl)benzene-1,3-diol (8) (novel), [(2´E)-3´,7´- dimethylocta-2´,6´-dien-1-yl]-2,2,7-trimethyl-2H-chromen-5-ol (9); P. arifolia, two phenolic polyketide [(2E)-3,7-dimethylocta-2,6-dien-1-yl]-5-methyl-2-(3-methylbut-2- en-1-yl)benzene-1,3-diol, also isolated from P. oreophila (10) (novel); P. urocarpa, the two phenolic polyketides 5-methyl-2-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1- yl]benzene-1,3-diol (11) and 2,4-dihydroxy-6-methyl-3-[(2E,6E)-3,7,11-trimethyldodeca- 2,6,10-trien-1-yl]benzoic acid (12); P. nitida, the phenylpropanoid apiole 1-allyl-3,6- dimethoxy-10,11-benzodioxole (13); the chromenes methyl 7-hydroxy-2,2,5-trimethyl- 2H-chromene-6-carboxylate (14) and methyl 7-methoxy-2,2,5-trimethyl-2H-chromene-6- carboxylate (15). The polyketide 2-hydroxy-4,6-dimethoxyacetophenone, the major compound in P. glabella leaves, had its biosynthetic origin investigated using acetyl-CoA and malonyl-CoA as precursors. The enzymatic activity of polyketide synthase was optimized to pH, incubation time, temperatures and substrate saturation, in addition to the analysis of circadian variation activity. The amplifications of putative PKS genes was based on primers from AviM gene of Streptomyces viridochromogenes that express for orsellinic acid. The sequencing will enable the identification of such regions and also to study the homology to fungi PKS

O espaço intergênico (IGS) juntamente com suas subunidades flanqueadoras (cpcB) e (cpcA) do operon do ficocianina foi usado para identificar linhagens de cianobactérias. Dentro do domínio Bacteria somente as cianobactérias possuem o operon da ficocianina e a região cpcBA-IGS é suficientemente variável para diferenciar linhagens desses microrganismos. No presente estudo 25 linhagens de cianobactérias isoladas de diversos locais brasileiros foram caracterizadas usando a seqüência cpcBA-IGS. DNA genômico foi extraído das ordens Chroococcales (oito linhagens), Oscillatoriales (duas linhagens), Nostocales (onze linhagens) e Stigonematales (quatro linhagens). Os oligonucleotídeos iniciadores PCβF/PCαR, específicos para a seqüência cpcBA-IGS, foram usados para amplificar fragmentos de DNA de aproximadamente 685 pb. Os produtos da PCR foram clonados, seqüenciados e as seqüências foram comparadas pela análise BLAST. Todas as seqüências de Microcystis e também as seqüências de Radiocystis fernadoi SPC736, Planktothrix mougeotii SPC788, Geitlerinema splendidum SPC923, Microchaete investiens CENA64 e Gloeotrichia UFV-B2 mostraram identidades com seqüências do GenBank. Entretanto, nenhuma identidade foi encontrada para as seqüências restantes. As relações filogenéticas das seqüências de cpcBA-IGS foram investigadas junto com outras seqüências de cianobactéria do Genbank usando a análise “Neighbour Joining”. A topologia da árvore foi congruente com outras árvores de cianobactérias, com exceção de todas as seqüências sem identidades no GenBank, as quais formaram um agrupamento separado. Os dados das seqüências de cpcBA-IGS analisadas confirmam que as cianobactéria heterocitadas formam um grupo monofilético. Estudos anteriores realizados com linhagens de cianobactéria mostraram que estes microrganismos são uma fonte rica de produtos naturais. No presente estudo conduzido com 59 linhagens de cianobactérias, sendo a maioria isolada de ambientes brasileiros, isto foi confirmado. Para alcançar esse objetivo, dois conjuntos de iniciadores degenerados foram usados para produzir seqüências amplificadas por PCR das sintetases de peptídeos não-ribossômicos (NRPSs), e de sintases policetídeos (PKSs) modulares, as quais são enzimas multifuncionais envolvidas na produção de produtos naturais. O sistema híbrido NRPS/PKS também foi amplificado por PCR usando uma combinação de iniciadores de NRPS e de PKS. Essa abordagem molecular mostrou a presença de genes de NRPS e de PKS em 93% e 81% linhagens de cianobactérias, respectivamente. Genes de NRPS/PKS foram encontrados em 87% das cianobactérias examinadas. Numa tentativa de atribuir funções a oito fragmentos de PKS identificados por PCR, estas seqüências foram clonadas, seqüenciadas e analisadas filogeneticamente. As seqüências de PKSs da Microcystis aeruginosa NPCD1 e Fischerella CENA62 mostraram correlação com a síntese de sideróforo e de microcistina, respectivamente. Todas as 59 linhagens foram analisadas para a produção do microcistinas e 20 linhagens apresentaram resultados positivos. Para a maioria das linhagens potencialmente produtoras de microcistinas os produtos de PCR esperados de NRPS, PKS e NRPS/PKS foram amplificados. A produção de sideróforos foi testada em 28 linhagens e somente cinco produziram resultados positivos. Em três linhagens produtoras de sideróforos todos os três sistemas moleculares analisados estavam presentes. Estes resultados serão altamente valiosos na exploração futura de cada peptídeo dessas cianobactérias e para a elucidação da bioatividade de tais produtos naturais. / The intergenic spacer (IGS) together with its flanking subunits  (cpcB) and  (cpcA) of the phycocyanin operon has been used to identify cyanobacterial strains. Within the Bacteria domain only cyanobacteria present phycocyanin operon and the cpcBA-IGS region is variable enough to differentiate strains of these microorganisms. In the present study 25 cyanobacterial strains isolated from several Brazilian locations were characterized using the cpcBA-IGS sequence. Genomic DNA was extracted from the orders Chroococcales (eight strains), Oscillatoriales (two strains), Nostocales (eleven strains) and Stigonematales (four strains). The primers PCβF/PCαR targeting the cpcBA-IGS sequence were used to amplify DNA fragments of approximately 685 bp. The PCR products were cloned, sequenced and the sequences were compared by BLAST analysis. All Microcystis sequences and also sequences from Radiocystis fernadoi SPC736, Planktothrix mougeotii SPC788, Geitlerinema splendidum SPC923, Microchaete investiens CENA64 and Gloeotrichia UFV-B2 showed identities with sequences from GenBank. However, no identities were found for the remaining sequences. Phylogenetic relationships of the cpcBA-IGS sequences were investigated together with other cyanobacterial sequences from Genbank using the Neighbour Joining analysis. The tree topology was congruent with previous cyanobacterial trees, except for all sequences with no identities in the GenBank, which formed a separated cluster. The cpcBA-IGS sequences analysis data confirm that heterocyte-forming cyanobacteria are a monophyletic group. Previous studies carried out with cyanobacterial strains showed that these microorganisms are a rich source of natural products. This has been confirmed in the present study conducted with 59 cyanobacterial strains, with the majority of them isolated from Brazilian environment. To reach this goal, two sets of degenerate primers were used to generate PCR amplification sequences of nonribosomal peptide synthetases (NRPSs) and modular polyketide synthases (PKSs), which are multifunctional enzymes implicated in natural products production. Also, NRPS/PKS hybrid system was PCR amplified by using a combination of NRPS and PKS primers. This molecular approach revealed the presence of NRPS and PKS genes in 93% and 81% cyanobacterial strains, respectively. NRPS/PKS genes were found in 87% of cyanobacteria examined. In an attempt to attribute functions to eight PCR identified PKS fragments, these sequences were cloned, sequenced and phylogenetically analyzed. PKSs sequences of Microcystis aeruginosa NPCD1 and Fischerella CENA62 showed correlation with the synthesis of siderophore and microcystin, respectively. All 59 strains were analyzed for microcystin production and 20 strains presented positive results. For the majority of potentially producing-microcystin strains expected PCR products of NRPS, PKS and NRPS/PKS were amplified. The siderophores production was tested in 28 strains and only five gave positive results. In three producing-siderophore strains all three molecular systems analyzed were present. These results will be highly valuable for further exploring each of these cyanobacterial peptides and for elucidating the bioactivity of such natural products.

Orientador: Luciana Gonzaga de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-19T08:47:55Z (GMT). No. of bitstreams: 1 Cruz_PedroLuisRochada_M.pdf: 2656746 bytes, checksum: 52eb866ff62a51e20e65bc5b01101aeb (MD5) Previous issue date: 2011 / Resumo: Actinobactérias são fontes importantes para a descoberta de novas moléculas com atividades biológicas destacadas. A este grupo pertencem Streptomyces, micro-organismos que possuem dentre outros, dois grupos de enzimas multimodulares conhecidas como policetídeo sintase (PKS) e peptídeo não ribossomal sintetase (NRPS) que catalisam a produção de policetídeos e peptídeos não ribossomais respectivamente. A biossíntese destas moléculas se dá geralmente seguindo uma relação linear entre o gene, a enzima e a estrutura da molécula. Desta forma, com o conhecimento das sequências do gene é possível prever a molécula a ser produzida e sua manipulação amplia a possibilidade de obter novas moléculas. Neste sentido foi adotada uma estratégia para o mapeamento dos genes biossintéticos sem a necessidade do sequenciamento extensivo do micro-organismo com o objetivo de prever o tipo de policetídeo e peptídeo não ribossomal produzido por linhagens de Streptomyces isoladas de Citrus ssp. Com o objetivo de conhecer os metabólitos produzidos por estes micro-organismos foi feito também o cultivo destes em meios contendo fontes de nutrientes variadas e o monitoramento de metabólitos presentes nos extratos utilizando técnicas de cromatografia líquida acoplada com espectrometria de massas. Ensaios em placas permitiram a visualização da produção de sideróforos e a atividade antibiótica dos meios de cultivo / Abstract: Actinobacteria are important sources of new molecules with biological activities. Streptomyces are the most important genera studied. Such microorganisms carries out among a variety of biosynthetic enzimes, two main groups known as polyketide synthase (PKS) and non ribosomal peptide synthetase (NRPS), both catalyzing the biosynthesis of polyketide and non ribosomal peptides respectively. PKS and NRPS biosynthesis follows a collinear relationship among the gene cluster, the enzyme and the secondary metabolite. Therefore, the knowledge of the gene sequences allows to find new molecules. In this work we adopted a strategy to map the biosynthetic genes without the need of extensive whole genome sequencing in order to predict the metabolite produced by Streptomyces strains isolated from Citrus ssp. The strains were also cultivated and the metabolite production monitored by liquid chromatography coupled with mass spectrometry. In addition, the biological activity of these medium was estimated using qualitative biochemical assays / Mestrado / Quimica Organica / Mestre em Química

Polyketide secondary metabolites constitute a structurally-diverse and clinically-important family of natural products. The wide range of biological activities represented by these substrates have contributed to therapeutic agents with annual sales exceeding $20B USD. Large multi-domain proteins called polyketide synthases (PKSs) use simple building blocks to generate highly-oxygenated and stereochemically-rich frameworks with astonishing selectivity. These substrates often feature rigidifying biases imposed by macrocyclic lactones and substituted heterocycles, which can impact their bioactive conformation. The work of this dissertation combines synthetic chemistry and biochemistry to investigate chemoenzymatic production of macrocyclic polyketide natural products. Research focused on validating a transannular oxa-conjugate addition strategy to assembly 2,6-cis-tetrahydropyran (THP) ring systems, as demonstrated by synthesis of the macrocyclic core to neopeltolide. Ultimately, we wish to apply this chemistry to de novo PKS pathways for rapid, reliable, and sustainable production of THP-bearing products like neopeltolide, and toward building SAR libraries. Additionally, a second study probed the specificity of the macrolactonizing thioesterase (TE) domain from the 6-deoxyerythronolide B (DEBS) biosynthetic pathway. This pathway is the paradigm for type-I PKS systems, and is responsible for producing the macrolide core of erythromycin. Our on-going research evaluates the limits of promiscuity within this specific catalytic domain, to characterize the structural elements required to accurately predict macrolactonization. The long-term goal of this study is to assess the potential applicability of DEBS TE as a generalized cyclization biocatalyst for combinatorial biochemistry and chemoenzymatic research.

Abstract Hypericum perforatum L. (St. John’s wort) is a medicinal plant widely utilized for the treatment of depression. The antidepressant activity is mainly attributed to the phenolic compounds hypericins and hyperforins, which also have a wide range of other pharmacologically interesting properties. The biosynthetic routes leading to hypericins and hyperforins are poorly understood, although a polyketide pathway including type III polyketide synthases (PKSs) has been suggested to be involved. Furthermore, a gene called hyp-1 is assumed to attend to the final stages of the hypericin biosynthesis. In the present work, the biosynthesis of hypericins and hyperforins in H. perforatum was further studied by focusing on the elucidation of the precursors and genes involved. The incorporation of isotopically labelled branched-chain amino acids into hyperforins was investigated as well as the possibilities to enhance the production of hyperforins in H. perforatum in vitro cultures by feeding them with amino acid precursors. Furthermore, two novel cDNAs encoding for type III PKSs were isolated from H. perforatum. The functions of these new genes, designated HpPKS1 and HpPKS2, as well as the role of hyp-1 were elucidated by comparing their expression with the levels of hypericins and hyperforins in H. perforatum tissues. The enzymatic activity of the recombinant HpPKS2 protein was also analyzed. To study Hyp-1 at a protein level, a protein extraction method was optimized for tissues of Hypericum species. The results show the incorporation of valine and isoleucine into the acyl side chain of hyperforin and adhyperforin, respectively. Through the biotransformation of the amino acid precursors, it is possible to enhance the levels of adhyperforin, but not hyperforin, in H. perforatum shoot cultures, which demonstrates the tight regulation of the hyperforin biosynthesis. A correlation between HpPKS1 expression and hyperforins was detected in H. perforatum tissues. The localization of HpPKS2 mRNA in dark glands in which hypericins accumulate as well as the octaketide synthase activity of the recombinant HpPKS2 suggest that HpPKS2 is associated with possible co-operating tailoring enzymes in the biosynthesis of hypericins. The presence of both hyp-1 mRNA and Hyp-1 protein in distinct places compared with hypericins in H. perforatum tissues does not support the idea that Hyp-1 would be involved in the biosynthesis of hypericins in dark glands, although mobility of the Hyp-1 protein was shown to be possible. The present thesis extends knowledge about the biosynthesis of hypericins and hyperforins in H. perforatum by providing new candidate genes for their biosynthesis and by identifying precursors for hyperforins. Moreover, new information was obtained about the role of hyp-1 in H. perforatum.

The analysis of Gene Regulatory Networks (GRNs) is essential to understanding the complete process of embryo development. Elucidating every gene regulatory circuit from maternal regulatory inputs all the way to the activation of differentiation gene batteries is an important step in increasing our understanding of developmental biology. In this work I study the cis-regulatory architecture of a pigment cell differentiation gene, polyketide synthase (SpPks) in the sea urchin Strongylocentrotus purpuratus. SpPks encodes an enzyme that is responsible for the biosynthesis of the sea urchin pigment echinochrome in larval pigment cells. The analysis of the promoter of a differentiation gene will lead to identifying the direct upstream regulators and ultimately to elucidating the structure of the upstream gene regulatory network, which is mostly uncharacterized. From previous studies the transcription factors SpGcm and SpGatae are predicted to be positive regulators of SpPks. Here, I identify a minimal 1kb promoter region containing putative DNA-binding sites for both GCM and GATAE that is able to recapitulate the expression of SpPks. I further show by mutagenesis that a putative DNA-binding site for GCM located 1,179 base pairs upstream of the start of transcription is a direct target for the positive cis-regulation of SpPks. Quantitative analysis of the transcriptional regulatory function of the GCM-mutagenized construct suggests that GCM is not necessary for the start of SpPks transcription but is required for its maintenance. Several GATA E binding sites have been identified within the minimal promoter for SpPks by means of consensus sequence. My analysis suggests that GATA E may be a direct positive regulator and could potentially be required for the onset of transcription of SpPks, though further experimentation will be necessary to characterize the exact regulatory function of GATA E.

No description available.

No description available.

Alternaria brassicicola is a necrotrophic pathogen that causes black spot disease on virtually all cultivated Brassicas, A. brassicicola is renowned for its ability to prodigiously produce secondary metabolites. To test the hypothesis that secondary metabolites produced by A. brassicicola contribute to pathogenicity, we identified seven nonribosomal peptide synthetases (NPSs) and 10 polyketide synthases (PKSs) in the A. brassicicola genome. The phenotype resulting from knockout mutations of each PKS and NPS gene was investigated with an emphasis on discovery of fungal virulence factors. A highly efficient gene disruption method using a short linear double stranded DNA construct with minimal elements was developed, optimized, and used to functionally disrupt all NPS and PKS genes in A. brassicicola. Three NPS and two PKS genes, and one NPS-like gene appeared to be virulence factors based upon reduced lesion development of each mutant on inoculated green cabbage and Arabidopsis compared with the wild-type strain. Furthermore some of the KO mutants exhibited developmental phenotypic changes in pigmentation and conidiogenesis. To further characterize the roles of several genes of interest in A. brassicicola development and pathogenesis, the genes AbNPS2, AbPKS9, and NPS-like tmpL were selected for in-depth functional analysis. We provide substantial evidence that the AbNPS2-associated metabolite is involved in conidial cell wall construction, possibly as an anchor connecting two cell wall layers. We also characterized a biosynthetic gene cluster harboring the AbPKS9 gene and demonstrated that this cluster is responsible for the biosynthesis of depudecin, an inhibitor of histone deacetylases and a minor virulence factor. Finally, we demonstrated that a NPS-like protein named TmpL is involved in a filamentous fungi-specific mechanism for regulating levels of intracellular reactive oxygen species during conidiation and pathogenesis in both plant and animal pathogenic fungi. Collectively our results indicate that small molecule nonribosomal peptides and polyketides in A. brassicicola play diverse, but also fundamental, roles in fungal development and pathogenesis. / Ph. D.