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This thesis is regarded with the biomimetic total synthesis of phenolic natural products and describes two different projects. Chapter 1 introduces phenolic natural products, the most important biosynthetic pathways for their formation and some examples of relevant biomimetic syntheses. A short introduction to dimeric and pseudo-dimeric natural products can also be found in this chapter. Specific introductions can be found at the start of Chapters 2 and 3. Chapter 2 describes the total synthesis of a dimeric thymol derivative isolated from Arnica sachalinensis (which we have named “thymarnicol”). Inspired by the biosynthesis proposed by Passreiter and co-workers, we tested and confirmed the feasibility of a key hetero-Diels–Alder dimerisation step. During our investigations, we gained significant new insights into the origin and reactivity of thymarnicol. The final oxidative cyclisation has been found to occur spontaneously upon exposure to visible light in air. Chapter 3 discusses our efforts to develop a divergent biomimetic synthetic strategy towards a family of prenylated phenylpropanoid natural products isolated from Illicium genus plants. Our first biomimetic approach revealed the chemical instability of our proposed key intermediates. Therefore, a revised approach was trialled, allowing the total synthesis of a small set of natural products and related structures. We envisage that this strategy could be exploited by accessing a large number of members of this family of compounds.

Les acides chlorogéniques (CGAs) représentent une famille d'esters formés d'un dérivé de l'acide cinnamique conjugué à l'acide quinique ou shikimique. Ces métabolites secondaires produits par la voie des phénylpropanoides sont largement répandus chez les végétaux terrestres et sont une source majeure d'antioxydants alimentaires. Les esters hydroxycinnamoyl-CoA sont les précurseurs des CGAs et d'autres composés phénoliques tels que les lignines. Ces intermédiaires activés sont synthétisés à partir d'un acide hydroxycinnamique et du coenzyme A par la 4-coumarate CoA ligase (4CL) appartenant à la superfamille des enzymes formant des adénylates. Nicotiana tabacum 4CL2 a été utilisée pour la production d'esters et sa structure a été résolue par remplacement moléculaire. Deux gènes codant pour des hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransférases chez Coffea canephora ont été clonés. CcHCT et CcHQT, qui appartiennent à la superfamille des acyltransférases acyl-CoA-dépendantes, ont été surexprimées dans E. coli et purifiées à homogénéité. L'analyse par diffraction aux rayons X de cristaux de CcHCT a permis de déterminer sa structure par remplacement moléculaire. Un modèle a été dérivé par homologie de séquence pour CcHQT afin de proposer les déterminants de la préférence pour l'acide quinique ou shikimique. Des modélisations moléculaires ont été réalisées afin d'identifier les résidus potentiellement impliqués dans les intéractions enzyme-substrat. L'analyse par chromatographie liquide haute performance des réactions enzymatiques ont montré que ces enzymes sont capables de synthétiser l'acide 5-O-caféoylquinique mais aussi le diester 3,5-O-dicaffeoylquinique, qui est un composé majeur du grain de café avant mûrissement. La production de variants par mutagenèse dirigée a permis l'identification de résidus importants pour la catalyse des réactions de mono- et de diacylation. L'approche combinée de la biologie structurale et de l'enzymologie s'avère particulièrement utile pour mieux comprendre le rôle de HCT et HQT. / Chlorogenic acids (CGAs) represent a family of esters formed between a cinnamic acid derivative and quinic or shikimic acid. CGAs are secondary metabolites produced via the phenylpropanoid pathway by higher plants and are a major source of dietary antioxidants. Hydroxycinnamoyl-CoA esters are the precursors for CGAs and other phenolic compounds such as lignins. These activated intermediates are synthesized from a hydroxycinnamic acid and coenzyme A by 4-coumarate CoA ligase (4CL), which belongs to the adenylate-forming enzyme superfamily. Nicotiana tabacum 4CL2 was used to produce hydroxycinnamoyl-CoA esters and its structure was solved by molecular replacement. Two genes encoding hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferases from Coffea canephora were cloned. CcHCT and CcHQT, which belong to the acyl-CoA-dependent acyltransferase superfamily, were overexpressed in E. coli and purified to homogeneity. X-ray diffraction analysis of CcHCT crystals resulted in a structural solution by molecular replacement. A homology model was derived for CcHQT in order to propose some determinants of the preference for quinic or shikimic acid. Docking experiments were carried out in order to identify potential residues involved in enzyme-substrate interactions. High performance liquid chromatography analysis of enzymatic reactions showed that these enzymes are capable of synthesizing 5-O-caffeoylquinic acid but also the diester 3,5-O-dicaffeoylquinic acid, which is a major component of the coffee grain before ripening. The production of variants by site-directed mutagenesis enabled the identification of residues important for catalysis of the mono- and diacyltransfer reactions. The combined approach of structural biology and enzymology provides molecular insights into the role of HCT and HQT in CGA biosynthesis.

The plant phenylpropanoid pathway is initiated from deamination of phenylalanine to form cinnamic acid followed by hydroxylation and methylation of the aromatic ring to generate a variety of phenolic compounds including lignin monomers, flavonoid compounds and sinapate esters. The incorporation of phenylpropanoid metabolism served as a key step in the early land-colonization of plants from aqueous environment since phenolic compounds play important roles in plant development and abiotic/biotic stress responses. Lignin is a heteropolymer of hydroxycinnamyl alcohols that are derived from the major branch of plant phenylpropanoid pathway. The main function of lignin is to enhance the strength of plant cell wall and waterproof the vascular system for long-distance transportation of water and solutes. In addition, lignin is also involved in protecting plants against pathogen attack. My Ph.D. research is to investigate how lignin biosynthesis contributes to plant immunity. The results showed that the expression of major lignin biosynthetic genes was induced upon host fungal pathogen infection. Moreover, a mutant disrupted in the lignin gene F5H1 showed enhanced susceptibility when challenged with several fungal pathogens. F5H1 encodes a ferulic acid 5-hydroxylase that is uniquely present in angiosperm plants, leading to the biosynthesis of syringyl lignin monomer, which is not present in gymnosperm plants. Subsequent research demonstrated that f5h1 mutation impaired the penetration (pre-invasion) resistance but did not impact post-invasion resistance. Furthermore, the pathogen-induced expression of lignin genes was independent of well-characterized defensive signaling pathways, and regulated by a novel regulating mechanism. F5H1 contributes to pmr2-mediated resistance but acts independently of other molecular components of penetration resistance including PEN1, PEN2, and PEN3. In contrast to f5h1, a knockout mutant of flavonoid pathway gene chalcone isomerase (CHI/TT5) showed enhanced resistance to host anthracnose pathogen Colletotrichum higginsianum in a salicylic acid (SA)-dependent manner. Taken together, our results for the first time provide genetic evidence demonstrating that lignin biosynthetic gene F5H1 plays critical roles in plant penetration resistance and that an uncharted pathway in flavonoid metabolism confers an SA-dependent resistance pathway in Arabidopsis.

The fungus Fusarium graminearum (Schwabe) causes Gibberella ear rot in maize, resulting in accumulation of harmful mycotoxins in the grain. Disease severity and pericarp/aleurone dehydrodiferulic acid content are negatively correlated. Furthermore, quantitative trait locus mapping (QTL) identified colocalization between QTL for both traits. A candidate gene approach was employed to identify the genes responsible for the observed colocalization. Candidate genes selected on the basis of their putative involvement in various aspects of cell wall DFA accumulation were mapped in silico using the maize genome sequence. Polymorphisms were discovered in putative genes and converted to molecular markers. The in silico mapping effort was successful in predicting map locations of the analyzed sequences, and the segregation of certain marker alleles could explain variation for Gibberella ear rot severity and pericarp-aleurone DFA content.

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-09-12T12:50:11Z No. of bitstreams: 1 arquivototal.pdf: 1631298 bytes, checksum: 709662241f9195898b65fdb8752da174 (MD5) / Made available in DSpace on 2017-09-12T12:50:11Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1631298 bytes, checksum: 709662241f9195898b65fdb8752da174 (MD5) Previous issue date: 2016-11-23 / 2-allylphenol is a phenylpropanoid widely marketed in China under the name Yinguo. It presents structural similarity to ginkgol the isolated compound from ginkgo fruit and has fungicidal activity already reported in the literature, however its effect in painful process has not been studied yet. This work aims to investigate the antinociceptive activity of 2-allylphenol in experimental models of pain in mice. Firstly, it was carried out the determination of the lethal dose 50 (LD50), in order to establish safe doses for subsequent tests. Then methodologies were performed to evaluate the antinociceptive activity. 2-allylphenol (25, 50, 75 e 100 mg/kg, i.p.) reduced the number of writhes, when compared to the control group. In the formalin test, in the doses 75 e 100 mg/kg, 2-allylphenol reduced the licking paw time on neurogenic (0-5 min) and inflammatory phase (15-30 min). In the hot plate test, which is sensible and specific to drugs that act by supraspinal mechanisms, 2-allylphenol did not change the latency in the paw withdrawal. While the test of nociception induced by glutamate, the 100mg/kg 2-allylphenol dose reduced the licking paw time. Based on these results we propose that the antinociceptive action of 2-allylphenol may be modulating pain via both peripherally as centrally in spinal levels. In an attempt to elucidate the mechanism of action involved in the antinociceptive effect of 2-allylphenol were used pharmacological tools in the formalin test. The antinociception produced by the 2-allylphenol was significantly blocked in animals pretreated with caffeine (10 mg/kg, s.c.), only in the second phase of the test, indicating the involvement of adenosinergic system. The antinociceptive effect of the 2-allylphenol, however, was not reversed by naloxone (non-selective antagonist of opioid receptors, 5 mg/kg, s.c.) and glibenclamide (K+ ATP channel blocker, 10 mg/kg, i.p.), suggesting that 2-allylphenol does not act by these mechanisms. / O 2-alilfenol é um fenilpropanóide amplamente comercializado na China sob o nome de Yinguo. Apresenta similaridade estrutural ao ginkgol composto isolado do fruto ginkgo. Possui atividade fungicida já relatada na literatura, porém sua ação em processos dolorosos nunca foi estudada. O presente estudo investigou o efeito do 2-alilfenol, pela via intraperitoneal, em modelos experimentais de dor em camundongos. Inicialmente, foi realizada a pesquisa da dose letal 50 (DL50) do fenilpropanóide, no intuito de estabelecer doses seguras para os testes subsequentes. Em seguida, foram realizadas metodologias para avaliar a atividade antinociceptiva. O 2-alilfenol (25, 50, 75 e 100 mg/kg, i.p.) reduziu o número de contorções, quando comparado ao grupo controle. No teste da formalina, utilizando as doses 75 e 100 mg/kg, o 2-alilfenol reduziu o tempo de lambida da pata na fase neurogênica (0-5 min) e na fase inflamatória (15-30 min). No teste da placa quente, que é sensível e específico para drogas que atuam por mecanismos supra-espinhais, o 2-alilfenol não alterou a latência na retirada da pata. Enquanto que no teste da nocicepção induzida por glutamato, a dose de 100mg/kg do 2-alilfenol reduziu o tempo de lambida da pata. A partir destes resultados podemos propor que a ação antinociceptiva do 2-alilfenol pode estar modulando a via da dor a tanto perifericamente quanto centralmente em níveis espinhais. Na tentativa de elucidar o mecanismo de ação envolvido no efeito antinociceptivo do 2-alilfenol foram usadas ferramentas farmacológicas no teste da formalina. A antinocicepção produzida pelo 2-alilfenol foi significativamente bloqueada em animais pré-tratados com cafeína (10 mg/kg, s.c.), apenas na segunda fase do teste, indicando o envolvimento do sistema adenosinérgico. O efeito antinociceptivo do 2-alilfenol, contudo, não foi revertido pela naloxona (antagonista não seletivo dos receptores opioides, 5 mg/kg, s.c.) e pela glibenclamida (bloqueador dos canais de K+ ATP, 10 mg/kg, i.p.), sugerindo que o 2-alilfeno não atua por esses mecanismos.

<p>Plants naturally deposit a significant amount of carbon towards lignin, a polymer that imparts mechanical strength to cell walls but impedes our utilization of the polysaccharides in lignocellulosic biomass. Genetic engineering of lignin has demonstrated profound success in improving the processing of the biomass. Lignin is derived from the phenylpropanoid pathway, the architecture of which is well understood based upon the biochemical and genetic studies conducted to date. In contrast, we lack a systematic and quantitative view of the factors that determine carbon flux into and within this branched metabolic pathway in plants. To explore the control of carbon allocation for phenylalanine and lignin biosynthesis, we have developed a kinetic model of the pathway in Arabidopsis to test the regulatory role of several key enzymatic steps. We first established a ¹³C isotope feeding system for the measurement of flux using excised wild-type Arabidopsis stems. The excised stems continued to grow and lignify in our feeding system. When ring ¹³C₆-labeled phenylalanine ([¹³C₆]-Phe) was supplied to excised stems, isotope label was rapidly incorporated into soluble intermediates and lignin. Using this approach, we then analyzed metabolite pool sizes and isotope abundances of the pathway intermediates in a time course from stems fed with [¹³C₆]-Phe of different concentrations, and used these data to parameterize a kinetic model constructed with Michaelis-Menten kinetics. Our model of the general phenylpropanoid pathway captured the dynamic trends of metabolite pools <i>in vivo</i>and predicted the metabolic profiles of an independent feeding experiment. Based on the model simulation, we found that subcellular sequestration of pathway intermediates is necessary to maintain lignification homeostasis when metabolites are over-accumulated. Both the measurements and simulation suggested that theavailability of substrate Phe is one limiting factor for lignin flux in developing stems. This finding indicates new gene targets for lignin manipulation in plants. To extend our kinetic model to simulate flux distribution in response to genetic perturbations, we conducted an RNA-sequencing experiment in wild type and 13 plants with modified lignification, and integrated the transcriptional data with the metabolic profiles. We found that the biosynthesis of Phe and lignification are tightly coordinated at transcriptional level. The coregulation of the shikimate and phenylpropanoid pathways involves transcriptional and post-translational regulatory mechanisms to maintain pathway homeostasis. Our results also indicate that induction of Phe supply and enhancement of PAL activity are both effective strategies to increase carbon flux into the phenylpropanoid network.</p><p>In this interdisciplinary project, we have taken various system biology approaches to understand metabolic flux towards lignin, the second most abundant carbon sink in nature. We have combined isotope labeling aided flux measurements and mathematical simulation, and have integrated metabolome data with transcriptome profiles. The experiments and analysis have been conducted in both wild-type Arabidopsis and those with perturbed lignification. The novel work not only provides insight into our knowledge of phenylpropanoid metabolism, but also creates a framework to systematically assemble gene expression, enzyme activity, and metabolite accumulation to study metabolic fluxes, the ultimate functional phenotypes of biochemical networks.</p>

Searches for sequence similarity against a previously isolated and known ABA regulated partial geranium cDNA on GenBank and TIGR databases gave results indicating sequence similarities to both firefly luciferase and 4-coumarate:CoA Ligase (4-CL). To determine whether or not this geranium cDNA, and a root mRNA from Arabidopsis that hybridized with the geranium cDNA, is 4-CL, specific Arabidopsis 4-CL primers were used for expression analysis by RT-PCR of Arabidopsis 4-CL mRNA from Arabidopsis root tissue. The expression pattern of the Arabidopsis 4-CL mRNA was identical with the expression pattern of the mRNA that hybridized with the geranium cDNA. This matching expression pattern gives very strong evidence that our geranium cDNA is 4-CL like and that Arabidopsis 4-CL is downregulated during water stress by ABA. This is significant in that it has not previously been reported. Northern analysis and PCR product sequence analysis further confirmed this expression data.

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Industrial hemp (Cannabis sativa) is a highly productive crop that is well suited to cultivation in Canada. To better understand the development of bast (phloem) fiber secondary walls and to facilitate reverse genetics screening for improved germplasm, I undertook two sets of microarray experiments. The first compared transcript expression in stem peels at three positions along the length of the stem. The second set of microarray experiments compared transcript expression in adjacent tissue layers along the radial axis of the stem. The transcripts that were enriched in fiber-producing tissues in both studies were consistent with a dynamic program of cell wall deposition. Detailed qRT-PCR analysis of specific lignification genes identified the best targets for reverse genetics. Finally, as a first step towards establishing a virally induced gene silencing (VIGS) system, I identified viruses that produced visual symptoms of infection, although qRT-PCR failed to confirm the infection / Plant Biology