Natural Products from Antarctic Colonial Ascidians of the Genera Aplidium and Synoicum: Variability and Defensive Role

Ascidians have developed multiple defensive strategies mostly related to physical, nutritional or chemical properties of the tunic. One of such is chemical defense based on secondary metabolites. We analyzed a series of colonial Antarctic ascidians from deep-water collections belonging to the genera Aplidium and Synoicum to evaluate the incidence of organic deterrents and their variability. The ether fractions from 15 samples including specimens of the species A. falklandicum, A. fuegiense, A. meridianum, A. millari and S. adareanum were subjected to feeding assays towards two relevant sympatric predators: the starfish Odontaster validus, and the amphipod Cheirimedon femoratus. All samples revealed repellency. Nonetheless, some colonies concentrated defensive chemicals in internal body-regions rather than in the tunic. Four ascidian-derived meroterpenoids, rossinones B and the three derivatives 2,3-epoxy-rossinone B, 3-epi-rossinone B, 5,6-epoxy-rossinone B, and the indole alkaloids meridianins AG, along with other minoritary meridianin compounds were isolated from several samples. Some purified metabolites were tested in feeding assays exhibiting potent unpalatabilities, thus revealing their role in predation avoidance. Ascidian extracts and purified compound-fractions were further assessed in antibacterial tests against a marine Antarctic bacterium. Only the meridianins showed inhibition activity, demonstrating a multifunctional defensive role. According to their occurrence in nature and within our colonial specimens, the possible origin of both types of metabolites is discussed.

Natural Products from Antarctic Colonial Ascidians of the Genera Aplidium and Synoicum: Variability and Defensive Role

Ascidians have developed multiple defensive strategies mostly related to physical, nutritional or chemical properties of the tunic. One of such is chemical defense based on secondary metabolites. We analyzed a series of colonial Antarctic ascidians from deep-water collections belonging to the genera Aplidium and Synoicum to evaluate the incidence of organic deterrents and their variability. The ether fractions from 15 samples including specimens of the species A. falklandicum, A. fuegiense, A. meridianum, A. millari and S. adareanum were subjected to feeding assays towards two relevant sympatric predators: the starfish Odontaster validus, and the amphipod Cheirimedon femoratus. All samples revealed repellency. Nonetheless, some colonies concentrated defensive chemicals in internal body-regions rather than in the tunic. Four ascidian-derived meroterpenoids, rossinones B and the three derivatives 2,3-epoxy-rossinone B, 3-epi-rossinone B, 5,6-epoxy-rossinone B, and the indole alkaloids meridianins AG, along with other minoritary meridianin compounds were isolated from several samples. Some purified metabolites were tested in feeding assays exhibiting potent unpalatabilities, thus revealing their role in predation avoidance. Ascidian extracts and purified compound-fractions were further assessed in antibacterial tests against a marine Antarctic bacterium. Only the meridianins showed inhibition activity, demonstrating a multifunctional defensive role. According to their occurrence in nature and within our colonial specimens, the possible origin of both types of metabolites is discussed.

Natural Products from Antarctic Colonial Ascidians of the Genera Aplidium and Synoicum: Variability and Defensive Role

Ascidians have developed multiple defensive strategies mostly related to physical, nutritional or chemical properties of the tunic. One of such is chemical defense based on secondary metabolites. We analyzed a series of colonial Antarctic ascidians from deep-water collections belonging to the genera Aplidium and Synoicum to evaluate the incidence of organic deterrents and their variability. The ether fractions from 15 samples including specimens of the species A. falklandicum, A. fuegiense, A. meridianum, A. millari and S. adareanum were subjected to feeding assays towards two relevant sympatric predators: the starfish Odontaster validus, and the amphipod Cheirimedon femoratus. All samples revealed repellency. Nonetheless, some colonies concentrated defensive chemicals in internal body-regions rather than in the tunic. Four ascidian-derived meroterpenoids, rossinones B and the three derivatives 2,3-epoxy-rossinone B, 3-epi-rossinone B, 5,6-epoxy-rossinone B, and the indole alkaloids meridianins A-G, along with other minoritary meridianin compounds were isolated from several samples. Some purified metabolites were tested in feeding assays exhibiting potent unpalatabilities, thus revealing their role in predation avoidance. Ascidian extracts and purified compound-fractions were further assessed in antibacterial tests against a marine Antarctic bacterium. Only the meridianins showed inhibition activity, demonstrating a multifunctional defensive role. According to their occurrence in nature and within our colonial specimens, the possible origin of both types of metabolites is discussed.

Total synthesis and antiproliferative activity screening of (±)-aplicyanins A, B and E and related analogs

The first total synthesis of the indole alkaloids ()-aplicyanins A, B and E, plus seventeen analogs, all in racemic form is reported. Modifications to the parent compound included changing the number of bromine substituents on the indole, the groups on the indole nitrogen (H, Me or OMe), and/or the oxidation level of the heterocyclic core tetrahydropyrimidine. Each compound was screened against three human tumor cell lines, and fourteen of the newly synthesized compounds showed considerable cytotoxicity. The assay results were used to establish structure-activity relationships. These results suggest that the acetyl group moiety on the imine nitrogen, and the bromine at position 5 of the indole, are both critical to activity.

Alcalóides indólicos isolados de espécies do gênero Aspidosperma (Apocynaceae)

Species from genus Aspidosperma (Apocynaceae) are popularly employed to treat various diseases. This genus is characterized by the occurrence of indole alkaloids. Taking into account the various biological activities attributed to these alkaloids, the description of the chemical diversity in genus Aspidosperma is important. A review of simple carbolinic alkaloids isolated from species of various genera was published in 1979. In 1987, it was published another one dealing with the relationships between the chemical structures of the indole alkaloids and the evolution of Aspidosperma species. This work updates the information about the indole alkaloids isolated from Aspidosperma species.

Alcaloides indólicos de cascas de Aspidosperma vargasii e A. desmanthum

Fractionation of extracts resulted in isolation and identification of indole alkaloids ellipticine and N-methyltetrahydroellipticine from A. vargasii and aspidocarpine from A. desmanthum. Identification of these compounds was achieved based on IR, MS, ¹H, 13C and 2-D NMR data and comparison to data in the literature.

A validated method using RP-HPLC for quantification of reserpine in the Brazilian tree Rauvolfia sellowii Müll. Arg. (Apocynaceae)

This study describes a simple, fast and reproducible method using RP-HPLC-UV, in a gradient system, for quantification of reserpine in Rauvolfia sellowii stem bark. The analysis were carried out on a C18 column; mobile phase was water and acetonitrile, and separations were carried out in 10 min, flow rate of 1.0 mL min-1, 25 ºC and 268 nm. The validation data showed that the method was specific, accurate, precise and robust. Results were linear over a range of 0.625-40.0 μg mL-1, and the mean recovery was 95.1%. The amount of reserpine found in the dried stem bark was 0.01% (m/m).

NOVEL ALKALOID FROM Rauvolfia capixabae(APOCYNACEAE)

A new sarpagine-type alkaloid, Na-methylrauflorine (1), was isolated from Rauvolfia capixabaetogether with isoreserpiline (2),Nb-oxide-isoreserpiline (3), ajmalicine (4), perakine (5) and vinorine (6) alkaloids. These compounds were characterized based on their spectral data basis, mainly one- (1H, 13C, APT) and two-dimensional(1H-1H-COSY, 1H-1H-NOESY, HMQC and HMBC) NMR, and mass spectra, also involving comparison with data from the literature.

The Catharanthus roseus 16-hydroxytabersonine O-methyltransferase involved in vindoline biosynthesis /

Madagascar periwinkle (Catharanthus roseus) produces the well known and remarkably complex dimeric anticancer alkaloids vinblastine and vincristine that are derived by coupling vindoline and catharanthine monomers. This thesis describes the novel application of carborundum abrasion (CA) technique as a tool for large scale isolation of leaf epidermis enriched proteins. This technique was used to facilitate the purification to apparent homogeneity of 16-hydroxytabersonine-16-0-methyltransferse (l60MT) that catalyses the second step in the 6 step pathway that converts tabersonine into vindoline. This versatile tool was also used to harvest leaf epidermis enriched mRNAs that facilitated the molecular cloning of the 160MT. Functional expression and biochemical characterization of recombinant 160MT enzyme showed that it had a very narrow substrate specificity and high affinity for 16-hydroxytabersonine, since other closely related monoterpene indole alkaloids (MIAs) did not act as substrates. In addition to allowing the cloning of this gene, CA technique clearly showed that 160MT is predominantly expressed in Catharanthus leaf epidermis, in contrast to several other OMTs that appear to be expressed in other Catharanthus tissues. The results provide compelling evidence that most of the pathway for vindoline biosynthesis including the 0- methylation of 16-hydroxytabersonine occurs exclusively in leaf epidermis, with subsequent steps occurring in other leaf cell types. Small molecule O-methyltransferases (OMTs) (E.C. 2.1.1.6.x) catalyze the transfer of the reactive methyl group of S-adenosyl-L-methionine (SAM) to free hydroxyl groups of acceptor molecules. Plant OMTs, unlike their monomeric mammalian homologues, exist as functional homodimers. While the biological advantages for dimer fonnation with plant OMTs remain to be established, studies with OMTs from the benzylisoquinoline producing plant, Thalictrum tuberosum, showed that co-expression of 2 recombinant OMTs produced novel substrate specificities not found when each rOMT was expressed individually (Frick, Kutchan, 1999) . These results suggest that OMTs can fonn heterodimers that confer novel substrate specificities not possible with the homodimer alone. The present study describes a 160MT model based strategy attempting to modify the substrate specificity by site-specific mutagenesis. Our failure to generate altered substrate acceptance profiles in our 160MT mutants has lead us to study the biochemical properties ofhomodimers and heterodimers. Experimental evidence is provided to show that active sites found on OMT dimers function independently and that bifunctional heterodimeric OMTs may be fonned in vivo to produce a broader and more diverse range of natural products in plants.

Localization and functional characterization of iridoid biosynthetic genes in Catharanthus roseus /

Catharanthus roseus is the sole biological source of the medicinal compounds vinblastine and vincristine. These chemotherapeutic compounds are produced in the aerial organs of the plant, however they accumulate in small amounts constituting only about 0.0002% of the fresh weight of the leaf. Their limited biological supply and high economical value makes its biosynthesis important to study. Vinblastine and vincristine are dimeric monoterpene indole alkaloids, which consists of two monomers vindoline and catharanthine. The monoterpene indole alkaloids (MIA's) contain a monoterpene moiety which is derived from the iridoid secologanin and an indole moiety tryptamine derived from the amino acid tryptophan. The biosynthesis of the monoterpene indole alkaloids has been localized to at least three cell types namely, the epidermis, the laticifer and the internal phloem assisted parenchyma. Carborundum abrasion (CA) technique was developed to selectively harvest epidermis enriched plant material. This technique can be used to harvest metabolites, protein or RNA. Sequencing of an expressed sequence tagged (EST) library from epidermis enriched mRNA demonstrated that this cell type is active in synthesizing a variety of secondary metabolites namely, flavonoids, lipids, triterpenes and monoterpene indole alkaloids. Virtually all of the known genes involved in monterpene indole alkaloid biosynthesis were sequenced from this library.This EST library is a source for many candidate genes involved in MIA biosynthesis. A contig derived from 12 EST's had high similarity (E'^') to a salicylic acid methyltransferase. Cloning and functional characterization of this gene revealed that it was the carboxyl methyltransferase imethyltransferase (LAMT). In planta characterization of LAMT revealed that it has a 10- fold enrichment in the leaf epidermis as compared to the whole leaf specific activity. Characterization of the recombinant enzyme revealed that vLAMT has a narrow substate specificity as it only accepts loganic acid (100%) and secologanic acid (10%) as substrates. rLAMT has a high Km value for its substrate loganic acid (14.76 mM) and shows strong product inhibition for loganin (Kj 215 |iM). The strong product inhibition and low affinity for its substrate may suggest why the iridoid moiety is the limiting factor in monoterpene indole alkaloid biosynthesis. Metabolite profiling of C. roseus organs shows that secologanin accumulates within these organs and constitutues 0.07- 0.45% of the fresh weight; however loganin does not accumulate within these organs suggesting that the product inhibition of loganin with LAMT is not physiologically relevant. The limiting factor to iridoid and MIA biosynthesis seems to be related to the spatial separation of secologanin and the MIA pathway, although secologanin is synthesized in the epidermis, only 2-5% of the total secologanin is found in the epidermis while the remaining secologanin is found within the leaf body inaccessable to alkaloid biosynthesis. These studies emphasize the biochemical specialization of the epidermis for the production of secondary metabolites. The epidermal cells synthesize metabolites that are sequestered within the plant and metabolites that are secreted to the leaf surface. The secreted metabolites comprise the epidermome, a layer separating the plant from its environment.

Strychnos L. da América do Sul e Central

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Abordagem fitoquímica, determinação da atividade antiplasmódica in vitro e avaliação preliminar da toxicidade do extrato hidroetanólico das cascas de Aspidosperma excelsum Benth (Apocynaceae)

A malária é uma doença causada por protozoários do gênero Plasmodium. O tratamento da malária está se tornando cada vez mais difícil com a expansão dos casos de parasitas resistentes aos fármacos utilizados na terapêutica. Neste contexto, produtos isolados a partir de plantas têm dado importante contribuição, representando importante fonte para a obtenção de novos fármacos antimaláricos. A atividade antiplasmódica de alcalóides de origem vegetal tem sido amplamente relatada na literatura. Plantas da família Apocynaceae, ricas em alcalóides indólicos, apresentam amplas propriedades medicinais e algumas espécies do gênero Aspidosperma já demonstraram potencial antimalárico. Assim, este trabalho teve como objetivo realizar uma abordagem fitoquímica, avaliar a atividade antiplasmódica in vitro e a toxicidade preliminar do extrato hidroetanólico concentrado das cascas de A. excelsum, nativa da Região Amazônica, onde é usada tradicionalmente para tratar várias enfermidades, inclusive malária. A atividade antiplasmódica in vitro de diferentes concentrações do extrato e frações alcaloídica e metanólica foi avaliada em culturas de P. falciparum W2 pela percentagem de inibição da parasitemia e determinação da concentração inibitória média (CI₅₀) em intervalos de 24, 48 e 72 h. O ensaio de citotoxicidade do extrato e fração alcaloídica foi realizado em fibroblastos L929 de camundongo pelo método do MTT e o teste de toxicidade aguda oral do extrato foi realizado de acordo com o Procedimento de Dose Fixa adotado pela OECD com pequenas adaptações. A prospecção fitoquímica revelou a presença de saponinas, açúcares redutores, fenóis e taninos e alcalóides e estes foram confirmados em quantidades significativas na fração alcaloídica extraída com clorofórmio (C2). Através de cromatografia em camada delgada e cromatografia líquida de alta eficiência do extrato, foi caracterizada a presença do alcalóide indólico ioimbina. O extrato e as frações apresentaram atividade antiplasmódica in vitro. O extrato apresentou a melhor atividade em 24 h (CI₅₀= 5,2 ± 4,1 μg/mL), indicando uma boa atividade esquizonticida. Apenas a fração alcaloídica C2 apresentou uma pequena, porém significativa citotoxicidade (concentrações superiores a800 μg/mL). O extrato não só não apresentou citotoxicidade como também nenhum sinal evidente de toxicidade aguda oral na dose de 5000 mg/mL. Os resultados obtidos indicam que o extrato de Aspidosperma excelsum Benth apresenta promissor potencial antimalárico, merecendo estudos mais detalhados sobre sua atividade antiplasmódica, com vistas no isolamento de compostos ativos e elucidação de seu(s) mecanismo(s) de ação.

Total Synthesis of (+)-trans-Trikentrin A

Several syntheses have already been reported for cis-trikentrins and herbindoles, which are indole alkaloids unsubstituted at the C2 and C3 positions that bear a trans-1,3-dimethylcyclopentyl unit. Herein, we describe the first asymmetric and stereoselective synthesis of the more challenging trans-trikentrin A as its naturally occurring isomer. Different approaches were investigated and the strategy of choice was a combination of an enzymatic kinetic resolution and a thallium(III)-mediated ring contraction. The antiproliferative activities of the natural product and related intermediates have been tested against human tumor cell lines, leading to the discovery of new compounds with potent antitumor activity.

Molecular cloning, heterologous expression and characterization of Strictosidine Glucosidase from Rauvolfia serpentina cell suspension cultures

Die vorliegende Arbeit hatte zum Ziel, die enzymatische Deglucosylierung von Strictosidin in Zellsuspensionskulturen von Rauvolfia serpentina zu charakterisieren.Ein Verfahren zur Isolierung und Reinigung von Strictosidin aus pflanzlicher Zellkulturen wurde entwickelt. Zwei somatische Hybridzellkulturen zwischen R. serpentina und Rhazya stricta wurden als potenzielle Quelle dieses Glucoalkaloides untersucht. Der Sekundärstoffwechsel der pflanzlichen Zellen wurde mit Methyljasmonat induziert und 15 Stoffe wurden identifiziert, u. a. das neue Indolalkaloid 3-Oxo-rhazinilam. Die Gehaltsänderung von 7 Indolalkaloiden nach Behandlung mit Methyljasmonat wurde untersucht.Deglucosylierung von Strictisidin bei in E. coli exprimierter Raucaffricin Glucosidase wurde detektiert.Die Strictosidin Glucosidase kodierende cDNA wurde aus R. serpentina Zellsuspensionskulturen cloniert und in E. coli exprimiert. Das Enzyme wurde mit Hilfe des Inteintages gereinigt und seine Eigenschaften wurden untersucht, u. a. optimale Temperatur und pH Wert und Substratspezifität.Die Produkte von der enzymatischen Strictosidinhydrolyse wurden als Cathenamin (unter normalen Bedingungen) und Sitsirikin und Isositsirikin (im Gegenwart von Reduktoren) identifiziert. Das neue Indolalkaloid 3-Isocorreantin A wurde nach der enzymatischen Deglucosylierung von Dolichantosid (Nß-Methylstrictosidin) gebildet.

Reinigung und Coexpression von Enzymen der Raubasin-Biosynthese

Die vorgestellten Arbeiten bezüglich der Biosynthese von pflanzlichen Indolalkaloiden können in einen molekularbiologischen und einen proteinche-mischen Teil aufgegliedert werden. Im molekularbiologischen Abschnitt stand die Entwicklung eines Vektorsystems im Vordergrund, das die gleichzeitige Expression mehrerer Enzyme in bakteriellen Kulturen erlaubte. Hierfür konnte zunächst die cDNA der Strictosidin-Synthase aus Rauvolfia serpentina in den Expressionsvektor pQE-70 einkloniert und das Protein aktiv exprimiert werden. Bevor es zum Einbau des Strictosidin-β-D-Glucosidase-Gens –ebenfalls aus Rauvolfia serpentina– kam, musste dessen Aktivität mit einer vorgeschalteten Ribosomenbindestelle sichergestellt werden. Diese zusätzliche Binderegion wurde an das 5’-Ende der cDNA angefügt, um die ungestörte Expression des Enzyms im späteren Coexpressions-System zu gewährleisten. Im Hinblick auf weiterführende Arbeiten in Bezug auf die komplette in vitro-Synthese bereits bekannter Alkaloide, wie z.B. das antiarrhythmisch wirkende Ajmalin oder das antihypertensiv wirkende Heteroyohimbin-Alkaloid Raubasin, wurde die ursprüngliche multiple-clonig-site des verwendeten Expressionsvektors pQE-70 um 27 zusätzliche Restriktionsschnittstellen (verteilt auf 253 bp) erweitert. Nach erfolgreicher Ligation der Strictosidin-β-D-Glucosidase-cDNA mit vorgeschalteter Ribosomenbindestelle an das 3’-Ende des Strictosidin-Synthase-Gens gelang die heterologe Coexpression beider Enzyme in einer homogenen Suspensionskultur des E. coli-Expressionsstamms M15. Dafür wurde das Vektorkonstrukt pQE-70bh-STR-RBS-SG entwickelt. Das Endprodukt der anschließenden enzymatischen Umsetzung von Tryptamin und Secologanin wurde über das Zwischenrodukt Strictosidin gebildet und konnte als Cathenamin identifiziert werden. Im proteinchemischen Teil der Dissertation wurde die Reinigung einer Cathenamin-Reduktase aus Zellsuspensionskulturen von Catharanthus roseus RC mit dem Ziel der partiellen Bestimmung der Aminosäuresequenz bearbeitet. Das gesuchte Enzym wandelte in einer NADPH-abhängigen Reaktion das Edukt Cathenamin in Raubasin um. Des weiteren wurde untersucht, wie viele Enzyme insgesamt an der Umwandlung von Cathenamin zu Raubasin und den eng verwandten Produkten Tetrahydroalstonin und 19-Epi-Raubasin beteiligt waren. Unter Anwendung eines hierfür entwickelten säulenchromatographischen Protokolls gelang die Reinigung einer Raubasin-bildenden Reduktase, deren Teilsequenz jedoch noch nicht bestimmt werden konnte. Die Anzahl der beteiligten Enzyme bei der Ausbildung von Raubasin, Tetrahydroalstonin und 19-Epi-Raubasin konnte auf mindestens zwei beziffert werden.