The role of the labile methyl groups of betaine in the transmethylation reactions in the barley plants was investigated using carbon-14 methyl labelled betaine. The N-methyl groups of N-methyl tyramine, hordenine and choline were found to arise from betaine. Betaine was also administered to castor bean seedlings and the alkaloid ricinine Isolated. It was found that Its N- and 0- methyl groups did not arise from betaine methyl. The origin of the methylenedioxy and N-methyl groups of the alkaloid protopine was also investigated by the tracer technique. Carbon-14 methyl labelled L- methionine, carbon-14 methyl labelled choline and carbon- 14 labelled sodium formate were fed to Dicentra species and the extent and position of labelling of the protopine molecule determined by degradation / Thesis / Master of Science (MS)

No / Cryptolepine, a naturally occurring indoloquinoline alkaloid used as an antimalarial drug in Central and Western Africa, has been found to bind to DNA in a formerly unknown intercalation mode. Evidence from competition dialysis assays demonstrates that cryptolepine is able to bind CG-rich sequences containing nonalternating CC sites. Here we show that cryptolepine interacts with the CC sites of the DNA fragment d(CCTAGG)2 in a base-stacking intercalation mode.

No / This article reports the structural elucidation of the Alangium alkaloid, tubulosine (1) on the basis of systematic 2D-NMR analyses (DEPT, COSY, TOCSY, NOESY, ROESY, HMQC and HMBC). The data obtained allowed the unambiguous assignment of all proton and carbon signals in 1 for the first time.

Lycorin gilt bei vielen Amaryllidaceae als Hauptalkaloid und die Aufnahme dieser Pflanzen ist eine häufige Vergiftungsursache bei Mensch und Tier. Als Hauptsymptome infolge dieser Pflanzenvergiftungen werden Nausea und Emesis genannt, aber systematische Untersuchungen zu diesen biologischen Effekten, zum Wirkmechanismus und zur Pharmakokinetik von Lycorin, das als auslösendes Agens angenommen wird, existieren bislang nicht. In der vorliegenden Arbeit werden die Zusammenhänge zwischen verabreichter Lycorin-dosis und Lycorin-induzierter Nausea und Emesis, die Beeinflussbarkeit dieser emetischen Effekte durch etablierte Antiemetika und die Pharmakokinetik von Lycorin in einem cross-over und vehikel-kontrollierten Design in vivo untersucht. Die Studie wurde an elf Beagle-Hunden beider Geschlechter durchgeführt. Die Lycorin-induzierten emetischen Effekte wurden quantifiziert und über Videoaufzeichnungen zeitnah dokumentiert. Nausea wird hierbei mittels eines Scoring-Systems quantifiziert, während die Parameter Latenzzeit, Dauer und Anzahl der Brechakte zur Beurteilung der Emesis herangezogen werden. Die subkutane Applikation von Lycorin induziert, beginnend ab einer Dosis von 0,5 mg/kg KGW Nausea und Vomitus. Eine statistische Signifikanz ist allerdings erst ab 1,0 mg/kg und ein maximaler emetischer Effekt bei einer Dosis von 2 mg/kg (ED100) zu verzeichnen. Die Ergebnisse zeigen eine Korrelation zwischen applizierter Lycorin-Dosis und Nausea-Score sowie der Anzahl der Brechakte. Lycorin-induzierte Nausea und Emesis sind in den vorliegenden Untersuchungen selbstlimitierend und dauern maximal 2,5 Stunden an. Lycorin weist in den untersuchten Dosierungen von 0,25 mg/kg bis 2,0 mg/kg eine lineare Plasmakinetik auf. Nach subkutaner Gabe werden maximale Plasmakonzentrationen (Cmax) nach 0,5 h gemessen, die mittlere Plasma-Halbwertszeit beträgt 0,67 h nach subkutaner, respektive 0,51 h nach intravenöser Applikation. Die errechnete orale Bioverfügbarkeit beträgt ca. 40 %. Das Auftreten von Nausea und Emesis, sowie deren Verlauf decken sich weitestgehend mit dem Verlauf der Lycorinkonzentration im Plasma. In keiner der untersuchten Dosisstufen sind blutchemische oder hämatologische Abweichungen aufgetreten. Um Rückschlüsse auf die Zielstrukturen von Lycorin und somit auf den emetischen Wirkungsmechanismus der Lycorin-induzierten Emesis und Nausea zu gewinnen, wurden die Hunde jeweils mit Diphenhydramin, Maropitant, Metoclopramid, Ondansetron oder Scopolamin vorbehandelt. Diese therapeutisch etablierten Antiemetika besitzen eine selektive Rezeptoraffinität und entfalten ihre antiemetische Wirkung über einen Antagonismus an histaminergen H1- (Diphenhydramin), dopaminergen D2- (Metoclopramid), muskarinergen M1-3- (Scopolamin), serotoninergen 5-HT3- (Ondansetron) oder Neurokinin-1-Rezeptoren (NK1) (Maropitant). Durch die Bindung des jeweiligen Antiemetikums an die spezifischen Rezeptoren, soll die anschließende Bindung von Lycorin an den gleichen Rezeptoren verhindert oder reduziert werden, was sich in einer Reduktion oder Abwesenheit von Nausea und Emesis auswirkt. Die Vorbehandlung mit Ondansetron ist mit einer signifikanten Verminderung der Anzahl der Brechakte verbunden und durch die Vorbehandlung mit Maropitant kann Lycorin-induzierte Emesis komplett verhindert werden. Einzig Ondansetron reduziert darüber hinaus den Ausprägungsgrad der Nausea und verlängert die Latenzzeit bis zum Auftreten von Vomitus, was eine Beteiligung von 5-HT3 Rezeptoren bei lycorin-induzierter Nausea nahe legt. Histaminerge (H1), dopaminerge (D2) und muskarinerge (M1-3) Rezeptoren sind vermutlich nicht an Lycorin-induzierter Nausea und Emesis beteiligt. Die Befunde der vorliegenden Arbeit weisen darauf hin, dass Lycorin bei Vergiftungen mit Pflanzen oder Pflanzenteilen, die zu den Amaryllidaceae gehören, eine entscheidende Bedeutung für die klinische Symptomatik und den Verlauf von Intoxikationen hat. Nach den Ergebnissen dieser Arbeit sind eine prädominierende Beteiligung von NK1- und eine etwas geringer ausgeprägte Beteiligung von 5-HT3-Rezeptoren im emetischen Wirkmechanismus wahrscheinlich. Somit erscheint die therapeutische Anwendung von Maropitant beim Hund (und evtl. Apreptitant beim Menschen) und/oder Ondansetron zur symptomatischen Behandlung anhaltender Nausea und Emesis bei Pflanzenvergiftungen mit Amaryllidacaen bei denen die Wirkung von Lycorin dominiert, wissenschaftlich begründet und klinisch von Vorteil gegenüber anderen antiemetischen Prinzipien zu sein.

Neotyphodium species, endophytic fungi associated with cool-season grasses, enhance host fitness and stress tolerance, but also produce biologically active alkaloids including ergot alkaloids associated with fescue toxicosis in grazing animals. One approach to reduce fescue toxicosis is to manipulate genes in the ergot alkaloid pathway. The gene, dmaW, encoding the first pathway-specific step in ergot alkaloid biosynthesis, was cloned previously from Claviceps spp. and its function was demonstrated by expression in yeast. Putative homologs have been cloned from Neotyphodium coenophialum (from tall fescue) and Neotyphodium sp. Lp1 (from perennial ryegrass). In order to confirm the function of dmaW in ergot alkaloid production, dmaW in Neotyphodium sp. isolate Lp1 was knocked out by gene replacement. The dmaW knockout mutant produced no detectable ergovaline or simpler ergot alkaloids. Complementation with Claviceps fusiformis dmaW restored ergovaline production. These results confirmed that the cloned endophyte gene was dmaW, and represented the first genetic experiments to show the requirement of dmaW for ergot alkaloid biosynthesis. Neotyphodium coenophialum, endophyte of the grass tall fescue (Lolium arundinaceum) has two homologs of dmaW. Considering the possible field applications in future, the Cre/lox site-specific recombination system was chosen because of the potential to sequentially knock out both homologs and obtain marker-free dmaW mutants of N. coenophialum. One homolog, dmaW-2, was disrupted by marker exchange, and the marker was eliminated by Cre, thus demonstrating the application of Cre/lox system in N. coenophialum to eliminate a marker gene. The dmaW-2 knockout did not eliminate ergovaline production, indicating that the dmaW-1 was probably also active in N. coenophialum. A putative ergot alkaloid biosynthesis gene cluster was identified in Claviceps purpurea and C. fusiformis. C. purpurea and C. fusiformis produce different subsets of ergot alkaloids. Identification of nine common genes between them suggests the possible role of these genes in the early part of the ergot alkaloid biosynthetic pathway.

The electron rich and aromatic character of the indole group allows for a wide range of oxidative and substitution reactions, creating a versatile platform for generating structurally diverse molecules. This thesis explores enzyme and synthetic chemistries that act upon indoles and related molecules. Chapter 1 describes the results of in vivo studies of RebC, an enzyme that plays a pivotal role in the biosynthesis of the indolocarbazole alkaloid rebeccamycin. A homologous enzyme, StaC, exists in the biosynthetic pathway for staurosporine, a related indolocarbazole. Structural differences between the RebC and StaC active sites were hypothesized to play a pivotal role in determining the oxidation state in the corresponding natural products. Sequence alignment of RebC and StaC with homologous enzymes from related indolocarbazole biosynthetic pathways revealed six non-conserved residues in the active site. Three RebC variants were generated by replacement of all six, four, or two specific residues with their StaC counterparts. It was demonstrated that only two substitutions, F216V and R239N, are required to convert the specificity of RebC to that of StaC. Analysis of the structure of the RebC bound to a putative reaction intermediate supports the importance of F216 and R239 in catalysis. Based on these results, contrasting mechanisms for RebC and StaC are proposed to account for their differing specificities. Chapter 2 describes a synthetic approach to primarily heterocyclic analogues of lycogarubin C. Suzuki coupling of appropriately functionalized 3,4-dibromopyrrole or 3,4-bis(trifluoromethanesulfonyl)pyrrole was effective for numerous π-excessive five-membered heterocyclic-3-boronic acids. The optimized conditions were less effective for cross-couplings involving heteroaromatic-2-boronic acids, π-deficient heteroaromatic boronic acids, and heteroaromatic boropinacolate esters. Oxidative cyclization of the 3,4-bis(thiophen-3-yl)pyrrole and 3,4-bis(benzothiophen-3-yl)pyrrole to give analogues of the corresponding indolocarbazoles was demonstrated. Chapter 3 describes preliminary results on the development of regioselective C-5 and C-7 indole metalation tactics of indole-6-carboxamides, in order to provide new functionalized indoles. The use of an indole C-2 silicon protection strategy in combination with a sterically bulky C-6 N,N-di-isopropyl carboxamide directed metalation group overcame undesired side reactions observed with the analogous N,N-diethyl indole-6-carboxamide, affording the C-5 and C-7 substituted products in 40% and 13% yields, respectively. / Thesis (Ph.D, Chemistry) -- Queen's University, 2013-02-13 11:14:49.599

A proposta deste trabalho experimental in vitro foi avaliar se a incorporação de 1% - em peso - do alcalóide teobromina (Sigma Aldrich, Darmstadt, Alemanha) ao cimento de ionômero de vidro (CIV) convencional (GC Gold Fuji 9, GC Corp, Japão) tem a capacidade de alterar as propriedades físico-químicas desse material. Para tanto, dois grupos experimentais foram propostos: G1 - CIV convencional e G2 - CIV com adição de teobromina. Foram confeccionados 160 discos de CIV de acordo com as instruções do fabricante, utilizando matrizes circulares. Para analisar as mencionadas propriedades, os discos foram submetidos a testes específicos, de acordo com as normas da International Standard Organization (ISO) para cada uma das propriedades. Discos de 15mmx1mm de diâmetro foram utilizados para as provas de sorção (n=5) e de solubilidade (n=5), com o auxílio de balança analítica, dissecadores e estufa a 23 e 37ºC por várias semanas. O ensaio de microdureza foi realizado em amostras (n=20) de 15mm×1mm submetidas a cinco edentações, com carga de 25 gramas e 30 segundos, à temperatura ambiente. Para a avaliação da cor, discos (n=20) de 15mm×1mm foram submetidos ao espectrofotômetro, adotando-se a guia colorimétrica da Comissão internacional de Iluminação (CIE). No ensaio de resistência flexural, os espécimes (n=60) de 12mm×1mm foram armazenados em estufa a 37ºC durante 24 horas para posterior analise na máquina de ensaios universal. Para avaliar a influência da teobromina adicionada ao CIV na formação de biofilme por Streptococcus mutans,, sobre os corpos da prova (n=40) de 12mm×1mm biofilmes foram desenvolvidos. As cepas de S.mutans foram cultivadas em Tryptic Soy Agar (TSA, Difco) a 37°C. Também a dosagem de flúor foi avaliada, em discos de prova (n=10) armazenados em saliva artificial e submersos em solução TISAB para posterior análise com eletrodo de flúor e obtenção da curva da liberação dessa substância. Os dados obtidos nos testes de sorção e solubilidade, microdureza, cor e resistência flexural foram submetidos à análise de variância ANOVA um fator e ao teste de Tukey para comparação entre os grupos, adotando-se 5% de nível de significância (p<0,05). O ensaio da influência da teobromina adicionada ao CIV na quantidade de biofilme de Streptococcus mutans formado e o teste de dosagem de flúor foram submetidos à análise de variância two-way ANOVA e ao teste de Tukey, com nível de significância de 5% para comparação entre os grupos experimentais. O segundo fator avaliado nestes dois ensaios foi o tempo. Os resultados não revelaram alteração da sorção e da solubilidade no CIV que recebeu teobromina (p>0,05). A microdureza aumentou com a adição de teobromina ao CIV (p<0,05). Não houve alteração de cor do CIV que recebeu teobromina (p>0,05). A resistência na flexão biaxial diminuiu quando da adição de teobromina ao CIV (p<0,05). Já a quantidade de biofilme formado foi menor em G2 (p<0,05). Em relação à liberação de flúor, observou-se que a adição de teobromina não altera essa propriedade do CIV (p>0,05). Com base em tais achados, conclui-se que a adição de teobromina a 1% ao cimento do ionômero de vidro convencional não produz alterações significativas nas propriedades desse material, podendo até mesmo otimizar algumas dessas propriedades. Ainda assim, estudos adicionais sobre o assunto devem ser realizados. / The purpose of this experimental in virtro work, was to evaluate if the incorporation of the 1% in weight of an alkaloid: theobromine (Sigma Aldrich Darmstadt, Germany) to the conventional glass ionomer cement (GIC) (GC Gold Fuji 9; GCC Japan Corp) has the capacity to change the properties physicochemical of this material. Whereby, it was proposed two experimental groups: G1 - Conventional GIC and G2 GIC incorporated with theobromine. According to the instructions of the creator, there were created 160 specimens, for which it was used circular matrices. To analyze the mentioned properties, the specimens were analyzed under the International Standard Organization (ISO) for each property. It was used matrices of 15mmx1mm diameter for the sorption (n=5) solubility (n=5) tests. With the help of an analytical balance, desiccators and a stove at 23º and 37° for various weeks; it was made essays of micro firmness using 20 matrices of 15mmx1mm, were submitted to five indentations with a charge of 26 grams and 30 seconds in environmental temperature. For the color evaluation it was used 20 matrices of 15mmx1mm, that were submitted to a spectrophotometer following the colorimetric guide of the International Commission of Illumination (CIE). An essay of biaxial flexural strength was made in 60 matrices of 12mmx1mm that were stored in a stove at 37º for 24 hours, for a later analysis in a universal testing machine. To evaluate the influence of the theobromine incorporated to the glass ionomer cement (GIC) in the creation of biofilm Streptococcus mutans, there were developed 40 matrices of 12mmx1mm of biofilm. The strains of the Streptococcus mutans were cultivated in Tryptic Soy-Agar at 37°. It was also evaluated the fluorine release capacity using 10 test matrices stored in artificial saliva and submerged in TISAB substance for a later analysis with an fluorine electrode, to obtain the release curve of that substance. The obtained information from the test of sorption, solubility, micro strength, color and biaxial flexural strength, were submitted to an analysis of variance ANOVA one factor and Tukey\'s test for a comparison between groups assuming the 5% level of significance (p <0,05). The essay of the influence of the theobromine added to the GIC in the amount of biofilm Streptococcus mutans formed and the test of fluorine release were submitted to the Bidirectional Variance Analysis ANOVA and Tukey\'s test, with a significant level of 5% to the comparison between experimental groups; the second factor evaluated in this two essays was time. The results didn\'t show an alteration in the sorption and solubility in the GIC that received theobromine (p >0,05). The micro strength increased with the addition of the theobromine to the GIC (p< 0,05). There were not any alterations in the color of the GIC that received theobromine (p >0.05). The resistance to the biaxial flexural strength decreased when the theobromine was added to the GIC (p <0.05). The amount of formed biofilm was less in the G2. In relation to the fluorine release it was observed that the addition of theobromine does not change the properties of GIC (p >0.05). Based on these findings, it is concluded that the addition of theobromine in 1% to the conventional glass ionomer cement, does not produce significant changes over this material properties and also, it can optimize some of these properties. However, more studies should be done about this topic.

A ipalbidina é um alcaloide indolizidínico com propriedades analgésica e antirradicais livres. Este alcaloide possui como fonte natural a Ipomoea alba L., uma espécie de dama-da-noite, sendo isolado das suas sementes na forma de aglicona da ipalbina (ligada à D-glicose). A ipalbidina possui estrutura química relativamente simples, porém sua síntese na forma enantiomericamente pura se apresenta como um desafio sintético. Dentre as inúmeras rotas de síntese da ipalbidina, apenas quatro são enantiosseletivas. Neste trabalho de dissertação é apresentada uma nova estratégia sintética visando a preparação da S-(+)-ipalbidina. A estratégia tem como etapas chaves: uma reação de olefinação (Wittig e Horner-Wadsworth-Emmons); a preparação de uma diazocetona e de &alpha;-clorocetonas; e a conversão destas no esqueleto indolizidínico por uma reação de ciclização. Como material de partida para a reação de olefinação foi empregado o S-prolinal protegido (Boc e Cbz). Este aminoaldeído foi empregado como fonte do centro estereogênico e de um dos anéis presentes na estrutura final. A partir das reações de olefinação, foram obtidos compostos carbonílicos &alpha;, &beta;-insaturados que, em seguida, sofreram redução da dupla ligação (28-94%). A partir dos produtos hidrogenados, foram preparados ácidos carboxílicos (66-83%) que, em seguida, foram convertidos na diazocetona (32%) e nas &alpha;-clorocetonas (34-70% a partir de ilídeos &beta;-ceto sulfoxônios). As &alpha; -clorocetonas também foram preparadas a partir de ésteres com bons rendimentos (60-70%). O esqueleto indolizidínico foi obtido a partir da reação de ciclização da diazocetona e das &alpha; -clorocetonas, porém os rendimentos não foram satisfatórios e a metodologia deve ser melhorada. Uma &alpha;-clorocetona mais simples foi reduzida ao álcool correspondente. A &beta;-cloridrina obtida foi convertida no aminoálcool cíclico (indolizidina monoidroxilada, 16%). A preparação deste aminoálcool cíclico sugere que a metodologia desenvolvida é promissora. A nova estratégia sintética pode levar à síntese da (+)-ipalbidina em seis ou sete etapas (uma rota curta). A funcionalização da &alpha;-clorocetona e o melhoramento do método a partir da diazocetona podem também levar à síntese outras indolizidinas. / Ipalbidine is an indolizidine alkaloid with analgesic and antioxidant properties. This alkaloid is isolated from the seeds of Ipomoea alba L., as the aglycone of ipalbine (associated to the D-glucose). The ipalbidine has a relatively simple chemical structure, but its synthesis in the enantiomerically pure form is a synthetic challenge. Although a great number of synthetic routes of ipalbidine have been reported, only four are enantioselective. This dissertation reports new studies toward the synthesis of (+)-ipalbidine. The key steps of the synthetic strategy are: olefination reaction (Wittig or Horner-Wadsworth-Emmons); preparation of a diazoketone and &alpha; -chloroketones; and the cyclization reaction from the diazoketone and &alpha; -chloroketones. A (Boc and Cbz) protected-S-prolinal was used as the initial reagent. This amino aldehyde was employed as the chiral pool and it provides one the rings necessary to build the indolizidine skeleton. A Wittig reaction (88-94%) and a Horner-Wadsworth-Emmons reaction (60-70%) were evaluated for the first step of the synthesis. &alpha;, &beta;-Unsaturated carbonyl compounds were prepared from the olefination reactions and, after that, double bond reduction was carried out under some hydrogenation conditions (28-90%). The carboxylic acids were prepared from the hydrogenated compounds (66-83%). The carboxylic acids were used to prepare the diazoketone (32%) and the &alpha; -chloroketones (34-70% from sulfoxonium ylides). The cyclization reaction from the diazoketone and &alpha; -chloroketones afforded the indolizidine skeleton, but the yields were not satisfactory. The reaction conditions, in this case, need to have to be improved. The &alpha; -chloroketones were also prepared from esters in good yields (60-70%). A simple &alpha; -chloroketone was reduced to the corresponding &beta;-chlorohydrin. After the preparation of this &beta;-chlorohydrin, the cyclization afforded a cyclic amino alcohol (a mono hydroxylated indolizidine, in 16% yield). These results suggest a promising methodology. The present strategy on the synthesis of (+)-ipalbidine can provide this alkaloid in six or seven steps (a short route) and can also be applicable to the syntheses of other indolizidine alkaloids.

Drug discovery is reliant on new developments in natural product chemistry as well as advances in chemical synthesis. The interconnectivity and interdependence of natural and synthetic investigation in drug discovery is evident. The chemical exploration reported herein elaborates the relationship between natural product chemistry and chemical synthesis. Of particular interest are chemicals from organisms residing in less accessible environments, particularly Antarctica and endophytic microbial communities. Degradation via reductive ozonolysis of palmerolide A, a macrocyclic polyketide isolated from the Antarctic tunicate Synoicum adareanum, and subsequent synthetic preparation of the resulting polyols (1,2,6-hexanetriol and 1,2,3,6-hexanetetraol) led to a revision in the absolute configuration of the bioactive natural product (7R, 10R, 11R to 7S, 10S, 11S). A partial synthesis of palmerolide A (C3-14) was completed using Grubb’s 2nd generation catalyst to couple fragments formed using the previously developed methodology from the degradation study. Isolation of indole-pyrimidine containing alkaloids meridianins A, B, C, and E from the Antarctic tunicate Synoicum sp. prompted a synthetic investigation of psammopemmin A, a related alkaloid from the Antarctic sponge Psammopemma sp. resulting in reassignment of the structure of psammopemmin A to that of meridianin A. Both meridianin A and psammopemmin A were synthesized through a Suzuki coupling of the same 4-indolol nucleophile to the apposite pyrimidine electrophile. Several synthetic 3-pyrimidylindole analogs were also prepared and investigated for central nervous system, antimalarial, and cytotoxic activity. Chemical investigation of extracts from mangrove fungal endophytes that displayed antimalarial properties in vitro resulted in the isolation of several potent but cytotoxic and cytostatic compounds: cytochalasin D, roridin E, and 12,13-deoxyroridin E.

A variety of heterocyclic alcohols were coupled with silyl ketene acetals and other [pi]-nucleophiles in the presence of trimethylsilyl trifluoromethanesulfonate to provide an array of substituted [beta]-heteroaryl propionates, including those with contiguous quaternary centers. This reaction also proceeds with high diastereoselectivity when the [pi]-nucleophile bears a chiral auxiliary. The formal synthesis of N-methylwelwitindolinone C isothiocyanate, a densely functionalized alkaloid with the ability to reverse multiple drug resistance, was completed featuring several key transformations. The first key transformation consisted of coupling a heterocyclic alcohol with a silyl ketene acetal to give a highly functionalized intermediate. Next, a palladium catalyzed enolate arylation followed by an intramolecular allylic alkylation successfully constructed the bicyclo(4.3.1)decane backbone of the N-methylwelwitindolinone C isothiocyanate. / text