This thesis describes investigations directed towards developing a novel synthetic route to the natural products perophoramidine and the communesins, with particular emphasis placed on the formation of the two vicinal all-carbon quaternary centres contained in these molecules. Chapter 1 introduces perophoramidine and the communesin group of natural products and explains how they are related to the calycanthaceous alkaloids. The isolation of perophoramidine and the communesins is outlined and their biosynthesis is discussed. Specific structural features of these natural products are highlighted before established synthetic strategies are reviewed. Chapter 1 concludes by proposing a novel synthetic route for the synthesis of perophoramidine and the communesins that involves a Claisen rearrangement in the indolo[2,3-b]quinoline system as a key step. Chapter 2 describes model studies on the proposed Claisen rearrangement in an attempt to form a quaternary centre in the indolo[2,3-b]quinoline system. These initial studies did not result in the generation of the desired quaternary centre. However, a detailed understanding of the reactions that occur leads to the design of a new model substrate. Chapter 3 describes studies on the revised model system that result in the formation of the desired quaternary centre using a Claisen rearrangement. The differences between the two systems are discussed before an investigation into the scope of the rearrangement is described. Chapter 3 concludes by describing an investigation into a protecting group strategy that would by required with this synthetic route. Chapter 4 describes investigations into the formation of the second vicinal quaternary centre using a model system. The synthetic routes investigated lead to two separate methods for the formation of the desired quaternary centre. Chapter 5 describes investigations into the effect a C-10 substituent has on the Claisen rearrangement. Additionally, an asymmetric version of the Claisen rearrangement is examined. Chapter 5 culminates in the preparation of an intermediate relevant to an asymmetric synthesis of the communesins.

<p>QCR 20161026</p>

<p>N-Heterocycles are ubiquitous in biologically active natural products and pharmaceuticals. Yet, new syntheses and modifications of N-heterocycles are continually of interest for the purposes of expanding chemical space, finding quicker synthetic routes, better pharmaceuticals, and even new handles for molecular labeling. There are several iterations of molecular labeling; the decision of where to place the label is as important as of which visualization technique to emphasize. </p><p>Piperidine and indole are two of the most widely distributed N-heterocycles and thus were targeted for synthesis, functionalization, and labeling. The major functionalization of these scaffolds should include a nitrogen atom, while the inclusion of other groups will expand the utility of the method. Towards this goal, ease of synthesis and elimination of step-wise transformations are of the utmost concern. Here, the concept of electrophilic amination can be utilized as a way of introducing complex secondary and tertiary amines with minimal operations.</p><p>Molecular tags should be on or adjacent to an N-heterocycle as they are normally the motifs implicated at the binding site of enzymes and receptors. The labeling techniques should be useful to a chemical biologist, but should also in theory be useful to the medical community. The two types of labeling that are of interest to a chemist and a physician would be positron emission tomography (PET) and magnetic resonance imaging (MRI). </p><p>Coincidentally, the 3-positions of both piperidine and indole are historically difficult to access and modify. However, using electrophilic amination techniques, 3-functionalized piperidines can be synthesized in good yields from unsaturated amines. In the same manner, 3-labeled piperidines can be obtained; the piperidines can either be labeled with an azide for biochemical research or an 18F for PET imaging research. The novel electrophiles, N-benzenesulfonyloxyamides, can be reacted with indole in one of two ways: 3-amidation or 1-amidomethylation, depending on the exact reaction conditions. Lastly, a novel, hyperpolarizable 15N2-labeled diazirine has been developed as an exogenous and versatile tag for use in magnetic resonance imaging.</p> / Dissertation

O fenômeno da bio- e quimiluminescência tem atraído o interesse da comunidade científica nas últimas décadas não só pelo seu inerente interesse acadêmico, mas também devido as incontáveis aplicações analíticas que dele têm surgido. A maior parte do trabalho acadêmico que tem sido desenvolvido está relacionado ao estudo do mecanismo de geração de estados excitados e a eficiência de desativação radiativa. Por outro lado, do ponto de vista das aplicações tecnológicas, as metodologias para análise de enzimas, drogas e metabólitos, aplicadas à imunologia, microbiologia, medicina forense, etc., que se baseiam em quimiluminescência, estão entre as mais utilizadas em procedimentos de rotina em laboratórios. O desenvolvimento de substratos e, conseqüentemente, novas técnicas quimiluminescentes tem se tornado cada vez mais importante devido a alta sensibilidade desses ensaios, tipicamente equivalente ou melhor do que aqueles que utilizam rótulos radioativos. Esta tese apresenta o desenvolvimento de novas metodologias quimiluminescentes para a determinação de atividade enzimática. O princípio químico é a geração de peróxidos cíclicos instáveis, conhecidos como 1,2-dioxetanos, após a hidrólise de substratos específicos, catalisada pela enzima objeto de estudo. Anéis dioxetânicos são conhecidos pela sua propriedade de gerar produtos em estados eletronicamente excitados quando decompostos. A emissão de luz pode ser relacionada à atividade enzimática. Foi desenvolvido o substrato (fosfato dissódico de 2-metil-1-propenila, NA-MPP) (I)), capaz de produzir o composto 2-metil-1-propen-1-ol quando hidrolisado via a ação catalítica das enzimas fosfatase alcalina (ALP) ou fosfatase ácida (ACP). Este enol é oxidado, sob ação catalítica da enzima peroxidase de raiz forte (HRP), gerando acetona em estado excitado triplete. A emissão de luz direta ou sensibilizada da acetona excitada pode ser correlacionada a atividade enzimatica da ALP ou ACP. A determinação da atividade dessas enzimas livres ou ligadas em anticorpos (conjugados ALP-IgG) tem grande aplicação em tecnologias de diagnóstico, seja como um marcador de diversas doenças, seja como uma sonda em ensaios imuno-enzimáticos (EIA). A sensibilidade alcançada com este substrato foi de 10-15 mols de ALP, 0,0027 unid. de ACP e diluições de até 300.000 de um conjugado (ALP-IgG) por ensaio. Também foi possível correlacionar a atividade de ALP à velocidade de consumo do oxigênio dissolvido no meio de reação, que é uma característica dessa oxidação. Partindo do mesmo princípio delineado no parágrafo anterior, desenvolveu-se um composto para determinação de proteases. Para isso, o composto N-etil-N-(2-metil-1-propenil)benzenamida (II) foi preparado, pois a clivagem de sua ligação amídica geraria uma enamina, que também pode ser oxidada pela ação catalítica da HRP. No entanto, nossos estudos mostraram que este composto não é reconhecido como substrato das proteases. Tomando como base a bem conhecida característica de gerar uma fraca emissão de luz quando derivados indólicos são oxidados por agentes oxidantes clássicos, como KMnO4, K2S2O4, etc., foi estudado o potencial quimiluminescente de alguns derivados indólicos quando submetidos ao sistema HRP/H2O2/O2. Como era esperado, detectou-se quimiluminescência de baixa intensidade para a maioria dos derivados indólicos. Também neste caso a clivagem do anel indólico, via um intermediário dioxetânico, parece ser a responsável pela emissão observada na maioria dos compostos testados. Além disso, a oxidação do composto 2-metilindol (III) mostrou uma eficiência de quimiluminescência com cerca de 3 ordens de grandeza maior que os demais derivados. Verificou-se que o comportamento diferenciado desse composto estava relacionado à exclusiva formação de um composto secundário. A estrutura desse composto foi parcialmente atribuída ao 2,2\'-dimetil-2,2\'-diindoxil. Então, utilizando o 2-metilindol como substrato, desenvolveu-se uma metodologia analítica para determinação de HRP livre ou ligada em anticorpos (conjugados HRP-IgG). Assim como no caso da enzima ALP, conjugados do tipo HRP-IgG são largamente utilizados em EIA. Também com base nas características quimiluminescentes de \'alfa\'-hidroperóxi-cetonas quando submetidas a um forte meio alcalino, desenvolveu-se um potencial substrato para análise de esterases. A hidrólise catalisada por esterase de 2-peracetoxiadamantano-2-carboxialdeído (IV) geraria um \'alfa\'-hidroperóxi-aldeído, que por um ataque nucleofílico intramolecular, levaria a um intermediário dioxetânico. Este composto mostrou-se instável, gerando quimiluminescência mesmo na ausência da enzima. Este fato inviabilizou o seu uso como planejado. / The bio- and chemiluminescent phenomena have attracted the scientists attention in the last decades not only because its inherent academic interests, but also due the uncounted analytical applications that it has originated. Most of the academic work was devoted to the study of the mechanism responsible for the generation of the excited states and the efficiency of radiative deactivation. On the other hand, the technological developments pointed to methodologies for enzyme, drug, and metabolite determination applied to immunological, microbiology, forensic science, etc., based on chemiluminescence, which are already among the most applied techniques in routine laboratory procedures. The development of chemiluminescent substrates has become increasingly important due to their high sensitivity, typically equivalent to or better than assays using radioactive labels. This thesis reports the development of new chemiluminescent methodologies for enzymatic activity determination. The chemical basis is the generation of unstable cyclic peroxides, called 1,2-dioxetanes, upon hydrolysis of specific substrates catalyzed by the target enzyme. Dioxetanes rings are known by their properties to generate electronically excited products upon decomposition. The light emission can be related to enzymatic activity. It was developed a substrate (dissodium 2-methyl-1-propenyl phosphate) (Na-MPP) (I) able to produce 2-methyl-1-propen-1-ol when catalytically hydrolyzed by alkaline (ALP) or acid (ACP) phosphatases enzymes. This enol is oxidized, upon horseradish peroxidase (HRP) action, yielding acetone in triplet excited state. The direct or sensitized light emission of the excited acetone can be correlated to enzymatic activity of ALP or ACP. The activity of this enzyme, free or bound to antibody (ALP conjugates), is widely used in diagnostic technologies, either as a direct marker of several diseases or as an enzymatic probe in enzyme immunoassays (EIA). The sensibility reached with this substrate was 10-15 mols to ALP, 0,0027 u/mL to ACP and dilutions up to 300.000 of ALP-IgG per assay. Since the HRP system consumes dissolved oxygen during the oxidation of the enol, ALP quantification may be performed by following the oxygen uptake rate. By applying the same principle above delineated, it was synthesized a compound for proteases activity determination. Thus, the compound N-ethyl-N-(2-methylpropen-1-yl)benzenamide (II) was prepared, since its hydrolysis would lead to an enamine , which is known to be oxidized via HRP with light emission. However, our studies showed that II is not recognized as a substrate by proteases. Owning to the well known weak emission elicited when indole derivatives are oxidized by classical oxidants like KMnO4, K2S2O4, etc., it was studied the chemiluminescent potential when indoles are submitted to the HRP/H2O2/O2 oxidant system. Indeed, weak chemiluminescence was detected for almost all derivatives. Likewise, the oxidation of 2,3-bond of indoles, through a dioxetane intermediate leading to an open-ring product, seems responsible for this emission. Furthermore, the oxidation of 2-methylindole (III) showed a chemiluminescence efficiency about 3 orders of magnitude higher. It was observed that the high chemiluminescent yield was related to exclusive formation of a secundary product. Its structure was partially attributed to 2,2\'-dimethyl-2,2\'-diindoxil. Thus, using 2-methylindole as substrate was possible to develop an analytical procedure to quantify HRP activity, free or bound to antibodies (conjugates HRP-IgG). In EIA the enzymes HRP and ALP are the most important labels. From the also known chemiluminescent characteristics of \'alpha\'-hidroperoxy-ketones, when submitted to strong alkaline medium, it was developed a potential substrate to esterases. The esterase catalyzed hydrolysis of 2-acetylperoxiadamantane-2-carboxaldeyde (IV) would generate an \'alpha\'-hidroperoxy-aldeyde which, by an intramolecular nucleofilic attack, would lead to a dioxetane intermediate. This compound showed to be unstable and it generated chemiluminescence in the absence of the enzyme. This fact impaired its use as planned.

Metabólitos secundários são produzidos por alguns grupos vegetais e são essenciais nas diferentes estratégias de adaptação às adversidades ambientais, atuando na proteção e comunicação das plantas, sendo responsivos a diversos fatores bióticos e abióticos. Entre as diversas categorias de metabólitos secundários, os alcalóides apresentam principal função relacionada à defesa contra herbívoros; atuam também na proteção contra patógenos e na interação química com outras plantas (alelopatia). Alcalóides monoterpenos indólicos (MIAs) são uma classe de alcalóides de origem biossintética mista, e apresentam propriedades farmacológicas conhecidas (e.g. MIAs de Catharanthus roseus e Rauwolfia serpentina). MIAs provenientes de algumas espécies de Psychotria do Sul do Brasil são descritos como agentes antioxidantes, antimutagênicos, ansiolíticos, antidepressivos, antipsicóticos e analgésicos, apresentando grande potencial farmacológico. N,β-D-glicopiranosil vincosamida (GPV) é o alcalóide majoritário de Psychotria leiocarpa (Rubiaceae – APG III), apresentando estrutura semelhante a alguns alcalóides bioativos de Psychotria da região, com a peculiaridade de ser N-glicosilado. No presente trabalho, foi avaliado o efeito de dano mecânico e aplicação de jasmonato sobre o acúmulo de GPV no contexto de um possível papel do alcalóide em respostas à herbivoria, além de propriedades antioxidantes do composto. O teor de GPV se manteve constante após a aplicação dos tratamentos, ao longo de todo o experimento. Portanto, a estratégia de acúmulo deste alcalóide segue o padrão de fitoanticipina. No ensaio de dano mecânico os teores de compostos fenólicos também foram monitorados e se mantiveram constantes. Ensaios de herbivoria utilizando dois modelos generalistas e um especialista, não constataram eficácia do GPV na proteção contra estes predadores. Ensaios de atividade contra oxigênio singleto, ânions superóxido, radicais hidroxil e peróxido de hidrogênio revelaram ampla atividade antioxidante, com alguns resultados similares ao controle positivo (Trolox, um análogo da vitamina E). Os resultados obtidos neste trabalho, juntamente com dados existentes da literatura para metabólitos correlatos, sugerem uma função fundamentalmente antioxidante de MIAs de Psychotria, atuando como um modulador de estresse oxidativo. / Some plants groups accumulate secondary metabolites, which may play a major role in different strategies to deal with environmental challenges, being responsive to several biotic and abiotic factors and functioning as protection and communication agents. Among secondary metabolites, alkaloids play a major role as anti-feedant agents and are also involved in pathogen protection and chemical interaction (allelopathy). Monoterpene indole alkaloids (MIAs) are derived from two distinct biosynthetic pathways and possess well known pharmacological properties (e.g. MIAs from Catharanthus roseus and Rauwolfia serpentina). MIAs from Southern Brazilian Psychotria have been characterized as antioxidant, antimutagenic, ansyolitic, antidepressive, antipsychotic and analgesic agents, therefore bearing relevant pharmacological potential. N,β-D-glucopyranosil vincosamide (GPV) is the major alkaloid from Psychotria leiocarpa (Rubiaceae - APG III) and its structure, besides being additionally glycosylated in the N indol ring, is similar to a few bioactive alkaloids from native Psychotria species. In the present work, the effects of wounding and jasmonate application on GPV accumulation, and also antioxidant properties, were evaluated in the context of a potential role of the alkaloid in herbivory responses. GPV content remained constant after treatments, at all times of exposure. Therefore, GPV seems to present a phytoanticipin-like accumulation pattern. In the mechanical wounding assay, phenolic compounds content was also monitored and remained constant. In two herbivory assay models, a generalist and a specialist, GPV was not efficient to prevent herbivore feeding. Singlet oxygen, superoxide anions, hydroxyl radicals and hydrogen peroxide assays showed GPV has broad antioxidant activity, in some cases with activity equivalent to the positive control (Trolox, a vitamin E analog). The results obtained in this work, together with published results from our research group, strongly suggest an antioxidant role for Psychotria MIA alkaloids, which may act as oxidative stress modulators.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2014-10-09T15:01:20Z No. of bitstreams: 2 Dissertação - Aline Pereira Moraes - 2013.pdf: 2375025 bytes, checksum: edebdd82c526094db969a19e855e1017 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-10-09T15:30:41Z (GMT) No. of bitstreams: 2 Dissertação - Aline Pereira Moraes - 2013.pdf: 2375025 bytes, checksum: edebdd82c526094db969a19e855e1017 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-10-09T15:30:41Z (GMT). No. of bitstreams: 2 Dissertação - Aline Pereira Moraes - 2013.pdf: 2375025 bytes, checksum: edebdd82c526094db969a19e855e1017 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-05-03 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The use of natural products and their synthetic derivatives has been a relevant strategy in the development of novel medicines. Phytochemical studies and synthesis of natural-product-based libraries are primordial in the search for therapeutic agents. Previous phytochemical studies of genus Psychotria (Rubiaceae) have resulted in the identification of polypyrrolidine indole and monoterpenoid indole alkaloids, which present a broad range of biological activities, such as antifungal and inhibition of monoamine oxidases (MAOs) A e B, that are related to neurodegenerative diseases. This study aims to evaluate the phytochemical composition of the aerial parts of Psychotria sp. collected in Brazilian Cerrado. Fractionation of the crude extract by column chromatography on silica gel and Sephadex led to isolation of three known indole alkaloids: bahienoside A, desoxycordifoline and desoxycordifolinic acid. Additionally, thiohydantoins and thioureas were synthesized from amino acids and R- (+)-limonene, in order to assess the cooperative effect of indole nucleus combined with terpene, thiourea and thiohydantoin units. Preliminary tests showed that desoxycordifoline, hydantoin (20) and thiohydantoin necrostatin-1 (14) inhibit the enzyme MAO-A higher than 80% at concentrations of 175 mM, 100 mM, 386 mM, respectively. Also, the polar extracts of the leaves of Psychotria sp. showed antioxidant activity with IC50 < 50 mg.mL-1 measured by DPPH free radical scavenging assay. / O uso de produtos naturais e seus derivados sintéticos tem sido uma relevante estratégia no desenvolvimento de novos medicamentos. Estudos fitoquímicos e derivatização de produtos naturais são fundamentais na busca por protótipos de fármacos. Estudos fitoquímicos anteriores do gênero Psychotria (Rubiaceae) resultaram na identificação de alcaloides indólicos monoterpênicos e pirroloindólicos. Essas classes de compostos são associadas a um amplo espectro de atividades biológicas, tais como antifúngica e de inibição de enzimas monoaminoxidases A e B (MAOs) relacionadas a doenças neurodegenerativas. Assim, esse trabalho teve como objetivo realizar o estudo fitoquímico das partes aéreas de Psychotria sp., presente no cerrado goiano. O fracionamento do extrato bruto por cromatografia em coluna em sílica gel e Sephadex resultou no isolamento de três alcaloides indólicos conhecidos: bahienosida A, desoxicordifolina e ácido desoxicordifolínico. Neste trabalho, tiohidantoínas e tioureias derivadas do R-(+)-limoneno e de aminoácidos, tais como o triptofano, foram sintetizadas, com o intuito de avaliar o efeito cooperativo do núcleo indólico combinado com as unidades terpênica, tioureia e tiohidantoína na ação antifúngica e de inibição das enzimas MAOs. Testes preliminares mostraram que desoxicordifolina, hidantoína (20) e tiohidantoína necrostatina-1 (14) inibem a enzima MAO-A acima de 80% nas concentrações de 175 μM, 100 μM, 386 μM, respectivamente. Ainda, os extratos polares das folhas de Psychotria sp mostraram atividade antioxidante com CI50 < 50 mg.mL-1 pelo método de captura dos radicais DPPH.

Faschingbauer J.: Cytotoxic and cholinesterase inhibitory activity of extracts from selected species of the Centaurea L. genus. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany, Hradec Králové, 2019. During the screening of biologically active secondary metabolites of plants carried out at the Department of Pharmaceutical Botany FAF UK, selected taxa of the genus Centaurea (Asteraceae) were investigated. This study is focused on a basic phytohemical research of extracts prepared from Centaurea cyanus, Centaurea jacea, Centaurea scabiosa, Centaurea pseudophrygia, Centuarea stoebe, Centaurea solstitialis a Centaurea benedicta. Extracts were prepared for evidence of the proof reactions of TLC and MS analysis (EI, ESI) to clarify a potential presence of alkaloids. EtOAc and ethanol extracts were evaluated for potential inhibitory activity against human erythrocyte acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BChE) and cytotoxicity against selected 9 tumor lines. C. cyanus alkaloid extract had interesting cholinesterase activity which selectively inhibited BChE (IC50 BChE = 22.62 ± 3.62 μg / ml, IC50 AChE = 221.50 ± 44.56 g / ml). Other EtOAc extracts of selected Centaurea species were considered inactive (IC50 > 100 μg/ml)....

Depuis le début des années 1990, l'hétéroannélation de Larock est apparue comme une méthode de choix pour obtenir, en une seule étape, des indoles 2,3-disubstitués. Cependant, bien qu'efficace, certains inconvénients restaient associés à cette stratégie comme l'utilisation d'un système catalytique homogène associé à l'emploi de sels. Pour cette raison, nous avons développé une nouvelle méthodologie sans sels ou additifs par catalyse hétérogène. Différents catalyseurs commerciaux ou faciles d'accès (Pd/C ou [Pd]/NaY) ainsi que de nouveaux complexes au palladium immobilisés sur SBA-15 ont été testés. De façon surprenante, ces nouvelles conditions ont permis une activation de 2-bromoanilines et ce, sans l'emploi d'additifs. Une autre méthode d'obtention de ces hétérocycles a émergé ces dernières années et se pose comme une alternative aux couplages traditionnels : l'arylation d'indoles. Malgré de nombreuses améliorations, aucun travail ne décrivait une procédure permettant une arylation complémentaire C2 ou C3 d'indoles libres avec un système catalytique unique. Nous avons développé un tel système, basé sur une pallado-catalyse dans l'eau. Cette stratégie permet d'atteindre des sélectivités C2/C3 ou C3/C2 élevées avec de bons rendements isolés par un simple contrôle du couple {base/halogénure d'aryle}.Enfin, dans le cadre de nos travaux visant la synthèse par catalyse hétérogène de produits à haute valeur ajoutée, nous avons initié des études sur l'obtention de pyrroloiminoquinones. La méthode consistant en une catalyse hétérogène monotope en seulement deux grandes étapes devrait permettre un accès rapide à de nombreux dérivés bioactifs isolés d'organismes marins.

Graduation date: 2006

3-Methyl indole (3-MI) is a malodorant in food and animal waste and Clostridium scatologenes ATCC 25775 is the model organism for the study of 3-MI production. 3-MI is an anaerobic degradation product of L-tryptophan and can cause pulmonary disorders and death in cattle and goats. To elucidate the 3-MI biosynthesis pathway and the underlying genes, it is necessary to develop a system to allow genetic modification in Clostridium scatologenes ATCC 25775. Bacteriophages and transposons are useful tools to achieve this goal. Isolation of Clostridium scatologenes ATCC 25775 bacteriophage was attempted by prophage induction and enrichments using environmental sources. To induce prophages, cultures of Clostridium scatologenes ATCC 25775 were exposed to an effective concentration of mitomycin C at 2μg/ml and 5μg/ml. Induction with temperature was performed at 42ºC and 55ºC. Bacteriophage liberation, determined by a decrease in optical density was not observed in response to mitomycin C or by different growth temperatures. Nineteen environmental samples were tested for the presence of a bacteriophage that could infect Clostridium scatologenes ATCC 25775. The first cycle of enrichments suggested a decrease in cell density, consistent with the presence of a bacteriophage but this was not observed in further iterations. Plaque assays were performed to confirm the presence of phage, but no plaques were observed. Although, different experimental conditions were tested, a transducing bacteriophage capable of infecting Clostridium scatologenes ATCC 25775 was not isolated. Transposons have been successfully used to generate mutants in Clostridium difficle. Therefore, we attempted to introduce transposons Tn5 and Tn916 into Clostridium scatologenes ATCC 25775 using electroporation. Transposon mutagenesis using Tn916 did not yield antibiotic resistant colonies. In contrast, commercially available transposon Tn5 gave antibiotic resistant colonies. However, further screening of the colonies using transposon specific primers in PCR reactions, did not yield any PCR product. We were unsuccessful in developing a genetic modification system in Clostridium scatologenes ATCC 25775 using bacteriophage or transposons.