La réaction de cycloaddition entre un azoture et un alcyne catalysée par le cuivre (I) (CuAAC) pour former un 1,2,3-triazole 1,4-disubstitué est très utilisée dans de nombreux domaines de la chimie. Cette réaction a très vite été utilisée en synthèse peptidique notamment du fait du caractère isostère du noyau triazole et de la liaison amide. Toutefois l'impact de l'insertion d'un triazole sur la structure secondaire de peptides n'a été que faiblement exploré. Ainsi, pour étudier l'effet d'un tel remplacement nous avons choisi deux peptides modèles structurés le premier modèle est un peptide linéaire de la famille des peptaibols et le second est un peptide cyclique la tamandarine B. Dans un premier temps, nous avons optimisé une voie de synthèse de peptaibols que nous avons appliqué à l'alaméthicine F50/5 et à un analogue de la bergofungine D. Pour cela, nous avons utilisé une synthèse peptidique en phase solide sous irradiation micro-ondes en profitant d'un cocktail de réactifs efficaces contenant du diisopropylcarbodiimide comme agent de couplage et de l'oxyma. Cette méthode de synthèse a été étendue à l'obtention d'analogues silylés de l'alamethicine en substituant dans différentes positions l'acide aminobutyrique par un résidu hydrophobe et encombré : la bis-triethylsilyl-dipropylglycine (TES-Dpg). Dans la deuxième partie nous avons développé la synthèse de dipeptides à motif triazole. Puis nous avons défini les conditions réactionnelles nécessaires pour leur utilisation en SPPS et synthétisé des peptides contenant plusieurs motifs triazoles. Ces dipeptides ont ensuite été utilisés pour réaliser un scan triazole sur les deux peptaibols modèles. Les études structurales par dichroïsme circulaire, RMN et les tests biologiques de différents analogues nous permettent de conclure que le triazole affecte la structure secondaire des peptaibols et par conséquence induit une perte d'activité. Ainsi il apparaît que le concept de triazole comme isostère de la liaison peptidique doit être employé avec prudence. Dans l'optique de comprendre l'impact de triazole sur l'activité et la structure secondaire de peptides cycliques, nous avons généré des analogues simplifiés de la tamandarine B un depsipeptide cyclique d'origine marine. Nous rapportons les résultats préliminaires de cette étude. / The cycloaddition reaction between an azide and an alkyne catalyzed by copper (I) (CuAAC) to form a 1,2,3-triazole 1,4-disubstituted is widely used in many areas of chemistry. This reaction was rapidily used in peptide synthesis because of the isosteric nature between the triazole ring and the amide bond. However, impact of the insertion of a triazole on the secondary structure of peptides was only scarcely explored. Thus, to study the effect of such a replacement we chose two models of structured peptides. The first models are linear peptides of the peptaibols family and the second is the cyclic peptide tamandarin B.First, we have optimized a peptaibols synthesis that was applied to alamethicin F50 / 5 and to an analog of bergofungin D. For this we used a solid phase peptide synthesis under microwave irradiation taking advantage of the efficient cocktail consisting of diisopropylcarbodiimide as a coupling agent and Oxyma. This synthesis has been extended to silylated analogues of alamethicin by substituting in different positions the aminobutyric acid by the hydrophobic and crowded residue: bis-triethylsilyl-dipropylglycine (Dpg-TES).In the second part we have developed the synthesis of dipeptides containing a triazole motif. Then we defined the reaction conditions necessary for their use in SPPS and synthesized peptides containing several triazoles rings. These dipeptides were then used to perform a triazole scan on the two peptaibols models. The structural studies by circular dichroism, NMR and biological tests of various analogs allow us to conclude that the triazole affect the secondary structure of peptaibols and consequently induces a loss of activity. Thus it appears that the concept of triazole as isosteric of the peptide bond should be used with caution.In order to understand the triazole impact on the activity and the secondary structure of cyclic peptides, we generated simplified cyclic analogues of tamandarin B a depsipeptide of marine origin. We report the preliminary results of this study.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-01-27T14:53:54Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - João Paulo Cabral de Brito - 2014.pdf: 977293 bytes, checksum: f8d05d4259ea9bbd904419bbd7674cf9 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-01-28T13:20:49Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - João Paulo Cabral de Brito - 2014.pdf: 977293 bytes, checksum: f8d05d4259ea9bbd904419bbd7674cf9 (MD5) / Made available in DSpace on 2015-01-28T13:20:49Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação - João Paulo Cabral de Brito - 2014.pdf: 977293 bytes, checksum: f8d05d4259ea9bbd904419bbd7674cf9 (MD5) Previous issue date: 2014-08-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Trichoderma genus corresponds to free-living filamentous fungi, including species able to act as biological control agents against pathogenic fungi. It is believed that this ability is due to the synergy of several mechanisms, including a wide variety of secondary metabolites produced by these organisms. Among them, there is the production of peptaibols, an antibiotic peptide group characterized by the presence of non-proteinogenic amino acids such as α-aminoisobutyrate (Aib), presence of N-terminal modifications and amino alcohols in the C-terminal region. These peptaibols have amphipathic nature, allowing the formation of ion channels in lipid membranes, which are believed to be related to its antimicrobial activity. This study aimed to draw a profile of production and identify of secreted peptaibols from Trichoderma asperellum TR356 strain. The fungi was grown in TLE with 0.3% glucose medium for 5 days, 120 rpm in the dark. Liquid medium filtrate was used as metabolites source. These extracts were subjected to high performance liquid chromatography (HPLC) and subsequent analysis by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF). The results indicate the production of two classes of peptaibols for this T. asperellum strain. Primary structure of two Asperelines (A and E) and five Trichotoxins (T5D2, T5E, T5F, and T5G 1717A) has been elucidated. Most of these peptaibols had been previously described in T. viride and T. asperellum marine strains. This is the first report of some of these compounds being produced by a T. asperellumstrain from soil. Future analysis will be necessary to elucidate the three-dimensional structures and their activity against pathogens. / O gênero Trichoderma corresponde a fungos filamentosos, de vida livre, que inclui espécies capazes de agir como agentes de controle biológicoeficazes contra fungos fitopatogênicos. Acredita-se que esta capacidade é devida a sinergia de vários mecanismos, incluindo a ampla variedade de metabólitos secundários produzidos por estes organismos. Dentre eles, há a produção de peptídeos antibióticos conhecidos como “peptaibols”, compostos caracterizados pela presença de aminoácidos não proteinogênicos como α-aminoisobutirato (Aib), e por possuírem modificações N-terminais e presença de amino álcoois na região C-terminal. Estes peptaibols apresentam natureza anfipática, permitindo a formação de canais iônicos nas membranas lipídicas, o que se acredita estar relacionado com sua atividade antimicrobiana. Este trabalhoteve como objetivo traçar um perfil de produção e identificar peptaibols secretados pela linhagem de Trichoderma asperellum TR356. O isolado foi crescido em meio TLE contendo 0,3% de glicose, por 5 dias, 120 rpm no escuro. O sobrenadante filtrado foi utilizado como fonte para extração de metabólitos. Estes extratos foram submetidos a cromatografia líquida de alta eficiência (CLAE) e posterior análise pela técnica de espectrometria de massas por ionização e dessorção a laser assistida por matriz (MALDI-TOF). Os resultados indicam a produção de duas classes de peptaibols por esta linhagem de T. asperellum. Foram elucidadas as estruturas primárias de duas Asperelinas (A e E), e de cinco Trichotoxinas (T5D2, T5E, T5F, T5G e 1717A). A maior parte destes peptaibols havia sido descrito anteriormente em linhagens de T. viride e linhagens de T. asperellum marinhas. Este é o primeiro relato de alguns destes compostos sendo produzidos por uma linhagem de T. asperellum proveniente do solo. Futuras análises serão necessárias para a elucidação de suas estruturas tridimensionais e atividade destes compostos contra fitopatógenos.